dichloromethane , diethyl ether , 
 methanol , and toluene were dried in - house using a pure solv - md solvent 
 purification system . all of the other reagents used in the synthesis 
 were purchased from sigma - aldrich except l - alanine isopropyl 
 ester . reactions 
 were monitored with analytical tlc on silica gel 60-f254 precoated 
 aluminum plates and visualized under uv ( 254 nm ) and/or with p nmr spectra . nmr data were recorded on a bruker av300 , 
 aviii300 , av400 , aviii400 , or drx500 spectrometer in the deuterated 
 solvents indicated , and the spectra were calibrated on residual solvent 
 peaks . chemical shifts (  ) are quoted in ppm , and j values are quoted in hz . in reporting spectral data , the following 
 abbreviations were used : s ( singlet ) , d ( doublet ) , t ( triplet ) , q 
 ( quartet ) , dd ( doublet of doublets ) , and m ( multiplet ) . hplc was carried 
 out on a dionex summit p580 quaternary low pressure gradient pump 
 with a built - in vacuum degasser using a summit uvd 170s uv / vis multichannel 
 detector . analytical 
 separations used a flow rate of 1 ml / min , semipreparative used a flow 
 rate of 3 ml / min , and preparative used a flow rate of 20 ml / min . all 
 tested compounds had a purity of 95% as shown by hplc or elemental 
 analysis ( see supporting information ) . furfurylamine ( 0.54 ml , 6.2 mmol ) and et3n were 
 added dropwise to a suspension of 6-chloropurine riboside 10 ( 600 mg , 2.1 mmol ) in etoh ( 30 ml ) . the resulting yellow solution was evaporated under reduced 
 pressure to yield the crude mixture as a cream paste . this was washed 
 with et2o ( 3  25 ml ) and filtered to give the product 
 as a white crystalline solid ( 679 mg , 93% ) . h nmr ( 400 
 mhz , meod )  8.26 ( 2h , s , h-2/h-8 ) , 7.43 ( 1h , dd , j = 1.9 , 0.9 hz , ch2aro - h ) , 6.34 ( 1h , 
 dd , j = 3.2 , 1.8 hz , ch2aro - h ) , 6.31 ( 1h , dd , j = 3.2 , 0.9 hz , ch2aro - h ) , 5.95 ( 1h , d , j = 6.4 hz , 
 1-h ) , 4.80 ( 2h , s , nh2ch2 ) , 4.74 ( 1h , dd , j = 6.4 , 5.1 hz , 2-h ) , 
 4.31 ( 1h , dd , j = 5.1 , 2.5 hz , 3h ) , 4.16 
 ( 1h , q , j = 2.5 hz , 4-h ) , 3.81 ( 2h , ddd , j = 57.3 , 12.6 , 2.6 hz , 5-h2 ) ; c nmr ( 100 mhz , meod ) 152.0 ( c-8/c-2 ) , 151.6 ( ch2aro - c ) , 149.6 , 147.8 ( arn - c ) , 142.0 ( ch2aro - ch ) , 140.3 ( c-8/c-2 ) , 119.9 ( arn - c ) , 110.0 ( ch2aro - ch ) , 106.9 
 ( ch2aro - ch ) , 89.9 ( 1-c ) , 86.8 
 ( 4-c ) , 74.0 ( 2-c ) , 71.3 ( 3-c ) , 62.1 ( 5-c ) , 
 36.7 ( nhch2 ) . ms - esi ( m / z ) : c15h17n5o5 [ m + na ] 370.1 . l - alanine 
 methyl ester hydrochloride ( 400 mg , 2.9 mmol ) was dissolved in anhydrous 
 ch2cl2 ( 20 ml ) under an inert atmosphere . following 
 this , phenyl phosphorodichloridate ( 0.53 ml , 2.9 mmol ) was added 
 dropwise over 15 min . et3n ( 0.77 ml , 5.8 mmol ) was then 
 added at 78 c , and the mixture was stirred for 30 min . the solution was then allowed to warm to room temperature over 2 h. 
 solvent was then removed under reduced pressure and the remaining 
 white precipitate suspended in anhydrous et2o ( 50 ml ) . this was filtered and the filtrate evaporated under reduced pressure , 
 yielding the crude product as a light brown oil . purification via 
 flash column chromatography gave the pure product as a colorless oil 
 ( 729 mg , 91% ) ( eluent 7:3 etoac / hexane ) . p nmr ( 120 mhz , 
 cdcl3 )  7.94 , 7.62 ; h nmr ( 400 mhz , 
 cdcl3 )  7.457.33 ( 2h , m , ar - h ) , 7.317.21 ( 3h , m , ar - h ) , 4.624.52 
 ( 1h , m , n - h ) , 4.264.14 ( 1h , m , chch3 ) , 3.79 , 3.77 ( 2h , 2 s , och3 ) , 1.51 ( 3h , dd , j = 7.1 , 4.1 hz , chch3 ) ; c nmr ( 100 mhz , cdcl3 )  
 173.4 ( coch3 ) , 150.1 ( ar - cop ) , 130.3 , 126.4 , 121.0 ( ar - ch ) , 53.3 ( och3 ) , 51.1 ( chch3 ) , 20.9 ( chch3 ) . prepared as 
 described for 6 using l - alanine tert - butyl ester hydrochloride ( 400 mg , 2.20 mmol ) , phenyl phosphorodichloridate 
 ( 0.30 ml , 2.20 mmol ) , and et3n ( 0.60 ml , 4.4 mmol ) . product 7 was obtained as a pale yellow oil , which was used in sequential 
 steps without further purification ( 803 mg , 114% ) . p 
 nmr ( 120 mhz , cdcl3 )  8.26 , 7.84 ; h 
 nmr ( 400 mhz , cdcl3 )  7.447.32 ( 2h , m , ar - h ) , 7.307.20 ( 3h , m , ar - h ) , 4.28 
 ( 1h , br s , nh ) , 4.123.98 ( 1h , m , chch3 ) , 1.561.34 ( 3h , m , chch3 , 9h , m , ocbu ) ; c nmr ( 100 mhz , cdcl3 )  171.9 ( cocobu ) , 149.9 ( ph - cop ) , 130.0 , 
 126.1 , 120.1 ( ph - ch ) , 82.9 ( ocbu ) , 51.0 ( chch3 ) , 28.1 ( ocbu ) , 20.8 ( chch3 ) . prepared as 
 described for 6 using l - alanine benzyl ester 
 hydrochloride ( 500 mg , 2.32 mmol ) , phenyl phosphorodichloridate 
 ( 0.35 ml , 2.32 mmol ) , and et3n ( 0.63 ml , 4.64 mmol ) . product 8 was obtained a pale yellow oil ( 505 mg , 95% ) . p nmr ( 120 mhz , cdcl3 )  7.85 , 7.49 ; h nmr ( 400 mhz , cdcl3 )  7.407.32 ( m , 5h , 
 ph - h ) , 7.287.21 ( m , 5h , ph - h ) , 5.21 ( 2h , d , j = 6.1 hz , och2ph ) , 4.314.15 ( 2h , 2 m , chch3 ) , 1.52 ( 3h , dd , j = 6.6 , 2.4 hz chch3 ) ; c nmr ( 100 mhz , cdcl3 )  172.6 ( cooch2ph ) , 149.7 ( ph - c ) , 135.0 ( ph - c ) , 130.0 , 128.7 , 128.6 , 
 128.4 , 126.0 , 120.6 ( ph - ch ) , 67.6 ( och2ph ) , 50.8 ( chch3 ) , 20.6 
 ( chch3 ) . prepared as 
 described for 6 using l - alanine isopropyl ester 
 hydrochloride ( 700 mg , 4.18 mmol ) , phenyl phosphorodichloridate 
 ( 0.62 ml , 4.18 mmol ) , and et3n ( 1.13 ml , 8.36 mmol ) . p nmr ( 120 mhz , cdcl3 )  8.10 , 7.80 ; h nmr ( 400 mhz , cdcl3 )  7.467.32 ( 2h , m , 
 ar - h ) , 7.307.19 ( 3h , m , ar - h ) , 5.165.00 ( 1h , m , ochpr ) , 4.504.30 ( 1h , m , n - h ) , 4.224.03 
 ( 1h , m , chch3 ) , 1.521.47 ( 3h , 
 m , chch3 ) , 1.321.22 ( 6h , m , ochpr ) ; c nmr 
 ( 100 mhz , cdcl3 )  172.2 ( coch3 ) , 149.9 ( ar - cop ) , 130.0 , 126.1 , 120.7 ( ar - ch ) , 69.8 ( ochpr ) , 50.8 ( chch3 ) , 21.8 ( 2c , ochpr ) , 20.6 ( chch3 ) . kr 2 ( 200 mg , 0.60 mmol ) was 
 suspended in anhydrous thf ( 15 ml ) under an inert atmosphere . to this , bumgcl ( 0.09 ml , 0.68 mmol ) was then added dropwise 
 over 15 min . the resulting solution was stirred for 10 min , and following 
 this , 6 ( 241 mg , 0.86 mmol ) in anhydrous thf ( 1.5 ml ) 
 was then added dropwise over 10 min . the mixture was left to stir 
 for 18 h. meoh ( 2 ml ) was then added to quench the reaction before 
 solvent was removed under reduced pressure to leave the crude product 
 as a pale yellow oil . this was purified via flash column chromatography 
 and then preparative tlc to yield the final product as a white solid 
 ( 34 mg , 10% ) ( eluent 3:97 to 5:95 meoh / ch2cl2 ) . p nmr ( 120 mhz , meod )  3.84 , 3.69 ; h nmr ( 400 mhz , meod )  8.27 ( 1h , s , arn - ch ) , 8.23 , 8.20 ( 1h , 2 s , arn - ch ) , 7.43 ( 1h , m , ch2aro - h ) , 7.30 ( 2h , t , j = 
 7.9 hz , ph - h ) , 7.227.12 ( 3h , m , ph - h ) , 6.34 ( 1h , dd , j = 3.4 , 1.8 hz , ch2aro - h ) , 6.32 ( 1h , d , j = 
 2.8 hz , ch2aro - h ) , 6.04 ( 1h , t , j = 5.0 hz , 1-h ) , 4.80 ( 2h , br s , nhch2 ) , 4.704.60 ( 1h , m , 2h ) , 4.464.30 
 ( 3h , m , 3h/5h2 ) , 4.294.23 ( 1h , 
 m , 4-h ) , 3.953.77 ( 2h , 2 m , chch3 ) , 3.61 ( 3h , s , och3 ) , 1.301.27 
 ( 3h , m , chch3 ) ; c nmr ( 100 
 mhz , meod )  175.5 ( cooch3 ) , 154.0 
 ( arn - ch ) , 153.4 ( aro - c ) , 152.1 ( ph - c ) , 150.3 ( arn - c ) , 143.4 ( aro - ch ) , 140.7 ( arn - ch ) , 130.7 , 126.2 , 121.9 ( ph - ch ) , 120.9 ( arn - c ) , 111.4 , 108.2 ( aro - ch ) , 90.0 ( 1-c ) , 84.4 ( 4-c ) , 75.4 ( 2-c ) , 
 71.6 ( 3-c ) , 67.3 ( 5-c ) , 52.7 ( och3 ) , 51.4 ( chch3 ) , 38.4 ( nhch2 ) , 20.3 ( chch3 ) . hrms - esi ( m / z ) : calcd for c25h29n6o9nap [ m + na ] 611.1631 , found 611.1633 . calcd 
 for c25h29n6o9p : c 51.0 , 
 h 4.97 , n 14.3% . prepared 
 as described for compound 11 using kr 2 ( 200 
 mg , 0.60 mmol ) , nmi ( 0.22 ml , 3.00 mmol ) , and 7 ( 552 
 mg , 1.80 mmol ) . the crude product was purified via flash column chromatography 
 to yield the final product as a white solid ( 52 mg , 14% ) ( eluent 3:97 
 to 5:95 meoh / ch2cl2 ) . p nmr ( 120 
 mhz , meod )  3.91 , 3.84 ; h nmr ( 400 mhz , meod )  
 8.22 ( 1h , s , arn - ch ) , 8.22 , 8.19 ( 1h , 2 s , arn - ch ) , 7.43 ( 1h , dd , j = 1.9 , 0.9 hz , ch2aro - h ) , 7.357.26 ( 2h , m , ph - h ) , 7.217.10 ( 3h , m , ph - h ) , 6.34 
 ( 1h , dd , j = 3.2 , 1.9 hz , ch2aro - h ) , 6.326.30 ( 1h , m , ch2aro - h ) , 6.056.02 ( 1h , m , 1-h ) , 4.79 ( 2h , s , nhch2 ) , 4.65 ( 1h , t , j = 5.2 hz , 
 2h ) , 4.484.30 ( 3h , m , 3-h/5h2 ) , 4.294.24 ( 1h , m , 4-h ) , 3.823.75 ( 1h , m , 
 chch3 ) , 1.39 ( 9h , s , bu - h ) , 1.23 ( 3h , dd , j = 7.1 , 1.2 hz , chch3 ) ; c nmr ( 100 mhz , meod )  172.9 ( co ) , 154.3 
 ( arn - c ) , 152.6 ( arnc - h ) , 151.8 ( aro - c ) , 150.7 ( phcop ) , 148.6 ( arn - c ) , 142.0 ( aroc - h ) , 139.3 ( arnc - h ) , 
 129.4 , 124.7 , 120.0 ( phc - h ) , 119.2 ( arn - c ) , 110.0 , 106.8 ( aroc - h ) , 88.6 ( 1-c ) , 83.0 
 ( 4-c ) , 81.2 ( ocbu ) , 74.0 ( 2-c ) , 
 70.2 ( 3-c ) , 65.7 ( 5-c ) , 50.8 ( chch3 ) , 37.1 ( nhch2 ) , 26.8 ( 3c , ocbu ) , 19.1 ( chch3 ) . hrms - esi ( m / z ) : 
 calcd for c28h35n6o9nap 
 [ m + na ] 653.2101 , found 653.2104 . found : c 53.4 , h 5.6 , 
 n 13.5 . prepared as described for compound 11 using 2 ( 250 mg , 0.72 mmol ) , nmi ( 0.29 ml , 
 3.6 mmol ) , and 8 ( 765 mg , 2.16 mmol ) . the crude product 
 was purified via flash column chromatography to yield the final product 
 as a white solid ( 60 mg , 13% ) ( eluent 3:97 to 5:95 meoh / ch2cl2 ) . p nmr ( 120 mhz , meod )  3.89 , 
 3.64 ; h nmr ( 400 mhz , meod )  8.26 ( 1h , s , arn - ch ) , 8.20 , 8.17 ( 1h , 2 s , arn - ch ) , 7.41 
 ( 1h , d , j = 1.8 , ch2aro - h ) , 7.327.24 ( 7h , m , ph - h ) , 7.217.06 
 ( 3h , m , ph - h ) , 6.32 ( 1h , dd , j = 
 3.3 , 1.9 hz , ch2aro - h ) , 6.29 ( 1h , d , j = 3.2 hz , ch2aro - h ) , 6.03 
 ( 1h , t , j = 4.9 hz , 1-h ) , 5.184.98 
 ( 2h , 2 m , ch2ph ) , 4.77 ( 2h , br s , nhch2 ) , 4.674.57 ( 1h , m , 2-h ) , 4.434.33 
 ( 3h , m , 3-h/4-h/5-h ) , 4.334.20 ( 1h , 
 m , 5-h ) , 4.023.85 ( 1h , m , chch3 ) , 1.25 ( 3h , dd , j = 19.4 , 7.0 hz , chch3 ) ; c nmr ( 100 mhz , meod )  
 174.8 ( cooch2ph ) , 155.8 ( arn - c ) , 153.9 ( arn - ch ) , 152.0 ( ph - c ) , 
 150.2 ( arn - c ) , 143.3 ( aro - ch ) , 140.6 
 ( arn - ch ) , 139.0 ( ph - ch ) , 137.1 ( ph - c ) , 130.7 , 129.2 , 128.8 , 126.2 , 121.9 , 121.3 ( ph - ch ) , 121.0 ( arn - c ) , 111.4 , 108.2 ( aro - ch ) , 89.9 ( 1-ch ) , 84.3 ( 4-c ) , 75.4 ( 2-ch ) , 
 71.4 ( 3-ch ) , 67.7 ( ch2ph ) , 67.1 
 ( 5-c ) , 51.5 ( chch3 ) , 38.2 ( nhch2 ) , 20.4 ( chch3 ) . hrms - esi ( m / z ) : calcd for c31h33n6o9nap [ m + na ] 687.1944 , found 687.1942 . found : c 55.95 , h 5.2 , n 12.6 . 
 calcd for c31h33n6o9p : 
 c 56.0 , h 5.0 , n 12.65% . prepared as described for compound 11 using 2 ( 250 mg , 0.72 mmol ) , nmi ( 0.29 ml , 
 3.60 mmol ) , and 9 ( 660 mg , 2.16 mmol ) . the crude product 
 was purified via flash column chromatography to yield the final product 
 as a white solid ( 185 mg , 42% ) ( eluent 3:97 to 5:95 meoh / ch2cl2 ) . p nmr ( 120 mhz , cdcl3 )  
 3.02 , 2.86 ; h nmr ( 400 mhz , cdcl3 )  
 8.27 ( 1h , s , arn - ch ) , 7.98 , 7.94 ( 1h , 2 s , arn - ch ) , 7.30 ( 1h , dd , j = 2.5 , 1.4 hz , ch2aro - h ) , 7.15 ( 2h , t , j = 
 7.7 hz , ph - h ) , 7.07 ( 2h , d , j = 
 7.7 hz , ph - h ) , 7.00 ( 1h , m , ph - h ) , 6.74 ( 1h , br s , nhch2aro ) , 6.25 ( 2h , 
 t , j = 1.7 hz , ch2aro - h ) , 5.98 ( 1h , t , j = 4.4 hz , 1-h ) , 5.38 ( 1h , 
 br s , o - h ) , 4.964.85 ( 1h , m , ochpr ) , 4.78 ( 2h , br s , nhch2aro ) , 4.574.25 
 ( 6h , m , nhchch3 , 2-h , 3-h , 
 4-h , 5-h2 ) , 3.943.81 ( 1h , m , nhchch3 ) , 1.24 ( 3h , t , j = 7.2 
 hz , nhchch3 ) , 1.191.05 ( 6h , m , 
 ochpr ) ; c nmr ( 100 mhz , 
 cdcl3 )  172.9 ( co ) , 154.0 ( arnc ) , 152.6 ( arnc - h ) , 151.4 ( aro - c ) , 150.2 ( ph - c ) , 148.5 ( arnc ) , 142.0 ( aro - ch ) , 138.6 ( arnc - h ) , 
 129.4 ( ph - ch ) , 124.7 ( ph - ch ) , 119.8 
 ( ph - ch ) , 119.6 ( arnc ) , 110.6 ( aro - ch ) , 107.3 ( aro - ch ) , 88.9 ( 1-c ) , 
 83.1 ( 4-c ) , 74.5 ( 2-c ) , 70.4 ( 3-c ) 68.9 ( ochpr ) , 65.8 ( 5-c ) , 
 50.0 ( nhchch3 ) , 37.2 ( nhch2aro ) , 21.4 ( ochpr ) , 20.5 ( nhchch3 ) . hrms - esi 
 ( m / z ) : calcd for c27h34n6o9p [ m + h ] 617.2125 , 
 found 617.2144 . hplc ch3cn / h2o 0:100 to 100:0 
 in 30 min ,  = 254 nm , tr = 16.51 , 
 16.69 min . protide 14 ( 5.0 mg ) 
 was dissolved in acetone - d6 ( 0.15 ml ) , 
 followed by addition of trizma buffer ( 0.30 ml , ph 7.4 ) . after recording a control p nmr spectrum containing protide in acetone - d6 and buffer , defrosted cathepsin a ( 0.1 mg 
 dissolved in 0.15 ml of trizma buffer ) was added to the mixture . a p nmr was then run immediately after the addition and then 
 at even time intervals over 11 h. the sample was then analyzed by p nmr after 24 and 48 h. all p nmr spectra recorded 
 at 22 c ( 1 ) . protide 14 ( 5.0 mg ) 
 was dissolved in dmso - d6 ( 0.10 ml ) and 
 d2o ( 0.15 ml ) . two control spectra were recorded , one 
 containing protide 14 ( 5.0 mg ) in dmso - d6 ( 0.10 ml ) and d2o ( 0.15 ml ) and the other 
 containing defrosted human serum ( 0.3 ml ) , dmso - d6 ( 0.10 ml ) , and d2o ( 0.15 ml ) . following this , 
 a previously defrosted serum ( human or mouse ) ( 0.30 ml ) spectra were recorded 
 at 30 min after the addition and then at even time intervals over 
 11 h. procedure was adapted from slusarczyk 
 et al . to protide 12 ( 5 
 mg ) in methanol - d4 ( 0.25 ml ) was added 
 acidic buffer , ph 1 ( prepared from equal parts of 0.2 m hcl and 0.2 
 m kcl ) . the sample was then subjected to p nmr experiments 
 at 37 c , and the spectra were recorded every 20 min over 12 
 h. pc windows 7 with intel core i7 - 4790/3.6ghz 
 microprocessor , 16 gb ram , and 64 bit operating system was used to 
 execute the computational studies . docking was performed applying 
 the molecular modeling modules , namely , omega2 , fred , and vida provided 
 by openeye scientific software ( http://www.eyesopen.com ) . the three - dimensional crystal structure 
 of the cocrystallized amp with the human hint1 was retrieved from 
 the protein data bank ( pdb code 1kpf ) , and the 
 active site was subsequently identified on the basis of the bound 
 ligand . fred ( fast 
 rigid exhaustive docking ) implements a rigid docking approach to fit 
 these conformers into the predefined binding site and rank the poses 
 by scoring functions . the vida module was then used 
 to visualize and inspect the docked poses within the receptor s 
 active site and to identify the main interacting residues . flp - in t - rex hek293 cells stably expressing 
 pink1-flag wild - type were generated previously . cells were maintained in dmem ( dulbeco s modified 
 eagle medium ) supplemented with 10% ( v / v ) fetal bovin serum , 2 mm l - glutamine , 100 u / ml penicillin , and 0.1 mg / ml streptomycin , 
 plus 15 g / ml blasticidin and 100 g / ml hygromycin at 
 37 c under a 5% co2 atmosphere . on day 0 , cells were 
 seeded in dmem . one day 1 , cells were transiently transfected with 
 wild type or s65a parkin using polyethylenimine ( polysciences ) according 
 to the manufacturer s instruction . one day 2 , pink1-flag overexpression 
 was induced by adding 0.1 g / ml doxycycline in dmem for 24 h 
 before treating cells with compounds or mitochondrial uncoupler , cccp , 
 as indicated in the figure legends . this was carried 
 out as we reported previously.lysis buffer used : 50 mm tris - hcl ( ph 7.5 ) , 1 mm edta , 1 
 mm egta , 1% ( w / v ) triton , 1 mm sodium orthovanadate , 10 mm sodium 
 glycerophosphate , 50 mm sodium fluoride , 10 mm sodium pyrophosphate , 
 0.25 m sucrose , 0.1% ( v / v ) 2-mercaptoethanol , 1 mm benzamidine , 0.1 
 mm pmsf , and protease inhibitor cocktail ( roche ) . antibodies 
 used : mouse monoclonal anti - pink1 antibody ( human pink1 residues 
 125539 ) was raised by dundee cell products , anti - vinculin 
 and anti - gapdh antibodies were obtained from cell signaling technology , 
 anti - parkin phospho - serine 65 rabbit monoclonal antibody was raised 
 by epitomics in collaboration with the michael j. fox foundation for 
 research , and anti - parkin mouse monoclonal antibody was obtained from 
 santa cruz .
since loss of function 
 mutations of pink1 lead to early onset parkinson s 
 disease , there has been growing interest in the discovery of small 
 molecules that amplify the kinase activity of pink1 . 
 we herein report 
 the design , synthesis , serum stability , and hydrolysis of four kinetin 
 riboside protides . 
 these protides , along with kinetin riboside , activated 
 pink1 in cells independent of mitochondrial depolarization . 
 this highlights 
 the potential of modified nucleosides and their phosphate prodrugs 
 as treatments for neurodegenerative diseases .