cyclic adenosine 5-diphosphoribose 
 ( cadpr , 1 , figure 1 ) , discovered 
 by lee et al .
in 1987 , is one of the principal 
 second messenger molecules that mobilize intracellular ca in a different way to the well - established d - myo - inositol 1,4,5-trisphosphate ( ins(1,4,5)p3 ) , by gating the ryanodine receptor . the cadpr / ca signaling system is active in diverse mammalian cellular 
 systems such as cardiac muscle , acinar cells , and plant cells .
cadpr is a metabolite of nicotinamide adenine 
 dinucleotide ( nad ) and is produced enzymatically by adp - ribosyl 
 cyclases ( adprc ) .
its structure was fully characterized by lee et 
 al . as a cyclic 18-membered dinucleotide 
 featuring two glycosidic bonds and a pyrophosphate linkage .
several 
 excellent reviews dealing with the chemistry of cadpr and the cadpr / ca signaling system have appeared in recent years
cadpr is readily hydrolyzable at the n1 glycosidic bond linkage to give adpr in both neutral aqueous solution 
 and under physiological conditions , thus rendering 
 chemical synthesis of analogues challenging .
the main choice has been 
 between total chemical synthesis and 
 a chemoenzymatic approach developed earlier and 
 modeled on the biosynthesis of cadpr from nad . by combination 
 of both approaches ,
a large number of cadpr analogues have been synthesized 
 over the years and their ca release activities examined 
 in several systems , such as sea urchin egg homogenate ( suh ) and t 
 cells inter alia .
modification at the 8-position with 8-amino and 
 8-bromo groups in particular has been shown to convert cadpr from 
 an agonist to an antagonist , although at high concentrations this does not apparently 
 hold .
however , 8-substituted cyclic adenosine 
 5-diphosphocarbocyclic ribose ( 8-x cadpcr ) analogues developed 
 by shuto were reported to be agonists 
 in sea urchin egg homogenates , indicating that 8-substitution alone 
 may not be responsible for antagonist activity .
3-o - methyl cadpr was also found to have antagonist properties , 
 at least in suh .
the hydrolysis - resistant 
 7-deaza 8-bromo cadpr has found several biological applications as a cell permeant competitive antagonist .
analogues with structural modifications 
 of the  northern  ribose showed agonist activities in t cells , including those with more 
 radical changes . recently
, we have also 
 pioneered a synthesis of novel cadpr derivatives that are highly stable 
 both chemically and enzymatically .
the 8-bromo n1-cyclic 
 inosine 5-diphosphoribose ( 8-br - n1-cidpr ) 
 thus obtained proved to be a novel agonist of ca release 
 in intact cells .
the opposed bioactivities of 
 the classical antagonist 8-br - cadpr and 8-br - cidpr thus show that 
 the smallest of structural changes ( i.e. , nh2  
 c = o at c-6 , as with changes at c-8 and c-3 ) can transform 
 a cadpr antagonist into an agonist . furthermore , the enhanced stability 
 of 8-br - cidpr allows direct chemical modification at the 8-position , 
 including to the parent n1-cidpr , ( 2 , figure 1 ) , which is roughly equipotent to 
 cadpr in permeabilized t cells .
this 
 stable analogue was recently cocrystallized with the wild - type ectoenzyme 
 cd38 , a multifunctional enzyme responsible for the formation and metabolism of cadpr , providing insight into substrate binding and the catalytic process .
the chemoenzymatic approach relies on 
 the selectivity of aplysia cyclase that , while promiscuous , 
 may not always recognize the required nad analogue as 
 substrate .
moreover , while nad analogues generally cyclize 
 at n1 , some are hydrolyzed to the corresponding linear 
 adpr analogue .
another exception , ethenonicotinamide adenine 5-dinucleotide , 
 in which the n1 position is substituted , cyclizes 
 through the n7 nitrogen by adprc .
nhd , a close analogue of nad but 
 bearing a 6-keto group on the purine ring rather than an amino group , 
 cyclizes at n7 , giving the fluorescent , biologically 
 inactive n7-cidpr , as indeed does the guanosine congener 
 ngd ( figure 2 ) .
6-thio nhd ( where a 6-mercaptopurine replaces the hypoxanthine ring 
 of nhd ) has yet to be explored as a potential substrate 
 for adp - ribosyl cyclase .
since the only modifications at position 
 6 in the cyclic nucleotide explored to date ( nh2 and c = o ) 
 result in major changes in biological activity , we anticipated that 
 the 6-thio ( c = s ) derivative in particular would be of significant 
 interest .
nad analogues with substituents 
 at c-6 such as sme , nh(ch2)6nh2 or 
 h all fail to cyclize when incubated with adprc .
the simple n - methylated analogue , 6-n - methyl nicotinamide adenosine 5-dinucleotide ( 6-nme - nad ) has , however , not yet been investigated .
n1-cidpr was prepared via both 8-br - nhd and 
 8-br - n1-cidpr to bypass the problem encountered with n7 cyclization .
to the best of our knowledge , even a total synthesis was never attempted for such a class of compound , 
 presumably because of the difficulty of incorporating an intact 
ribose into the molecule . in order to further investigate the requirements 
 for n1 versus n7 cyclization inter 
 alia
, we report here syntheses of both 6-nme - nad and 6-thio 
 nhd and an investigation of their cyclization behavior .
6-nme adenosine 7 was earlier synthesized 
 from 1-n - methyladenosine via a dimroth rearrangement 
 that involves an opening and recyclization of the adenine ring .
we synthesized 7 by a different 
 synthetic route that could provide wider flexibility for other modifications 
 longer term .
6-chloropurine 4 was first activated with 
 tms - triflate , then condensed with tetraacetyl ribose 5 to produce the 6-chloro protected nucleoside 6 in its 
  configuration ( scheme 1 ) .
treatment 
 with methylamine hydrochloride followed by acetate removal with methanolic 
 ammonia produced 6-nme adenosine 7 in 
 good yield .
phosphorylation was achieved by using the established 
 pocl3/tep method but , in contrast to other nucleosides , 
 both the 5-monophosphate 8a and 3,5-bisphosphate 8b were formed as a mixture .
the 
 desired monomorpholidate 9a was then easily isolated 
 by ion - exchange chromatography and condensed with -nmn to form the target 6-nme - nad10 .
reagents and conditions : 
 ( i ) tmsotf , dbu , mecn , 60 c , 1 h ; ( ii ) methylamine hydrochloride , 
 dcm / etoh / et3n , 60 c , overnight ; ( iii ) nh3/meoh , 0 c , 3 h ; ( iv ) pocl3 , tep , h2o , 
 0 c , 3 h ; ( v ) pph3 , dipyridyl disulfide , morpholine , 
 room temp , 2 h ; ( vi ) -nmn , mncl2 in 
 formamide , room temp , 48 h ; ( vii ) aplysia californica , 25 mm hepes ( ph 7.4 ) , room temp . incubation 
 of 6-nme - nad10 with aplysia cyclase generated only the hydrolyzed product 6-nme - adpr 11 , confirmed by h nmr 
 spectroscopy and a molecular ion of 572 in the electrospray mass spectrum . 
 the latter is in accord with the structure of a linear nucleotide 
 rather than the cyclic 6-nme - cadpr , as it differs 
 by 17 mass units for the additional hydroxyl group .
moreover , the 
 presence of two nmr doublets at 5.24 and 5.14 ppm represents a typical 
 anomeric proton pattern for the terminal ribose hydroxyl in the  
 and  configurations .
this result is perhaps not too surprising 
 based upon another report of the hydrolysis of c-6 substituted nad analogues , but these had more radical changes .
it was initially assumed that cyclization of 6-nme - nad is most likely blocked by the steric 
 hindrance brought about by the free rotation of the methyl group . 
 however , an early review reported that 
 the compound with an h at c-6 rather than an amino group is apparently 
 also unable to cyclize , indicating that the cyclization catalyzed 
 by aplysia cyclase might ( not surprisingly ) involve 
 the participation of the c-6 amino group
. it may be possible that 
 the steric hindrance of the methyl group disturbs the electrophilic 
 attack at the n1 position and therefore blocks the 
 cyclization .
it seems likely that the steric bulk of the nme might 
 interfere with the approach to the known covalent e179 enzyme 
to occur requires the formation of the 6-nme - nad  enzyme covalent complex , which allows water to attack 
 the ribosyl c-1 rather than n1
. another explanation 
 could be interference with the actual binding of 6-nme - nad to aplysia cyclase by reducing 
 the h - bonding sites in the active site .
nhd was first reported by atkinson et al . and synthesized by coupling 6-thio imp ( 14 ) with nicotinamide mononucleotide with dicyclohexylcarbodiimide , 
 a method developed by todd et al . the 
 low yield and lack of structural data in the initial report prompted 
 us to investigate a new , reliable and more modern , route to 6-thio nhd .
the key compound is 14 , which was previously 
 synthesized mostly in the 1960s more or less successfully .
we initially reasoned that the synthesis of 14 should 
 be easily accomplished by selective phosphorylation of 6-thioinosine 12 by adaptation of a published method that we have used very successfully on a wide range of 
 nucleosides .
however , treatment of 6-thioinosine with pocl3 in tep was unsuccessful because of the poor solubility of the starting 
 nucleoside in tep ( scheme 2 ) . an isopropylidene 
 protecting group at the 2,3-hydroxyls was then considered 
 to have the double advantage of solving the solubility issue and being 
 removable during the phosphorylation procedure , which is carried out 
 under acidic conditions .
indeed , hplc analysis of the quenched 
 reaction showed a complex mixture of products that could not be isolated 
 by reverse - phase chromatography .
we reasoned that there was a mixture 
 of protected and deprotected material as well as products phosphorylated 
 at the 5-oh , the sulfur atom , or both , since the sulfur is 
 more nucleophilic than the oxygen .
phosphoramidite chemistry 
 has been reported on 2,3-o - isopropylidene-6-thioinosine 13(41 ) using n , n - diisopropyl - di - tert - butylphosphoramidite 
 as a phosphitylating reagent .
both tert - butyl and isopropylidene protecting groups could then 
 be cleaved under acidic conditions .
however , in our hands the phosphorylation 
 step could not be repeated , as the starting material is insoluble 
 in most organic solvents except dmf and dmso , and therefore only the 
 starting material was recovered after 24 h with no sign of product .
reagents and conditions : 
 ( i ) dimethoxypropane , acetone , hclo4 , 20 min , room temp ; 
 ( ii ) pocl3 , tep , h2o , 0 c , 1 h ; ( iii ) 
 dinitrofluorobenzene , et3n , mecn , room temp , 1 h ; ( iv ) 
 diisopropyl - di - tert - butylphosphoramidite , tetrazole , 
 dcm , room temp , 1 h , then mcpba , 78 c , 10 min ; ( v ) 10% 
 mercaptoethanol in mecn + 1% dipea , room temp , 1 h ; ( vi ) 50% aq tfa , 
 room temp , overnight ; ( vii ) morpholine , pph3 , dipyridyl 
 disulfide , dmso , room temp , 1 h ; ( viii ) -nmn , 0.2 
 m mncl2 in formamide , room temp , overnight .
thio - substituted nucleotides are particularly useful in 
 molecular biology , as they have been incorporated into oligonucleotides 
 by chemical methods and used for postsynthetic modification . during the former process , the sulfur is always protected to avoid 
 unnecessary side reactions during phosphitylation and oxidation of 
 the phosphite . since the synthesis of 
 6-thio imp
is hampered by the lack of solubility of 6-thioinosine , 
 protecting the sulfur should further solve the solubility issue encountered . 

various protecting groups have been used such as cyanoethyl , which 
 can be removed by treatment with dbu .
although the cyanoethyl group 
 has been used extensively , we decided to utilize the 2,4-dinitrophenyl 
 ( dnp ) group , as previous studies have shown that it can easily be 
 removed with mercaptoethanol under very mild alkaline conditions .
2,3-o - isopropylidene-6-thioinosine 13 was prepared in a very high yield ( 86% over five steps ) 
 using published methods starting from inosine 12 .
protection of the sulfur was achieved by treatment of 2,3-o - isopropylidene-6-thioinosine with triethylamine and 2,4-dinitrofluorobenzene . 
 the yellow product 15 obtained after flash chromatography 

could then be selectively phosphitylated at the 5-hydroxyl 
 using the tert - butyl protected phosphitylating reagent 
 in high yield to give 16 .
removal of the dnp protecting 
 group was accomplished by treatment with mercaptoethanol under mild 
 alkaline conditions .
careful purification by flash chromatography 
 led to a very clean product in 75% yield .
next , both the isopropylidene 
 and tert - butyl protecting groups were removed very 
 cleanly by treatment with 50% aqueous tfa to generate the desired 
 6-thio imp 14 in 86% yield , without further purification . 

this clearly is the advantage of the dnp group , as an ion - exchange 
 purification step is avoided during which , from our experience , some 
 product often gets unavoidably lost .
14 was thus obtained 
 in a satisfying 48% yield after a four - step procedure from 2,3-o - isopropylidene-6-thioinosine . with 6-thio imp in 
 hand
the synthesis of the pyrophosphate was achieved 
 using a procedure initially reported by moffatt and later improved by lee et al .
that relies on the coupling of a sugar phosphate with a nucleotide 
 phosphoromorpholidate in the presence of a lewis acid .
we have previously 
 used this method to successfully generate various nad and 
 nhd analogues in relatively high yield .
6-thio imp was first activated using a combination of morpholine / dipyridyl 
 disulfide and triphenylphosphine to yield the morpholidate that was 
 then condensed with -nmn with mncl2 as 
 lewis acid .
a p nmr shift of around 10 ppm clearly 
 indicated the formation of a pyrophosphate linkage .
purification on 
 reverse - phase chromatography afforded the desired 6-thio nhd17 as the sole product .
there are several possible outcomes for this reaction ( figure 3 ) , i.e. , formation of 6-thio n1-cidpr , 
 6-thio n7-cidpr as for the oxo congener , 6-thio idpr 
 ( 6-thioinosine 5-diphosphate ribose ) , and it is possible to 
 envisage cyclization on to the sulfur to give the 6-thio - s - cidpr . 

the new product displayed a molecular ion of 557 in the es mass spectrum , which ruled out the formation of both the hydrolyzed 
 product 6-thio idpr and surprisingly also the n7 
 cyclized product 6-thio n7-cidpr , for which a mass 
 of 575 and 558 , respectively , would be expected.h nmr 
 spectroscopy showed a chemical shift of 5.2 ppm for h-2 , a 
 typical value for a nucleotide in the syn conformation , 
 which also ruled out the formation of 6-thio n7-cidpr . 
 additionally , a c-6 c chemical shift of 175 ppm is typical 
 of a thione form ( thiol , 155 ppm ) , therefore ruling out the 
 putative sulfur - cyclized product 6-thio s - cidpr as 
 a possibility .
crucially , a ghmbc spectrum showed cross - peaks between 
 the h-2 proton of the purine ring (  9.32 ppm ) and the anomeric 
 carbon c-1 of the  northern  ribose (  
 94.9 ppm ) and between the anomeric proton h-1 (  6.65 
 ppm ) with the carbon c-2 (  144.9 ppm ) and c-6 (  175.5 
 ppm ) of the nucleobase ( figure 3 ) . finally , 
 the p 
p coupling constant for the 
 pyrophosphate linkage in 3 showed j = 
 11.8 hz , similar to that of cadpr and cidpr ( 13.5   and 12.5   hz , respectively ) , whereas the p 
p coupling of the linear idpr generally has a much higher frequency 
 ( 20 hz ) .
these analytical data thus provide evidence of a 
 successful and surprising cyclization of 6-thio nhd into 
 the corresponding 6-thio n1 cidpr 3 ( figure 4 ) .
h / c correlations based on ghmbc 
 spectrum supporting the formation of the n1c1 bond . the photochemical properties of 6-thio n1-cidpr 3 were also examined . while adenine 
 and guanosine based nucleotides have an absorbance maximum of around 
 260 nm , replacement of the oxygen by a sulfur atom shifts the uv absorbance 
 spectrum to 320340 nm .
the uv spectrum in water exhibited intense absorption at 320 nm ( max ) with an extinction coefficient (  ) of 18  600 
 mol dm cm ( data 
 not shown ) .
more interestingly , 
 6-thio cidpr displays fluorescent properties in water at room temperature . 
 when it is excited at 335 nm ( just above the maximum uv absorption ) , 
 an emission spectrum with a single peak at 415 nm was observed ( see supporting information ) . in contrast ,
no fluorescence 
 was observed by the related n1-cidpr ( data not shown ) . 

thus far , only n7-cyclized dinucleotides such as n7-cidpr , n7-cgdpr , and etheno cyclic adp 
 diphosphoribose have been demonstrated to be fluorescent , and this 
 property was used to rule out the formation of n7-cyclic 
 product when no fluorescence was observed . to the best of our knowledge , 6-thio n1-cidpr is 
 therefore the first fluorescent n1-cyclized cadpr 
 analogue and this property may find applications in cadpr binding 
 protein biochemistry .
there have been reports that 
 the ca - release activity and antagonism of cadpr analogues may be linked to their conformation in solution . 

the major puckering mode of cadpr is c2 endo in the n9 ribose moiety with a syn conformation 
 about the n9 glycosidic bond .
the furanose ring is in equilibrium between c2 endo / c3 
 endo forms , and the ratio can be calculated from h nmr 
 data following the equation [ c2 endo ] = [ j1,2/(j1,2 + j3,4 ) ]  100 .
also , the h-2 chemical shift can be 
 used as an indicator of glycosidic bond conformation .
therefore , from our nmr data , 6-thio cidpr exhibits a 74% c2 
 endo puckering with a syn conformation about the 
 glycosidic bond ( table 1 ) , which is consistent 
 with that observed by both cadpr and n1-cidpr .
the cyclization 
 of nad analogues usually takes place at the n1 position of the adenine ring .
however , nad analogues , 
 which have their n1 position blocked ( e.g. , etheno 
 nad ) , cyclize at n7 . if n1 is free but is electronically deactivated 
 ( e.g. , in 2-fluoro nad ) and 
 the purine is still adenine , then this seems to be correctly aligned 
 in the active site but the substrate is hydrolyzed rather than cyclized 
 at n7 .
the enzyme can also cyclize nhd , ngd , and nxd to their respective n7 cyclized dinucleotide n7-cidpr , n7-cgdpr , and n7-cxdpr . in those cases , the most nucleophilic n7 nitrogen in guanine / hypoxanthine
is often invoked to rationalize 
 cyclization at this position . however , 
 nucleophilicity is not the only factor to consider .
we previously 
 made correlations showing that the conformation about the glycosidic 
 bond of nhd analogues appears to play a role in their 
 cyclization .
we argued that a syn conformation about the glycosidic bond is key for a 
 successful cyclization at n1 , i.e. , that the enzyme 
 seems to utilize the linear precursor in its prearranged conformation 
 ( at least in the case of the hypoxanthine series and presumably the 
 guanosine series as well ) .
indeed , nhd ( anti ) cyclizes at n7 ( product also in anti conformation ) , 8-x - nhd ( syn ) cyclizes 
 at n1 ( products in syn conformation ) , 
 and 7-deaza nhd ( anti and no possibility 
 to cyclize at n7 ) hydrolyzes to the linear 7-deaza 
 idpr . however , the nad analogues 
 cyclize at n1 regardless of their conformation ; nad ( anti ) , 8-x nad ( syn ) , and 7-deaza nad ( anti ) cyclize at n1 , all forming products in their syn conformation . 

when 6-thio nhd is used as a substrate , the enzyme converts 
 it into 6-thio n1-cidpr , therefore suggesting that 
 the enzyme utilizes this substrate in the same way as nad but clearly differently from nhd , although nhd and 6-thio nhd are structurally very similar .
have intensively investigated the structures of both aplysia adp - ribosyl cyclase and human cd38 to unravel the 
 catalytic mechanism of the nad cyclization and cadpr hydrolysis 
 reactions .
recently , they suggested that the cyclization reaction 
 of nad analogues occurs through a four - step sequence where 
 both residues tyr-81 and phe-174 play an instrumental role by stabilizing 
 the nucleobase through -stacking interactions in a folded conformation 
 so that cyclization can occur .
mutagenesis confirmed 
 that phe-174 was likely to be responsible for folding the linear substrate 
 in the correct conformation .
however , this is only true for adenine - based 
 substrates ; mutagenesis did not affect the cyclization of ngd to n7-cgdpr .
therefore , it is likely that 
 two residues are responsible for the different base cyclization sites 
 ( n1 versus n7 ) and that residue 
 phe-174 may also be involved in the cyclization of 6-thio nhd to 6-thio n1-cidpr . with the help 
 of molecular modeling , we investigated further how nad cyclization to cadpr may occur .
the ligand in the ribo-2-f - nadadprc structure ( pdb code 3i9o ) was manually 
 manipulated by rotating individual bonds to approximate the position 
 the adenine would have to be in to attack c-1 and form the n1-cyclized product ( figure 5 ) . in 
 order for nad to cyclize at n1
, the adenine 
 must rotate around the n9/c-1 bond to adopt 
 the syn configuration .
it can then approach the c-1 
 by stacking with trp-140 and having the 6-amino group forming at least 
 one hydrogen bond to glu-98 to further orient and hold the adenine 
 in position .
this proposed mechanism has some support from the kinetic 
 data for glu-98 mutants , as indeed mutation of this residue was shown 
 to reduce cyclase activity .
nhd ( or ngd ) would be unable to form the hydrogen bonds 
 to glu-98 , and the lack of stabilization may partly explain the lack 
 of cyclization at n-1 .
two views of a model 
 of the position of the adenine of the intermediate from ribo-2-f - nadadprc complex immediately prior ring closure .
the model was built by rotating 
 individual bonds in the pdb 3i9o structure ( ribo-2-f - nadadprc ) 
 using the schrdinger software running under maestro , version 9.0.111 .
the color scheme for the ligand is as follows : pink , carbon ; 
 blue , nitrogen ; red , oxygen ; white , hydrogen ; light blue , fluorine ; 
 orange , phosphorus .
the color scheme for the residues is the same 
 as for the ligand except the carbons are green .
it is intuitively clear that in 
 order for 6-thio nhd to cyclize , the enzyme should be 
 capable of stabilizing it in its thiol form , although the mercaptopurine 
 base normally exists predominantly in its thione form in solution . in the case where the protonation state plays 
 a role in the reaction mechanism , the pka values of n1 , n7 , and x1 ( nh2 , oh , sh ) and x2 ( = o,=nh ,
= s ) were calculated 
 computationally ( figure 6 ; see also supporting information ) .
the calculations were 
 done on the nucleoside ( adenosine , inosine , and 6-thioinosine ) and 
 each respective tautomer ; we presume that it is reasonable to propose 
 that these values would follow the same trend for the corresponding 
 nad / nhd analogue .
three of the possible ionization states , the 
 program was unable to calculate a pka , 
 presumably because they are too extreme . at physiological ph ,
most 
 of these ionizable groups are going to be either fully protonated 
 or deprotonated , the exception being for the oxygen - containing compound 
 ( see supporting information ) . in the a 
 tautomer
the x1 ionization ( oh  o ) has a pka of 6.56 , and in the b tautomer 
 the n1 ionization ( nh  n ) has a pka of 8.67 .
these two values are within the range 
 of physiological ph , which means that at this ph there will be a mixture 
 of four tautomeric forms for the oxygen - containing compound ( figure 7 ) while there will be only two forms of the nitrogen- 
 and sulfur - containing compound . presumed different forms of the oxygen 
 containing compound that may be present at physiological ph .
previously , we argued that the deciding interaction 
 through the adenine ring was via hydrogen bond donation from the base , 
 since the hypoxanthine base has only hydrogen acceptor character in 
 its keto form .
however , the 6-thiopurine base has virtually no hydrogen 
 bond acceptor or donor character , which may imply that hydrogen bonding 
 is unimportant at c-6 and that cyclization is influenced by other 
 factors such as the size of the substituent ( the sulfur atom is larger 
 than the nitrogen and oxygen atoms ; see supporting 
 information ) , the conformation about the glycosidic bond ( for 
 the oxygen containing nucleotides ) , electronic properties , or a combination 
 of these .
it may be that an h - bonding interaction with the enzyme 
 locks the purine of nhd in the anti form , 
 leading to n7 cyclization
. however , this interaction 
 could be very weak or nonexistent with the 6-thiopurine , allowing 
 it to rotate to facilitate n1 cyclization . in the 
 case of 8-br nhd the strong orienting effect of the bromo 
 group may be enough to overcome such h - bonding .
protein crystallography 
 with such a ligand may reveal those interactions as important for 
 catalysis to occur .
the ca release activity of 
 the newly synthesized 6-thio cidpr 3 was evaluated fluorimetrically 
 in permeabilized jurkat t cells , and the results are shown in figure 8 . because of the intrinsic fluorescence of 6-thio 
 cidpr , fluo3 was used as a ca indicator to avoid any 
 possible interference . at low concentration , 6-thio cidpr 3
ca release was , however , observed at significantly higher concentration 
 ( 100 m and above , figure 8a and figure 8b ) . in comparison with n1-cidpr 2 ,
weaker agonist activity was observed for 6-thio cidpr 3 ; however , in the current series of experiments the permeabilized 
 cell preparations responded also somewhat more weakly to the naturally 
 occurring cadpr 1 . regarding antagonism by 6-thio cidpr 3 a concentration - dependent effect on cadpr and
n1-cidpr 2 induced ca release was observed 
 ( figure 8c ) . while the ic50 for 
 cadpr induced ca release was between 30 and 100 m , 
 a somewhat better inhibition of ca release induced by n1-cidpr 2
we thus demonstrate here that a simple modification from 
 a c = o to c = s bond at c-6 weakens agonist activity but 
 enhances antagonist activity in jurkat t cells .
antagonist activity 
 was specifically strong when n1-cidpr 2 was used to trigger ca release , indicating that the 
 oxygen group in cidpr can be replaced at lower 6-thio cidpr 3 concentrations compared to the situation where ca release was induced by cadpr ( figure 8c ) . 
 the most likely explanation for this differential effect of 6-thio 
 cidpr 3 is differences in protonation pattern and hydrogen 
 bonding interactions .
the amino group in cadpr can be a hydrogen bond 
 donor or acceptor , whereas the oxygen group in cidpr is a good hydrogen 
 bond acceptor .
obviously , 6-thio cidpr 3 competes more 
 effectively with cidpr for binding to the cadpr receptor .
taken 
 together , these results show that replacement of the keto moiety by 
 a more hydrophobic thione does interfere with the functional consequences 
 of binding of the ligand to its receptor .
there is an obvious role 
 in the c-6 group in antagonizing ca release , if not directly 
 then through subtle conformational effects or other .
it may well be 
 that a change from a hypoxanthine to a mercaptopurine results in a 
 different protonation pattern and hydrogen bonding interactions .
the 
 amino group in cadpr can be a hydrogen bond donor or acceptor , whereas 
 the oxygen group in cidpr is a good hydrogen bond acceptor , as opposed 
 to the sulfur atom .
a c2-endo / syn conformation 
 is obviously not the sole factor that governs agonist / antagonist activity , 
 since all three nucleotides cadpr , cidpr , and 6-thio cidpr have the 
 same global conformation , although one can suggest that a c3-endo / anti conformation ( as in n7-cidpr and n7-cgdpr , table 1 ) would most likely 
 lead to inactive compounds .
it is also well - known that the hydrophobicity 
 of sulfur leads to differences in the associated water shell .
thus , 
 it may be that differential arrangements of additional water molecules 
 in the receptor binding site can influence the receptor machinery .
although surprising , this is not the first time that ca release has been observed at higher concentrations .
indeed , we previously 
 reported that the classical and widely used antagonists such as 8-bromo 
 cadpr and 8-amino cadpr also show such unexpected agonist activity 
 at high concentration .
one of the reasons 
 invoked to explain this earlier unobserved effect is that there may 
 be two previously unrecognized different sites of action , such as 
 an additional lower affinity nucleotide binding site , which causes 
 ca mobilization distinct from the high affinity cadpr 
 site .
this would not have been noticed with the parent cidpr , since 
 it only acts as a potent agonist , but is again revealed with the structurally 
 related 6-thio cidpr .
6-thio - cidpr - induced ca release and effect 
 of 6-thio - cidpr on cadpr and cidpr - induced ca release 
 in permeabilized jurkat t cells .
jurkat t cells were permeabilized , 
 and [ ca ] was measured in the presence of fluo-3 , atp , 
 and an atp regenerating system as detailed in the experimental section .
( b ) concentration  response curve of 
 6-thio - cidpr induced ca release .
results represent ca increase over baseline (  values ) expressed as mean 
  sd ( n = 47 ) of single tracings .
( c ) 
 the inhibitory effect of 6-thio - cidpr was estimated by previous addition 
 of 6-sh - cidpr and subsequent addition of either cidpr or cadpr .
since 
 6-thio - cidpr also elicited a weak agonist effect on its own ( a , b ) , 
 calculation of its antagonist effect on cadpr ( 30 m ) or cidpr 
 ( 30 m ) was carried out by subtracting the corresponding agonist 
 data ( data in part b ) .
response curves represent 
 the mean  sd ( n = 3 ) of single tracings .
a 
 one - phase exponential decay was used to fit curves ; r was 
 0.6486 for cadpr and 0.7107 for cidpr .
the c-6 position in cadpr may therefore be revealed 
 as an attractive new site to imbue antagonistic properties in a cadpr 
 analogue .
its proximity to the crucial n1c1 linkage 
 makes it likely that structural modifications could lead to modulation 
 of activity of cadpr binding proteins . as such
, it will be of interest 
 to develop new ways to approach the synthetic problem of c-6 modification . 

although the present result has been achieved somewhat unexpectedly 
 via a chemoenzymatic route , it seems most likely that greater flexibility 
 in this regard will be offered by total synthetic approaches in the 
 future .
the ca release activity of 
 the newly synthesized 6-thio cidpr 3 was evaluated fluorimetrically 
 in permeabilized jurkat t cells , and the results are shown in figure 8 . because of the intrinsic fluorescence of 6-thio 
 cidpr , fluo3 was used as a ca indicator to avoid any 
 possible interference . at low concentration , 6-thio cidpr 3
ca release was , however , observed at significantly higher concentration 
 ( 100 m and above , figure 8a and figure 8b ) . in comparison with n1-cidpr 2 ,
weaker agonist activity was observed for 6-thio cidpr 3 ; however , in the current series of experiments the permeabilized 
 cell preparations responded also somewhat more weakly to the naturally 
 occurring cadpr 1 . regarding antagonism by 6-thio cidpr 3 a concentration - dependent effect on cadpr and
n1-cidpr 2 induced ca release was observed 
 ( figure 8c ) . while the ic50 for 
 cadpr induced ca release was between 30 and 100 m , 
 a somewhat better inhibition of ca release induced by n1-cidpr 2
we thus demonstrate here that a simple modification from 
 a c = o to c = s bond at c-6 weakens agonist activity but 
 enhances antagonist activity in jurkat t cells .
antagonist activity 
 was specifically strong when n1-cidpr 2 was used to trigger ca release , indicating that the 
 oxygen group in cidpr can be replaced at lower 6-thio cidpr 3 concentrations compared to the situation where ca release was induced by cadpr ( figure 8c ) . 
 the most likely explanation for this differential effect of 6-thio 
 cidpr 3 is differences in protonation pattern and hydrogen 
 bonding interactions .
the amino group in cadpr can be a hydrogen bond 
 donor or acceptor , whereas the oxygen group in cidpr is a good hydrogen 
 bond acceptor .
obviously , 6-thio cidpr 3 competes more 
 effectively with cidpr for binding to the cadpr receptor .
taken 
 together , these results show that replacement of the keto moiety by 
 a more hydrophobic thione does interfere with the functional consequences 
 of binding of the ligand to its receptor .
there is an obvious role 
 in the c-6 group in antagonizing ca release , if not directly 
 then through subtle conformational effects or other .
it may well be 
 that a change from a hypoxanthine to a mercaptopurine results in a 
 different protonation pattern and hydrogen bonding interactions .
the 
 amino group in cadpr can be a hydrogen bond donor or acceptor , whereas 
 the oxygen group in cidpr is a good hydrogen bond acceptor , as opposed 
 to the sulfur atom .
a c2-endo / syn conformation 
 is obviously not the sole factor that governs agonist / antagonist activity , 
 since all three nucleotides cadpr , cidpr , and 6-thio cidpr have the 
 same global conformation , although one can suggest that a c3-endo / anti conformation ( as in n7-cidpr and n7-cgdpr , table 1 ) would most likely 
 lead to inactive compounds .
it is also well - known that the hydrophobicity 
 of sulfur leads to differences in the associated water shell .
thus , 
 it may be that differential arrangements of additional water molecules 
 in the receptor binding site can influence the receptor machinery . although surprising , this is not the first time that ca release has been observed at higher concentrations . indeed , we previously 
 reported that the classical and widely used antagonists such as 8-bromo 
 cadpr and 8-amino cadpr also show such unexpected agonist activity 
 at high concentration .
one of the reasons 
 invoked to explain this earlier unobserved effect is that there may 
 be two previously unrecognized different sites of action , such as 
 an additional lower affinity nucleotide binding site , which causes 
 ca mobilization distinct from the high affinity cadpr 
 site .
this would not have been noticed with the parent cidpr , since 
 it only acts as a potent agonist , but is again revealed with the structurally 
 related 6-thio cidpr .
6-thio - cidpr - induced ca release and effect 
 of 6-thio - cidpr on cadpr and cidpr - induced ca release 
 in permeabilized jurkat t cells .
jurkat t cells were permeabilized , 
 and [ ca ] was measured in the presence of fluo-3 , atp , 
 and an atp regenerating system as detailed in the experimental section .
( b ) concentration  response curve of 
 6-thio - cidpr induced ca release .
results represent ca increase over baseline (  values ) expressed as mean 
  sd ( n = 47 ) of single tracings .
( c ) 
 the inhibitory effect of 6-thio - cidpr was estimated by previous addition 
 of 6-sh - cidpr and subsequent addition of either cidpr or cadpr .
since 
 6-thio - cidpr also elicited a weak agonist effect on its own ( a , b ) , 
 calculation of its antagonist effect on cadpr ( 30 m ) or cidpr 
 ( 30 m ) was carried out by subtracting the corresponding agonist 
 data ( data in part b ) .
response curves represent 
 the mean  sd ( n = 3 ) of single tracings .
a 
 one - phase exponential decay was used to fit curves ; r was 
 0.6486 for cadpr and 0.7107 for cidpr .
the c-6 position in cadpr may therefore be revealed 
 as an attractive new site to imbue antagonistic properties in a cadpr 
 analogue .
its proximity to the crucial n1c1 linkage 
 makes it likely that structural modifications could lead to modulation 
 of activity of cadpr binding proteins . as such
, it will be of interest 
 to develop new ways to approach the synthetic problem of c-6 modification . 

although the present result has been achieved somewhat unexpectedly 
 via a chemoenzymatic route , it seems most likely that greater flexibility 
 in this regard will be offered by total synthetic approaches in the 
 future .
in summary , we present a synthetic focus 
 upon enzymatic c-6 structural modifications with a view to their enzymatic 
 incorporation into cadpr .
the substituted n6-nad analogue 6-nme - nad , upon incubation 
 with aplysia cyclase , hydrolyzes to the linear product 
 6-nme - adpr , presumably because of steric hindrance 
 and/or reduction of hydrogen bonding interaction with the enzyme . 

surprisingly , this analogue cyclizes at n1 in a similar manner to nad , to give the novel 
 fluorescent cadpr analogue 6-thio n1-cidpr , although 
 the parent oxo - congener nhd cyclizes at n7 .
a mechanistic study implies a more complex explanation than simply 
 a hydrogen bonding interaction , as the enzyme recognizes this thio 
 derivative as a normal substrate , although it is structurally closer 
 to nhd than it is to nad .
biological evaluation 
 in permeabilized jurkat t cells reveals that this compound is decreased 
 in its agonist activity relative to cidpr but possesses new antagonist 
 activity against both cadpr- and cidpr - induced ca release , 
 showing that substitution of the oxygen of the hypoxanthine ring for 
 sulfur interferes with the functional consequences of ligand binding 
 to its receptor . like two
antagonist activity 
 in cadpr analogues has thus now been demonstrated in compounds substituted 
 at the 8-position of the purine , the 3-hydroxyl group of the 
  southern  ribose , and now for the first time in a compound 
 with a c-6 structural modification .
both the fundamental causes of 
 antagonism and the switch to agonism at higher concentration in some 
 cases thus seem highly complex and seem to defy a simple explanation . 

the present study demonstrates , however , that future synthetic efforts 
 to facilitate further c-6-structural modification should be a fruitful 
 enterprise in elucidating cadpr sar and defining useful new tools 
 for dissection of its signaling pathway .
all reagents and solvents were of commercial 
 quality and were used without further purification unless described 
 otherwise .
triethylamine and morpholine were dried over potassium 
 hydroxide , distilled , and then stored over potassium hydroxide pellets . 

all h , c , and p nmr spectra of final compounds were collected in d2o on a jeol delta machine at 270 mhz ( h ) or 109 
 mhz ( p ) or on a varian mercury - vx system at 400 mhz ( h ) or 100 mhz ( c ) .
all h and c nmr assignments are based on gcosy , ghmbc , ghmqc , and dept 
 experiments .
abbreviations for splitting patterns are as follows : 
 br , broad ; s , singlet ; d , doublet ; t , triplet ; m , multiplet .
uv spectra 
 were collected in aqueous solution on a perkin - elmer lambda ez 201 
 or lambda 3b spectrophotometer .
high resolution time - of - flight mass 
 spectra were obtained on a bruker daltonics microtof mass spectrometer 
 using electrospray ionization ( esi ) .
hplc analyses were carried out 
 on a waters 2695 alliance module equipped with a waters 2996 photodiode 
 array detector ( 210350 nm ) .
the chromatographic system consisted 
 of a hichrom guard column for hplc and a phenomenex synergi 4 m 
 max - rp 80a column ( 150  4.60 mm ) , with elution at 1 ml / min with 
 the following ion - pair buffer : 0.17% ( m / v ) cetrimide and 45% ( v / v ) 
 phosphate buffer ( ph 6.4 ) in meoh .
the purities of all phosphate containing 
 compounds were determined by hplc , and they were in all cases over 
 95% .
all other compounds were analyzed using a phenomenex gemini column 
 5 m c18 ( 150 mm  4.6 mm ) , with elution at 1 ml / min with 
 a mecn / h2o gradient ( 565% over 20 min ) .
preparative 
 chromatography was performed on a pharmacia biotech gradifrac system 
 equipped with a peristaltic p-1 pump and a fixed wavelength uv-1 optical 
 unit ( 280 nm ) .
the following purification methods were employed : lichroprep 
 rp-18 equilibrated with 0.05 m teab buffer ( ph 6.06.4 ) , gradient 
 of 0.05 m teab buffer against mecn at 5 ml / min , q - sepharose washed 
 with h2o , gradient of 1 m teab buffer ( ph 7.17.6 ) 
 against h2o at 5 ml / min , and ag mp-1 washed with h2o , gradient 150 mm tfa against h2o at 3 ml / min . 

cadpr , n1-cidpr , 
 and 6-thio n1-cidpr were built using sybyl - x 1.1.1 , 
 and the volumes were calculated .
the pka values were calculated using the sparc online calculator http://sparc.chem.uga.edu / sparc/. fluo-3 was purchased from molecular 
 probes .
atp , creatine phosphate , egta , tris , and nacl were provided 
 from sigma aldrich .
jurkat t - lymphocytes ( subclone jmp ) were 
 cultured as described previously at 37 
 c in the presence of 5% co2 in rpmi 1640 medium containing 
 glutamax i and hepes ( 25 mm ) and supplemented with 7.5% ( v / v ) ncs 
 ( newborn calf serum ) , 100 units / ml penicillin , and 100 g / ml 
 streptomycin .
permeabilized cells were prepared as described , and the ca concentration was measured by the 
 use of fluo-3 . in brief
, cells were permeabilized in the presence 
 of saponin ( 55 g / ml ) for 20 min in an intracellular buffer 
 ( 20 mm hepes , 110 mm kci , 2 mm mgci2 , 5 mm kh2po4 , 10 mm naci , ph 7.2 ) at 37 c .
an aliquot containing 
 1  10 cells was transferred to a cuvette , and fluorescence 
 was measured in a hitachi f-2000 spectrofluorometer ( excitation 504 
 nm , emission 524 nm ) at 37 c in the presence of fluo-3 ( 1 m ) 
 with continuous stirring .
reuptake of ca into stores 
 was achieved by addition of atp ( 1 mm ) , creatine phosphate ( 20 mm ) , 
 and creatine kinase ( 20 u / ml ) . at the end of each experiment ,
the 
 free ca concentration was calibrated by addition of cacl2 and subsequently by addition of egta / tris and calculated 
 by using the following equation : [ ca ] = kd(f  fmin)/(fmax  f ) 
 where fmin is the fluorescence intensity 
 in the absence of ca , fmax is the fluorescence intensity of the ca - saturated indicator , f is the fluorescence during the measurement , and kd is the dissociation constant of fluo-3 .
kd of fluo-3 ( 503 nm ) was determined on the basis 
 of the calcium calibration buffer kit ( molecular probes ) for intracellular 
 buffer containing 1 mm atp , 20 mm creatine phosphate , and 20 u / ml 
 creatine kinase at 37 c , ph 7.06 .
vigorously stirred 
 solution of 6-chloropurine ( 500 mg , 3.23 mmol ) , -d - ribofuranose 1,2,3,5-tetraacetate ( 926 mg , 2.91 mmol ) , and dbu ( 1.3 
 ml , 8.70 mmol ) in dry mecn ( 22 ml ) was added tmsotf ( 2.12 ml ) at 0 
 c under an argon atmosphere .
the reaction mixture was heated 
 at 60 c for 1 h and carefully quenched by addition of a saturated 
 solution of nahco3 ( 100 ml ) .
the crude compound was extracted 
 with dcm ( 3  100 ml ) , and the organic layers were combined , 
 dried over mgso4 , and evaporated under reduced pressure . 

the yellow residue obtained was further purified by flash column chromatography , 
 eluting with dcm  meoh , 20:1 , to give the desired compound as 
 a yellow oil ( 960 mg , 77% ) .
h ( cdcl3 , 270 mhz ) 
  8.76 ( s , 1h , h-8 ) , 8.28 ( s , 1h , h-2 ) , 6.21 ( d , 1h , j1,2 = 5.2 hz , h-1 ) , 
 5.92 ( dd , 1h , j2,3 = 5.4 
 hz and j2,1 = 5.2 hz , 
 h-2 ) , 5.62 ( dd , 1h , j3,2 = 5.4 hz and j3,4 = 
 4.9 hz , h-3 ) , 4.37 ( m , 3h , h-4 and h-5 ) , 2.14 
 ( s , 3h , ch3 ) , 2.10 ( s , 3h , ch3 ) , and 2.07 ( s , 
 3h , ch3 ) .
the title compound was synthesized by adaptation of a literature 
 protocol . to a suspension of 6-chloroadenosine 
 triacetate 6 ( 800 mg , 1.94 mmol ) and methylamine hydrochloride 
 ( 573 mg , 8.42 mmol ) in a mixture of dcm ( 20 ml ) and ethanol ( 4.2 ml ) 
 was added triethylamine ( 3.8 ml ) .
tlc analysis indicated that the starting 
 material was completely consumed and three more polar spots were given . 

the reaction mixture was transferred into a pressure tube with triethylamine 
 ( 4 ml ) , methylamine hydrochloride ( 500 mg , 7.35 mmol ) , and ethanol 
 ( 4 ml ) .
the mixture was heated at 60 c for 6 h and left at room 
 temperature for 2 days .
tlc of the mixture indicated only one major 
 spot at rf = 0.25 ( dcm  meoh , 10:1 ) . 

the solvent was removed and the crude compound was purified by flash 
 column chromatography , eluting with dcm  meoh , 30:1 , to give 
 a mixture of the desired compound and the methylamine hydrochloride . 

pure title compound was obtained as a white solid by washing the resulting 
 mixture with milli - q water ( 420 mg , 67% ) .
h ( dmso - d6 , 270 mhz )  8.35 ( s , 1h , h-8 ) , 8.24 
 ( brs , 1h , h-2 ) , 7.81 ( brs , 1h , nh ) , 5.91 ( d , 1h , j1,2 = 5.0 hz , h-1 ) , 5.61 ( m , 1h , 
 2-oh ) , 5.42 ( m , 1h , 3-oh ) , 4.67 ( m , 1h , h-2 ) , 
 4.314.18 ( m , 4h , h-3 , h-4 , h-5 ) , 2.95 
 ( brs , 3h , ch3n ) , 1.95 ( s , 3h , ch3 ) ; hrms ( es ) calcd for c13h18n5o5 324.1308 ( mh ) , found 324.1302 .
a suspension of 6-n - methyladenosine 5-acetate 
 ( 400 mg , 1.23 mmol ) in a saturated methanolic ammonia ( 50 ml ) was 
 stirred at room temperature for 3 h , after which the solvent was removed 
 in vacuo .
the residue obtained was purified by flash column chromatography , 
 eluting with dcm  methanol , 10:1 , to produce the title compound 
 as a white solid ( 300 mg , 86% ) ; mp 178180 c ; h ( dmso - d6 , 270 mhz , d2o shake ) 
  8.31 ( s , 1h , h-8 ) , 8.20 ( brs , 1h , h-2 ) , 5.85 ( d , 1h , j1,2 = 6.4 hz , h-1 ) , 
 4.57 ( dd , 1h , j2,1 = 6.4 
 hz and j2,3 = 5.2 hz , h-2 ) , 
 4.12 ( dd , 1h , j3,2 = 5.2 
 hz and j3,4 = 3.0 hz , 
 h-3 ) , 3.97 ( app.q , 1h , j4,5a = j4,5b = j4,3 = 3.0 hz , h-4 ) , 3.64 ( dd , j5a,5b = 12.9 hz and j5a,4 = 3.0 hz , 1h , h-5a ) , 
 3.55 ( dd , 1h , j5b,5a = 
 12.9 hz and j5b,4 = 3.0 
 hz , h-5b ) , and 2.94 ( brs , 3h , ch3n ) ; hrms ( es ) calcd for [ m + h ] c11h16n5o4 282.1202 ( mh ) , found 282.1201 . a suspension 
 of 7 ( 110 mg , 0.39 mmol , dried in vacuo at 100 c 
 for 2 h ) in triethyl phosphate ( 1.4 ml ) , was heated strongly with 
 a heat gun for 5 min . to the resulting clear solution
were added pocl3 ( 0.3 ml , 3.20 mmol ) and h2o ( 1 l ) at 0 
 c , and the mixture was stirred for 1 h. the reaction mixture 
 was quenched by addition of ice ( 15 ml ) .
the resulting solution was 
 extracted with cold ethyl acetate ( 5  20 ml ) .
the aqueous layer 
 was neutralized with naoh ( 5 m ) and loaded onto a reverse phase column , 
 eluting with a gradient of 030% mecn against 0.05 m teab . 

fractions containing the desired compound were pooled , evaporated 
 and excess teab was coevaporated with meoh ( 3 ) to give a mixture 
 of title compound 8a ( 150 mg , 0.20 mmol , 51% ) and the 
 bis - phosphate 8b .
h ( d2o , 270 
 mhz )  8.17 ( s , 1h , h-8 ) , 7.71 ( s , 1h , h-2 ) , 5.83 ( d , 1h , j1,2 = 5.7 hz , h-1 ) , 
 4.57 ( m , 1h , h-2 ) , 4.37 ( m , 1h , h-3 ) , 4.25 ( m , 1h , 
 h-4 ) , 4.03 ( m , 2h , h-5 ) and 2.78 ( m , 3h , ch3n ) ; p ( d2o , 109 mhz )  1.12 ( s ) . bisphosphate 8b : h ( d2o , 270 mhz )  8.13 ( s , 
 1h , h-8 ) , 7.81 ( s , 1h , h-2 ) , 6.06 ( d , 1h , j1,2 = 4.5 hz , h-1 ) , 5.25 ( m , 1h , h-2 ) , 5.05 ( m , 1h , 
 h-3 ) , 4.75 ( m , 1h , h-4 half overlap with hod signal ) , 
 4.55 ( m , 2h , h-5 ) , and 2.80 ( m , 3h , ch3 ) .
a suspension of 6-n - methyl - amp and the bisphosphate as above 8a/8b ( 0.20 mmol , calculated by h nmr integration ) in dry 
 dmso ( 0.9 ml ) was evaporated with dry dmf ( 3  2 ml ) . to the 
 residue were added in sequence triphenylphosphine ( 280 mg , 1.07 mmol ) , 
 morpholine ( 0.15 ml , 1.72 mmol ) , and dipyridyl disulfide ( 235 mg , 
 1.07 mmol ) .
the resulting yellow solution was stirred at room temperature 
 for 4 h , after which a solution of sodium iodide in acetone ( 0.2 m , 
 15 ml ) was added and the resulting precipitate was filtered and washed 
 with acetone .
the crude product was further purified by ion - exchange 
 chromatography , eluting with a gradient of 050% 1 m teab against 
 milli - q water .
the solvent was evaporated in vacuo and excess teab 
 was coevaporated with meoh ( 3 times ) to give the title compound as 
 its triethylammonium salt ( 93 mg , 78% ) .
hplc , tr = 2.8 min at 254 nm ; uv ( h2o ) max 266.4 nm ; h ( d2o , 270 mhz )  8.23 ( s , 
 1h , h-8 ) , 7.98 ( s , 1h , h-2 ) , 5.93 ( d , 1h , j1,2 = 4.9 hz , h-1 ) , 4.68 ( m , 1h , h-2 ) , 4.42 ( m , 1h , 
 h-3 ) , 4.26 ( m , 1h , h-4 ) , 3.94 ( m , 2h , h-5 ) , 
 3.44 ( m , 4h , 2  ch2o ) , 2.91 ( m , 3h , ch3n ) , and 2.80 ( m , 4h , 2  ch2n ) ; p ( d2o , 109 mhz )  8.10 ( s ) ; hrms ( es ) c15h24n6o7p 431.1444 calcd 
 for mh , found 431.1441 . to a mixture of 9 ( 56 mg , 95 mol ) , -nmn ( 55 mg , 165 mol ) , and mgso4 ( 38 mg , 317 
 mol ) was added a solution of mncl2 in formamide 
 ( 0.2 m , 1.18 ml ) .
the resulting suspension was stirred at room temperature 
 for 48 h under a nitrogen atmosphere , and the reaction mixture was 
 quenched by dropwise addition of mecn ( 2 ml ) .
the yellow precipitate 
 was filtered , washed with acetone , and dissolved in small amount of 
 milli - q water .
the aqueous solution was treated with chelex 100 ( sodium 
 form ) to remove any residual manganese and purified by reverse - phase 
 chromatography , eluting with a gradient of 0.05 m teab against mecn . 

the title compound was isolated as a glassy solid in the triethylammonium 
 form ( 51 mg , 64% ) .
hplc , tr = 3.4 min 
 at 254 nm ; uv ( h2o ) max 262.5 nm ( /dm mol cm 14  560 ) ; h ( d2o , 400 mhz )  9.25 ( s , 1h , hn-2 ) , 9.07 ( d , 1h , j6,5 = 5.9 hz , hn-6 ) , 8.71 ( d , 1h , j4,5 = 8.2 hz , 

hn-4 ) , 8.26 ( s , 1h , h-8 ) , 8.09 ( dd , 1h , j5,4 = 8.2 hz and j5,6 = 5.9 
 hz , hn-5 ) , 7.97 ( s , 1h , h-2 ) , 5.99 ( d , 1h , j1,2 = 5.5 hz , h-1 ) , 5.91 ( d , 1h , j1,2 = 5.9 hz , h-1 ) , 
 4.70 ( dd , 1h , j2,1 = 5.9 
 hz and j2,3 = 5.5 hz , 
 h-2 ) , 4.48 ( m , 1h , h-4 ) , 4.44 ( m , 2h , h-3 
 and h-3 ) , 4.37 ( m , 1h , h-2 ) , 4.324.16 ( m , 
 5h , h-4 , h-5 , and h-5 ) , and 2.93 ( ch3n ) ;
p ( d2o , 109 mhz )  10.73 
 ( brs ) ; hrms ( es ) calcd for c22h28n7o14p2 676.1169 [ m  
 h ] , found 676.1158 . to a 
 solution of 10 ( 20 mg , 24 mol ) in hepes buffer 
 ( 25 mm , ph 7.4 , 60 ml ) was added aplysia adp - ribosyl 
 cyclase ( 80 l ) .
the resulting solution was stirred at room 
 temperature until rp - hplc indicated that all the starting material 
 had reacted .
the solution was diluted , and then product was purified 
 by ion - exchange chromatography , eluting with a gradient of 050% 
 1 m teab buffer against milli - q water .
the appropriate fractions were 
 collected , evaporated and excess teab was removed by coevaporating 
 with meoh ( 3 times ) to give the compound as a glassy solid in its 
 triethylammonium form ( 10 mg , 59% ) .
hplc , tr = 11.8 min at 254 nm ; uv ( h2o ) max 265.1 
 nm ( /dm mol cm 13  780 ) ; h ( d2o , 270 mhz )  
 8.37 ( s , 1h , h-8 ) , 8.10 ( s , 1h , h-2 ) , 6.02 ( d , 1h , j1,2 = 5.9 hz , h-1 ) , 5.24 ( d , 0.3h , j1,2 = 4.0 hz , h-1 ) , 5.14 ( m , 0.7h , h-1 ) , 4.703.94 
 ( m , 10h , h - ribose ) , and 2.87 ( m , 3h , ch3n ) ; p ( d2o , 109 mhz )  10.6 ( brs ) ; hrms ( es ) calcd for c16h24n5o14p2 572.0795 [ m  h ] , found 572.0792 . to a suspension of 13 ( 80 mg , 0.236 
 mmol ) in dry mecn ( 5 ml ) were added triethylamine ( 197 l , 1.416 
 mmol ) and dinitrofluorobenzene ( 36 l , 0.283 mmol ) .
the resulting 
 solution was stirred at room temperature for 1 h after the solvent 
 was removed under reduced pressure and the residue obtained was purified 
 by flash chromatography on silica gel ( etoac / hexane , 1:1 ) to yield 
 the desired product as a yellow foam ( 96 mg , 83% ) .
hplc , tr = 14.6 min at 260 nm ; h ( 270 mhz , cdcl3 )  8.98 ( d , 1h , j = 2.5 hz , ar - h ) , 
 8.65 ( s , 1h , h-2 ) , 8.38 ( dd , 1h , j = 8.8 and 2.5 
 hz , ar - h ) , 8.16 ( s , 1h , h-8 ) , 7.97 ( d , 1h , j = 8.8 
 hz , ar - h ) , 5.94 ( d , 1h , j1,2 = 4.7 hz , h-1 ) , 5.215.17 ( m , 1h , h-2 ) , 5.125.08 
 ( m , 1h , h-3 ) , 4.554.54 ( m , 1h , h-4 ) , 3.983.76 
 ( m , 2h , h-5 ) , 1.64 ( s , 3h , ch3 ) , and 1.37 ( s , 3h , 
 ch3 ) ; hrms ( es ) calcd for c19h19n6o8s 4910.0980 ( mh ) , found 
 491.0957 ; rf = 0.3 ( etoac / hexane , 6:4 ) .
to a solution of 15 ( 70 mg , 0.138 
 mmol ) in dry dcm ( 3 ml ) were added tetrazole ( 20 mg , 0.276 mmol ) and n , n - diisopropyl - di - tert - butylphosphoramidite ( 66 l , 0.208 mmol ) .
the reaction mixture 
 was stirred at room temperature for 1 h , after which time tlc analysis 
 ( hexane / etoac , 6:4 ) indicated conversion of starting material to a 
 single phosphite .
the mixture was cooled to 78 c , and 
 mcpba ( 47 mg , 0.276 mmol ) was added .
after 20 min , 10% aqueous na2so3 ( 10 ml ) was added and the mixture was warmed 
 to room temperature .
the organic layer was separated and washed with 
 a saturated solution of nahco3 ( 15 ml ) and brine ( 15 ml ) , 
 dried ( na2so4 ) , filtered , and evaporated under 
 reduced pressure to leave an oil which was purified by column chromatography 
 on silica gel ( hexane / etoac , 1:1 ) to give the title compound 16 as a yellow oil ( 85 mg , 90% ) .
hplc , tr = 12.1 min at 260 nm ; h ( 270 mhz , cdcl3 )  8.98 ( d , 1h , j = 2.5 hz , ar - h ) , 8.74 ( s , 
 1h , h-2 ) , 8.35 ( d , 1h , j = 2.5 hz , ar - h ) , 8.33 ( s , 
 1h , h-8 ) , 7.92 ( d , 1h , j = 8.8 hz , ar - h ) , 6.23 ( d , 
 1h , j1,2 = 2.7 hz , h-1 ) , 
 5.33 ( dd , 1h , j2,3 = 6.0 
 hz and j2,1 = 2.7 hz , 
 h-2 ) , 5.04 ( dd , 1h , j3,2 = 6.0 hz and j3,4 = 
 2.4 hz , h-3 ) , 4.554.54 ( m , 1h , h-4 ) , 4.164.12 
 ( m , 2h , h-5 ) , 2.03 ( s , 3h , ch3 ) , 1.63 ( s , 3h , ch3 ) ,
1.44 ( s , 9h , bu ) , and 1.40 
 ( s , 9h , bu ) ; p ( decoupled , 
 109 mhz , cdcl3 )  9.3 ( s ) ; hrms ( es ) calcd for c27h36n6o11ps 683.1895 ( mh ) , found 683.1871 ; rf = 0.14 ( hexane / etoac , 6:4 ) .
16 ( 80 mg , 0.116 mmol ) was stirred in a solution of 
 mecn ( 10 ml ) containing 10% ( v / v ) 2-mercaptoethanol ( 1 ml ) and 1% 
 dipea ( 0.1 ml ) for 1 h. water ( 15 ml ) and dcm ( 30 ml ) were then added . 

the organic layer was washed several times with water to remove the 
 excess 2-mercaptoethanol .
the organic phase was dried , filtered , and 
 evaporated in vacuo to leave an oil which was purified by column chromatography 
 on silica gel ( dcm / acetone , 7:3 ) to give the title compound as a white 
 waxy solid ( 45 mg , 75% ) .
h ( 270 mhz , cdcl3 ) 
  8.38 ( s , 1h , h-2 ) , 8.36 ( s , 1h , h-8 ) , 6.14 ( d , 1h , j1,2 = 2.2 hz , h-1 ) , 
 5.52 ( dd , 1h , j2,3 = 6.1 
 hz and j2,1 = 2.1 hz , 
 h-2 ) , 5.04 ( dd , 1h , j3,2 = 6.1 hz and j3,4 = 
 2.5 hz , h-3 ) , 4.564.54 ( m , 1h , h-4 ) , 4.234.17 
 ( m , 2h , h-5 ) , 1.63 ( s , 3h , ch3 ) , 1.44 ( s , 18h , 
 2  bu ) , and 1.42 ( s , 3h , ch3 ) ; p ( decoupled , 109 mhz , cdcl3 )  
 9.3 ( s ) ; hrms ( es ) calcd for c21h34n4o7ps 517.1880 ( mh ) , found 
 517.1863 .
2,3-o - isopropylidene-5-o-(di - tert - butylphosphoramidite)-6-thioinosine 
 ( 35 mg , 0.067 mmol ) was stirred in a 50% aqueous tfa solution ( 2 ml ) 
 at room temperature for 24 h. the solvent was removed under reduced 
 pressure and coevaporated several times with meoh to remove any residual 
 tfa .
the residue was dissolved in water and worked up with etoac ( 2 
  10 ml ) .
the aqueous layer was evaporated to dryness to produce 
 the desired monophosphate 14 as a glassy solid ( 21 mg , 
 86% ) .
hplc , tr = 2.9 min at 320 nm ; h ( 270 mhz , d2o )  8.94 ( s , 1h , h-2 ) , 8.23 
 ( s , 1h , h-8 ) , 6.05 ( d , 1h , j1,2 = 3.9 hz , h-1 ) , 4.60 ( dd , 1h , j2,3 = 4.7 hz and j2,1 = 
 3.9 hz , h-2 ) , 4.41 ( app t , 1h , j = 5.0 hz , 
 h-3 ) , 4.27 ( dd , 1h , j4,3 = 5.0 hz and j4,5a = 
 2.5 hz , h-4 ) , 4.17 ( ddd , 1h , j5a,5b = 11.8 hz , j5a , p = 4.4 hz , and j5a,4 = 2.5 hz , h-5a ) , and 4.05 
 ( ddd , 1h , j5a,5b = 11.8 
 hz , j5a , p = 5.5 hz , and j5a,4 = 2.8 hz , h-5b ) ; p ( decoupled , 109 mhz , d2o )  0.51 ( s ) ; hrms ( es ) calcd for c10h14n4o7ps 365.0315 ( mh ) , found 365.0310 .
14 ( 13 mg , 
 0.036 mmol ) was dissolved in dry dmso ( 1 ml ) and coevaporated with 
 dry dmf ( 5  3 ml ) .
the residue was dissolved in dmso ( 400 l ) , 
 to which were added morpholine ( 16 l , 0.187 mmol ) , dipyridyl 
 disulfide ( 19 mg , 0.089 mmol ) , and triphenylphosphine ( 24 mg , 0.089 
 mmol ) , at which point the solution became bright yellow .
it was stirred 
 for 1 h at room temperature , after which hplc analysis showed completion 
 of the reaction .
precipitation of the product occurred by dropwise 
 addition of a solution of nai in acetone ( 0.1 m , 8 ml ) .
the resulting 
 precipitate was filtered , washed with acetone , and dried ( p 6.7 ppm and hplc , tr = 5.9 min 
 at 320 nm ) .
it was then reacted with -nmn ( 17 mg , 
 0.050 mmol ) and mgso4 ( 11 mg , 0.092 mmol ) in a 0.2 m solution 
 of mncl2 in formamide ( 0.35 ml ) at room temperature overnight , 
 after which hplc analysis showed completion of the reaction ( tr(-nmn ) = 2.1 min and tr(6-sh - nhd ) = 7 min ) .
the precipitate was filtered , dissolved in milli - q , 
 and applied to a reverse - phase column , eluting with a gradient of 
 mecn in 0.05 m teab .
further treatment with chelex 100 to remove any 
 paramagnetic particles afforded the desired dinucleotide 17 as a glassy solid ( 10 mg , 15.2 mol , 31% from 6-thio imp ) . 

hplc , tr = 6.9 min at 320 nm ; h ( 270 mhz , d2o )  9.17 ( s , 1h , hn2 ) , 
 8.96 ( d , 1h , j6,5 = 6.1 hz , hn6 ) , 8.65 ( d , 1h , j4,5 = 7.7 hz , hn4 ) , 8.30 ( s , 1h , h-2 ) , 8.08 ( s , 1h , h-8 ) , 8.027.96 
 ( m , 1h , hn5 ) , 6.03 ( d , 1h , j1,2 = 6.5 hz , h-1 ) , 5.86 ( d , 1h , j1,2 = 5.8 hz , h-1 ) , 4.65 ( app t , 1h , j2,1 = j2,3 = 5.5 hz , h-2 ) , and 4.414.07 ( m , 9h , hsugar ) ; p ( decoupled , 109 mhz , d2o )  10.5 
 ( d , j = 19.7 ) and 10.8 ( d , j = 19.7 ) ; hrms ( es ) calcd for c21h27n6o14p2s 681.0776 ( mh ) , found 681.0752 ; uv ( h2o , ph 7.3 ) max 322 nm ( /dm mol cm 20  320 ) .
17 ( 13.5 mol ) was incubated 
 with aplysia cyclase ( 150 l ) in a 0.1 m nahco3 buffer ( 30 ml , ph 7.6 ) at room temperature .
after 30 h , hplc 
 analysis showed total consumption of starting material and formation 
 of a new large peak at 11 min .
the water was evaporated to a minimum , 
 and the residue was applied on a rp-18 column , eluting with a gradient 
 of mecn in 0.05 m teab ( 065% over 300 ml ) .
the residue was passed through 
 a small chelex column previously washed with milli - q water to bring 
 the ph down to 8 .
it 
 was then lyophilized to afford the desired cyclic dinucleotide as 
 its sodium salt ( 7 mol , 52% ) .
hplc , tr = 10.7 min at 320 nm ; h ( 270 mhz , d2o )  9.32 ( s , 1h , h-2 ) , 8.29 ( s , 1h , h-8 ) , 6.65 ( br s , 1h , 
 h-1 ) , 5.99 ( d , 1h , j1,2 = 6.3 hz , h-1 ) , 5.20 ( dd , 1h , j2,1 = 6.3 hz and j2,3 = 
 5.0 hz , h-2 ) , 4.64 ( dd , 1h , j3,2 = 5.0 hz and j3,4 = 
 2.2 hz , h-3 ) , and 4.594.07 ( m , 8h , h-4 , h-5 , 
 h-2 , h-3 , h-4 , and h-5 ) ; p ( decoupled , 109 mhz , d2o )  9.2 ( d , ab 
 system , j = 11.8 hz ) , 10.5 ( d , ab system , j = 11.8 hz ) ; hrms ( es ) calcd for c15h19n4o13p2s 557.0150 
 ( mh ) , found 557.0161 ; uv ( h2o , ph 7.2 ) . 

 two nicotinamide adenine dinucleotide ( nad+ ) analogues modified at the 6 position of the purine ring were synthesized , 
 and their substrate properties toward aplysia californica adp - ribosyl cyclase were investigated . 
 6-n - methyl 
 nad+ ( 6-n - methyl nicotinamide adenosine 
 5-dinucleotide 10 ) hydrolyzes to give the linear 
 6-n - methyl adpr ( adenosine 5-diphosphoribose , 11 ) , whereas 6-thio nhd+ ( nicotinamide 6-mercaptopurine 5-dinucleotide , 17 ) generates a cyclic dinucleotide . 
 
 surprisingly , nmr correlation spectra confirm this compound to be 
 the n1 cyclic product 6-thio n1-cidpr 
 ( 6-thio cyclic inosine 5-diphosphoribose , 3 ) , 
 although the corresponding 6-oxo analogue is well - known to cyclize 
 at n7 . in jurkat t cells , unlike the parent cyclic 
 inosine 5-diphosphoribose n1-cidpr 2 , 
 6-thio n1-cidpr antagonizes both cadpr- 
 and n1-cidpr - induced ca2 + release but 
 possesses weak agonist activity at higher concentration . 3 is thus identified as the first c-6 modified cadpr ( cyclic adenosine 
 5-diphosphoribose ) analogue antagonist ; it represents the 
 first example of a fluorescent n1-cyclized cadpr 
 analogue and is a new pharmacological tool for intervention in the 
 cadpr pathway of cellular signaling . 
Introduction
Results and Discussion
Pharmacology
Conclusion
Experimental Section