heat - shock proteins 
 are a highly conserved family of molecular 
 chaperones that facilitate the folding , stability , and cellular localization 
 of their substrate proteins .
up - regulation 
 of the pathways associated with the heat - shock response has been implicated 
 in a number of disease areas , including cancer .
recent focus has been on the inhibition of the molecular 
 chaperone heat - shock protein 90 ( hsp90 ) using adenosine triphosphate 
 ( atp ) competitive inhibitors , an approach that has resulted in considerable 
 success as several compounds have now entered clinical trials . the heat - shock protein 70 ( hsp70 )
family of molecular 
 chaperones represents another potential target for small - molecule 
 mediated antagonism of the heat - shock response pathway .
the hsp70 
 isoform , heat - shock cognate 70 ( hsc70 ) , is ubiquitously expressed 
 in tissues , while the inducible isoform , heat - shock protein 72 ( hsp72 ) , 
 is largely expressed in response to stress , including treatment with 
 hsp90 inhibitors , and aids cell survival through inhibition of several 
 apoptotic pathways .
we have previously 
 shown that dual knockdown of these two hsp70 isoforms in human colon 
 and ovarian tumor cell lines results in apoptosis , which was in contrast 
 with nontumorigenic cell lines where apoptosis was not observed , indicating 
 a potential therapeutic window for hsp70 inhibitors . to execute their refolding activity , the hsp70 proteins 
 utilize 
 the hydrolysis of atp to adenosine diphosphate ( adp ) and inorganic 
 phosphate ( adp / pi ) in a complex catalytic cycle involving 
 a number of protein conformational changes and through a process which 
 is tightly regulated by various cochaperones such as the heat - shock 
 protein 40 ( hsp40 ) proteins and the nucleotide exchange factor bag 
 family molecular chaperone regulator 1 ( bag1 ) protein .
while this complexity presents numerous opportunities to 
 antagonize the refolding activity of hsp70 , the clearest strategy 
 remains atp - competitive binding of inhibitors to the conserved nucleotide - binding 
 domain of the protein .
there remains only one published chemotype which displays 
 atp - competitive submicromolar inhibition of hsp70 and has been shown 
 to be effective in cellular assays , a chemotype derived from adenosine 
 ( figure 1 ) .
the affinity 
 of three known hsp70 inhibitors derived from adenosine and measured 
 by spr , see ref ( 7 ) for details .
the atpase domain of 
 hsp70 is a member of the actin atpase family 
 of proteins , a target class which has delivered very little success 
 in the discovery of high affinity ligands .
a recent study to assess the potential 
 of the hsp70-atp binding site for antagonism with small molecules 
 using sitemap described the target as 
  difficult  , while a separate 
 analysis using a fragment - based screening approach returned a very 
 low hit rate ( 0.4% ) , a result generally 
 associated with low ligandability .
several 
 studies into the biochemical mechanism of hsp70 refolding activity 
 and atp hydrolysis have demonstrated that the atp binding site of 
 hsp70 in solution is highly flexible in nature , undergoing numerous 
 conformational changes . with the 
 challenge of finding atp - competitive hit matter against 
 hsp70 hindering the potential development of inhibitors for this important 
 target , we sought to investigate the binding mechanism of adenosine - derived 
 ligands to the atp site of hsp70 .
the aim was to improve our understanding 
 of how high affinity ligands bind to this region of the protein so 
 that this knowledge could be applied to future inhibitor design .
the slow 
 turnover of atp by hsp70 , and the potent product inhibition by adp / pi , means that using functional 
 assays is a challenge for the characterization of hsp70 ligand binding . 

therefore , we focused on surface plasmon resonance ( spr ) as a biophysical 
 method to assess the affinity of ligands .
unfortunately , full - length 
 human hsp72 gave poor spr data in our hands , displaying erratic and 
 difficult to interpret sensorgrams .
therefore , the nucleotide - binding 
 domain ( nbd ) of human hsc70 ( hsc70-nbd residues 1381 ) was used in all spr experiments .
adenosine 1 is a relatively weak ligand for hsc70-nbd , displaying a 
 pkd= 3.95  0.01 ( kd = 110 m , n = 3 ) , when measured by spr , but we decided to use 
 this compound as a starting point for our investigations into the 
 binding mechanisms of this chemotype to the hsp70 proteins .
we began 
 by analyzing the importance of the ribose motif to the binding affinity 
 of adenosine 1 . removing either the 2- or 3-hydroxyl 
 groups from the sugar motif or changing 
 their relative and absolute stereochemistry resulted in no measurable 

removal of either the 
 6-amino group or the 3-nitrogen of the adenine ring also resulted 
 in the loss of all measurable affinity .
these results demonstrate 
 the importance of the ribose motif and the adenine aminopyrimidine 
 motif to binding of adenosine - derived ligands to the hydrophilic region 
 of the protein .
in contrast , removal of the 5-hydroxyl was 
 well tolerated , as compound 2 retained its affinity in 
 the spr assay with a pkd = 3.88  
 0.02 ( kd = 130 m , n = 3 ) ( figure 2 and supporting information table 1 ) .
the heteroatoms 
 and stereochemistry highlighted in red are important for affinity 
 to hsp70 and could not be replaced by hydrogen or ch or the stereochemistry 
 changed .
it has previously been shown by 
 massey et al . that addition of 
 a primary or secondary amine to the 8-position of adenosine resulted 
 in a significant increase in affinity for these ligands . in our hands ,
8-aminoadenosine 3 gave a pkd = 5.16  0.01 ( kd = 7.0 m , n = 3 ) , a 
 16-fold increase in affinity when compared to adenosine 1 .
we decided to investigate this substituent in order to better understand 
 its role in the improved affinity of these compounds ( table 1 ) .
all results 
 are quoted as the geometric 
 mean  standard error of the mean ( sem ) of three independent 
 experiments unless otherwise stated , pkd = log10(kd(m ) 
  10 ) .
methyl substitution 
 on the 8-amino group ( entry 2 ) gave ligand 4 and resulted 
 in a 2.5-fold drop in affinity , while 
 dimethyl analogue 5 ( entry 
 3 ) displayed a kd greater than 1 mm .
replacement 
 of the 8-amino substituent with a methoxy group to give 6 ( entry 4 ) also resulted in a complete loss of activity .
this dramatic 
 effect on binding suggested that one of the hydrogens of the 8-amino 
 group was involved in hydrogen bonding . to assess
whether an intramolecular 
 hydrogen bond to the 5-hydroxyl was important for affinity , 
 we removed this group to give 7 ( entry 5 ) .
however , only 
 a 1.6-fold drop in affinity was observed with analogue 7 when compared with 8-n - methylaminoadenosine 4 ( entry 2 ) .
these results suggest that an intramolecular 
 hydrogen bond between the 8-amino substituent and the 5-hydroxyl 
 group can not rationalize the increased affinity observed with the 
 8-aminoadenosine series .
we speculated 
 that the role of the 8-amino substituent as a hydrogen bond donor 
 is through interactions with the cyclic ribose oxygen to stabilize 
 a gauche conformation of adenosine , which facilitates binding by reducing 
 the energy barrier to the initial binding event .
finally , we sought to investigate the role of the 
 imidazole ring 
 of adenosine to the binding of these ligands to hsp70 ( table 2 ) .
all compounds 
 were purchased from 
 the relevant commercial suppliers and used without further purification .
all results are quoted as the 
 geometric 
 mean  sem of three independent experiments unless otherwise 
 stated , pkd = log10(kd(m )  10 ) .
we screened three 
 commercially available bacterial natural products , 
 all based on the replacement of the purine of adenosine with a pyrrolopyrimidine 
 scaffold , exchanging the nitrogen at the 7-position with carbon . comparing this 
 change in scaffold to adenosine 1 ( entry 1 ) : the more 
 lipophilic derivative tubercidin 8 ( entry 2 ) displayed 
 a 4-fold improvement in affinity , substitution at the 7-postion was 
 well tolerated , with the nitrile derivative toyocamycin 9 ( entry 3 ) displaying comparable affinity to adenosine 1 , and the primary amide derivative sangivamycin 10 ( entry 
 4 ) gave a 35-fold increase in affinity at 3.3 m .
with 
 knowledge of the increased affinity observed with the pyrrolopyrimidine 
 scaffold in hand , we planned to combine the scaffold hop with the 
 improved affinity previously described for 8-amino substitution of 
 the adenosine scaffold .
8-amino substitution of tubercidin proved 
 synthetically intractable due to the absence of an electron - withdrawing 
 group at the 7-position , making aromatic substitution at the 8-position 
 challenging .
therefore , we focused our efforts on the synthesis of 
 8-aminotoyocamycin 12 and 8-aminosangivamycin 13 ( scheme 1 ) .
tribenzoyl intermediate 11 was prepared in 
 four steps 
 and 20% yield using a previously described procedure . despite repeated attempts ,
however , we were successful 
 using methylamine as a nucleophile to give 8-n - methylaminotoyocamycin 12 , which underwent in situ deprotection of the benzoyl groups 
 under the reaction conditions .
treatment of intermediate 11 with basic hydrogen peroxide resulted in hydrolysis of the nitrile 
 group , and subsequent addition of methylamine gave 8-n - methylaminosangivamycin 13 in low yield and moderate 
 purity .
8-n - methylaminotoyocamycin 12 gave a pkd = 5.47  0.02 
 ( kd = 3.3 m , n = 3 ) against hsc70-nbd .
this result represented a 27-fold improvement 
 in activity compared to toyocamycin 9 ( table 2 , entry 1 ) and a 5-fold increase 
 in affinity when compared to the corresponding 8-n - methyladenosine 12 ( table 1 , entry 2 ) .
kd = 30 m , n = 1 ) , which is a 7-fold drop 
 in affinity compared to sangivamycin 10 ( table 2 , entry 3 ) .
we rationalized 
 this structure  activity relationship ( sar ) through the potential 
 effect of the 7-substituent on the hydrogen bonding ability of the 
 key 8-n - methylamino group .
the primary amide substituent 
 of 13 can form an intramolecular hydrogen bond with the 
 8-n - methylamino group to give a resonance stabilized 
 six - membered intramolecular hydrogen bond , which would mask the hydrogen bond donor effect of the 8-n - methylamino substituent and block the binding of this 
 ligand . to develop the toyocamycin scaffold
further , we sought 
 to introduce 
 a benzylic substituent to the 8-position amine .
previously , massey 
 et al . have shown that n - benzyl substitution at the 
 8-amino position could improve the affinity of adenosine derived inhibitors 
 of hsp70 ( figure 1 ) . because our toyocamycin derived scaffold 12 had displayed a 5-fold improvement in affinity compared 
 to the corresponding adenosine scaffold 4 , we hypothesized 
 that addition of an 8-n - benzyl substituent to toyocamycin 
 would result in an improved chemotype for hsp70 inhibition .
8-n - benzyltoyocamycin 14 was prepared via a similar 
 method to 12 using benzylamine as a nucleophile . for 
 comparison ,
8-n - benzyladenosine 15 was 
 prepared in one step via a literature procedure from commercially 
 available 8-bromoadenosine ( scheme 2 ) .
8-n - benzyladenosine derivative 15 gave a pkd = 5.84  0.02 
 ( kd = 1.5 m , n = 3 ) , representing 
 a 12-fold improvement in binding affinity compared to the 8-n - methyladenosine analogue 4 ( table 1 , entry 2 ) ; however , the 8-n - benzyltoyocamycin analogue 14 gave a pkd = 5.56  0.02 ( kd = 2.8 m , n = 3 ) , representing little 
 change in affinity compared to 8-n - methyltoyocamycin 
 derivative 12 despite the increase in molecular weight 
 and lipophilicity . even though toyocamycin 9 was apparently 
 an improved scaffold , when compared to adenosine , for the inhibition 
 of hsp70 , we had been unable to develop the compounds beyond a micromolar 
 affinity ligand .
we believed that improving our understanding of the 
 complex sar surrounding the nucleoside core and lipophilic 8-n - benzyl substituent through analysis of ligand / hsp70 co - crystal 
 structures would be crucial to the discovery and development of small - molecule 
 inhibitors of hsp70 . in 1995 ,
kinetic 
 studies by mckay 
 and ha using changes in tryptophan fluorescence indicated that the 
 hsp70 isoform , hsc70 , undergoes a number of conformational changes 
 during its catalytic cycle . the energy 
 released from atp hydrolysis drives subsequent conformational changes 
 in the substrate binding domain , allowing hsp70 to carry out its refolding 
 function on client proteins .
however , mckay and ha discovered that 
 atp and adp have two distinct binding mechanisms . while adp binds 
 and then dissociates in an apparent single - step mechanism with slow - off 
 kinetics , the binding of atp is a two - step process , requiring an additional 
 conformational change of the nucleotide - binding domain prior to atp 
 hydrolysis . because it had been demonstrated that the two nucleotide 
 substrates of hsp70 can jump between binding mechanisms , we hypothesized 
 that the complexity surrounding the apparent sar of the nucleoside 
 derived hsp70 inhibitors was due to a change in mechanism between 
 a one - step and an induced conformational change two - step mechanism , 
 even if kinetically this would be difficult to observe . to investigate
whether a two - step binding 
 model is consistent with the observed nucleoside sar , we decided to 
 probe hsp70 ligand binding by x - ray crystallography .
there is currently 
 no crystal structure of full - length human hsp70 ; therefore , we focused 
 our crystallography efforts on the nbd of the human hsp70 isoform 
 hsp72 ( hsp72-nbd ) . in our hands ,
adp 16 gave an 
 affinity of pkd = 6.49 ( kd = 0.32 
 m , n = 1 ) when measured by spr against the 
 hsc70-nbd .
the co - crystal structure of 
 adp / pi bound to the hsp72-nbd has previously been solved and analysis of this structure clearly revealed 
 a closed conformation of the protein .
the two -helices flanking 
 the sides of the atp binding site , closed around the adenosine motif 
 of adp , forming multiple hydrogen bonds with the ribose and adenine 
 moieties of adp ( figure 3 ) .
pdb 3atu , important hydrogen 
 bonding interactions , with their distances in  , and residues 
 are indicated . the key salt - bridge interaction between lysine-56 and 
 glutamic acid-268 was measured at 2.8  ( 2 sf ) .
only selected 
 residues are shown and solvent has been omitted for clarity . from analysis of this structure , 

the two phosphate groups of adp 
 form multiple hydrogen bonds within the phosphate binding region of 
 the hsp72-nbd .
it is also clear that the hsp72-nbd would need to undergo 
 a conformational change in order for adp / pi to dissociate , 
 resulting in the previously described slow - off kinetics .
it has been suggested previously that the closed 
 conformation is apparently stabilized by the formation of a solvent 
 exposed salt - bridge between glutamic acid-268 and lysine-56 .
intrigued by the formation of this intramolecular 
 interaction , we then sought to generate a co - crystal structure with 
 the bacterial natural product sangivamycin 10 ( kd = 3.3 m ) ( figure 4 ) .
pdb 5aqz , key 
 hydrogen bonding interactions and their distances in  are indicated . 

the crystal structure 
 of sangivamycin 10 revealed 
 a similar hydrogen bonding framework to the adenosine motif of the 
 adp / pi structure ( figure 3 ) .
the pyrrolopyrimidine ring interacts with serine-275 
 and the 2- and 3-hydroxyls of the ribose interact 
 with lysine-271 .
however , in contrast to the adp / pi hsp72-nbd 
 structure , sangivamycin 10 co - crystallized in a more 
 open conformation .
the two -helices of the nucleotide binding 
 domain are no longer in close proximity , and the solvent exposed salt - bridge 
 between glutamic acid-268 and lysine-56 is absent .
it is this difference 
 in the hsp70-nbd conformation which we hypothesize is an important 
 factor in the complexity of sar observed for nucleoside - derived inhibitors . 

the binding of both adp / pi and sangivamycin 10 are dominated by the formation of multiple hydrogen bonds to key 
 residues in the nucleoside binding cleft but the crucial difference 
 between the two ligands is their ability to induce and stabilize the 
 closed conformation of the hsp72-nbd .
it is this ligand - driven induced 
 conformational change that leads to high affinity for hsp70 .
the hsp72-nbd 
 closed conformation observed in the adp / pi bound structure 
 is presumably brought about by the interactions of the -phosphate 
 group with the two glycine - rich loops of the phosphate binding region . 

we speculated that this conformational change could force water molecules 
 from the nucleotide binding domain cleft , strengthening the many hydrogen 
 bond contacts surrounding the adenosine due to the more hydrophobic 
 environment .
the absence of the phosphate groups , and in particular 
 the -phosphate group , in sangivamycin 10 means 
 there is no induced conformational change so the affinity is only 
 dependent on the multiple hydrogen bonds ( figure 5 ) . induced open and induced closed conformations 
 of hsp72 .
pdb 3atu and 5aqz , 
 the copper - colored 
 structure represents the co - crystal structure of adp / pi bound to the hsp72-nbd in the induced closed conformation due to 
 interactions of the phosphate groups through hydrogen bonds with the 
 glycine rich loops and stabilized by the salt - bridge .
the gray - colored 
 structure represents the co - crystal structure of sangivamycin 10 bound to the hsp72-nbd .
the overlay shows sangivamycin 10 bound to a more open nbd conformation , in which the formation 
 of the glu - lys salt - bridge is not possible .
only selected residues 
 are shown and solvent has been omitted for clarity . mimicking phosphate groups with druglike ligands 
 represents a significant challenge in medicinal chemistry . however ,
if it were possible to induce the closed 
 conformation of the hsp70 nucleotide binding domain via an alternative 
 mechanism , then mimicking the -phosphate would be unnecessary . 

because the solvent exposed salt - bridge between glutamic acid-268 
 and lysine-56 was the only additional enthalpic intradomain interaction 
 that we observed in the hsp70-nbd closed conformation structure , when 
 compared with the more open sangivamycin 10 structure , 
 we hypothesized that a ligand stabilizing this interaction would stabilize 
 the closed conformation of hsp70 , leading to increased affinity .
we 
 therefore proposed that the previously described potent 8-n - benzyladenosine derived ligands were able induce a conformational 
 change in hsp70 but via a lipophilic mechanism , which would be more 
 amenable to drug discovery . to investigate this hypothesis , we synthesized 
 the known quinoline derived hsp70 inhibitor 17 in one 
 step and 68% yield using our previously described method ( scheme 3 ) .
although several 8-n - substituted hsp70 inhibitors 
 have been described in the literature , the 8-n - quinoline adenosine derived ligand 17 was chosen because it was reported to be the highest affinity 
 ligand which was only substituted at the 8-position . in our hands ,
17 gave a pkd = 6.14  0.01 
 ( kd = 0.72 m , n = 3 ) against hsc70-nbd when measured by spr , which was consistent 
 with the data reported in the literature .
8-n - quinoline aminoadenosine derived ligand 17 was then submitted to our co - crystallization protocol with 
 hsp72-nbd ( figure 6 ) .
co - crystallization of 8-n - quinoline aminoadenosine 
 derived ligand 17 bound to hsp72 .
pdb 5ar0 , the hsp72 structure 
 clearly demonstrates the formation the key salt - bridge when co - crystallized 
 with 8-n - quinolineadenosine 17 .
co - crystallization of 17 with the hsp72-nbd showed 
 the nbd in a closed conformation ( figure 5 ) , nearly identical to the conformation of 
 the hsp72-nbd in the adp / pi 16-bound structures ( figure 6 ) . to our knowledge , 
 this is the first example of a co - crystal structure demonstrating 
 a non - nucleotide ligand binding to the closed conformation of hsp70 . 

the similarity of the respective hsp72 conformations is consistent 
 with a potential role of the induced conformational change in enhancing 
 the affinity of the 8-n - benzyl nucleoside derived 
 hsp70 ligands .
however , the mechanism by which the inhibitor induced 
 conformational change occurs must be distinctly different from the 
 mechanism of nbd closure upon nucleotide binding because the quinoline 
 moiety of 17 forms no interactions within the phosphate 
 binding region . to rationalize the induced conformational change 

observed with 17 , we further analyzed the closed structure 
 of hsp72-nbd 
 to identify the key binding interactions .
8-n - quinoline 
 adenosine 17 displays a similar hydrogen bonding network 
 with the same key residues observed in both adp / pi and 
 sangivamycin 10 , with an additional solvent exposed water 
 molecule bound to the quinoline nitrogen .
however , in contrast with 
 the more open conformation observed in the hsp72-nbd sangivamycin 10 complex , the key salt - bridge between glutamic acid-268 
 and lysine-56 , which is present in the nucleotide - bound hsp70-nbd 
 structures , is clearly formed in the 17-bound hsp72-nbd 
 structure ( figure 5 ) . in the adp / pi/ hsc70-nbd co - crystal structure , the 
 salt - bridge is solvent exposed , weakening its effect ( figure 7 ) . by contrast , in the 17-bound hsp72-nbd co - crystal structure , 

the quinoline moiety is able to form a -stack with arginine-272 , 
 although this interaction is solvent exposed so is only likely to 
 be weak ; the result is to place the quinoline 
 group directly in front of the salt - bridge .
because the quinoline 
 is highly lipophilic , this creates a more lipophilic environment surrounding 
 the salt - bridge , protecting it from water and strengthening the interaction , 
 which leads to increased affinity for 8-n - benzyl 
 aminonucleoside derived ligands .
we propose 
 the binding mechanism of these ligands to hsp70 is analogous to a 
  door and latch  .
the initial binding event is similar 
 for all nucleoside derived ligands of hsp70 and is dominated by hydrogen 
 bonds to serine-275 and lysine-271 .
the nucleotide binding domain 
 is then able to close around the ligand but for this process to be 
 favorable , when balanced by the entropy cost of restricting the conformational 
 freedom of the protein , it must be stabilized by the ligand .
adp can 
 achieve this through interactions with the phosphate binding region , 
 while quinoline ligand 17 stabilizes the key salt - bridge 
 through hydrophobic desolvation .
sangivamycin 10 has 
 neither of these substituents so predominately binds to the open conformation .
overlay 
 of adp / pi and 8-n - quinoline 
 adenosine 17 co - crystal structures with hsp72 .
pdb 3atu and 5ar0 , overlay of the 
 adp / pi - hsp72 co - crystal structure ( copper ) and the 8-n - quinoline adenosine 17-hsp72 co - crystal structure 
 ( gray ) .
only selected residues are shown and solvent has been omitted 
 for clarity . to test this hypothesis , 
 and to generate more active ligands of hsp70 for further development , 
 we synthesized a series of 8-n - benzylaminoadenosine 
 analogues using our previously described method ( table 3 ) .
all results are 
 quoted as the geometric 
 mean  sem of three independent experiments unless otherwise 
 stated , pkd = log10(kd(m )  10 ) .
the 8-n - benzyl derivative 15 , as 
 shown previously , gave a pkd = 5.84  
 0.02 ( kd = 1.4 m , n = 3 ) when measured by spr against hsc70-nbd .
the weaker activity 
 observed with the benzyl group 15 compared to quinoline 17 we rationalized was due to its less efficient desolvation 
 of the glutamic acid / lysine salt bridge by the smaller lipophilic 
 group .
therefore , we decided to add a number of lipophilic substituents 
 to assess whether we could improve the desolvation effect , promote 
 the induced closed conformation , and improve the affinity of the ligands .
para - chloro - substitution 
 gave adenosine derivative 18 ( entry 3 ) with a pkd = 6.55  0.01 ( kd = 0.28 m , n = 9 ) , a 5-fold improvement 
 in affinity compared to compound 15 and a 60-fold improvement 
 compared to 8-n - methylaminoadenosine 4 ( table 1 , entry 2 ) . 

although the kinetics of the these ligands binding to hsp70 were at 
 the limit of what could be accurately measured by spr , analysis of 
 the off - rate did reveal that the quinoline ligand 17 and 
 the para - chlorobenzyl derivative 18 possessed 
 measurably slow off - rates compared to sangivamycin 10 , whose half - life was too short to be observed with this technique 
 ( see supporting information for details ) .
similar improvements were also observed for 
 the para - fluoro derivative 19 ( entry 
 4 , pkd = 6.34  0.01 , kd = 0.46 m , n = 3 ) and para - methyl derivative 20 ( entry 5 , pkd = 6.53  0.01 , kd = 0.30 m , n = 3 ) . because the crystal 
 structure of this ligand class shows that the benzylic moiety resides 
 in the cleft formed by the two -helices in the nucleotide binding 
 domain of hsp70 , it is unlikely that the para - lipophilic 
 substituents interact with hsp70 via a lipophilic pocket , as the group 
 is essentially solvent exposed .
this effect could not be explained 
 by an increase in the overall lipophilicity of the ligand to exploit 
 the nonspecific hydrophobic effect because dichlorobenzyl derivative 21 ( entry 6 , pkd = 5.45  
 0.01 , kd = 3.5 m , n = 3 ) displayed a significant drop in affinity .
the atp binding site of hsp70 is a challenging region of the protein 
 to target with small molecules due to its hydrophilic nature and high 
 flexibility . to target the nucleotide binding domain it is important 
 to understand the conformational changes that this region of the protein 
 undergoes .
using protein / ligand x - ray crystallography , we have demonstrated 
 that non - nucleotide ligands of hsp70 can induce conformational changes 
 in the protein and that these changes can play an important role in 
 the binding of hsp70 inhibitors . in solution , kinetic studies suggest 
 that this protein undergoes a number of conformational changes of 
 not just the nucleotide binding domain but also the substrate - binding 
 domain .
also , interactions between these 
 two domains and the role of cochaperones in these conformational changes 
 have yet to be addressed .
better understanding of the flexibility 
 of hsp70 and its effect on the affinity of ligands will contribute 
 to better assay design and more efficient inhibitor optimization .
unless otherwise 
 stated , reactions were conducted in oven - dried glassware under an 
 atmosphere of nitrogen using anhydrous solvents .
thin layer chromatography 
 ( tlc ) was performed on precoated aluminum sheets of silica ( 60 f254 
 nm , merck ) and visualized using short - wave uv light .
flash column 
 chromatography was carried out on merck silica gel 60 ( partial size 
 4065 m ) .
h nmr spectra were recorded on 
 bruker amx500 ( 500 mhz ) spectrometers using an internal deuterium 
 lock .
chemical shifts are quoted in parts per million ( ppm ) using 
 the following internal references : cdcl3 ( h 7.26 ) , 
 meod ( h 3.31 ) , and dmso - d6 ( h 
 2.50 ) .
signal multiplicities are recorded as singlet ( s ) , doublet 
 ( d ) , triplet ( t ) , quartet ( q ) , multiplet ( m ) , doublet of doublets 
 ( dd ) , doublet of doublet of doublets ( ddd ) , apparent triplet ( app 
 t ) broad ( br ) , or obscured multiplet ( obs m ) .
c nmr 
 spectra was recorded on bruker amx500 spectrometers at 126 mhz using 
 an internal deuterium lock .
chemical shifts are quoted to 0.01 ppm , 
 unless greater accuracy was required , using the following internal 
 references : cdcl3 ( c 77.0 ) , meod ( c 49.0 ) , 
 and dmso - d6 ( c 39.5 ) .
high resolution 
 mass spectra were recorded an agilent 1200 series hplc and diode array 
 detector coupled to a 6210 time - of - flight mass spectrometer with dual 
 multimode apci / esi source .
analytical separation was carried out on 
 a merck purospher star rp-18 , 30 mm  4 mm column using a flow 
 rate of 1.5 ml / min in a 4 min gradient elution , uv detection was at 
 254 nm . all compounds were > 95% purity by hplc analysis unless 
 otherwise 
 stated .
to a solution of 8-bromoadenosine ( 0.051 g , 0.15 
 mmol ) 
 in etoh ( 1.5 ml ) was added methylamine solution ( 33% solution in etoh , 
 0.25 ml , 3.0 mmol ) , and the mixture was heated to 80 c for 12 
 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 8:2 ) to give the desired compound as a white solid 
 ( 0.031 g 71% ) ; h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 6.95 ( d , j = 4.8 hz , 1h ) , 6.57 ( s , 
 2h ) , 5.92 ( dd , j = 6.0 , 4.0 hz , 1h ) , 5.86 ( d , j = 7.3 hz , 1h ) , 5.26 ( d , j = 6.7 hz , 1h ) , 
 5.16 ( d , j = 4.0 hz , 1h ) , 4.68 ( td , j = 7.0 , 5.3 hz , 1h ) , 4.12 ( ddd , j = 5.6 , 4.1 , 2.1 
 hz , 1h ) , 3.97 ( d , j = 2.3 hz , 1h ) , 3.703.58 
 ( m , 2h ) , 2.89 ( d , j = 4.5 hz , 3h ) ; c ( 126 
 mhz , dmso - d6 ) 152.86 , 152.52 , 150.28 , 
 148.89 , 117.64 , 86.99 , 86.13 , 71.42 , 71.20 , 62.15 , 29.59 .
( m + h ) requires 297.1306 , found 297.1299 . to a solution of 8-bromoadenosine ( 0.064 g , 0.19 
 mmol ) 
 in etoh ( 1.9 ml )
was added dimethylamine ( 40% solution in water , 0.42 
 ml , 3.70 mmol ) , and the mixture was heated to 80 c for 12 
 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 9:1 ) to give the desired product as a white solid 
 ( 0.027 g , 47% ) ; h ( 500 mhz , meoh ) 8.05 ( d , j = 1.1 hz , 1h ) , 5.91 ( d , j = 7.5 hz , 1h ) , 5.16 ( dd , j = 7.5 , 5.3 hz , 1h )
, 4.36 ( dd , j = 5.2 , 
 1.4 hz , 1h ) , 4.14 ( d , j = 1.9 hz , 1h ) , 3.86 ( dd , j = 12.6 , 2.3 hz , 1h ) , 3.73 ( dd , j = 12.6 , 
 2.7 hz , 1h ) , 3.03 ( s , 6h ) ; c ( 126 mhz , meod ) 157.19 , 153.92 , 
 149.92 , 149.22 , 116.86 , 89.06 , 86.97 , 71.96 , 71.79 , 62.86 , 41.89 . 
 hrms ( esi ) c12h19n6o4 ( m 
 + h ) requires 311.1462 , found 311.1464 . to a solution of 8-bromoadenosine ( 0.11 g , 0.33 
 mmol ) in meoh ( 4 ml ) was added naome ( 0.18 g , 3.3 mmol ) , and the mixture 
 was stirred at room temperature for 12 h. after this time , 
 the solvent was removed under reduced pressure and the resulting residue 
 was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 9:1 ) to give the desired product as a yellow oil ( 0.025 
 g , 26% ) .
h ( 500 mhz , meod ) , 8.06 ( s , 1h ) , 5.91 ( d , j = 7.1 hz , 1h ) , 4.93 ( dd , j = 7.0 , 5.2 
 hz , 1h ) , 4.33 ( dd , j = 5.2 , 2.0 hz , 1h ) , 4.21 ( s , 
 3h ) , 4.164.09 ( m , 1h ) , 3.86 ( dd , j = 12.6 , 
 2.5 hz , 1h ) , 3.71 ( dd , j = 12.6 , 2.9 hz , 1h ) ; c 
 ( 126 mhz , meod ) 155.19 , 154.01 , 150.10 , 148.34 , 115.35 , 87.65 , 86.82 , 
 72.43 , 71.55 , 62.62 , 56.69 . hrms ( esi ) c11h16n5o5
( m + h ) requires 298.1146 , 
 found 298.1152 . to a solution of 5-deoxyadenosine ( 0.024 g , 0.096 
 mmol ) in 
 dioxane
( 0.48 ml ) and water ( 0.48 ml ) was added khpo4 ( 0.065 
 g , 0.29 mmol ) and bromine ( 0.023 g , 0.14 mmol ) as a solution in water 
 ( 0.50 ml ) , and the mixture was stirred for 15 min .
the mixture was 
 then quenched satd sodium thiosulfate solution ( 5.0 ml ) , extracted 
 with etoac , dried ( mgso4 ) , and the solvent removed under 
 reduced pressure .
the product from the previous 
 step ( 0.032 g , 0.097 mmol ) was dissolved in 2 m menh2 in 
 etoh ( 1.9 ml ) , and the mixture was heated to 70 c for 12 
 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was purified by silica gel chromatography eluting with 2 m meoh / nh3:etoac ( 1:9 ) to give the desired product as a white solid 
 ( 0.009 g , 33% over two steps ) ; h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 6.75 ( q , j = 4.5 hz , 
 1h ) , 6.45 ( s , 2h ) , 5.58 ( d , j = 4.2 hz , 1h ) , 5.23 
 ( d , j = 5.0 hz , 1h ) , 5.09 ( q , j = 
 5.0 hz , 1h ) , 4.99 ( d , j = 5.7 hz , 1h ) , 4.144.04 
 ( m , 1h ) , 3.83 ( app p , j = 6.2 hz , 1h ) , 2.88 ( d , j = 4.5 hz , 3h ) , 1.24 ( d , j = 6.3 hz , 3h ) ; 
 c ( 126 mhz , dmso - d6 ) 152.97 , 152.91 , 
 150.19 , 149.12 , 117.95 , 88.07 , 79.10 , 74.87 , 71.05 , 29.73 , 18.96 . 
 hrms ( esi ) c11h17n6o3 ( m 
 + h ) requires 281.1357 , found 281.1354 .
( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl 
 dibenzoate 11 ( 120 mg , 0.17 mmol ) was dissolved in 2 
 m methylamine in ethanol ( 5.0 ml ) , and the reaction was heated in 
 a microwave reactor at 140 c for 1 h. after this time , the mixture 
 was cooled to room temperature and the solvent removed under reduced 
 pressure .
the resulting residue was purified by silica gel chromatography 
 with the biotage sp1 purification system ( column , 
 10+s ; flow rate , 15 ml / min ; gradient starting with 
 100% dcm from 0 to 1 cv then from 100% dcm to 2% nh4oh/18% 
 meoh/80% dcm from 1 cv to 21 cv ) to give the desired compound as a 
 white solid ( 0.045 g , 80% yield ) ; h ( 500 mhz , meod ) 3.22 ( s , 
 3h ) , 3.833.84 ( m , 2h ) , 4.14 ( q , j = 1.9 hz , 
 1h ) , 4.27 ( dd , j = 5.6 , 2.0 hz , 1h ) , 4.65 ( dd , j = 7.6 , 5.6 hz , 1h ) , 6.29 ( d , j = 7.9 
 hz , 1h ) , 8.01 ( s , 1h ) ; c ( 126 mhz , meod ) 29.55 , 56.92 , 61.31 , 
 70.91 , 71.19 , 86.12 , 87.35 , 101.19 , 118.75 , 148.27 , 149.87 , 151.26 , 
 154.16 . hrms ( esi ) c13h17n6o4 ( m + h ) requires 321.1306 , found 321.1303 . to a solution of ( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl 
 dibenzoate 11 ( 87 mg , 0.13 mmol ) in thf ( 1.0 ml )
was 
 added hydrogen peroxide ( 33% , 1.0 ml ) and 1 m naoh ( 1.0 ml ) .
the reaction 
 was stirred at room temperature for 18 h before 1 m hcl was 
 added until the mixture reached ph 6 , when the solvent was removed 
 under reduced pressure .
the resulting residue was dissolved in 2 m 
 methylamine in ethanol ( 5.0 ml ) , and the reaction was heated to 120 
 c in a microwave reactor for 50 min . after this time , the mixture 
 was cooled to room temperature and the solvent removed under reduced 
 pressure .
the resulting residue was purified by silica gel chromatography 
 using the biotage sp1 purification system ( column , 10+s ; flow rate , 
 15 ml / min ; gradient starting with 100% dcm from 0 to 1 cv then from 
 100% dcm to 2% nh4oh/18% meoh/80% dcm from 1 cv to 21 cv ) 
 to give the desired product as a yellow solid ( 0.009 g , 10% yield ) ; 
 h ( 500 mhz , meod ) 2.89 ( s , 3h ) , 3.78 ( dd , j = 12.2 , 1.9 hz , 1h ) , 3.86 ( dd , j = 12.2 , 2.2 hz , 
 1h ) , 4.18 ( q , j = 1.7 hz , 1h ) , 4.33 ( dd , j = 5.5 , 1.3 hz , 1h ) , 4.85 ( m , 1h ) , 6.12 ( d , j = 7.9 hz , 1h ) , 8.02 ( s , 1h ) ; c ( 126 mhz , meod ) 35.35 , 62.23 , 
 71.62 , 72.28 , 86.70 , 87.49 , 98.51 , 101.02 , 146.72 , 147.31 , 150.61 , 
 157.30 , 167.61 .
( 2r,3r,4r,5r)-2-(4-amino-6-bromo-5-cyano-7h - pyrrolo[2,3-d]pyrimidin-7-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl 
 dibenzoate 11 ( 31 mg , 0.045 mmol ) was dissolved in etoh 
 ( 2.0 ml ) , and benzylamine ( 29 mg , 0.27 mmol ) was added to the solution . 

the reaction was heated in a microwave reactor at 160 c for 
 1 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was dissolved in methanol ( 0.5 ml ) , and 0.5 m sodium methoxide ( 1.0 
 ml ) was added .
the reaction was stirred at room temperature for 1 
 h , then the solvent was removed under reduced pressure .
the resulting 
 residue was purified by silica gel chromatography with the biotage sp1 purification system ( column , 10+s ; 
 flow rate , 15 ml / min ; gradient starting with 100% dcm from 0 to 1 
 cv then from 100% dcm to 2% nh4oh/18% meoh/80% dcm from 
 1 cv to 21 cv ) to give the desired compound as a white solid ( 0.007 
 g , 39% yield ) ; h ( 500 mhz , meod ) 3.803.86 ( m , 2h ) , 
 4.18 ( q , j = 1.8 hz , 1h ) , 4.28 ( dd , j = 5.6 , 1.7 hz , 1h ) , 4.73 ( dd , j = 7.8 , 5.7 hz , 
 1h ) , 4.78 ( d , j = 10.3 hz , 2h ) , 6.37 ( d , j = 7.9 hz , 1h ) , 7.257.27 ( m , 1h ) , 7.337.36 
 ( m , 2h ) , 7.417.43 ( m , 2h ) , 8.01 ( s , 1h ) ;
c ( 126 mhz , 
 meod ) 46.28 , 61.40 , 71.14 , 71.29 , 86.27 , 86.81 , 87.38 , 101.06 , 118.30 , 
 126.72 , 126.98 , 128.25 , 138.41 , 148.25 , 149.94 , 150.10 , 154.22 . hrms 
 ( esi ) c19h21n6o4
( m + 
 h ) requires 397.1619 , found 397.1614 . to a solution of 8-bromoadenosine ( 0.061 g ,
0.18 
 mmol ) 
 in n - butanol ( 1.76 ml ) was added pr2net ( 0.092 ml , 0.53 mmol ) and benzylamine ( 0.038 ml , 0.35 
 mmol ) , and the mixture was heated to 120 c for 16 h. 
 after this time , the mixture was cooled to room temperature and the 
 resulting residue was purified by silica gel chromatography eluting 
 with etoac : meoh ( 95:5 ) to give the desired product as a white solid 
 ( 0.017 g , 26% ) ; h ( 500 mhz , meod ) 8.00 ( s , 1h ) , 7.457.38 
 ( m , 2h ) , 7.377.32 ( m , 2h ) , 7.287.23 ( m , 1h ) , 6.11 
 ( d , j = 7.7 hz , 1h ) , 4.81 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.69 ( d , j = 15.8 hz , 1h ) , 
 4.64 ( d , j = 15.8 hz , 1h ) , 4.30 ( dd , j = 5.6 , 1.6 hz , 1h ) , 4.18 ( q , j = 1.9 hz , 1h ) , 3.85 
 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.79 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 152.12 , 151.99 , 149.65 , 
 148.63 , 138.75 , 128.11 , 126.68 , 126.52 , 116.56 , 87.29 , 86.41 , 71.64 , 
 71.52 , 61.75 , 45.42 . hrms ( esi )
c17h21n6o4 ( m + h ) requires 373.1619 , found 
 373.1623 . to a solution of 8-bromoadenosine ( 0.099 g , 0.29 
 mmol ) in n - butanol ( 2.9 ml )
was added quinolin-6-ylmethanamine 
 ( 0.045 g , 0.29 mmol ) and pr2net ( 0.15 ml , 0.85 
 mmol ) , and the mixture was heated to 120 c under n2 for 24 h. after this time , the mixture was cooled to room temperature 
 and the solvent removed under reduced pressure .
the resulting residue 
 was purified by chromatography eluting with etoac:2 m meoh / nh3 ( 80:20 ) to give the desired product as a white solid ( 0.008 
 g , 7% ) ; h ( 500 mhz , meod ) 8.83 ( dd , j = 4.4 , 
 1.7 hz , 1h ) , 8.37 ( dt , j = 8.4 , 1.2 hz , 1h ) , 8.04 
 ( d , j = 8.7 hz , 1h ) , 8.02 ( s , 1h ) , 7.95 ( d , j = 1.8 hz , 1h ) , 7.86 ( dd , j = 8.8 , 2.0 
 hz , 1h ) , 7.54 ( dd , j = 8.3 , 4.3 hz , 1h ) , 6.16 ( d , j = 7.6 hz , 1h ) , 4.924.83 ( obs m , 3h ) , 4.32 ( dd , j = 5.4 , 1.7 hz , 1h ) , 4.21 ( q , j = 1.9 
 hz , 1h ) , 3.87 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.80 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 151.98 , 
 151.94 , 149.72 , 149.52 , 148.44 , 146.69 , 137.89 , 136.94 , 129.30 , 128.46 , 
 127.90 , 125.07 , 121.35 , 116.59 , 87.38 , 86.47 , 71.70 , 71.53 , 61.75 , 
 45.29 . hrms ( esi ) c20h22n7o4 ( m + h ) requires 424.1728 , found 424.1725 . to a solution of 8-bromoadenosine ( 0.078 g , 0.23 
 mmol ) in n - butanol ( 2.3 ml ) was added pr2net ( 0.12 ml , 0.68 mmol ) and ( 4-chlorophenyl)methanamine 
 ( 0.055 ml , 0.45 mmol ) , and the mixture was heated to 120 c for 
 12 h. after this time , the mixture was cooled to room temperature 
 and the resulting residue was purified by silica gel chromatography 
 eluting with etoac : meoh ( 95:5 ) to give the desired product as a white 
 solid ( 0.033 g , 36% ) ; h ( 500 mhz , meod ) 8.00 ( s , 1h ) , 7.40 
 ( d , j = 8.3 hz , 2h ) , 7.35 ( d , j = 
 8.6 hz , 2h ) , 6.10 ( d , j = 7.5 hz , 1h ) , 4.79 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.66 ( d , j = 15.9 
 hz , 1h ) , 4.62 ( d , j = 16.0 hz , 1h ) , 4.30 ( dd , j = 5.4 , 1.7 hz , 1h ) , 4.18 ( q , j = 1.9 
 hz , 1h ) , 3.85 ( dd , j = 11.9 , 2.3 hz , 1h ) , 3.79 ( dd , j = 12.0 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 152.19 , 
 151.83 , 149.68 , 148.72 , 137.67 , 132.42 , 128.22 , 128.16 , 116.54 , 87.30 , 
 86.40 , 71.65 , 71.50 , 61.73 , 44.80 . hrms ( esi ) c17h20cln6o4 ( m+h ) requires 407.1229 , 
 found 407.1237 . to a solution of 8-bromoadenosine ( 0.048 g , 0.14 
 mmol ) in n - butanol ( 1.4 ml ) was added ( 4-fluorophenyl)methanamine 
 ( 0.032 ml , 0.28 mmol ) and pr2net ( 0.073 ml , 
 0.42 mmol ) , and the mixture was heated to 120 c for 12 
 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was purified by silica gel chromatography eluting with etoac : meoh 
 ( 9:1 ) to give the desired product as a white solid ( 0.043 g , 79% ) ; 
 h ( 500 mhz , meod ) 7.99 ( s , 1h ) , 7.50 ( dd , j = 8.3 , 5.3 hz , 1h ) , 7.447.38 ( m , 2h ) , 7.237.16 ( m , 
 1h ) , 7.097.04 ( m , 2h ) , 6.09 ( d , j = 7.6 hz , 
 1h ) , 4.78 ( dd , j = 7.6 , 5.4 hz , 1h ) , 4.65 ( d , j = 15.7 hz , 1h ) , 4.60 ( d , j = 15.7 hz , 
 1h ) , 4.29 ( dd , j = 5.4 , 1.6 hz , 1h ) , 4.17 ( q , j = 1.9 hz , 1h ) , 3.83 ( dd , j = 12.0 , 2.2 
 hz , 1h ) , 3.78 ( dd , j = 11.9 , 1.7 hz , 1h ) ; c 
 ( 126 mhz , meod ) 164.14 , 163.00 , 161.06 , 152.14 , 151.85 , 149.66 , 148.65 , 
 134.75 , 134.72 , 130.99 , 130.92 , 128.53 , 128.46 , 116.55 , 115.67 , 115.50 , 
 114.79 , 114.62 , 87.26 , 86.41 , 71.60 , 71.50 , 61.72 , 44.79 . hrms ( esi ) c17h20fn6o4 ( m + h ) requires 391.1525 , found 
 391.1517 . to a solution of 8-bromoadenosine ( 0.062 g , 0.18 
 mmol ) in n - butanol ( 1.79 ml ) was added p - tolylmethanamine ( 0.045 ml , 0.36 mmol ) and pr2net ( 0.093 ml , 0.54 mmol ) , and the mixture was heated to 120 c 
 for 48 h. after this time , the mixture was cooled to room 
 temperature and the solvent removed under reduced pressure .
the resulting 
 residue was purified by silica gel chromatography eluting with etoac : meoh 
 ( 9:1 ) to give the desired product as a white solid ( 0.042 g , 61% ) ; 
 h ( 500 mhz , meod ) 7.99 ( s , 1h ) , 7.28 ( d , j = 7.8 hz , 2h ) , 7.16 ( d , j = 7.7 hz , 2h ) , 6.09 ( d , j = 7.6 hz , 1h ) , 4.80 ( dd , j = 7.6 , 5.4 
 hz , 1h ) , 4.64 ( d , j = 15.6 hz , 1h ) , 4.58 ( d , j = 15.6 hz , 1h ) , 4.29 ( dd , j = 5.4 , 1.7 
 hz , 1h ) , 4.18 ( d , j = 1.9 hz , 1h ) , 3.84 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.78 ( dd , j = 11.9 , 
 1.7 hz , 1h ) , 2.32 ( s , 3h ) ; c ( 126 mhz , meod ) 152.08 , 152.01 , 
 149.64 , 148.59 , 136.39 , 135.64 , 128.70 , 126.57 , 116.57 , 87.27 , 86.40 , 
 71.61 , 71.50 , 61.74 , 45.26 , 19.70 . hrms ( esi ) c18h23n6o4
( m + h ) requires 387.1775 , 
 found 387.1779 . to a microwave vial
was added 8-bromoadenosine 
 ( 0.30 g , 0.87 mmol ) and 3,4-dichlorobenzylamine ( 0.29 ml , 2.2 mmol ) 
 in ethanol ( 1.7 ml ) .
the reaction mixture was concentrated , and the crude residue 
 was purified by silica gel column chromatography eluting with etoh : dcm 
 ( 3:7 ) to give the desired product as a white solid ( 0.28 g , 73% ) ; 
 h ( 500 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 
 7.707.53 ( m , 3h ) , 7.37 ( dd , j = 8.3 , 2.0 
 hz , 1h ) , 6.55 ( s , 2h ) , 5.995.85 ( m , 2h ) , 5.32 ( d , j = 6.6 hz , 1h ) , 5.18 ( d , j = 4.0 hz , 1h ) , 
 4.72 ( td , j = 7.0 , 5.3 hz , 1h ) , 4.56 ( d , j = 6.0 hz , 2h ) , 4.12 ( ddd , j = 5.5 , 3.9 , 
 1.9 hz , 1h ) , 3.99 ( q , j = 2.3 hz , 1h ) , 3.62 ( dt , j = 7.0 , 3.2 hz , 2h ) 3.453.35 ( obs .
m , 1h ) ; c 
 ( 126 mhz , dmso - d6 ) 153.01 , 151.48 , 150.25 , 
 149.17 , 141.70 , 131.27 , 130.86 , 129.66 , 129.58 , 128.05 , 117.38 , 87.00 , 
 86.30 , 71.53 , 71.38 , 62.23 , 44.76 . hrms ( esi ) c17h19cl2n6o4
( m + h ) requires 442.0867 , found 442.0859 . to a solution of 8-bromoadenosine ( 0.073 g , 0.21 
 mmol ) in n - butanol ( 4.2 ml )
was added ( 4-methoxyphenyl)methanamine 
 ( 0.083 ml , 0.63 mmol ) and pr2net ( 0.22 ml , 
 1.3 mmol ) , and the mixture was heated to 120 c for 12 
 h. after this time , the mixture was cooled to room temperature and 
 the solvent removed under reduced pressure .
the resulting residue 
 was purified by silica gel chromatography eluting with etoac : meoh 
 ( 95:5 ) to give the desired product as a white solid ( 0.049 g , 58% ) ; 
 h ( 500 mhz , meod ) 7.98 ( s , 1h ) , 7.32 ( d , j = 8.8 hz , 2h ) , 6.89 ( d , j = 8.8 hz , 2h ) , 6.07 ( d , j = 7.6 hz , 1h ) , 4.78 ( dd , j = 7.6 , 5.4 
 hz , 1h ) , 4.60 ( d , j = 15.3 hz , 2h ) , 4.55 ( d , j = 15.3 hz , 1h ) , 4.28 ( dd , j = 5.6 , 1.7 
 hz , 1h ) , 4.16 ( q , j = 1.9 hz , 1h ) , 3.83 ( dd , j = 12.0 , 2.3 hz , 1h ) , 3.78 ( s , 3h ) , 3.77 ( obs .
dd , j = 11.9 , 1.7 hz , 1h ) ; c ( 126 mhz , meod ) 158.96 , 
 152.09 , 151.98 , 149.63 , 148.59 , 130.63 , 127.94 , 116.59 , 113.49 , 87.27 , 
 86.38 , 71.61 , 71.48 , 61.73 , 54.26 , 45.05 . hrms ( esi ) c18h22n6nao5 ( m + na ) requires 
 425.1544 , found 425.1540 . all surface plasmon 
 resonance ( spr ) experiments
were carried out 
 on a biacore t100 enhanced to t200 sensitivity ( ge life sciences , 
 amersham place , uk ) .
amine coupling chemistry was used to immobilize 
 the truncated hsc70-nbd domain ( residues 1381 ) on a research 
 grade cm5 sensor chip .
the running buffer used in the immobilization 
 step consisted of 1 phosphate buffered saline ( 10 mm nahpo4/nah2po4 ph 7.4 , 2.7 mm kcl , 137 mm 
 nacl ) , and the chip surface was activated for 10 min using a 1:1 mixture 
 of 100 mm n - hydroxysuccinimide and 400 mm 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide . 

hsc70-nbd was injected at a concentration of 70 g / ml in a 10 
 mm acetate buffer ph 5.0 with 750 m adp as protection for the 
 active site lysines .
the reaction was monitored in real time and stopped 
 when a target immobilization level of 5000ru was obtained . 

finally , the surface was blocked via an injection of 1 m ethanolamine 
 at ph 8.5 for 7 min .
the flow rate was maintained at 10 l / min 
 for all the above procedures .
flow cell one was left unmodified and 
 used as the reference surface . following protein immobilization , 
 the running buffer
was changed to 1 phosphate buffered saline 
 containing 0.05% ( v / v ) tween20 and 5% ( v / v ) dmso in order to reduce 
 the nonspecific binding and increase solubility of the compounds .
all liquid handling was carried out using an echo 550 acoustic 
 liquid dispenser ( labcyte , dublin , ireland ) , and compounds were added 
 to 384-well polypropylene v - bottomed plates ( greiner , stonehouse , 
 uk ) , which were used as sample plates for the spr experiments . for 
 each compound ,
an eight - point dose response experiment was carried 
 out using a concentration range of 502000 m , 5200 
 m or 0.06252.5 m , depending on the potency of 
 the compound .
the experiments were performed at a flow rate of 30 l / min , 
 a sample injection time of 60 s , and a dissociation time of 120 s. 
 the cm5 surface was not regenerated between sample injections .
kd values were calculated from the 
 background normalized binding curve generated from the sensorgrams 
 under equilibrium conditions , using the 1:1 binding model in the biacore 
 software version 2 ( ge life sciences , amersham place , uk ) .
all results 
 are reported as the geometric mean  standard error of the mean 
 ( sem ) of at least three independent measurements unless otherwise 
 stated .
the 
 human hspa1a ( hsp72 ) gene was amplified from the image clone 
 3345864 ( accession no .
bc002453 ) by pcr using the respective forward 
 and reverse primers 5-gatcgaccatatggccaaagccgcggcga-3 
 and 5-acagaattcctaatctacctcctcaatgg-3. 
 the hspa1a gene was cloned into a ptwoe vector , which is a modified 
 version of pet-17b ( merck chemicals ltd . , nottingham , uk ) encoding 
 a n - terminal 6xhis - tag followed by a human rhinovirus 3c protease 
 cleavage site .
bl21-ai cells ( invitrogen , paisley , uk ) transformed 
 with the vector containing the hspa1a gene were grown in luria 
bertani 
 medium to an optical density at 600 nm of 0.6 and induced with 0.5 
 mm isopropyl--d-1-thiogalactopyranoside and 0.2% ( w / v ) 
 arabinose at for 16 h at 20 c .
cells were harvested by centrifugation 
 at 6000 rpm for 40 min at 4 c using an avanti centrifuge j-26xp 
 ( beckman coulter , high wycombe , uk ) with a jla 8.100 rotor .
cell pellets 
 were resuspended in 3 volumes of lysis buffer consisting of 25 mm 
 tris , 50 mm nacl , 5% ( v / v ) glycerol , 1 complete edta - free protease 
 inhibitors ( roche , basel , switzerland ) , 25 u / ml benzonase nuclease 
 ( merck chemicals ltd . ) at ph 7.5 .
cell lysis was performed by sonication 
 using a vibra - cell vcx500 ( sonics & materials inc . ,
newtown , ct , 
 usa ) with a 13 mm solid probe for 24 cycles of 5 s on , 55 s off with 
 amplitude set at 50% .
the lysate was clarified by centrifugation at 
 20000 rpm for 30 min at 4 c using an avanti centrifuge j-26xp 
 ( beckman coulter ) with a ja 25.50 rotor .
the supernatant was 
 passed through a 1.2 m syringe filter ( sartorius stedim , germany ) 
 and loaded onto a 5 ml histrap ff column ( ge healthcare , chalfront 
 st . giles , uk ) equilibrated in buffer a , comprising 25 mm tris , 50 
 mm nacl , ph 7.5 , and eluted with a gradient of 0100% buffer 
 b ( buffer a + 250 mm imidazole ) over 10 column volumes ( cv ) .
fractions 
 containing hsp72 were pooled , concentrated , and loaded onto a superdex 
 200 ( 16/60 ) size exclusion column ( ge healthcare ) equilibrated in 
 25 mm tris , 400 mm nacl , 2 mm edta 5% ( v / v ) glycerol , ph 7.5 , for 
 further purification .
fractions containing hsp72 were pooled and further 
 purified to remove contaminating nucleotides using a 6 ml resource 
 q column ( ge healthcare ) equilibrated in 20 mm tris , 2 mm edta , 5% 
 ( v / v ) glycerol , ph 7.5 . following a 10 cv wash with the same buffer , 

hsp72 was eluted using a gradient from 0 to 500 mm nacl over 6 cv 
 and loaded onto a superdex 200 16/60 column ( ge healthcare ) equilibrated 
 in a buffer containing 25 mm tris , 400 mm nacl , 15 mm edta , 5% ( v / v ) 
 glycerol , ph 7.5 .
the removal of contaminating nucleotides was followed 
 by measuring the ratio of absorbance at 260 and 280 nm ( a260/a280 ) using a nanodrop 
 nd-1000 uv spectrophotometer ( thermofisher , wilmington , de , usa ) . 
 samples with a260/a280 below 0.6 were regarded as nucleotide free .
purified 
 hsp72-nbd protein was thawed ; buffer exchanged into fresh 100 mm hepes 
 ph 7.5 and incubated with 5 mm of the inhibitor for 30 min on ice 
 prior to crystallization .
/ inhibitor co - crystals were grown 
 at 18 c in sitting drops by mixing equal volumes of protein 
 solution ( 512 mg / ml ) and precipitant solution containing 1728% 
 ( v / v ) peg3350 , 0.1 m hepes ph 7.5 , 2 mm mgcl2 , and 2 mm 
 nah2po4 .
co - crystals of approximate dimensions 
 100  100  300 m typically formed overnight . 

crystals were in liquid nitrogen , using 22.5% ( v / v ) ethylene glycol 
 for the co - crystals with sangivamycin 10 and 25% ( v / v ) 
 glycerol for compound 17 .
x - ray diffraction data 
 were collected at 100 k at diamond light source ( oxfordshire , uk ; 
 beamlines i041 and i24 ) .
all 
 data were imported to mtz format with pointless , then scaled and merged 
 with aimless and the ccp4 suite .
the structures were solved by molecular 
 replacement with phaser , with the public domain hsp72-nbd structure 
 1s3x as the search model after removal of all nonprotein atoms .
structures 
 were refined in iterative cycles of model building with coot and refinement 
 with buster .
the data collection and refinement 
 statistics are presented in supporting information table s15 , and for fo  fc electron density figures for all structural 
 data , see supporting information figure s14 .
 hsp70 is a molecular 
 chaperone and a key component of the heat - shock 
 response . because of its proposed importance in oncology 
 , this protein 
 has become a popular target for drug discovery , efforts which have 
 as yet brought little success . 
 this study demonstrates that adenosine - derived 
 hsp70 inhibitors potentially bind to the protein with a novel mechanism 
 of action , the stabilization by desolvation of an intramolecular salt - bridge 
 which induces a conformational change in the protein , leading to high 
 affinity ligands . 
 we also demonstrate that through the application 
 of this mechanism , adenosine - derived hsp70 inhibitors can be optimized 
 in a rational manner . 
Introduction
Results
and Discussion
Conclusions
Experimental Section