proteolytic activities play a decisive 
 role in all aspects of cellular 
 life , including cell division processes , metabolism and catabolism , 
 protein translocation , immune defense mechanisms , and more .
the human bacterial pathogen streptococcus 
 pyogenes ( or group a streptococcus ) is the causative 
 agent of a great variety of infections , ranging from mucocutaneous 
 infections of the throat and skin to life threatening conditions including 
 necrotizing fasciitis and streptococcal toxic shock syndrome .
more than 600 million people are estimated to 
 suffer from streptococcal pharyngitis alone , and mucocutaneous infections 
 cause substantial morbidity and considerable economic loss to society .
postinfectious sequelae include serious inflammatory 
 diseases such as acute rheumatic fever ( arf ) , rheumatic heart disease 
 ( rhd ) , and poststreptococcal glomerulonephritis ( psgn ) .
inflammatory 
 autoimmune diseases such as guttate psoriasis have also been associated 
 with streptococcal infections although 
 the underlying molecular mechanisms still remain to be solved .
s. pyogenes employs two papain - like 
 cysteine proteases to adapt to the dynamic environment in its human 
 host and to evade the human immune response : the classical streptococcal 
 cysteine protease speb and the immunoglobulin g ( igg ) degrading protease , 
 ides .
both enzymes adopt a canonical papain - like 
 structural fold and show , despite the lack of sequence similarity , 
 large structural similarities .
besides ides , also speb and papain have the ability 
 to cleave the igg heavy chain .
the speb cleavage site is identical 
 to ides cleavage at a defined site between glycine residues 236 and 
 237 , creating one f(ab)2 fragment and two identical 
 1/2fc fragments .
papain cleavage occurs at the peptide bond between histidine in 
 position 224 and threonine in position 225 of the hinge region of 
 igg , thereby generating two fab fragments and one fc fragment .
however , the proteases have distinguished substrate 
 recognition properties : speb and papain exhibit a broad proteolytic 
 activity and degrade or activate a wide variety of substrates .
ides , on the other side , is highly specific and recognizes only 
 igg as substrate .
furthermore , ides , in contrast to papain and other prokaryotic cysteine 
 proteases , including speb and the staphylococcal cysteine protease 
 stpa , is not inhibited by the classical 
 cysteine protease inhibitor e64 . this interesting property 

is explained by an unusually narrow active site cleft that does not 
 offer enough space to accommodate the p3 residue of e64 and thus points 
 to distinct substrate recognition properties .
given the essential role of ides in the evasion of igg mediated 
 immune responses , there is a high medical interest to identify specific 
 inhibitors for prokaryotic cysteine proteases .
furthermore , ides is 
 currently evaluated as a therapeutic agent to treat conditions in 
 which antibodies reacting against human antigens misdirect the human 
 immune response toward the body s own cells .
the efficient 
 removal of pathogenic igg is an important clinical challenge , and 
 several animal models have provided the proof of principle for the 
 use of ides as a therapeutic agent .
however , an ides specific 
 inhibitor would also allow the external control of proteolytic activity 
 in these applications , which might prove to be a valuable tool in 
 treatment .
however , because of the structural similarity of 
 papain - like proteases , 
 it is not a simple task to identify inhibitors that efficiently block 
 prokaryotic proteases without affecting several essential protease 
 functions in the human host .
compounds reported to inhibit ides , including 
 alkylating agents , z - lvg chn2 and tpck / tlck , are also efficient inhibitors of other cysteine proteases and do 
 not exhibit any selectivity toward ides .
recently , we showed that 
 tpck / tlck analogues containing aldehyde - based warheads act as reversible 
 inhibitors of ides , however their selectivity was not studied .
the rationale for the approach in the present 
 study was to identify specific inhibitors for ides based on the fact 
 that a noncovalent inhibitor lacking an electrophilic warhead would 
 have to depend on other specific interactions with the enzyme , which 
 therefore should increase the selectivity and thus harbor the potential 
 to be specific .
ides does only hydrolyze igg and neither synthetic 
 or natural peptides 
 containing the p4p1 subsites of the 
 igg hinge region , nor peptides with sequences covering the ides cleavage 
 site are cleaved by the protease . because 
 such peptide - based substrates are not hydrolyzed by ides ,
they have 
 in the present study instead been investigated for their putative 
 inhibitory capacity on the streptococcal cysteine proteases ides and 
 speb and also on papain .
the tested peptides were of different length , 
 from four up to eight amino acids , covering the p4p4 residues of igg .
in addition , a series of di- , tri- , and 
 tetrapeptide analogues based on the amino acid sequence of igg surrounding 
 the ides cleavage site have been synthesized and were tested for potential 
 inhibitory activity . in the analogues ,
one of the two glycine residues 
 at the cleavage site , gly236 or gly237 , was 
 replaced by a piperidine moiety , thus forming either pip236g- or gpip237-fragments ( figure 1 ) . in the synthesized analogues ,
a piperidine moiety replaces one 
 of the two glycine residues at the ides cleavage site .
thereby , a 
 new stereogenic center is introduced at different positions in the 
 two fragments ( marked with an asterisk ) .
the piperidine moiety can be inserted through a 
 short and efficient 
 synthetic route , and the strategy used allows further extension both n- and c - terminally to the desired lengths 
 of the peptide analogues .
the piperidine ring rigidifies the structure 
 of the analogues compared to the original peptide backbone , yet the 
 structure is flexible as the six - membered ring can adopt different 
 chair conformations .
a new stereogenic center is introduced at different 
 positions in the two fragments ( figure 1 ) , 
 and the environment around the scissile amide bond is changed by the 
 different polarity profiles of the analogues .
peptides and synthesized peptide analogues 
 were screened for their 
 ability to inhibit ides , speb , and papain .
interestingly , several 
 of the synthesized peptide analogues affected the activity of the 
 enzymes , however , different inhibitory profiles were observed for 
 the three cysteine proteases .
short and efficient 
 synthetic routes to the peptide analogues discussed in this study 
 have been developed .
the synthesis starts by formation of the new 
 stereogenic center via a reductive amination with n - protected 3-piperidone and either glycine or enantiopure l - proline .
the stereoisomers were separated and further modified or 
 used in peptide coupling reactions with enantiopure l - amino 
 acids to form the desired peptide analogues .
separation of the stereoisomers 
 made it possible to keep track of the absolute configuration of the 
 created stereogenic center throughout the synthesis .
further , no racemization 
 or epimerization were observed in any of the amino acid coupling reactions .
reductive amination of boc - protected 3-piperidone with ethyl glycinate 
 and nabh(oac)3 gave the racemate 1 in moderate 
 yield ( 62% ) ( scheme 1 ) .
reagents and conditions : 
 ( a ) 
 ethyl glycinate , nabh(oac)3 , acoh , molecular sieves , dichloromethane , 
 room temp , 20 h ( 62% ) ; ( b ) preparative hplc ; ( c ) ethyl glyoxylate , 
 nabh(oac)3 , acoh , dichloromethane , room temp , 4 h ( 56% 
 and 62% ) . to determine the absolute configuration 
 of the two enantiomers , 
 reference compounds were synthesized via reductive amination of ethyl 
 glyoxylate with ( r)- and ( s)-3-amino-1-boc - piperidine 
 to afford ( r)- and ( s)-1 , respectively , with known configuration at the stereogenic center 
 ( scheme 1 ) .
comparison of the retention times 
 from analytical hplc allowed an unequivocal assignment of the configuration 
 of the enantiomers isolated via preparative hplc .
unfortunately , the 
 synthesized reference compounds showed too low ee ( 90% and 86% , respectively ) to be used as starting material for 
 the synthesis of the analogues .
however , 2 g of ( rac)-1 was separated into ( r)- and ( s)-1 in 11 injections in sufficient amount 
 for the synthetic route to follow . as shown in scheme 2 , aminolysis of the 
 isolated ( r)- and ( s)-1 with ammonia in methanol afforded the amides ( r)- and ( s)-2 , respectively , in high 
 yields ( 92% and 90% ) without the need for purification .
the c nmr spectra showed a considerable broadening of the signals assigned 
 to the carbon atoms adjacent to the nitrogen in the piperidine ring , 
 indicating that the boc - group gives rise to rotamers .
removal of the 
 boc protecting group by tfa gave the glygly - nh2 analogues 
 ( r)- and ( s)-pipg ( 3 ) in quantitative yields .
furthermore , the coupling pattern 
 in the h nmr spectra implies that the piperidine ring 
 adopts one single chair conformation with the glycine amide residue 
 in an equatorial position .
reagents and conditions : 
 ( a ) 
 nh3 in methanol , room temp , 72 h ( 92 and 90% ) ; ( b ) tfa , 
 room temp , 12 h ( quant yields ) ; ( c ) cbz - osu , et3n , dichloromethane , 
 0 c  room temp , 17 h ( 82% and 84% ) ; ( d ) ( i ) tfa , room 
 temp , 16 h , ( ii ) boc - l - leu  h2o , edc , hobt , 
 dichloromethane , molecular sieves , et3n , room temp , 15 
 h ( 91% and 79% over two steps ) ; ( e ) nh3 in methanol , room 
 temp , 72 h ( 65% and 73% ) ; ( f ) ( i ) h2 , pd / c , ethanol , room 
 temp , 72 h , ( ii ) tfa , room temp , 4 h ( 84% and 75% over two steps ) ; 
 ( g ) ( i ) tfa , room temp , 16 h , ( ii ) boc - l - leu  h2o , edc , hobt , dichloromethane , molecular sieves , et3n , room temp , 15 h ( 88 and 91% , over two steps ) ; ( h ) nh3 in methanol , room temp , 168 h ( 63% and 86% ) ; ( i ) ( i ) h2 , pd / c , ethanol , 3 h , ( ii ) tfa , room temp , 14 h ( 90% and 92% over 
 two steps ) .
routes to the analogues of the 
 tripeptide leuglygly - nh2 , ( r)- and ( s)-lpipg ( 7 ) , and the tetrapeptide 
 leuleuglygly - nh2 , ( r)- and ( s)-llpipg ( 10 ) , are shown in scheme 2 . 

the isolated enantiomers ( r)- and ( s)-1 were protected using cbz - succinimide ( cbz - osu ) to 
 afford ( r)- and ( s)-4 , respectively , in high yields ( 82% and 84% ) .
the c 
 nmr spectra recorded at ambient temperature showed rotamers from both 
 the boc- and cbz - groups , however the signals coalesced in spectra 
 recorded at 50 c .
the enantiomers ( r)- and 
 ( s)-4 were boc - deprotected followed 
 by coupling with boc - l - leucine to afford high yields of ( r)- and ( s)-5 , respectively 
 ( 91% and 79% ) .
aminolysis afforded amides ( r)- and 
 ( s)-6 , which were deprotected to the 
 end products ( r)- and ( s)-lpipg ( 7 ) , respectively ( yields : 84% and 75% 
 over two steps ) .
boc - deprotection of ( r)- and ( s)-5 and coupling to boc - l - leucine 
 gave ( r)- and ( s)-8 , respectively , in high yields ( 88% and 91% ) .
the reaction times were rather long and purification was needed 
 which resulted in lower yields ( 63% and 86% ) .
finally , the cbz- and 
 boc - protecting groups were removed by catalytic hydrogenation ( pd / c ) 
 and tfa hydrolysis , respectively , to afford the end products ( r)- and ( s)-llpipg ( 10 ) in high yields ( 90% and 92% ) . the total yield for the 
 synthesis of ( r)- and ( s)-llpipg ( 10 ) over eight steps from ( r)- and ( s)-1 , respectively , was 37% 
 and 48% .
n - ethylated byproducts were formed 
 during the 
 cbz - deprotection to ( r)- and ( s)-lpipg ( 7 ) as well as ( r)- and 
 ( s)-llpipg ( 10 ) using catalytic 
 hydrogenation ( pd / c 10% ) .
isolation of the byproducts after prolonged 
 reaction time and subsequent tfa hydrolysis gave ( r)- and ( s)-llpipetg ( 11 ) ( scheme 3 ) . already after three hours , byproduct 
 formation was observed . to verify the structure of the byproduct
, 
 cbz - deprotected ( r)-9 was converted 
 to the corresponding ethylated product in 76% yield via reductive 
 amination using acetaldehyde and nabh(oac)3 ( reaction not 
 shown ) .
the nmr spectra of the synthesized compound and that of ( r)-llpipetg ( 11 ) were identical . 

it has been proposed that a pd - catalyzed oxidation of ethanol to acetaldehyde 
 can occur during the hydrogenation reaction .
the acetaldehyde forms an imine with the cbz - deprotected secondary 
 amine , and a subsequent reduction provides the ethylated byproduct .
reagents and conditions : 
 ( a ) 
 ( i ) h2 , pd / c , ethanol , room temp , 144 h ( 23 and 13% ) , ( ii ) 
 tfa in dichloromethane , room temp , 16 h ( 99% and 98% ) .
the unsaturated enamine byproduct 12 ( scheme 4 ) was collected from a reductive amination reaction 
 of 1-boc-3-piperidone with methyl glycinate performed in a highly 
 concentrated reaction mixture .
the opportunity to use the unsaturated 
 piperidine as a scaffold for the synthesis of analogues was taken . 

attempts to cbz - protect 12 using the conditions described 
 for ( s)-4 gave only unreacted starting 
 material back .
boc - deprotection by tfa in dichloromethane was performed , 
 but subsequent coupling with boc - l - leucine did not provide 
 satisfying results .
however , 12 was amidated by treatment 
 with nh3 in methanol and subsequent boc - deprotection gave pip(db)g ( 13 ) in 90% ( scheme 4 ) .
reagents and conditions : 
 ( a ) 
 nh3 in methanol , room temp , 72 h ( 93% ) ; ( b ) tfa / dichloromethane , 
 room temp , 12 h ( 97% ) .
the synthetic route 
 to the leuglyglypro - nh2 analogues 
 ( r)- and ( s)-lpipgp ( 18 ) , in which gly236 in igg is mimicked 
 by the piperidine moiety , is shown in scheme 5 . starting from the cbz - protected core compounds ( r)-and ( s)-4 , hydrolysis of the esters 
 in excellent yields ( 95% and 99% of ( r)- and ( s)-14 , respectively ) followed by coupling to l - proline amide afforded ( r)- and ( s)-15 , respectively , in high yields ( 82% and 
 78% ) . a small portion of ( r)- and ( s)-15 were deprotected to provide the glyglypro - nh2 analogues ( r)- and ( s)-pipgp ( 16 ) .
furthermore , the coupling 
 pattern in the h nmr spectra resembled the pattern seen 
 for ( r)- and ( s)-pipg ( 3 ) , implying that also here the monosubstituted piperidine 
 ring adopts a defined chair conformation .
to continue , removal of 
 the boc - protecting groups in ( r)- and ( s)-15 and subsequent couplings with boc - l - leucine 
 mediated by hobt and edc gave the epimers ( r)- and 
 ( s)-17 in moderate to high yields ( 64% 
 and 90% , respectively ) .
the lower yield is due to the need of two 
 purification steps . also here , the nmr spectra recorded at different 
 temperatures identified rotamers .
the h nmr spectrum of 
 ( r)-17 showed five doublets assigned 
 to the two methyl groups in the leucine side chain , implying that 
 the molecule adopts several different conformations at room temperature . 
 finally , the two protecting groups were removed as described above 
 to give the end products ( r)- and ( s)-lpipgp ( 18 ) in good yields ( 72% and 73% , 
 respectively ) both with excellent purities ( 100% ) when analyzed by 
 reversed phase hplc .
after the deprotection , the h nmr 
 spectrum of both ( r)- and ( s)-lpipgp ( 18 ) showed two doublets assigned to the 
 two nonequivalent methyl groups , as expected .
the total yield for 
 the synthesis of ( r)- and ( s)-lpipgp ( 18 ) over seven steps was 29% and 43% , 
 respectively .
reagents and conditions : 
 ( a ) 
 lioh 1 m aq , thf / meoh / h2o 3:1:1 , 0 c 30 min , room 
 temp , 3 h ( 95 and 99% ) ; ( b ) l - pro - nh2 , edc , hobt , 
 et3n , dichloromethane , room temp , 16 h ( 82% and 78% ) ; ( c ) 
 ( i ) h2 , pd / c , ethanol , room temp , 24 h , ( ii ) tfa , room 
 temp , 4 h ( quant yields over two steps ) ; ( d ) ( i ) tfa , room temp , 16 
 h , ( ii ) boc - l - leu  h2o , edc , hobt , dichloromethane , 
 molecular sieves , et3n , room temp , 15 h ( 64% and 90% over 
 two steps ) ; ( e ) ( i ) h2 , pd / c , ethanol , room temp , 48 and 
 24 h , ( ii ) tfa , room temp , 4 h ( 72% and 73% over two steps ) .
the corresponding tetrapeptide analogues in which 
 the piperidine 
 moiety replaces gly237 in igg ( ( r)- and 
 ( s)-lgpipp ( 23 ) ) were also 
 synthesized ( scheme 6 ) .
reductive amination 
 of boc - protected 3-piperidone with l - proline amide and nabh(oac)3 afforded 19 in 68% yield and a diastereomeric 
 ratio of 60:40 .
the diastereomers were separated by preparative hplc 
 to give ( r)- and ( s)-19 , and the absolute configurations were determined by chemical correlation .
reagents and conditions : 
 ( a ) 
 ( i ) l - pro - nh2 , nabh(oac)3 , hoac , dichloromethane , 
 room temp , 20 h ( 68% ) , ( ii ) preparative hplc ; ( b ) ( i ) tfa / dichloromethane , 
 room temp , ( ii ) boc - gly , edc , hobt , dichloromethane , 0 c  
 room temp , 15 h , 45 and 47% over two steps ) ; ( c ) tfa / dichloromethane , 
 room temp , 20 h ( quant yields ) ; ( d ) ( i ) tfa / dichloromethane , room 
 temp , 20 h ( quant yields ) , ( ii ) boc - l - leu , edc , hobt , et3n , dichloromethane , 0 c  room temp , 15 h ( 93% 
 and 78% ) ; ( e ) tfa / dichloromethane , room temp , 12 h ( quant yield and 
 98% ) .
boc - protected glycine was coupled to 
 each deprotected diastereomer 
 using edc and hobt to afford ( r)- and ( s)-20 in 45% and 47% yield , respectively .
deprotection 
 with tfa in dichloromethane gave quantitative yields of ( r)- and ( s)-gpipp ( 21 ) 
 as tfa salts .
a subsequent coupling to boc - protected l - leucine 
 gave ( r)- and ( s)-22 , respectively , in high yields ( 93% and 78% ) .
deprotection with tfa 
 in dichloromethane overnight afforded ( r)- and ( s)-lgpipp ( 23 ) in excellent yields . 

the total yield for the synthesis of ( r)- and ( s)-lgpipp ( 23 ) in five steps from 
 ( r)- and ( s)-19 was 
 42% and 36% , respectively .
eight different peptides based on the amino acid sequence of the 
 igg hinge region ( figure 2 ) were tested for 
 putative inhibitory activity toward ides .
the inhibitory activity was screened in a gel - electrophoresis 
 ( sds - page ) 
 based assay to detect igg cleavage products .
none of the peptides was found to be active as inhibitors of ides 
 ( data not shown ) .
thus , although the sequences of the tested peptides 
 are identical to the igg sequence around the ides cleavage site , these 
 short peptides did apparently not bind strongly enough to the enzyme 
 to affect enzymatic activity . in total 17 piperidine - containing analogues of the peptide sequences 
 gg , llgg , and lggp ,
all based on the ides cleavage site in human igg1 , 
 were tested for their inhibitory activity toward ides .
inhibitory 
 effects were screened by sds - page and quantified by surface plasmon 
 resonance spectroscopy ( figure 3 ) .
( a ) representative nonreducing sds - page analysis of igg 
 and igg 
 hydrolyzed by ides in the presence ( ( s)-pipg ( ( s)-3 ) ) or absence ( dmso ) of peptide 
 analogues .
scigg indicates single - chain cleaved igg ; f(ab)2 
 indicates double - chain cleaved igg .
( b ) representative surface plasmon 
 resonance spectroscopy curves of uncleaved igg ( control ) and cleaved 
 igg in presence of peptide analogues ( s)-pipg ( ( s)-3 ) and ( r)-gpipp ( ( r)-21 ) .
( s)-pipg ( ( s)-3 ) has greater inhibitory 
 activity than ( r)-gpipp ( ( r)-21 ) . whereas the corresponding peptides were inactive , 
 the peptide analogues 
 showed a significant inhibitory activity ( figure 4 ) .
the most potent inhibitors of ides activity are ( r)-pipgp ( ( r)-16 ) , ( s)-lpipgp ( ( s)-18 ) , ( s)-pipgp ( ( s)-16 ) , ( s)-pipg ( ( s)-3 ) , and ( r)-lpipg ( ( r)-7 ) . in general , peptide analogues 
 in which the piperidine moiety replaces the glycine corresponding 
 to g236 in igg ( pip236 g ) appear to be stronger 
 inhibitors than analogues with the piperidine moiety replacing g237 of igg ( gpip237 ) , independently of c - terminal or n - terminal amino acid extensions . for 
 example , ( r)-pipgp ( ( r)-16 ) is more active than ( r)-gpipp ( ( r)-21 ) and ( s)-lpipgp ( ( s)-18 ) is more active than ( s)-lgpipp ( ( s)-23 ) . the two ( r)- and ( s)-epimers of each analogue pair were tested , but no clear 
 trend how stereochemistry of the piperidine moiety affected potency 
 could be seen ( figure 4 ) .
ides activity in presence 
 of peptides and peptide analogues using 
 igg1 as substrate .
ides activity is expressed relative to a standard 
 curve obtained in absence of inhibitors .
( r ) and 
 ( s ) indicate the different stereoisomers of each 
 analogue ;  pip  represents the piperidine 
 moiety replacing glycine residues .
the enzyme activity was determined 
 for ides at a final concentration of 0.8 m in the presence 
 of 4.8  10 molar excess of peptides / peptide analogues .
the ethylated analogues ( r)- and 
 ( s)-llpipetg ( 11 ) have 
 also been tested , 
 both showing comparable inhibitory activity against ides as ( s)-llpipg ( ( s)-10 ) .
this implies that the secondary amine is not involved in hydrogen 
 bond donation and that there is space enough for an ethyl group .
interestingly , 
 the structural difference between lpipgp ( 18 ) and pipg ( 3 ) is two amino acids , but 
 they show similar inhibitory activity .
this indicates that the extra 
 amino acids are not contributing to more favorable interactions to 
 accomplish more efficient binding or are too flexible to gain favorable 
 binding energies .
thus , when evaluating ligand efficiency ( le ) , where 
 binding energy per atom is considered , ( s)-pipg ( ( s)-3 ) is the most favorable 
 peptide analogue in the series . the pka values for ( r)- and ( s)-pipg ( 3 ) and 
 ( r)- and ( s)-lpipgp ( 18 ) have been determined ( table 1 ) .
interestingly , the two pka values 
 differ considerably . for ( r)- and ( s)-pipg ( 3 ) ,
the acyclic amine bears the 
 electron withdrawing acetamide group , thus the piperidine nitrogen 
 would be expected to be protonated first . for ( r)- 
 and ( s)-lpipgp ( 18 )
, the 
 primary amine would be expected to have the pka of an amino acid amide , i.e. , between 8 and 9 , which is what 
 is found .
the acyclic secondary amines will not be protonated at physiological 
 ph , and the pip moiety is therefore able to act as a 
 glycine isostere .
determination of pka values performed by a screening method based on pressure - assisted 
 capillary electrophoresis and mass spectrometry .
prediction of pka values was 
 performed using the acd / laboratories software ( advanced chemistry 
 development , inc . ) .
ides exhibits pronounced 
 substrate specificity in comparison to other igg cleaving cysteine 
 proteases of the papain superfamily . still , 
 the extended structural similarity of papain - like proteases raised 
 the question whether piperidine containing peptide analogues could 
 have inhibitory capacity toward other igg cleaving proteases or in 
 fact harbor a potential specificity toward ides .
therefore , peptides 
 and peptide analogues were also screened for activity toward the streptococcal 
 cysteine protease speb and the family protease papain ( figure 5 ) .
inhibitory profile of peptides and peptide analogues on 
 the activity 
 of streptococcal cysteine protease speb ( a ) and family class protease 
 papain ( b ) .
( r ) and ( s ) indicate the different stereoisomers of each analogue ;  pip  represents the piperidine moiety replacing glycine 
 residues .
the enzyme activity was determined for papain at a final 
 concentration of 0.9 m and speb at final concentration of 0.28 
 m in the presence of 4.8  10 molar excess 
 of peptides / peptide analogues .
interestingly , very different inhibitory profiles 
 were obtained 
 that apparently distinguish the proteases from the profile obtained 
 for ides and also from each other ( figures 4 and 5 ) . in general , the inhibitory effect 
 of all the tested compounds was much less pronounced on speb compared 
 to inhibition of ides .
hardly any of the pip236g - fragment containing analogues had 
 any inhibitory effect on speb activity ( figure 5a ) .
in contrast to the effect observed on ides activity 
 ( figure 4 ) , the degree of inhibition was not 
 improved when testing llpipg ( 10 ) peptide 
 analogues in which gly236 was replaced with a piperidine 
 moiety . however , a clearly improved degree of inhibition could be 
 observed when testing the ethylated analogues ( r)- 
 and ( s)-llpipetg ( 11 ) . 

in addition , ( r)- and ( s)-lgpipp ( 23 ) containing a similar tertiary amine 
 fragment as the ethylated analogues also inhibited speb activity , 
 which is in contrast to the effect observed for ides .
thus , the absence 
 of the possibility of hydrogen bond donation and the rigid structure 
 of neighboring piperidine and proline rings appear to increase the 
 interaction between the lgpipp ( 23 ) analogues 
 and speb .
like speb also , papain exhibits a broad proteolytic 
 activity and degrades a wide variety of substrates , including igg . 

the analysis of putative inhibitory effects of peptides and piperidine - containing 
 analogues on papain revealed that the enzyme was clearly very sensitive 
 toward most compounds tested ( figure 5b ) .
in 
 analogy to speb , the positioning of the piperidine in the peptide 
 sequence favors a replacement of the glycine corresponding to g237 , resulting in the most efficient papain inhibitors in the 
 series , with ( r)-lgpipp ( ( r)-23 ) being the most active analogue . in summary , 

the most active inhibitors of ides , ( r)-pipgp ( ( r)-16 ) and ( s)-lpipgp ( ( s)-18 ) , are even more 
 active toward papain but show no inhibitory activity 
 toward speb .
however , both ( s)-pipg ( ( s)-3 ) and ( r)-lpipg ( ( r)-7 ) show promising selectivity 
 profiles as they are both potent inhibitors of ides but are hardly 
 affecting papain and do not inhibit speb .
these compounds were investigated 
 further to evaluate whether they might be a suitable starting point 
 for the development of specific ides protease inhibitors .
various 
 amounts of inhibitor and substrate were used to determine 
 the inhibition constant ( ki ) for these 
 two compounds . because a mechanism of competitive inhibition was postulated , 
 initial velocities were determined in the presence or absence of inhibitor . 

the binding affinity of ides for igg has been previously determined , 
 and a high affinity was observed with a kd of 2.5 m and a km for complete 
 enzymatic cleavage ranging from 6.8 to 18.9 m depending on 
 the igg subtype . because of the 
 high affinity substrate binding , a considerable 
 excess of inhibitor was expected to be necessary to affect initial 
 velocities and to successfully compete for igg .
the ki values for ( s)-pipg ( ( s)-3 ) and ( r)-lpipg ( ( r)-7 ) were determined to approximately 
 6.7 and 5.7 mm , respectively . although these concentrations are too 
 high for in vivo evaluation , they were surprisingly low considering 
 the high affinity of ides for igg .
importantly , initial velocities 
 of papain activity remained unchanged by the presence of the inhibitors 
 and therefore ki values could not be determined 
 reliably , but the values exceed those observed for ides at least 16267-fold , 
 respectively .
thus , the aminopiperidine containing analogues 
 ( s)-pipg ( ( s)-3 ) and ( r)-lpipg ( ( r)-7 ) exhibit effective and specific ides inhibitor properties 
 and will 
 serve as a starting point for further development of more potent inhibitors . 
 initially ,
an attempt to increase the rigidity of the compound will 
 be undertaken which hopefully will increase the potency of the inhibitor . 

a series of 17 piperidine - containing peptide 
 analogues have been 
 efficiently synthesized .
the peptide analogues were tested in inhibition 
 assays addressing cysteine protease activities with focus on the streptococcal 
 igg degrading enzyme ides .
several of the synthesized piperidine - containing 
 peptide analogues are active ides inhibitors , but the piperidine moiety 
 does not necessarily improve the inhibitory capacity of the analogues 
 toward other cysteine proteases .
to the best of our knowledge , these 
 are the first noncovalently bound inhibitors of ides , also showing 
 selectivity toward speb and papain .
starting materials and reagents 
 were purchased from sigma - aldrich or bachem and were used as such . 

analytical tlc was performed 
 on silica plated aluminum sheets ( grade 60 f254 , merck ) .
the spots 
 were visualized by treatment with a dip solution [ kmno4 ( 1.5 g ) , k2co3 ( 10 g ) , naoh ( 10% ) ( 1.25 ml ) 
 in water ( 200 ml ) ] , followed by heating .
h and c nmr spectra were recorded on a jeol eclipse 
 400 spectrometer at 400 and 100 mhz , respectively , at ambient temperature 
 and in cdcl3 if not otherwise stated .
c nmr 
 spectra were also recorded on a varian 500 spectrometer at 125 mhz . 

chemical shifts are reported in ppm , with the solvent residual peak 
 as internal standard ( chcl3  7.26 ,  77.0 , ch3oh  3.30 ,  49.0 ) .
dept , cosy , tocsy , and hsqc spectra were recorded 
 to validate structural assignments of the nmr signals .
separation 
 of the stereoisomers of 1 and 19 were performed 
 on a preparative hplc shimadzu system equipped with a chiralpak ad 
 250 mm  20 mm column ( daicel chemical co. , tokyo , japan ) with 
 a mobile phase consisting of hexane / ethanol 95:5 or hexane/2-propanol 
 90:10 at a flow rate of 15 or 10 ml / min , respectively .
chiral hplc analysis was performed on a varian 
 system equipped with a chiralpak ad 4.6 mm  250 mm column ( daicel 
 chemical co. , tokyo , japan ) at a flow rate of 1 ml / min .
analytical reversed phase hplc was performed 
 on a waters 2690 system ( photodiode array detector at 220 nm and flow 
 rate 1.0 ml / min ) equipped with a 250 mm  4.6 mm reprosil pur 
 c18aq column .
optical rotation was measured with a perkin - elmer 341 
 lc polarimeter at 20 c .
infrared spectra were recorded on a 
 perkin - elmer 16 pc ftir spectrometer , and only the major peaks are 
 listed .
the 
 purity of all tested compounds was determined using analytical reversed 
 phase hplc on a waters 2690 system ( photodiode array detection at 
 220 nm and a flow rate of 1.0 ml / min ) equipped with a 250 mm  
 4.6 mm reprosil pur c18aq column .
( s)- and ( r)-pipg ( 3 ) were further analyzed 
 using a reversed phase waters acquity uplc system equipped with a 
 bec c18 column , connected to a ms instrument with a waters sqd single 
 quadrupole detector .
all compounds showed a purity > 95% except 
 ( s)-pipg ( ( s)-3 ) which was 90% pure .
measurement of the aqueous dissociation 
 constant , 
 pka values , of ( r)- and 
 ( s)-lpipgp ( 18 ) and ( r)- and ( s)-pipg ( 3 ) were performed at astrazeneca , r&d mlndal , sweden .
nabh(oac)3 ( 5.3 g , 25.0 mmol ) was added to a solution of 1-boc - piperidin-3-one 
 ( 4.98 g , 25.0 mmol ) , acetic acid ( 2.25 g , 37.5 mmol ) , glycine ethyl 
 ester hydrochloride ( 3.5 g , 25.0 mmol ) , and molecular sieves ( 4.5 
 g ) in dichloromethane ( 100 ml ) .
the reaction mixture was stirred at 
 room temperature for 20 h , diluted with dichloromethane ( 100 ml ) , 
 and quenched by the addition of aqueous naoh ( 3 m ) ( 20 ml ) .
the combined 
 organic phases were dried ( na2so4 ) , filtered , 
 and concentrated in vacuo .
purification by flash chromatography ( heptane / ethyl 
 acetate 1:2 , rf = 0.18 ) gave ( rac)-1 as a transparent oil ( 4.4 g , 62% ) .
resolution of a portion 
 of the racemate ( 2.0 g ) by chiral preparative hplc gave the enantiomers 
 of 1 .
resolution of ( rac)-1 ( 2.0 g ) gave enantiomerically pure ( r)-1 ( 779 mg ) . analytical chiral hplc ( hexane/2-propanol 95:5 ) tr 9.4 min ; 95% purity , ee 100% .
h nmr  4.18 ( q , j = 7.1 hz , 2h ) , 3.803.73 
 ( m , 2h ) , 3.553.41 ( ab system , 2h ) , 2.862.51 ( m , 3h ) , 
 2.201.80 ( m , 2h ) , 1.731.64 ( m , 1h ) , 1.471.21 
 ( m , 2h ) , 1.44 ( s , 9h ) , 1.26 ( t , j = 7.1 hz , 3h ) .
c nmr  172.2 , 154.6 , 79.2 , 60.6 , 53.3 , 48.6 , 48.1 , 
 43.9 , 31.1 , 28.2 , 23.2 , 14.0 ; [ ]d 8.5 
 ( c 1.0 , chcl3 ) .
ir ( neat ) v 3515 , 3333 , 2976 , 2933 , 2860 , 1738 , 1691 , 1423 , 1157 cm .
hrms ( ft - icr - ms ) calcd for c14h26n2o4 [ m + h ] 287.1965 ; found 287.1962 .
resolution of ( rac)-1 ( 2.0 g ) gave enantiomerically pure ( s)-1 ( 802 mg ) .
analytical chiral hplc ( hexane/2-propanol 95:5 ) tr 8.7 min , 94% purity , ee 100% .
h nmr  4.18 ( q , j = 7.1 hz , 2h ) , 3.813.73 
 ( m , 2h ) , 3.513.37 ( ab system , 2h ) , 2.932.48 ( m , 3h ) , 
 2.031.80 ( m , 3h ) , 1.731.64 ( m , 1h ) , 1.491.21 
 ( m , 10h ) , 1.26 ( t , j = 7.1 hz , 3h ) .
c nmr  172.2 , 154.6 , 79.1 , 60.5 , 53.3 , 48.9 , 48.1 , 43.9 , 31.0 , 
 28.2 , 23.2 , 13.9 ; [ ]d + 8.5 ( c 1.0 , chcl3 ) .
ir ( neat ) v 3515 , 
 3333 , 2976 , 2933 , 2860 , 1738 , 1691 , 1423 , 1157 cm . hrms ( ft - icr - ms ) calcd for c14h26n2o4 [ m + h ] 287.1965 ; found 287.1962 .
both enantiomers of 1 were also obtained separately 
 by reductive alkylation of 3-amino-1-tert - butyloxycarbonylpiperidine 
 with defined stereochemistry .
ethyl glyoxylate in toluene ( 50% ) ( 0.5 ml , 2.5 
 mmol ) was added dropwise to a mixture of ( r)-3-amino-1-tert - butyloxycarbonylpiperidine ( 500 mg , 2.5 mmol ) and na2so4 ( 900 mg ) in dichloromethane ( 9 ml ) .
the reaction 
 mixture was stirred at room temperature for 2 h. nabh(oac)3 ( 529 mg , 2.5 mmol ) and acoh ( 225 mg , 3.74 mmol ) were added , and 
 the stirring continued for 90 min .
the mixture was diluted with dichloromethane 
 ( 20 ml ) and quenched by addition of aqueous naoh ( 3 m ) ( 8 ml ) .
the 
 aqueous phase was extracted with dichloromethane ( 1  20 ml ) . 

the combined organic phases were dried ( na2so4 ) and filtered and the solvent was removed in vacuo .
purification 
 by flash chromatography ( heptane / ethyl acetate 1:3 , rf = 0.17 ) gave ( r)-1 as 
 a transparent oil ( 410 mg , 60% ) .
analytical chiral hplc ( hexane/2-propanol 
 95:5 ) tr 9.3 min ; ee 90% . 

nmr data were in agreement with those reported for the pure enantiomer 
 ( r)-1 .
compound ( s)-1 was 
 synthesized from ( s)-3-amino-1-tert - butyloxycarbonylpiperidine ( 500 mg , 2.5 mmol ) using the same procedure 
 as described for ( r)-1 to afford ( s)-1 as a transparent oil ( 380 mg , 56% ) . analytical 
 chiral hplc ( hexane/2-propanol 95:5 ) tr 8.5 min ; ee 86% .
nmr data were in agreement with 
 those reported for the pure enantiomer ( s)-1 .
compound ( r)-2 was 
 synthesized from ( r)-1 ( 210 mg , 0.73 
 mmol ) and nh3 in methanol ( 7 n ) ( 4.2 ml ) 
 as described for ( s)-2 to give the product 
 as a white solid ( 174 mg , 92% ) .
h nmr  
 7.05 ( br s , 2h ) , 5.53 ( br s , 1h ) , 3.973.54 ( m , 2h ) , 3.383.25 
 ( ab system , 2h ) , 3.162.67 ( m , 2h ) , 2.56 ( app tt , j = 8.1 , 8.1 , 3.7 , 3.7 hz , 1h ) , 1.971.84 ( m , 1h ) , 1.731.63 
 ( m , 1h ) , 1.511.23 ( m , 2h ) , 1.45 ( s , 9h ) .
c nmr 
  175.1 , 154.6 , 79.2 , 53.7 , 49.5 , 48.7 , 43.8 , 30.9 , 28.1 , 22.9 ; 
 [ ]d 4.6 ( c 1.0 , chcl3 ) .
ir ( kbr ) v 3398 , 2977 , 
 2933 , 1683 , 1638 , 1423 , 1262 , 1180 , 1155 cm .
hrms 
 ( ft - icr - ms ) calcd for c12h23n3o3 [ m + h ] 258.1812 ; found 258.1809 .
nh3 in methanol ( 7 n ) ( 4.3 ml ) was added to 
 ( s)-1 ( 214 mg , 0.75 mmol ) , and the reaction 
 mixture was stirred at room temperature for 72 h. the solvent and 
 excess of reagent were removed in vacuo .
the remaining was trituated 
 with hexane / diethyl ether ( 1:1 ) ( 1 ml ) to give ( s)-2 as a white solid ( 173 mg , 90% ) .
analytical chiral 
 hplc ( hexane / ethanol 97:3 ) tr 76.0 min , 
 purity 100% .
h nmr  7.06 ( br s , 2h ) , 5.62 ( br s , 
 1h ) , 3.943.56 ( m , 2h ) , 3.393.24 ( ab system , 2h ) , 3.322.72 
 ( m , 2h ) , 2.56 ( app tt , j = 8.1 , 8.1 , 3.7 , 3.7 hz , 
 1h ) , 1.951.86 ( m , 1h ) , 1.731.57 ( m , 2h ) , 1.45 ( s , 
 9h ) , 1.391.23 ( m , 2h ) .
c nmr  175.0 , 154.5 , 
 79.1 , 53.6 , 49.4 , 48.6 , 43.7 , 30.8 , 28.0 , 22.8 ; [ ]d + 5.1 ( c 1.0 , chcl3 ) .
ir 
 ( kbr ) v 3398 , 2977 , 2933 , 1683 , 1638 , 1423 , 1262 , 
 1180 , 1155 cm .
hrms ( ft - icr - ms ) calcd for c12h23n3o3 [ m + h ] 258.1812 ; found 258.1810 .
deprotection of ( r)-2 ( 137 mg , 0.53 mmol ) using tfa ( 364 mg , 3.2 mmol ) was performed as 
 described for ( s)-pipg ( ( s)-3 ) to give the tfa salt of ( r)-pipg ( ( r)-3 ) as a white solid 
 ( 210 mg , quant ) .
h nmr ( cd3od )  4.003.88 
 ( ab system , 2h ) , 3.73 ( dd , j = 12.1 , 4.0 hz , 1h ) , 
 3.633.51 ( app tt , j = 11.4 , 11.4 , 4.1 , 4.1 
 hz , 1h ) , 3.40 ( app dd , j = 12.9 , 3.6 hz , 1h ) , 3.08 
 ( app t , j = 11.7 hz , 1h ) , 2.96 ( dt , j = 12.7 , 12.7 , 3.2 hz , 1h ) , 2.322.24 ( m , 1h ) , 2.142.04 
 ( m , 1h ) , 1.871.78 ( m , 1h ) , 1.71 ( app ddt , j = 12.5 , 12.5 , 3.6 hz , 1h ) .
c nmr ( cd3od ) 
  168.5 , 53.0 , 46.6 , 45.0 , 44.6 , 26.2 , 21.6 ; [ ]d 9.3 ( c 0.41 , ch3oh ) .
ir ( neat ) v 3399 , 3012 , 2822 , 1683 , 1436 , 1205 , 
 1130 cm . hrms ( ft - icr - ms ) calcd for c7h15n3o [ m + h ] 158.1288 ; found 
 158.1287 ; pka1 = 5.13 , pka2 = 8.46 . for nmr spectral assignment ,
tfa ( 375 mg , 3.3 mmol ) was added to ( s)-2 ( 141 mg , 0.55 mmol ) in dichloromethane ( 3 ml ) , and 
 the reaction mixture was stirred at room temperature for 12 h. the 
 solvent and excess of reagent were removed in vacuo to give the tfa - salt 
 of ( s)-pipg ( ( s)-3 ) as a white solid ( 210 mg , quant ) .
h nmr ( cd3od )  4.003.85 ( ab system , 2h ) , 3.763.65 
 ( m , 1h ) , 3.56 ( app tt , j = 11.4 , 11.4 , 3.7 , 3.7 , 
 1h ) , 3.443.34 ( m , 1h ) , 3.08 ( app t , j = 11.7 
 hz , 1h ) , 2.96 ( app dt , j = 12.8 , 12.8 , 3.3 hz , 1h , ) , 
 2.332.21 ( m , 1h ) , 2.152.01 ( m , 1h ) , 1.881.60 
 ( m , 2h ) .
c nmr ( cd3od )  168.4 , 53.0 , 
 46.6 , 45.0 , 44.6 , 26.2 , 21.6 ; [ ]d + 6.6 
 ( c 0.41 , ch3oh ) .
ir ( neat ) v 3383 , 3014 , 2824 , 1676 , 1432 , 1202 , 1130 cm . 
 hrms ( ft - icr - ms ) calcd for c7h15n3o [ m + h ] 158.1288 ; found 158.1287 .
compound ( r)-4 was synthesized from ( r)-1 ( 802 mg , 2.8 mmol ) and n-(benzyloxycarbonyloxy)succinimide 
 ( 698 mg , 2.8 mmol ) with et3n ( 312 mg , 3.1 mmol ) in dichloromethane 
 ( 11 ml ) as described for ( s)-4 to give 
 the product as a transparent oil ( 0.98 g , 82% ) .
analytical chiral 
 hplc ( hexane/2-propanol 95:5 ) tr 19.8 
 min , 98% purity , ee 100% .
h nmr ( rotamers ) 
  7.597.20 ( m , 5h ) , 5.19 and 5.10 ( rot , s , 2h ) , 4.343.72 
 ( m , 6h ) , 2.802.43 ( m , 3h ) , 2.031.87 ( m , 1h ) , 1.800.98 
 ( m , 1h ) , 1.431.17 ( m , 2h ) , 1.43 and 1.37 ( rot , s , 9h ) , 1.26 
 and 1.17 ( rot , t , j = 7.1 hz , 3h ) . c 
 nmr  169.7 , 155.8 , 155.1 , 154.5 , and 154.3 ( rot ) , 136.1 , 128.3 , 
 and 128.2 ( rot ) , 127.8 , 127.5 , 79.4 , 67.4 , and 67.1 ( rot ) , 60.9 , 53.1 , 
 and 52.9 ( rot ) , 47.0 and 46.5 , 45.6 and 45.3 , 43.2 , 28.7 , 28.1 , 24.4 , 
 13.9 ; [ ]d 14.9 ( c 1.0 , chcl3 ) .
ir ( neat ) v 3500 , 2976 , 
 2864 , 1749 , 1693 , 1416 , 1152 cm .
hrms ( ft - icr - ms ) 
 calcd for c22h32n2o6 [ m 
 + h ] 421.2333 ; found 421.2333 .
et3n ( 290 mg , 2.9 mmol ) in dichloromethane 
 ( 2 ml ) was added to a solution of ( s)-1 ( 745 mg , 2.6 mmol ) in dichloromethane ( 8 ml ) at 0 c , and n-(benzyloxycarbonyloxy)succinimide ( 648 mg , 2.6 mmol ) was 
 added in portions .
the reaction mixture was stirred at room temperature 
 for 17 h , diluted with dichloromethane ( 30 ml ) , and washed with brine 
 ( 2  15 ml ) .
the organic phase was dried ( na2so4 ) , filtered , and concentrated in vacuo .
purification by flash 
 chromatography ( heptane / ethyl acetate 2:1 , rf = 0.23 ) gave ( s)-4 as a transparent 
 oil ( 0.94 g , 84% ) .
analytical chiral hplc ( hexane/2-propanol 95:5 ) tr 18.7 min , 98% purity , ee 100% .
h nmr ( rotamers )  7.487.19 ( m , 5h ) , 5.18 and 
 5.11 ( rot , s , 2h ) , 4.313.79 ( m , 6h ) , 2.802.41 ( m , 
 3h ) , 2.041.89 ( m , 1h ) , 1.801.32 ( m , 12h ) , 1.27 and 
 1.17 ( rot , t , j = 7.1 hz , 3h ) .
c nmr 
  169.8 , 155.9 , 155.1 , 154.5 , and 154.4 ( rot ) , 136.2 , 128.3 , 
 and 128.2 ( rot ) , 127.8 , 127.6 , 79.5 , 67.5 , and 67.1 ( rot ) , 61.0 , 53.2 , 
 and 53.0 ( rot ) , 47.1 and 46.5 ( rot ) , 45.6 and 45.4 ( rot ) , 43.3 , 28.7 , 
 28.2 , 24.4 , 13.9 ; [ ]d + 14.0 ( c 1.0 , chcl3 ) .
ir ( neat ) v 3499 , 
 2976 , 2863 , 1747 , 1693 , 1415 , 1152 cm ; hrms ( ft - icr - ms ) 
 calcd for c22h32n2o6 [ m 
 + h ] 421.2333 ; found 421.2334 .
deprotection of ( r)-4 ( 870 mg , 2.07 mmol ) with tfa ( 2.0 ml , 
 27.0 mmol ) in dichloromethane ( 8 ml ) was performed as described for 
 ( s)-5 to give the boc - deprotected intermediate . 
 the crude intermediate and boc - l - leu  h2o ( 551 mg , 2.21 mmol ) were coupled as described for ( s)-5 using edc ( 424 mg , 2.21 mmol ) , hobt ( 299 mg , 2.21 
 mmol ) , and et3n ( 813 mg , 8.04 mmol ) in dichloromethane 
 ( 11 ml ) with molecular sieves ( 600 mg ) .
purification by flash chromatography 
 ( heptane / ethyl acetate 1:1 , rf = 0.2 ) 
 gave ( r)-5 as a transparent oil ( 1.0 
 g , 91% yield over two steps ) .
analytical chiral hplc ( hexane / ethanol 
 70:30 ) tr 7.5 min , 99% purity , de 97% .
h nmr ( cd3od )  7.417.25 
 ( m , 5h ) , 5.195.10 ( m , 1h ) , 5.06 ( s , 2h ) , 4.644.38 
 ( m , 2h ) , 4.233.84 ( m , 6h ) , 3.223.04 ( m , 1h ) , 3.032.91 
 ( m , 1h ) , 2.762.61 ( m , 1h ) , 2.552.41 ( m , 1h ) , 1.991.56 
 ( m , 2h ) , 1.551.07 ( m , 14h ) , 1.000.72 ( m , 6h ) . c nmr ( cd3od )  173.7 and 173.4 ( rot ) , 171.8 
 and 171.7 ( rot ) , 157.6 , 157.2 , 137.8 , 129.5 , 129.1 , 128.8 , 80.5 , 68.6 , 
 62.3 , 55.0 , 50.2 , 46.7 , 46.0 , 43.1 , 42.4 , and 42.1 ( rot ) , 29.0 , 28.7 , 
 26.0 , 25.4 , 23.6 , 22.2 , 14.4 ; [ ]d 31.5 
 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3423 , 2959 , 1753 , 1708 , 1642 , 1445 , 1205 cm . 

hrms ( ft - icr - ms ) calcd for c28h43n3o7 [ m + h ] 534.3174 ; found 534.3176 .
tfa ( 2.0 ml , 27.0 mmol ) 
 was added to ( s)-4 ( 950 mg , 2.26 mmol ) 
 in dichloromethane ( 8 ml ) and stirred at room temperature for 16 h. 
 the solvent and excess of reagents were removed in vacuo to give the 
 boc - deprotected intermediate .
boc - l - leu  h2o ( 620 mg , 2.49 mmol ) , edc ( 477 mg , 2.49 mmol ) , and hobt ( 336 mg , 
 2.49 mmol ) were stirred in dichloromethane ( 6 ml ) with molecular sieves 
 ( 600 mg ) at 0 c for 15 min . into this solution ,
the boc - deprotected 
 intermediate and et3n ( 9.14 mg , 9.04 mmol ) in dichloromethane 
 ( 5 ml ) were added and the reaction mixture was stirred at room temperature 
 for 15 h. the mixture was diluted with dichloromethane ( 20 ml ) and 
 washed with aqueous citric acid ( 10% ) ( 2  10 ml ) , saturated 
 aqueous nahco3 ( 2  10 ml ) , and brine ( 1  10 
 ml ) .
purification by flash 
 chromatography ( heptane / ethyl acetate 1:1 , rf = 0.18 ) gave ( s)-5 as a transparent 
 oil ( 950 mg , 79% yield over two steps ) .
analytical chiral hplc ( hexane / ethanol 
 70:30 ) tr 6.4 min , purity 100% .
h nmr ( cd3od )  7.497.22 ( m , 5h ) , 5.445.15 
 ( m , 1h ) , 5.07 ( s , 2h ) , 4.724.47 ( m , 2h ) , 4.243.65 
 ( m , 6h ) , 3.583.30 ( m , 1h ) , 3.122.81 ( m , 1h ) , 2.692.48 
 ( m , 1h ) , 2.472.23 ( m , 1h ) , 2.121.90 ( m , 2h ) , 1.891.10 
 ( m , 15h ) , 1.060.72 ( m , 6h ) .
c nmr ( cd3od )  ( 174.0 , 173.8 and 173.4 , rot ) , 171.8 , 171.6 , ( 157.7 , 
 157.0 and 156.8 , rot ) , 137.8 and 137.6 ( rot ) , 129.5 and 129.4 ( rot ) , 
 129.2 and 129.1 ( rot ) , 128.8 and 128.7 ( rot ) , 80.4 and 80.1 ( rot ) , 
 68.5 and 68.3 ( rot ) , 62.2 , 54.5 , and 54.3 ( rot ) , 50.4 , 48.0 , 46.4 , 
 and 46.2 ( rot ) , 45.8 , 30.1 , 29.7 , and 29.5 ( rot ) , 28.8 , 26.2 , 25.9 , 
 and 25.7 ( rot ) , 23.7 , 22.0 , 14.5 ; [ ]d + 25.7 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3425 , 2958 , 1754 , 1720 , 1640 , 1442 , 1204 cm . hrms ( ft - icr - ms ) calcd for c28h43n3o7 [ m + h ] 534.3174 ; found 534.3174 .
compound ( r)-6 was synthesized from ( r)-5 ( 200 mg , 0.38 mmol ) and nh3 in methanol ( 7 n ) ( 4.0 ml ) as described for ( s)-2 .
purification by flash chromatography ( dichloromethane / methanol 
 95:5 , rf = 0.09 ) gave ( r)-6 as a white solid ( 122 mg , 65% ) .
analytical chiral 
 hplc ( hexane / ethanol 60:40 ) tr 12.3 min , 
 95% purity , de 90% .
h nmr ( cd3od , 50 c )  7.397.23 ( m , 5h ) , 5.12 ( br s , 2h ) , 
 4.494.44 ( m , 1h ) , 4.113.80 ( m , 3h ) , 3.183.04 
 ( m , 1h ) , 3.032.89 ( m , 1h ) , 2.772.61 ( m , 1h ) , 2.562.40 
 ( m , 1h ) , 2.031.91 ( m , 1h ) , 1.851.27 ( m , 15h ) , 1.020.73 
 ( m , 6h ) .
c nmr ( cd3od , 50 c )  
 174.4 , 173.7 , and 173.4 ( rot ) , 157.6 , 137.8 , 129.5 , 129.1 , 128.8 , 
 80.5 , 68.7 , 54.9 , 50.2 , 47.6 , 46.7 , and 46.4 ( rot ) , 43.1 , 42.4 , 28.8 , 
 26.6 , 25.9 , 25.5 , 23.5 , 22.2 ; [ ]d 33.9 
 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3414 , 2956 , 1707 , 1640 , 1446 cm . hrms ( ft - icr - ms ) 
 calcd for c26h40n4o6 [ m 
 + h ] 505.3021 ; found 505.3021 .
compound ( s)-6 was synthesized from ( s)-5 ( 156 mg , 0.29 mmol ) and nh3 in methanol ( 7 n ) ( 4.0 ml ) as described for ( s)-2 .
purification by flash chromatography ( dichloromethane / methanol 
 95:5 , rf = 0.07 ) gave the product as a 
 white solid ( 108 mg , 73% ) .
analytical chiral hplc ( hexane / ethanol 
 60:40 ) tr 8.5 min , 100% purity .
h nmr ( cd3od , 50 c )  7.467.23 ( m , 
 5h ) , 5.12 ( br s , 2h ) , 4.494.38 ( m , 1h ) , 4.203.79 ( m , 
 3h ) , 3.263.10 ( m , 1h ) , 3.092.93 ( m , 1h ) , 2.772.62 
 ( m , 1h ) , 2.562.38 ( m , 1h ) , 2.051.61 ( m , 4h ) , 1.601.24 
 ( 12h ) , 1.000.75 ( m , 6h ) .
c nmr ( cd3od , 50 c )  174.4 , 173.8 , and 173.5 ( rot ) , 157.7 , 137.8 , 
 129.5 , 129.0 , 128.8 , 80.4 , 68.7 , 54.7 , 50.6 , 47.4 , 46.4 , 43.6 , 42.8 , 
 and 42.2 ( rot ) , 28.8 , 26.3 , 26.0 , 25.6 , 23.6 , 22.1 ; [ ]d + 13.2 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3421 , 2957 , 1703 , 1638 , 1456 cm .
hrms ( ft - icr - ms ) calcd for c26h40n4o6 [ m + h ] 505.3021 ; found 505.3022 .
compound ( r)-6 ( 115 mg , 0.23 mmol ) was deprotected as described for ( r)-llpipg ( ( r)-10 ) to give 
 the white tfa salt of ( r)-lpipg ( ( r)-7 ) ( 95 mg , 84% ) .
analytical reversed phase 
 hplc ( the mobile phase was 6.432.8% ch3cn in water 
 during 30 min , 0.085% tfa throughout ) tr 6.2 min , 100% purity .
h ( cd3od ) nmr  
 4.75 ( app d , 1h ) , 4.484.38 ( m , 1h ) , 3.983.86 ( m , 2h ) , 
 3.74 ( app d , 1h ) , 3.233.08 ( m , 2h ) , 2.85 ( app t , 1h ) , 2.322.18 
 ( m , 1h ) , 1.981.51 ( m , 6h ) , 1.050.95 ( m , 6h ) .
c nmr ( cd3od )  169.9 , 168.5 , 54.7 , 50.5 , 
 46.6 , 46.4 , 44.5 , 41.0 , 28.0 , 25.3 , 25.0 , 23.5 , 21.6 ; [ ]d 11.2 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3420 , 2967 , 1676 , 1202 , 1134 
 cm .
hrms ( ft - icr - ms ) calcd for c13h26n4o2 [ m + h ] 271.2129 ; 
 found 271.2128 . for nmr spectral assignment , see the supporting information . compound ( s)-6 ( 117 mg , 0.23 mmol ) was deprotected as described for ( r)-llpipg ( ( r)-10 ) but 
 with stirring for 14 h for the first deprotection .
the second deprotection 
 gave the white tfa salt of ( s)-lpipg ( ( s)-7 ) ( 87 mg , 75% ) .
analytical reversed 
 phase hplc ( the mobile phase was 6.432.8% ch3cn 
 in h2o for 30 min , 0.085% tfa throughout ) tr 6.6 min , 100% purity .
h ( cd3od ) 
 nmr  4.554.40 ( m , 1h ) , 4.25 ( app d , 1h ) , 4.003.85 
 ( m , 2h ) , 3.663.57 ( m , 1h ) , 3.503.42 ( m , 1h ) , 2.63 
 ( app t , 1h ) , 2.322.16 ( m , 1h ) , 2.031.93 ( m , 1h ) , 1.901.48 
 ( m , 6h ) , 1.070.94 ( m , 6h ) .
c nmr ( cd3od )  170.3 , 168.5 , 54.9 , 50.5 , 46.7 , 46,6 , 43.9 , 41.1 , 27.3 , 
 25.3 , 23.5 , 23.1 , 21,7 ; [ ]d + 14.8 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3418 , 
 2965 , 1676 , 1201 , 1134 cm .
hrms ( ft - icr - ms ) calcd 
 for c13h26n4o2 [ m + h ] 271.2129 ; found 271.2128 .
deprotection of ( r)-5 ( 649 mg , 1.22 mmol ) by tfa ( 1.0 ml , 13 mmol ) in dichloromethane 
 ( 6 ml ) were performed as described for ( s)-5 to give the boc - deprotected intermediate . the intermediate 
 and the second boc - l - leu  h2o ( 334 mg , 1.34 
 mmol ) were coupled as described for ( s)-5 with edc ( 257 mg , 1.34 mmol ) , hobt ( 181 mg , 1.34 mmol ) , et3n ( 493 mg , 4.87 mmol ) , and molecular sieves ( 200 mg ) in dichloromethane 
 ( 11 ml ) .
purification by flash chromatography ( heptane / ethyl acetate 
 1:1 , rf = 0.14 ) gave ( r)-8 as a transparent oil ( 690 g , 88% yield over two 
 steps ) .
analytical chiral hplc ( hexane / ethanol 70:30 ) tr 9.2 min , 100% purity .
h nmr ( cd3od )  7.437.25 ( m , 5h ) , 5.205.02 ( m , 2h ) , 5.004.85 
 ( m , 1h ) , 4.654.51 ( m , 1h ) , 4.504.38 ( m , 1h ) , 4.213.80 
 ( m , 8h ) , 3.213.03 ( m , 1h ) , 3.022.89 ( m , 1h ) , 2.792.61 
 ( m , 1h ) , 2.522.38 ( m , 1h ) , 1.981.35 ( m , 13h ) , 1.341.18 
 ( m , 2h ) , 1.161.09 ( m , 3h ) 1.040.81 ( m , 12h ) .
c nmr ( cd3od )  174.9 , 172.3 , and 172.1 
 ( rot ) , 171.6 and 171.4 ( rot ) , 157.5 , 157.2 , 157.0 , and 156.7 ( rot ) , 
 137.6 , 129.4 , 129.0 , 128.8 , 128.7 , 80.2 , 68.4 , and 68.3 ( rot ) , 62.2 
 and 62.1 ( rot ) , 54.2 , 54.5 , 49.6 , 46.5 , 45.9 , and 45.6 ( rot ) , 42.8 , 
 42.2 , and 41.9 ( rot ) , 28.7 , 26.4 , and 25.7 ( rot ) , 25.6 , 25.4 , 23.7 , 
 23.6 , 23.5 , 22.2 , 22.0 , 21.4 , 14.5 ; [ ]d 43.5 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3318 , 2957 , 1754 , 1709 , 1640 , 1445 , 1206 cm . hrms ( ft - icr - ms ) calcd for c34h54n4o8 [ m + h ] 647.4014 ; found 647.4015 .
deprotection of ( s)-5 ( 535 mg , 1.0 mmol ) with tfa ( 1.0 ml , 13 mmol ) and dichloromethane 
 ( 6 ml ) were performed as described for the synthesis of ( s)-5 to give the boc - deprotected intermediate . the intermediate 
 and the second boc - l - leu  h2o ( 275 mg , 1.10 
 mmol ) were coupled as described for the synthesis of ( s)-5 with edc ( 212 mg , 1.10 mmol ) , hobt ( 149 mg , 1.10 
 mmol ) , et3n ( 406 mg , 4.02 mmol ) , and molecular sieves ( 240 
 mg ) in dichloromethane ( 11 ml ) .
purification by flash chromatography 
 ( heptane / ethyl acetate 1:1 , rf = 0.24 ) 
 gave ( s)-8 as a transparent oil ( 590 
 g , 91% yield over two steps ) .
analytical chiral hplc ( hexane / ethanol 
 70:30 ) tr 5.6 min , 100% purity .
h nmr ( cd3od )  7.477.23 ( m , 5h ) , 5.285.02 
 ( m , 2h ) , 5.004.86 ( m , 1h ) , 4.674.50 ( m , 1h ) , 4.484.36 
 ( m , 1h ) , 4.233.97 ( m , 6h ) , 3.963.81 ( m , 1h ) , 3.743.62 
 ( m , 1h ) , 3.493.36 ( m , 1h ) , 3.273.17 ( m , 1h ) , 3.052.90 
 ( m , 1h ) , 2.732.60 ( m , 1h ) , 2.542.35 ( m , 1h ) , 2.141.08 
 ( m , 18h ) , 1.000.74 ( m , 12h ) .
c nmr ( cd3od )  174.8 , 172.6 , and 172.5 ( rot ) , 171.7 and 171.5 ( rot ) , 
 157.5 , 156.8 , and 156.5 ( rot ) , 137.6 and 137.5 ( rot ) , 129.3 , 129.1 , 
 and 128.9 ( rot ) , 128.7 , 128.6 , 80.2 , 68.5 , 68.3 , and 68.1 ( rot ) , 62.1 , 
 54.3 , 50.2 , 46.3 , 46.2 , 45.7 , 43.4 , 42.7 , 41.9 , 30.0 , 28.8 , 26.2 , 
 25.8 , 23.8 , 23.6 , 22.4 , 22.2 , 22.0 , 14.5 ; [ ]d 9.2 ( c 0.5 , ch3oh ) . 

ir ( kbr ) v 3319 , 2957 , 1753 , 1709 , 1641 , 1447 , 1205 
 cm .
hrms ( ft - icr - ms ) calcd for c34h54n4o8 [ m + h ] 647.4014 ; 
 found 647.4014 . compound ( r)-9 was synthesized from ( r)-8 ( 213 mg , 0.33 mmol ) and nh3 in methanol ( 7 n ) ( 2.0 ml ) as described for ( s)-2 , but the reaction mixture was stirred for 168 h. purification 
 by flash chromatography ( dichloromethane / methanol 95:5 , rf = 0.05 ) gave the product as a white solid ( 119 mg , 63% ) . 

analytical chiral hplc ( hexane / ethanol 70:30 ) tr 13.7 min , 100% purity .
h nmr ( cd3od ) 
  7.427.24 ( m , 5h ) , 5.205.05 ( m , 2h ) , 4.964.87 
 ( m , 1h ) , 4.664.52 ( m , 1h ) , 4.484.37 ( m , 1h ) , 4.143.84 
 ( m , 6h ) , 3.172.92 ( m , 2h ) , 2.732.59 ( m , 1h ) , 2.542.40 
 ( m , 1h ) , 2.031.91 ( m , 1h ) , 1.871.34 ( m , 14h ) , 1.000.84 
 ( m , 12h ) .
c nmr ( cd3od , 50 c )  
 175.0 , 174.4 , 172.6 , and 172.4 ( rot ) , 157.7 , 157.5 , 137.8 , 129.5 , 
 129.0 , 128.7 , 80.6 , 68.7 , 55.2 , 54.8 , 54.6 , 47.6 , 46.7 , 46.1 , 43.0 , 
 42.3 , 42.1 , 29.1 , 28.7 , 26.6 , 26.0 , 25.7 , 25.3 , 23.6 , 23.3 , 22.2 , 
 21.9 ; [ ]d 50.0 ( c 0.67 , ch3oh ) .
ir ( kbr ) v 3408 , 2956 , 
 1704 , 1638 , 1449 cm . hrms ( ft - icr - ms ) calcd for 
 c32h51n5o7 [ m + h ] 618.3861 ; found 618.3863 .
compound ( s)-9 was synthesized from ( s)-8 ( 394 mg , 0.61 mmol ) and nh3 in methanol ( 7 n ) ( 4.0 ml ) as described for ( r)-7 .
purification by flash chromatography ( dichloromethane / methanol 
 95:5 , rf = 0.13 ) gave ( s)-7 as a white solid ( 325 mg , 86% ) .
analytical chiral hplc ( hexane / ethanol 
 70:30 ) tr 8.4 min , 100% purity .
h nmr ( cd3od )  7.517.22 ( m , 5h ) , 5.275.06 
 ( m , 2h ) , 4.984.86 ( m , 1h ) , 4.664.50 ( m , 1h ) , 4.474.37 
 ( m , 1h ) , 4.233.79 ( m , 6h ) , 3.283.11 ( m , 1h ) , 3.062.91 
 ( m , 1h ) , 2.722.58 ( m , 1h ) , 2.542.38 ( m , 1h ) , 2.041.81 
 ( m , 2h ) , 1.801.25 ( m , 13h ) , 0.990.75 ( m , 12h ) .
c nmr ( cd3od , 50 c )  174.9 , 174.3 , 
 and 174.2 ( rot ) , 172.7 and 172.3 ( rot ) , 157.5 , 137.8 , and 137.7 ( rot ) , 
 129.4 , 128.9 , 128.7 , 80.5 , 68.6 , 56.4 , 54.7 , 47.3 , 46.4 , 46.1 , 43.5 , 
 42.8 , 42.1 , 42.0 , 29.1 , 28.8 , 26.2 , 25.8 , 25.7 , 23.6 , 23.4 , 22.2 , 
 22.0 ; [ ]d 9.5 ( c 0.67 , ch3oh ) .
ir ( kbr ) v 3407 , 2957 , 
 1707 , 1638 , 1450 cm . hrms ( ft - icr - ms ) calcd for 
 c32h51n5o7 [ m + h ] 618.3861 ; found 618.3861 .
compound ( r)-9 ( 282 mg , 
 0.46 mmol ) was deprotected as described for ( s)-lpipgp ( ( s)-18 ) but with stirring 
 for 3 h during the first deprotection step .
purification by flash 
 chromatography ( ethyl acetate / methanol 20:3 ) gave the cbz - deprotected 
 intermediate ( 189 mg ) .
further deprotection ( 180 mg ) was performed 
 as described for ( s)-lpipgp ( ( s)-18 ) with tfa / dichloromethane ( 1:10 ) ( 3 ml ) 
 and with stirring for 14 h to give the white tfa salt of ( r)-llpipg ( ( r)-10 ) ( 251 mg , 90% ) .
analytical reversed phase hplc ( the mobile phase 
 was 10.837.2% ch3cn in h2o for 30 min 
 with 0.1% tfa throughout ) tr 8.3 min , 
 100% purity .
h nmr ( cd3od )  4.934.82 
 ( m , 1h ) , 4.67 ( app d , 1h ) , 4.09 ( app d , 1h ) , 4.043.83 ( m , 
 3h ) , 3.823.64 ( m , 2h ) , 3.473.38 ( m , 1h ) , 3.263.09 
 ( m , 2h ) , 2.84 ( app t , 1h ) , 2.322.11 ( m , 1h ) , 1.991.41 
 ( m , 6h ) , 1.040.76 ( m , 12h ) .
c nmr ( cd3od )  172.8 , 170.7 , 168.6 , 55.6 , 54.8 , 52.7 , 46.7 , 46.4 , 44.5 , 
 41.8 , 41.3 , 28.0 , 25.7 , 25.1 , 25.0 , 23.5 , 23.1 , 21.9 , 21.7 ; [ ]d 13.6 ( c 1.0 , ch3oh ) . ir ( kbr ) v 3422 , 2964 , 1676 , 1202 , 1135 
 cm .
hrms ( ft - icr - ms ) calcd for c19h37n5o3 [ m + h ] 384.2969 ; 
 found 384.2967 . for nmr spectral assignment , see the supporting information . compound ( s)-9 ( 230 mg , 
 0.37 mmol ) was deprotected as described for ( r)-llpipg ( ( r)-10 ) to give the 
 white tfa salt of ( s)-llpipg ( ( s)-10 ) ( 209 mg , 92% ) . analytical reversed phase 
 hplc ( the mobile phase was 10.837.2% ch3cn in h2o for 30 min ) tr 8.60 min , 100% 
 purity .
h nmr ( cd3od )  4.834.77 
 ( m , 1h ) , 4.534.39 ( m , 1h ) , 4.133.86 ( m , 3h ) , 3.753.65 
 ( m , 1h ) , 3.633.45 ( m , 2h ) , 3.393.32 ( m , 1h ) 2.60 ( app 
 t , 1h ) , 2.312.14 ( m , 1h ) , 2.091.97 ( m , 1h ) , 1.941.81 
 ( m , 1h ) , 1.801.56 ( m , 5h ) , 1.551.39 ( m , 1h ) , 1.040.86 
 ( m , 12h ) .
c nmr ( cd3od )  173.0 , 171.3 , 
 168.7 , 56.1 , 55.2 , 52.7 , 47.1 , 46.9 , 44.1 , 41.6 , 41.3 , 27.5 , 25.8 , 
 25.2 , 24.4 , 23.5 , 23.2 , 22.1 , 21.8 ; [ ]d + 14.3 ( c 1.0 , ch3oh ) .
ir ( kbr ) v 3419 , 2966 , 1674 , 1202 , 1137 cm .
hrms 
 ( ft - icr - ms ) calcd for c19h37n5o3 [ m + h ] 384.2969 ; found 384.2966 . for recombinant ides expression 

an ides allele was pcr - amplified from genomic streptococcal 
 dna using primers 5-tgttaccatggatagtttttctgctaat-3 
 introducing a ncoi restriction site and 5-gctaggtaccttaattggtctgattccaactat-3 introducing an acc65i restriction site for subsequent cloning into plasmid 
 pettrx_1a .
ides was expressed in escherichia coli strain bl21 to obtain a his - tagged 
 trx - ides protein .
the his - trx tag was removed by enzymatic 
 cleavage with tev - protease in his - trap binding buffer ( 20 mm tris - hcl 
 ph8.0 , 150 mm nacl , 10% glycerol ) at 30 c for up to 20 h followed 
 by a second round of purification on ni - resin to remove 
 uncleaved fusion protein .
the collected 
 flow through , containing ides without tag , was purified by size exclusion 
 chromatography on a hiprep 16/60 sephacryl s-300 hr column ( ge healthcare ) . 

ides ( 10 
 l ) ( 1.83 m in pbs ) was mixed with peptide or peptide 
 analogue ( 3 l ) ( 30 mm in 50% dmso , 50 mm phosphate buffer ph 
 7.4 ) and incubated for 15 min at room temperature prior to the addition 
 of igg1 ( 10 l ) ( sigma i5154 , 8.07 m in 20 mm tris - buffered 
 saline ph 8.0 ) as substrate .
the reactions were incubated for 20 min 
 at 37 c before termination by the addition of iodoacetamide 
 ( 10 l , 16.5 mm ) ( sigma i1149 ) .
controls where ides had been replaced with pbs were included in duplicates 
 for each peptide / peptide analogue .
ki values were determined by accessing velocity curves 
 at different substrate concentrations in the presence or absence of 
 inhibitor .
inhibitor concentrations were 5 , 2 , and 0.5 mm for ( r)-lpipg ( ( r)-7 ) and 5 , 2.5 , and 1 mm for ( s)-pipg ( ( s)-3 ) .
for ides , repurified polyclonal 
 igg at concentrations of 150 , 100 , 50 , 20 , or 10 m , was used 
 in reactions with 2 m of ides .
reactions were analyzed by non- 
 reducing sds page gel analysis to allow measurements of the initial 
 substrate cleavage .
the enzyme velocity v was determined 
 according to the following equation:1for papain , n-benzoyl - dl - arginine 4-nitroanilide hydrochloride ( sigma ) 
 at concentrations of 6 , 3 , 1 , 0.5 , or 0.25 mm was used with 5 m 
 of papain and inhibitor .
ki nonlinear 
 regression ( curve fit ) for competitive inhibition and enzymatic kinetics 
 were calculated using graphpad prism version 5.04 for windows , graphpad 
 software , san diego california , www.graphpad.com .
surface 
 plasmon resonance spectroscopy assays were performed with 1042.5 ru 
 of protein a ( abcam ) covalently bound to a cm5 chip using nhs / edc 
 coupling according to the manufacturer s instructions .
one 
 part of the igg1/ides reaction was mixed with two parts water before 
 applying the sample on the protein a - cm5 chip .
run conditions on the 
 biacore x100 machine were 5 l / min flow rate , a 90 s sample 
 injection time , hbs - ep+ ( biacore ) as running buffer , and 20 mm glycine 
 ph 2.0 as regeneration solution of the chip .
report point for amount 
 of protein bound to the chip surface was retrieved at 190 s after 
 sample injection stop .
amount of igg1 cleavage was first plotted as 
 percentage decrease between pbs - control ( uncleaved igg1 ) and ides / igg1 
 samples . a standard curve for ides concentration was made without 
 the presence of peptide or peptide analogue .
the experimental data 
 obtained from assays with ides together with peptide / peptide analogue 
 experiments were plotted to the standard curve and presented as approximate 
 fraction of active ides at any given time point .
inhibition of the enzymatic activity 
 of papain by peptides and peptide analogues was determined by a colorimetric 
 assay .
papain ( fluka 76218 ) at a concentration of 1.23 m was 
 diluted in assay buffer ( 81 l ) ( 123 mm phosphate buffer ph 
 6.2 , 1.23 mm edta , 1.23 mm dtt ) , mixed with peptides or peptide analogues 
 ( 16 l ) ( 30 mm in 50% dmso , 50 mm phosphate buffer ph 6.2 ) and 
 incubated for 10 min at room temperature .
the final concentration 
 of papain during the experiment was 0.9 m . the chromogenic 
 papain substrate n-benzoyl - dl - arginine-4-nitroanilide hydrochloride ( 3 l ) ( sigma 
 b4875 , 100 mm in dmso ) was added , and the reaction was incubated for 
 2 h at 37 c before termination by the addition of glacial acetic 
 acid ( 10 l ) .
samples were run in duplicates on two separate 
 occasions . for purification of speb ,
hewitt 
 broth ( th ) ( bd biosciences ) at 37 c ; 5% ( v / v ) co2 .
the growth media was cleared by centrifugation and subjected to 
 ammonium sulfate precipitation ( 5080% ( w / v ) ) .
the resulting 
 pellet was dissolved in 1 pbs , dialyzed o / n at 4 c against 
 20 mm sodium acetate buffer , ph 5 .
the sample was further purified 
 on a hitrap sp ff anion exchange column ( ge healthcare ) , equilibrated 
 in 20 mm sodium acetate , ph 5 .
proteins were eluted by a linear nacl 
 gradient and speb eluted at approximately 0.2 m nacl .
the eluted protein 
 fraction was dialyzed o / n against 1 pbs and kept at 20 
 c .
briefly , speb 
 was activated with 2 mm dithiothreitol ( dtt ) for 30 min at 37 c 
 and mixed with 150 l of 1 mm substrate solution , n - benzoyl - proline - phenylalanine - arginine - p - nitroanilide 
 hydrochloride ( bpfa ) ( ph 4 ) ( sigma ) , in 0.1 m phosphate buffer ( ph 
 6 ) . the change in absorbance at od405
the 
 final concentration of speb during the experiment was 0.28 m 
 the amount of active speb was determined by active - site titration 
 using various amount of the cysteine protease inhibitor e-64 as previously 
 described . for inhibition assays ,
11 
 l of activated speb ( 1.12 m in pbs ) was mixed with 2 
 l of peptide or peptide analogue ( 30 mm in 50% dmso , 50 mm 
 phosphate buffer ph 7.4 ) and incubated for 15 min at room temperature 
 prior to the addition of substrate solution .
 a series of eight peptides corresponding to the amino 
 acid sequence 
 of the hinge region of igg and 17 newly synthesized peptide analogues 
 containing a piperidine moiety as a replacement of a glycine residue 
 were tested as potential inhibitors of the bacterial igg degrading 
 enzyme of streptococcus pyogenes , ides . 
 
 none of the peptides showed any inhibitory activity of ides , but several 
 piperidine - based analogues were identified as inhibitors . 
 two different 
 analysis methods were used : an sds - page based assay to detect igg 
 cleavage products and a surface plasmon resonance spectroscopy based 
 assay to quantify the degree of inhibition . to investigate the selectivity 
 of the inhibitors for ides , 
 all compounds were screened against two 
 other related cysteine proteases ( speb and papain ) . 
 the selectivity 
 results show that larger analogues that are active inhibitors of ides 
 are even more potent as inhibitors of papain , whereas smaller analogues 
 that are active inhibitors of ides inhibit neither speb nor papain . 
 
 two compounds were identified that exhibit high selectivity against 
 ides and will be used for further studies . 
Introduction
Results and Discussion
Conclusion
Experimental Section