Journal of Medicinal Chemistry 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.17−19 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,6 Z-LVG CHN26 and TPCK/TLCK,15 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.20 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 P4−P1 subsites of the IgG hinge region, nor peptides with sequences covering the IdeS cleavage site are cleaved by the protease.12 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 P −P′4 4 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). The piperidine moiety can be inserted through a short and efficient synthetic route, and the strategy used allows further 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). Article 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. The hydrogen bond acceptor/donor ability is also altered. 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. ■ RESULTS AND DISCUSSION Synthesis of Peptide Analogues. 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). The racemate was separated into the pure enantiomers using preparative HPLC. 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 13C 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. The 1H NMR spectra showed high purity for both compounds. Furthermore, the coupling pattern in the 1H NMR spectra implies that the piperidine ring adopts one single chair conformation with the glycine amide residue in an equatorial position. The suggested ring conformation is shown in the Supporting Information. 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 2550 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560