more than 5 million 
 people in the united states and 24 million worldwide are affected 
 by alzheimer s disease ( ad ) , a disease characterized by memory 
 loss and neurodegeneration . insoluble aggregates of the 42 
 and 40 amino acid long amyloid  peptides ( a42 and a40 , respectively ) are believed to be intimately 
 involved in the onset of ad . however , while amyloid plaques , the ultimate 
 product of a aggregation , have been proposed to promote neurodegeneration 
 and dementia , recent studies have shown 
 that the soluble a oligomers are more neurotoxic both in vitro 
 and in vivo . in addition , while a40 is present in larger amounts 
 in amyloid plaques , the longer and more hydrophobic a42 isoform has a higher tendency to generate neurotoxic oligomeric 
 species . interestingly , unusually high concentrations 
 of copper , iron , and zinc have been found within the amyloid deposits 
 in ad - affected brains . these metal ions 
 have been shown to affect a aggregation , as well as lead to 
 the formation of reactive oxygen species ( ros ) . although many studies have investigated the involvement 
 of metal ions in a aggregation and plaque formation , the molecular 
 mechanisms of metal a species interactions , especially 
 for the more neurotoxic a42 , are still not completely 
 understood . exley et al . have used fluorescence assays and transmission 
 electron microscopy ( tem ) to show that both sub- and superstoichiometric 
 concentrations of cu prevent aggregation of a42 into thioflavin t ( tht)-positive -sheet - rich amyloid 
 fibrils . we have also recently reported that cu ions slow down fibrillization of the a42 peptide 
 and stabilize the soluble a42 species , as observed 
 by native gel electrophoresis / western blotting , tem , cell viability 
 studies , and pulsed hydrogen / deuterium exchange mass 
 spectrometry studies . in this regard , 
 metal chelators have been shown to reduce metal - mediated a 
 aggregation , ros formation , and neurotoxicity in vitro . recent efforts to control abnormal ametal interactions 
 have focused on small molecules , bifunctional chelators ( bfcs ) , that 
 have binding affinity for both metal ions and a aggregates . however , the majority of these 
 bfcs have been studied for their effect on aggregation of the a40 peptide , and fewer studies have focused 
 on their interaction with the more neurotoxic a42 peptide . compounds that inhibit metal - mediated 
 a40 aggregation or promote disaggregation of amyloid 
 fibrils were shown to lead to increased cell viability . however , this approach may not 
 be optimal for the a42 peptide , given the increased 
 toxicity observed for soluble a42 oligomers . our current efforts are aimed at the development of bfcs that control 
 the metal neurotoxicity in vivo and take 
 into consideration the formation of neurotoxic soluble a42 oligomers and their proposed role in ad neuropathogenesis . 
 reported herein are two new bfcs , 2-[2-hydroxy-4-(diethylamino)phenyl]benzothiazole 
 ( l1 ) and 2-(2-hydroxy-3-methoxyhenyl)benzothiazole ( l2 ) , that were 
 designed following a previously reported strategy of incorporating 
 metal - binding atoms into the structural framework of an a-interacting 
 compound . l1 and l2 
 have a modified 2-(2-hydroxyphenyl)benzothiazole ( hpb ) molecular framework 
 reminiscent of the amyloid - binding fluorescent dye tht ( scheme 1 ) , which was shown to be promising in the design 
 of new a-binding compounds . the bifunctional character of the two compounds was confirmed 
 by metal - chelating and a-binding studies . the cu complexes of l1 and l2 were isolated and characterized spectroscopically 
 and by x - ray structural determination . the amyloid - binding properties 
 of l1 and l2 were explored by fluorescent titrations and by tht competition 
 assays . while l1 exhibits an acceptable binding affinity for a 
 fibrils , with the higher affinity binding site corresponding to a ki of 260 nm , l2 binds very strongly to a 
 fibrils with a ki of 15 nm . in addition , 
 l1 and l2 moderately inhibit a42 aggregation in 
 the absence of metal ions , yet they promote aggregation in the presence 
 of metal ions , as observed by native gel / western blotting and tem . in addition , l1 and l2 were able to reduce the cell toxicity of preformed 
 a42 oligomers as well as that of soluble copper - stabilized 
 a42 oligomers , which were shown recently to be neurotoxic . these promising properties of l1 and l2 make them suitable candidates 
 for further in vivo investigation . all reagents were purchased from commercial 
 sources and used as received unless stated otherwise . solvents were 
 purified prior to use by passing through a column of activated alumina 
 using an mbraun solvent purification system . all solutions and buffers 
 were prepared using metal - free millipore water that was treated with 
 chelex overnight and filtered through a 0.22 m nylon filter . h ( 300.121 mhz ) and c ( 75 mhz ) nmr spectra were 
 recorded on a varian mercury-300 spectrometer . chemical shifts are 
 reported in ppm and referenced to residual solvent resonance peaks . uv  vis spectra were recorded on a varian cary 50 bio spectrophotometer 
 and are reported as max , nm (  , m cm ) . electrospray ionization mass spectrometry 
 ( esi - ms ) experiments were performed using a bruker m - axis qtof mass 
 spectrometer with an esi source . esi - ms was provided by the washington 
 university mass spectrometry nih resource ( grant p41rr0954 ) , and elemental 
 analyses were carried out at intertek chemical and pharmaceuticals 
 testing and analysis services . suitable crystals of appropriate dimensions were mounted in a bruker 
 kappa apex ii ccd x - ray diffractometer equipped with an oxford cryostream 
 lt device and a fine - focus mo k radiation x - ray source (  
 = 0.71073  ) . preliminary unit cell constants were determined 
 with a set of 36 narrow frame scans . typical data sets consist of 
 combinations of  and  scan frames with a typical scan 
 width of 0.5 and a counting time of 1530 s / frame at 
 a crystal - to - detector distance of 4.0 cm . the collected frames 
 were integrated using an orientation matrix determined from the narrow 
 frame scans . apex ii and saint software 
 packages were used for data collection 
 and data integration . final cell constants were determined by the 
 global refinement of reflections from the complete data set . structure solutions and refinement 
 were carried out using the shelxtl - plus software 
 package . the structures were refined 
 with full - matrix least - squares refinement by minimizing w(fo  fc ) . all hydrogen atoms 
 were added in the calculated position and refined using appropriate 
 riding models ( afix m ) . vis 
 ph titrations were employed for determination of the acidity constants 
 of l1 and l2 and their stability constants with cu and 
 zn . for the acidity constants , solutions of bfcs ( 25 
 m for l1 and 50 m for l2 , 0.1 m nacl , ph 3 ) were titrated 
 with small aliquots of 0.1 m naoh at room temperature . at least 30 
 uv  vis spectra were collected in the ph 311 range . because of the limited solubility of l1 and l2 in water , methanol 
 ( meoh ) stock solutions ( 10 mm ) were used and titrations were performed 
 in a meoh water mixture in which meoh did not exceed 20% ( v / v ) . 
 similarly , the stability constants for the l1copper system 
 were determined by titrating a solution of l1 ( 25 m ) and cu(clo4)26h2o ( 12.5 m ) with small 
 aliquots of 0.1 m naoh at room temperature . the acidity and 
 stability constants were calculated using the hypspec computer program ( protonic software gmbh , u.k . ) . speciation plots of the compounds and their metal complexes 
 were calculated using the program hyss2009 ( protonic 
 software gmbh , u.k . ) . a monomeric films were prepared by dissolving 
 commercial a42 ( or a40 for a-fibril - binding 
 studies ) peptide ( keck biotechnology resource laboratory , yale university ) 
 in hfip ( 1 mm ) and incubating for 1 h at room temperature . this solution was then aliquoted out , and hfip 
 was allowed to evaporate overnight . the aliquots were vacuum - centrifuged , 
 and the resulting monomeric films were stored at 80 c . a fibrils were generated by dissolving monomeric a films 
 in dimethyl sulfoxide ( dmso ) , diluting into the appropriate buffer , 
 and incubating for 24 h at 37 c with continuous agitation ( the 
 final dmso concentration was < 2% ) . for metal - containing fibrils , 
 the corresponding metal ions were added before initiation of the fibrillization 
 conditions . for inhibition studies , bfcs ( 50 m , dmso stock 
 solutions ) were added to a solutions ( 25 m ) in the absence 
 or presence of metal salts ( cucl2 or zncl2 , 
 25 m ) and incubated for 24 h at 37 c with constant agitation . 
 for disaggregation studies , the preformed a fibrils in the 
 absence or presence of metal ions were treated with bfcs and further 
 incubated for 24 h at 37 c with constant agitation . for the 
 preparation of soluble a42 oligomers , a literature 
 protocol was followed . a monomeric film of a42 was dissolved in anhydrous dmso , followed by the addition 
 of dmem - f12 media [ 1:1 ( v / v ) , without phenol red ; invitrogen ] . the 
 solution ( 50100 m ) was incubated at 4 c for 24 
 h and then centrifuged at 10000 g for 10 min . all fluorescence 
 measurements were performed using a spectramax m2e plate reader ( molecular 
 devices ) . for tht fluorescence studies , samples were diluted to a 
 final concentration of 2.5 m a in phosphate - buffered 
 saline ( pbs ) containing 10 m tht and the fluorescence was measured 
 at 485 nm ( ex = 435 nm ) . for a-fibril - binding 
 studies , a 5 m a fibril solution was titrated with small 
 amounts of compound and their fluorescence intensity measured ( ex/em = 330/450 nm ) . for tht competition 
 assays , a 5 m a fibril solution with 2 m tht 
 was titrated with small amounts of compound and the tht fluorescence 
 measured ( ex/em = 435/485 nm ) . 
 for calculation of ki values , a kd value of 1.17 m was used for the binding 
 of tht to a40 fibrils . glow - discharged grids ( formar / carbon 300 mesh , electron 
 microscopy sciences ) were treated with a samples ( 25 m , 
 5 l ) for 23 min at room temperature . excess solution 
 was removed using filter paper , and grids were rinsed twice with water 
 ( 5 l ) . grids were stained with uranyl acetate ( 1% w / v , water , 
 5 l ) for 1 min , blotted with filter paper , and dried for 15 
 min at room temperature . images were captured using a fei g2 spirit 
 twin microscope ( 6080 kv , 650097000 magnification ) . all gels , 
 buffers , membranes , and other reagents were purchased from invitrogen 
 and used as directed except where otherwise noted . the gel was transferred 
 to a nitrocellulose membrane in an ice bath , and the protocol was 
 followed as suggested except that the membrane was blocked overnight 
 at 4 c . after blocking , the membrane was incubated in a solution 
 ( 1:2000 dilutions ) of 6e10 anti - a primary antibody ( covance ) 
 for 3 h. invitrogen s western breeze chemiluminescent kit was 
 used to visualize the bands . an alkaline / phosphatase antimouse secondary 
 antibody was used , and the protein bands were imaged using a fujifilm 
 las-1000ch luminescent image analyzer . mouse neuroblastoma neuro2a ( n2a ) cell lines 
 were purchased from the american type culture collection ( atcc ) . cells 
 were grown in dmem/10% fbs , which is the regular growth media for 
 n2a cells . n2a cells were plated to each well of a 96-well plate ( 2.5 
  10/well ) with dmem/10% fbs . after 1 h , the reagents ( 20 m 
 a42 species , compounds , and metals ) were added . 
 because of the poor solubility of the compounds in water or media , 
 the final amount of dmso used was 1% ( v / v ) . after an additional incubation 
 of 40 h , the alamar blue solution was added in each well and the cells 
 were incubated for 90 min at 37 c . the absorbance was measured 
 at 570 nm ( control optical density = 600 nm ) . for these toxicity studies , three types of a42 species were tested : freshly 
 made monomeric a42 ( ma42 ) , a42 oligomers ( oa42 ) , and a42 fibrils ( fa42 ) . these a42 species 
 were prepared as described above . a mixture of 2-aminothiophenol ( 2.5 g , 20 mmol ) and ( diethylamino)salicylaldehyde 
 ( 3.86 g , 20 mmol ) in dmso ( 20 ml ) was heated with stirring at 150 
 c for 6 h under dinitrogen . then it was cooled to 0 c 
 for 8 h , and the orange solid crystallized out , was collected by filtration , 
 and was washed with hexane ( 2.97 g , yield 50% ) . h nmr 
 ( cdcl3 ) :  7.85 ( d , 1h , arh ) , 7.81 ( d , 1h , arh ) , 
 7.46 ( d , 1h , arh ) , 7.43 ( d , 1h , arh ) , 7.30 ( d , 1h , phenol h ) , 6.30 
 ( d , 1h , phenol h ) , 6.27 ( s , 1h , phenol h ) , 3.41 ( q , 2h , ch2ch3 ) , 1.22 ( t , 3h , ch2ch3 ) . c nmr ( cdcl3 ) :  
 169.78 , 159.98 , 152.43 , 151.52 , 132.08 , 130.04 , 126.42 , 124.35 , 121.40 , 
 121.18 , 105.96 , 104.34 , 98.053 , 44.72 , 12.86 . vis [ max , nm , (  , m cm ) ] : in mecn , 262 ( 10300 ) , 380 ( 43000 ) ; in pbs , 272 ( 9500 ) , 375 ( 18000 ) , 
 405 ( 19500 ) . calcd for [ m + h ] : m / z 299.1218 . found : m / z 299.1225 . a mixture of o - vanilin ( 2.0 g , 0.0131 mol ) and 2-aminothiophenol ( 1.64 
 g , 0.0131 mol ) in ethanol ( 15 ml ) was refluxed with stirring for 24 
 h. then it was cooled to room temperature and poured in water . the 
 sticky yellow solid was crystallized from meoh to obtain yellow crystals 
 ( 1.15 g , yield 37% ) . h nmr ( cdcl3 ) :  
 8.02 ( d , 1h , arh ) , 7.91 ( d , 1h , arh ) , 7.52 ( t , 1h , arh ) , 7.42 ( t , 
 1h , arh ) , 7.33 ( d , 1h , arh ) , 7.01 ( d , 1h , arh ) , 6.99 ( t , 1h , arh ) , 
 3.97 ( s , 3h , och3 ) . c nmr ( cdcl3 ) :  169.28 , 151.50 , 148.93 , 148.19 , 148.17 , 132.59 , 126.70 , 
 125.56 , 122.16 , 121.45 , 119.98 , 119.14 , 116.71 , 114.06 , 56.22 . vis 
 [ max , nm , (  , m cm ) ] : in mecn , 307 ( br , 10200 ) , 340 ( sh , 5500 ) ; in pbs , 
 305 ( 3500 ) , 340 ( sh , 1800 ) , 390 ( 800 ) . hrms . calcd for [ m + h ] : m / z 258.0588 . found : m / z 258.0584 . a solution of cucl22h2o ( 0.011 g , 0.084 
 mmol ) in mecn ( 2 ml ) was added to the stirring mixture of l1 ( 0.050 
 g , 0.167 mmol ) and et3n ( 0.036 g , 0.334 mmol ) in mecn ( 5 
 ml ) . the brown precipitate immediately obtained was filtered , washed 
 with ether , and dried under vacuum ( 0.040 g , yield 54% ) . vis 
 [ max , nm , (  , mcm ) ] : in mecn , 273 ( sh , 18000 ) , 391 ( 56800 ) , 404 ( sh , 
 50500 ) , 540 ( 900 ) . hrms . calcd for [ m + h ] : m / z 658.1497 . found : m / z 658.1492 . calcd for c34h34n4o2s2cu0.5h2o : c , 61.52 ; h , 
 5.25 ; n , 8.44 . found : c , 61.63 ; h , 6.11 ; n , 8.44 . a solution of zncl2 ( 0.011 g , 0.084 
 mmol ) in mecn ( 2 ml ) was added to the stirring mixture of l4 ( 0.050 
 g , 0.167 mmol ) and et3n ( 0.018 g , 0.167 mmol ) in mecn ( 5 
 ml ) . the red solution was kept for slow evaporation , and a few single 
 crystals were obtained within 2 days ( 0.052 g , yield 58% ) . vis 
 [ max , nm , (  , mcm ) ] : in mecn , 265 ( 19900 ) , 391 ( 51000 ) . calcd 
 for [ ( l1)zncl + h ] : m / z 397.01 . found : m / z 397.01 . satisfactory 
 elemental analysis data could not be obtained because of the presence 
 of a small amount of free ligand . a solution of cucl22h2o ( 0.013 g , 0.097 
 mmol ) in mecn ( 2 ml ) was added to the stirring mixture of l2 ( 0.050 
 g , 0.194 mmol ) and et3n ( 0.078 g , 0.776 mmol ) in meoh ( 10 
 ml ) . the brown precipitate immediately obtained was filtered , washed 
 with ether , and dried under vacuum ( 0.051 g , yield 91% ) . vis 
 [ max , nm , (  , mcm ) ] : in mecn , 398 ( 26200 ) , 515 ( sh , 800 ) , 700 ( 350 ) . calcd for [ m + na ] : m / z 598.0 . found : m / z 598.1 . calcd for c28h20n2o4s2cu0.5h2o : c , 57.47 ; h , 3.62 ; n , 4.79 . 
 found : c , 57.34 ; h , 3.62 ; n , 4.83 . herein we employed 
 the strategy of developing metal bfcs with amyloid - binding properties 
 as potential therapeutic agents for ad . the fluorescent dye tht is 
 well - known for its amyloid - binding properties because of the 2-phenylbenzothiazole 
 aromatic framework . previously , an iodinated hpb and a few related 
 compounds were shown to have potential as metal - chelating agents by 
 rodrguez - rodrguez et al . however , in that report , only the solution coordination properties 
 of those compounds were investigated in detail , while the a-binding 
 and antiaggregation properties were only qualitatively evaluated through 
 epifluorecense microscopy and turbidity assays . in contrast , in addition 
 to the solution coordination chemistry , we have also determined the 
 solid - state structure of the copper and zinc complexes of our compounds , 
 and we have also quantitatively determined their a-binding 
 affinities through fluorescence titrations . in addition , we have used 
 both tem and gel electrophoresis / western blotting to provide a complete 
 picture of the effect of our compounds on a aggregation and 
 formation of various sized a aggregates , in both the absence 
 and presence of metal ions . most importantly , we have correlated the 
 in vitro studies with cellular toxicity studies , thus making this 
 work a comprehensive study of these compounds in the context of developing 
 new compounds that can control the toxicity and aggregation of various 
 a species . we have also recently reported 2-phenylbenzothiazole / vanillin 
 derivatives as bfcs that showed a strong affinity for a fibrils . herein we designed two new molecules , l1 and 
 l2 , that contain a 2-phenylbenzothiazole moiety for a recognition 
 and oxygen- and nitrogen - atom donors needed for metal chelation . synthesis 
 of l1 and l2 was accomplished upon oxidative cyclization of the corresponding 
 arylaldehyde and 2-aminothiophenol ( scheme s1 in the si ) . these compounds exhibit 
 uv absorption in mecn at 260 and 380 nm for l1 and at 305 and 345 
 nm for l2 ( figures s1 and s2 in the si ) . while both l1 and l2 are 2-phenylbenzothiazole 
 derivatives , l1 exhibits low - intensity emission at 450 nm and a shoulder 
 at 525 nm , yet l2 shows a more intense emission at 525 nm in pbs ( figures 
 s3 and s4 in the si ) . the parent molecule hpb has emission properties similar 
 to those of l2 ( figure s4 in the si ) , which 
 suggests that having a diethylamino group in l1 strongly affects its 
 emission properties . another important aspect of the design 
 of new drug molecules for central nervous system diseases is their 
 ability to cross the blood brain barrier ( bbb ) . both l1 and l2 are small in size , and they 
 also satisfy lipinski s rules for crossing the bbb ( table 1 ) . the ability of a compound to cross the bbb can 
 be evaluated by determining the blood  brain partitioning parameter 
 log bb that is determined by taking into account the molecular weight 
 of the molecule , charge , lipophilicity , and its hydrogen - bonding capability . molecules having log bb values higher than 0.3 are generally 
 expected to cross the bbb . the calculated 
 log bb values for l1 and l2 are + 0.33 and + 0.087 , respectively , suggesting 
 that these bfcs are expected to cross the bbb . mw = molecular weight ; c log p = calculated octanol  water partition coefficient ; 
 hbd = hydrogen - bonding donor atoms ; hba = hydrogen - bonding acceptor 
 atoms ; psa = polar surface area ; log bb = 0.0148psa + 0.152 c log p + 0.130 ( lipinski , c. a. ; lombardo , 
 f. ; dominy , b. w. ; feeney , p. j. adv . drug delivery rev.1997 , 23 , 325 ; clark , d. e. ; pickett , s. d. drug discovery today2000 , 5 , 4958 ) . because both l1 and l2 contain functional groups that 
 can undergo protonation , the acidity constants ( pka ) were determined by uv  vis spectrophotometric 
 titrations . for both l1 and l2 , uv  vis titrations from ph 3.0 
 to 11.0 reveal several changes in the spectra ( figures 1 and 2 ) . for l1 , the best fit to the 
 data was obtained with pka values of 3.556(2 ) , 
 8.427(3 ) , and 10.108(2 ) ( table 2 ) . the first 
 and second pka values can be assigned 
 to deprotonation of the nitrogen atoms of the benzothiazole and diethylamino 
 groups , while the largest pka value is 
 likely due to phenol deprotonation . for l2 , the 
 best fit to the data was obtained with two pka values of 3.154(2 ) and 9.360(1 ) ( table 2 ) . these pka values can be assigned to 
 deprotonation of the nitrogen atoms of the benzothiazole and phenol 
 groups , respectively . variable - ph uv spectra of l1 ( 25 m , 25 c , i = 0.1 m nacl ) and the species distribution plot . 25 
 c , i = 0.1 m nacl ) and the species distribution 
 plot . the bfcs l1 and l2 were explored for their 
 ability to chelate metal ions . the stoichiometry of l1cu and l1zn complexes in solution was 
 determined by job s plot analysis . for l1cu , a break in the plot at 0.4 mole fraction 
 of cu suggests the formation of both 1:1 and 2:1 l1cu complexes in solution ( figure s4 in the si ) . for l1zn , the break occurs at 0.3 mole fraction of zn , suggesting the formation of a 2:1 l1zn complex 
 ( figure s5 in the si ) . by contrast to l1 , no job s plot analysis could 
 be performed for l2cu because of the limited 
 solubility of this complex , while no significant spectral change was 
 observed for the l2zn system . the cu complexes of l1 or l2 were synthesized by reacting cucl22h2o with the corresponding ligand in mecn in the 
 presence of an equivalent amount of et3n . the 2:1 complex 
 formation was confirmed by the esi - ms signal at m / z 658.1 corresponding to [ m + h ] , which 
 was further supported by x - ray structural determination ( see below ) . d transition at 700 nm and a 
 more intense band at 540 nm , while a phenolate - to - cu charge - transfer 
 band is observed at 400 nm ( figure s6 in the si ) . similarly , the uv  vis 
 spectrum of the l2cu complex 3 shows 
 characteristic d d transition bands at 700 and 515 nm as well 
 as a phenolate - to - cu charge - transfer band at 400 nm ( figure 
 s7 in the si ) . although a single crystal suitable for x - ray crystallography could 
 be obtained from the reaction mixture of l1 and zn , the 
 isolation of the zinc complexes of l1 or l2 did not lead to pure complexes 
 to allow for their complete characterization . spectrophotometric 
 titrations were also performed to determine the stability constants 
 and solution speciation of cu with l1 . the pka values of the ligands were included in the calculations , and the best fit to the data shows that l1 binds 
 cu with a stability constant of 11.99(2 ) for the m + 
 l = ml equilibrium and a value of 20.99(2 ) for the ml + l = ml2 equilibrium ( l represents the deprotonated form of l1 ) . in addition , the concentration of free cu ( pcu = log [ cufree ] ) can be determined 
 at specific ph values and total ion concentrations , with pcu values 
 of 6.7 and 8.2 being determined for the cu these pcu values are lower compared to 
 those for the analogous hpb compound reported previously , likely because of the strong electron - withdrawing 
 effect of the diethylamine group upon protonation . unfortunately , 
 the spectrophotometric titration of l2 in the presence of cu revealed few spectral changes at micromolar concentrations , while 
 the use of higher concentrations led to precipitation of the l2cu complex . however , it is expected that l2 exhibits a higher 
 binding affinity to cu than l1 because of the electron - donating o - methoxy group present in l2 . in addition , almost no changes 
 were observed in the spectra of both l1 and l2 upon the addition of 
 zn ; therefore , the corresponding stability constants 
 could not be determined . attempts were then made to determine the 
 apparent stability constants for the zn  l1 and zn a 
 interaction studies and exhibits a kd  
 10 m for zinc . in our case , the 
 addition of zincon to solutions of zn  l1 and zn in addition , l1 and l2 do not compete significantly with zincon for 
 zinc binding , even when present in large excess versus zincon , suggesting 
 that they exhibit affinities for zinc that are at least 10 times lower 
 than that of zincon . variable - ph uv spectra of the l1cu system ( [ l1 ] = 25 m ; [ cu ] = 12.5 m , 25 
 c , i = 0.1 m nacl ) and the species distribution 
 plot . to further characterize the metal - binding properties 
 of compounds l1 and l2 , single - crystal x - ray structures were determined 
 for complexes 13 ( figure 4 and tables s1s3 in the si ) . complex 1 affords 
 single crystals by et2o diffusion into a ch2cl2 solution of 1 that crystallized out in 
 the c2/c space group . an oxygen 
 atom and a nitrogen atom from two ligand molecules n1 bond lengths are 1.980(3 ) 
 and 1.876(2 )  , respectively , and the trans angles o1cu1o1#1 
 and n1#1cu1n1 are 153.3(2) and 154.2(2) , 
 respectively , with these parameters suggesting a distorted square - planar 
 geometry for cu . for complex 2 , the zn ion exhibits a distorted tetrahedral geometry and is coordinated 
 to the benzothiazole nitrogen and phenol oxygen atoms of l1 and two 
 chloride ions . the copper complex 3 afforded single crystals 
 by et2o diffusion into a ch2cl2 solution 
 and has structural properties similar to those of 1 , 
 yet it crystallized out in the p2/n space group . as shown for 1 , in 3 an oxygen 
 atom and a nitrogen atom from two ligand molecules coordinate to cu . n1 bond lengths are 1.873(4 ) 
 and 1.965(4 )  , respectively , and the trans angles o1cu1o1#1 
 and n1#1cu1n1 are 145.1(2) and 146.2(2) , 
 respectively , reflecting a distorted square - planar geometry for cu . all hydrogen atoms , counterions , 
 and solvent molecules are omitted for clarity . selected bond distances : 1 , cu1n1 1.980(3 ) , cu1o1 1.876(2 ) ; 2 , zn  n1 cl(2 ) 2.222(1 ) ; 3 , cu1n1/n2 1.965(4 ) , 
 cu1o1/o3 1.873(4 ) . both compounds 
 l1 and l2 contain a 2-phenylbenzothiazole fragment reminiscent of 
 the fluorescent dye tht used to detect the -sheet structure 
 of fibrillar a aggregates . in this context , 
 the affinity of l1 and l2 toward a fibrils was investigated 
 by fluorescence assays . these studies were performed with a40 fibrils , which form more homogeneous fibrillar structures 
 without any nonfibrillar aggregates . titration 
 of l1 with a fibrils was performed , and a saturation behavior 
 is observed . the data were fitted with a one - site binding model to 
 give a kd of 4.70  0.30 m 
 ( figure 5a ) , which suggests a moderate affinity 
 of l1 for the a fibrils . tht fluorescence competition assays 
 were also performed by the addition of l1 to a solution of a 
 fibrils in the presence of tht . competitive - binding curve 
 fitting yields a ki value of 260  
 40 nm ( figure 5b ) . it should be noted that 
 the ki value for l1 is smaller than the kd value , which is most likely due to the fact 
 that tht binds with different affinities to more than one site to 
 the a fibrils . as such , we propose 
 that l1 replaces tht only from the higher affinity binding site . by 
 constrast , l2 did not show any significant increase in the fluorescence 
 intensity in the presence of a fibrils yet replaces tht from 
 the a fibrils and exhibits a ki value of 15  10 nm , indicating a very strong binding affinity 
 to a fibrils ( figure 5c ) . ( a ) fluorescence 
 titration assay of l1 with a fibrils ( [ a ] = 5 m ; 
 ex/em = 330/450 nm ) . tht fluorescence 
 competition assays of a fibrils with ( b ) l1 and ( c ) l2 ( [ a ] 
 = 2 m ; [ tht ] = 1 m ; ex/em = 435/485 nm ) . to investigate the effect of l1 and l2 on a aggregation , we 
 performed both inhibition and disaggregation studies ( scheme 3 ) . importantly , the a42 peptide 
 was employed in these studies because it was shown to form neurotoxic 
 soluble a oligomers . for inhibition experiments , freshly prepared a42 fibrils were treated with metal ions , bfcs , or both , and the reactions 
 was monitored by tht fluorescence , native gel electrophoresis / western 
 blotting analysis , and tem . in inhibition experiments , a reduced tht fluorescence 
 was observed for the cu- and zn - containing 
 fibrils , as reported previously ( figure 6 ) . in the absence of metal ions , the presence of l1 leads to a low tht fluorescence intensity , while 
 l2 has a negligible effect on a42 fibrillization , 
 suggesting that l1 can inhibit fibrillization more efficiently than 
 l2 . in the presence of cu and either l1 or l2 , a very 
 low tht fluorescence intensity is observed , which could be due to 
 either limited a42 fibril formation or fluorescence 
 quenching by the paramagnetic cu ions . by comparison , the presence of zn and either l1 or l2 shows almost no 
 effect on the tht fluorescence intensity ( figure 6 ) . because the tht fluorescence assays do not show a complete 
 picture of the effect of bfcs on a42 aggregation , 
 native gel electrophoresis / western blotting analysis and tem were 
 employed in order to characterize in more detail the various a42 species formed during these aggregation studies . as such , 
 the a42 peptide forms well - defined a42 fibrils , as confirmed by tem ( figure 7 , panel 1 ) , while a42 aggregation in the presence 
 of cu shows the formation of a dramatically reduced number 
 of a42 fibrils ( figure 7 , 
 panel 2 ) , thus supporting the low tht fluorescence intensity observed 
 above . by comparison , aggregation of a42 in the 
 presence of zn leads to the formation of large amorphous 
 aggregates ( figure 7 , panel 3 ) . native gel / western 
 blotting analysis shows that aggregation of a42 in 
 the absence of metal ions also forms high - molecular - weight oligomers , 
 along with the insoluble aggregates that are observed at the top of 
 the blot because they do not enter the gel ( figure 7 , lane 1 ) . as reported previously , the presence of cu ions inhibits the formation of large 
 soluble and insoluble a42 aggregates ( figure 7 , lane 2 ) . normalized tht fluorescence of the inhibition 
 of a fibrillization , measured upon incubaton at 37 c 
 for 24 h. samples are as indicated on top of the lanes ( pbs ; [ a ] 
 = 25 m ; [ m ] = 25 m ; [ compound ] = 50 m ) . top : tem images of the inhibition of a42 aggregation by l1 and l2 , in the presence or absence of 
 metal ions ( [ a42 ] = [ m ] = 25 m ; 
 [ compound ] = 50 m ; pbs , 37 c , 24 h , scale bar = 500 nm ) . panels 
 and lanes are as follows : ( 1 ) a42 ; ( 2 ) a42 + cu ; ( 3 ) a42 + zn ; ( 4 ) a42 + l1 ; ( 5 ) a42 + l1 
 + cu ; ( 6 ) a42 + l1 + zn ; ( 7 ) a42 + l2 ; ( 8) a42 + l2 
 + cu ; ( 9 ) a42 + l2 + zn ; ( 10 ) mw marker . the presence of either 
 l1 or l2 in the absence of metal ions does not seem to significantly 
 inhibit a42 aggregation , although some morphological 
 changes were observed for the a42 fibrils ( figure 7 , panels 4 and 7 ) ; the observed decrease in the 
 tht fluorescence intensity may thus be due to these morphological 
 changes of the a42 aggregates . native gel / western 
 blotting analysis reveals a limited effect of l1 and l2 on a42 aggregation ( figure 7 , lanes 4 and 
 7 ) . interestingly , the presence of either l1 or l2 has a dramatic 
 effect on the cu - mediated oligomerization of a42 and promotes the formation of larger a42 aggregates , as observed by tem ( figure 7 , 
 panels 5 and 8) . importantly , native gel / western blotting analysis 
 reveals bands at the top of the gel , suggesting the presence of insoluble 
 a42 aggregates ( figure 7 , 
 lanes 5 and 8) , which were not observed in the presence of cu alone ( figure 7 , lane 2 ) . thus , compounds 
 l1 and l2 reduce the amount of soluble a42 oligomers 
 formed in the presence of cu by promoting the formation 
 of insoluble a42 aggregates . in addition , the presence 
 of either l1 or l2 does not dramatically affect the formation of insoluble 
 a42 aggregates in the presence of zn ( figure 7 , panels 6 and 9 ) , which is supported 
 by native gel / western blotting analysis showing no formation of high - molecular - weight 
 soluble a42 oligomers , although insoluble aggregates 
 are observed at the top of the gel ( figure 7 , lanes 6 and 9 ) . overall , these studies suggest that the presence 
 of either l1 or l2 , in both the presence or absence of metal ions , 
 does not promote the formation of soluble a42 oligomers . 
 because the soluble a42 oligomers are the most neurotoxic 
 species , these compounds are expected to control the neurotoxicity 
 of a42 species in the presence of metal ions ( vide 
 infra ) . although it was shown in previous studies that the a42 peptide is 
 more prone to form neurotoxic soluble a oligomers than the 
 a40 peptide , we have also investigated the effect 
 of l1 and l2 on the metal - mediated aggregation of a40 . tem analysis shows that a40 forms well - defined 
 fibrils either in the absence or presence of l1 or l2 ( figure s9 in 
 the si , panels 1 and 4 ) . importantly , the 
 presence of cu and zn does not inhibit a40 aggregation , yet they change 
 the morphology of the insoluble aggregates that become more amorphous 
 ( figure s9 in the si , panels 2 and 3 ) . moreover , in the presence of cu and either l1 or l2 , 
 a small amount of a40 fibrils are still formed ( figure 
 s9 in the si , panels 5 and 8) , while the 
 presence of zn and either l1 or l2 leads to the formation 
 of a larger amount of fibrillar or amorphous aggregates , respectively 
 ( figure s9 in the si , panels 6 and 9 ) . the tem results are also supported by native gel electrophoresis / western 
 blotting analysis ( figure s10 in the si ) , although this is less informative than in the case of the a42 peptide because of the lack of formation of an appreciable 
 amount of soluble a oligomers under any experimental conditions . therefore , because we are particularly interested in evaluating the 
 role of metal ions and bifunctional compounds in controlling the formation 
 of the neurotoxic soluble a oligomers , the a42 peptide was employed in the additional studies described below . the effect 
 of l1 and l2 on preformed a42 fibrils was also investigated 
 in both the absence and presence of metal ions ( scheme 3 ) . for disaggregation studies , a42 fibrils 
 were prepared by incubation for 24 h at 37 c , in both the presence 
 and absence of metal ions . then , l1 or l2 was added and further incubated 
 for an additional 24 h at 37 c . tem and native gel electrophoresis / western 
 blotting characterization was performed on all of these samples . first , 
 either l1 or l2 in metal - free conditions does not disaggregate the 
 preformed a42 fibrils to an appreciable extent . only some morphological changes were observed even after 24 h of incubation , 
 and a significant amount of a42 aggregates were 
 still observed by tem ( figure 8 , panels 4 and 
 7 ) . native gel electrophoresis / western blotting analysis reveals high - molecular - weight 
 soluble a42 species and insoluble aggregates , although 
 to a lesser extent in the presence of l1 ( figure 8 , lanes 4 and 7 ) . in the presence of cu , either 
 l1 or l2 partially inhibits the cu - promoted formation 
 of small soluble aggregates and allows for enhanced a42 aggregation ( figure 8 , panels 5 and 8 vs 
 panel 2 ) . this is supported by native gel / western blotting , which 
 shows that while there is a slightly increased amount of low - molecular - weight 
 ( 1080 kda ) soluble a42 species , more insoluble 
 aggregates are also observed at the top of the gel ( figure 8 , lanes 5 and 8) . in the presence of zn , either l1 or l2 seems to have a limited effect on the morphology 
 of the insoluble a42 aggregates , as observed by 
 both tem and western blotting ( figure 8 , panels / lanes 
 6 and 9 ) . overall , all of these in vitro studies suggest that compounds 
 l1 and l2 should control the formation of cu - stabilized 
 soluble a42 species and thus should control their 
 neurotoxicity ( vide infra ) . top : tem images of the disaggregation of a42 aggregation by l1 and l2 , in the presence or absence of 
 metal ions ( [ a42 ] = [ m ] = 25 m ; 
 [ compound ] = 50 m ; pbs , 37 c , 24 h for fibrilization 
 and an additional 24 h for disaggregation , scale bar = 500 nm ) . panels and lanes 
 are as follows : ( 1 ) a42 ; ( 2 ) a42 + cu ; ( 3 ) a42 + zn ; 
 ( 4 ) a42 + l1 ; ( 5 ) a42 + l1 + cu ; ( 6 ) a42 + l1 + zn ; ( 7 ) a42 + l2 ; ( 8) a42 + l2 + cu ; 
 ( 9 ) a42 + l2 + zn ; ( 10 ) mw marker . a species have been shown to be neurotoxic , the development of metal - binding compounds 
 that control the metal - dependent a toxicity is desired . in 
 this regard , the effect of l1 and l2 on the a42 neurotoxicity 
 in n2a cells was investigated using an alamar blue cell viability 
 assay . first , we examined the toxicity 
 of both l1 and l2 along with ethylenediaminetetraacetic acid ( edta ) 
 and cq at various concentrations ranging from 0.2 to 20 m ( figure 
 s11 in the si ) . both l1 and l2 show no appreciable cell toxicity ( > 80% cell survival ) 
 even up to 20 m concentration . by comparison , the clinically 
 tested compound cq was quite toxic to cells even at 2 m concentration , 
 while edta does not show any toxicity up to 20 m . for the preformed a42 fibrils , no significant 
 cell toxicity was observed in either the absence or presence of cu ( figure 9 , lanes 1 and 2 ) , supporting 
 the previously reported diminished toxicity of a42 fibrils . moreover , compounds l1 and l2 do not affect the cell viability when 
 added to the n2a cells in the presence of preformed a42 fibrils ( figure 9 , lanes 3 and 4 ) . even in 
 the presence of preformed a42 fibrils and cu , the cell viability is not affected by either l1 or l2 ( figure 9 , lanes 5 and 6 ) . by contrast , cq shows quite high 
 cell toxicity to cells in the presence of cu and a42 fibrils with a cell survival of only 26  3% ( figure 9 , lane 7 ) . thus , these cellular toxicity data suggest 
 that l1 and l2 do not lead to any cell toxicity in the presence or 
 absence of a42 fibrils and cu ions , 
 suggesting that these compounds do not disaggregate a42 fibrils to generate neurotoxic soluble a42 species . as shown previously , the preformed soluble 
 a42 oligomers show a limited cell survival of 45 
  6% ( figure 9 , lane 8) . also , we have 
 shown previously that while cu by itself is not toxic 
 to n2a cells , the presence of cu and either monomeric 
 a42 or soluble a42 oligomers exhibits 
 enhanced cell toxicity . however , the addition 
 of either l1 or l2 to the n2a cells along with the preformed a42 oligomers drastically reduces the neurotoxicity of soluble 
 a42 species to 100  15% and 103  15% 
 cell viability , respectively ( figure 9 , lanes 
 9 and 10 ) . even in the presence of cu , which has been 
 shown to stabilize the soluble a42 oligomers , l1 and l2 were able to eliminate the neurotoxicity 
 of soluble a42 oligomers ( figure 9 , lanes 9 and 10 ) . these results are particularly exciting 
 because , to the best of our knowledge , these are the first bfcs that 
 reduce the toxicity of soluble a42 oligomers , in 
 both the presence or absence of cu ions . these bfcs thus 
 represent promising lead compounds for the development of potential 
 therapeutic agents that can control the metal - mediated neurotoxicity 
 of soluble a42 species in ad and are currently the 
 focus of our ongoing in vivo studies . cell viability ( % dmso control ) of n2a 
 cells determined by the alamar blue assay , upon incubation with ( 1 ) 
 a42 fibrils , ( 2 ) a42 fibrils + 
 cu , ( 3 ) a42 fibrils + l1 , ( 4 ) a42 fibrils + l2 , ( 5 ) a42 fibrils + cu + l1 , ( 6 ) a42 fibrils + cu + l2 , ( 7 ) a42 fibrils + cu + cq , 
 ( 8) a42 oligomers , ( 9 ) a42 oligomers 
 + l1 , ( 10 ) a42 oligomers + l2 , ( 11 ) a42 oligomers + l1 + cu , and ( 12 ) a42 oligomers + l2 + cu . conditions : [ compound ] 
 = 20 m ; [ cu ] = 20 m ; [ a42 ] = 20 m . the t - test analysis reveals values 
 of p < 0.001 for the pairs of data sets marked 
 with asterisks . in 
 summary , two small bfcs , l1 and l2 , were developed to specifically 
 target the interactions of cu and zn ions 
 with the a42 peptide and to control the formation 
 of neurotoxic soluble a42 oligomers . the bifunctionality 
 of l1 and l2 ( i.e. , metal chelation and a interaction ) was 
 established through various spectroscopic studies such as spectrophotometric 
 titrations , structural characterization , and fluorescent assays . we 
 have previously shown that bfcs , which have high affinity for both 
 metal ions and a fibrils , can cause significant oligomerization 
 via disaggregation of a42 fibrils and lead to enhanced 
 neurotoxicity . however , the bfcs described 
 herein bind to metal ions and a fibrils only with moderate 
 affinity and are having the desired opposite effect of promoting fibrillization 
 of cu - stabilized soluble a42 oligomers , 
 and most importantly they drastically reduce the neurotoxicity of 
 soluble a42 species , in both the presence or absence 
 cu ions . because , to the best of our knowledge , these 
 bfcs are the first to control the metal - mediated neurotoxicity of 
 a42 oligomers , l1 and l2 are promising lead compounds 
 for the development of chemical agents that can control the neurotoxicity 
 of soluble a42 species in ad .
multifunctional metal chelators that 
 can modulate the amyloid  ( a ) peptide aggregation and 
 its interaction with metal ions such as copper and zinc hold considerable 
 promise as therapeutic agents for alzheimer s disease ( ad ) 
 . 
 however , specific rather than systemic metal chelation by these compounds 
 is needed in order to limit any side effects . 
 reported herein are 
 two novel small bifunctional chelators , 2-[2-hydroxy-4-(diethylamino)phenyl]benzothiazole 
 ( l1 ) and 2-(2-hydroxy-3-methoxyphenyl)benzothiazole ( l2 ) , in which 
 the metal - binding donor atoms are integrated within a molecular framework 
 derived from the amyloid - binding fluorescent dye thioflavin t ( tht ) . 
 the metal - binding properties of l1 and l2 were probed by ph spectrophotometric 
 titrations to determine their pka values 
 and the corresponding metal complex stability constants , and the isolated 
 metal complexes were structurally characterized . 
 the amyloid - fibril - binding 
 properties of l1 and l2 were investigated by fluorescence titrations 
 and tht competition assays . 
 interestingly , l1 and l2 do not lead to 
 the formation of neurotoxic a42 oligomers in the 
 presence or absence of metal ions , as observed by native gel electrophoresis , 
 western blotting , and transmission electron microscopy . 
 in addition , 
 l1 and l2 were able to reduce the cell toxicity of preformed a42 oligomers and of the copper - stabilized a42 oligomers . given their ability to reduce the toxicity of soluble 
 a42 and cu - a42 species , l1 and 
 l2 are promising lead compounds for the development of chemical agents 
 that can control the neurotoxicity of soluble a42 species in ad .