Journal of Medicinal Chemistry Scheme 6a Article aReagents 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%). (S)-LGpipP (23) in five steps from (R)- and (S)-19 was 42% and 36%, respectively. IgG-Based Peptides As Potential Inhibitors of IdeS. Eight different peptides based on the amino acid sequence of the IgG hinge region (Figure 2) were tested for putative inhibitory activity toward IdeS.24 Figure 2. Peptides tested for inhibitory activity. The inhibitory activity was screened in a gel-electrophoresis (SDS-PAGE) based assay to detect IgG cleavage products.25 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. Inhibitory Capacity of Peptide Analogues toward IdeS. 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 spectros- copy (Figure 3).15 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 (pip236G) 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 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). Differences in response units are indicated by arrows. (S)-pipG ((S)-3) has greater inhibitory activity than (R)-GpipP ((R)-21). tested, but no clear trend how stereochemistry of the piperidine moiety affected potency could be seen (Figure 4). 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 2553 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560