Recent characterization of multiple classes of functionalized azapeptides as effective covalent inhibitors of cysteine proteases prompted us to investigate O-acyl hydroxamates and their azapeptide analogues for use as activity-based probes (ABPs). We report here a new class of azaglycine-containing O-acylhydroxamates that form stable covalent adducts with target proteases. This allows them to be used as ABPs for papain family cysteine proteases. A second class of related analogues containing a novel O-acyl hydroxyurea warhead was found to function as covalent inhibitors of papain-like proteases. These inhibitors can be easily synthesized on solid support, which allows rapid optimization of compounds with improved selectivity and potency for a given target enzyme. We present here one such optimized inhibitor that showed selective inhibition of falcipain 1, a protease of the malaria-causing parasite, Plasmodium falciparum

Arastu-Kapur, Shirin

Bogyo, Matthew

Fonovic, Marko

Verhelst, Steven H.L.

Witte, Martin D.

ARTS repository - University of Groningen

   University of GroningenNovel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine ProteasesVerhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo,MatthewPublished in:ChemBioChemDOI:10.1002/cbic.200600001IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza PeptideInhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.https://doi.org/10.1002/cbic.200600001CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023               Supporting Information  © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006    © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006       Supporting Information  for  Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family Cysteine Proteases  Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  Marko Fonovic, and Matthew Bogyo*   Synthetic Procedures Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. After LCMS analysis had proved complete conversion, the reaction was quenced with methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA (10 equiv) in DMF were added followed by shaking overnight. Acylation was performed on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-trated under reduced pressure and purified by HPLC.  N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in methanol and the mixture was refluxed overnight, concentrated under reduced pressure and purified by silicagel column chromatography giving the title compound as a white solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-DMSO): δ=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 (s, 9H). {[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine (10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic layers were concentrated under reduced pressure, and the crude product was purified by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J = 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J = 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). ([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ= 10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 3H). ([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 3H). {((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 (m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). ([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 (quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 (s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 3H).  ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide (MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). {(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 (m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz).  Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with control dmso (upper) or MW6bio (lower). ∆mobs = 810 Dalton. ∆m for covalent modification of the enzyme with expulsion of the p-methoxybenzoate is 801.  ______________ [1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 

English

Novel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases

University of Groningen

University of GroningenNovel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine ProteasesVerhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo,MatthewPublished in:ChemBioChemDOI:10.1002/cbic.200600001IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza PeptideInhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.https://doi.org/10.1002/cbic.200600001CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-12-2025               Supporting Information  © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006    © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006       Supporting Information  for  Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family Cysteine Proteases  Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  Marko Fonovic, and Matthew Bogyo*   Synthetic Procedures Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. After LCMS analysis had proved complete conversion, the reaction was quenced with methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA (10 equiv) in DMF were added followed by shaking overnight. Acylation was performed on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-trated under reduced pressure and purified by HPLC.  N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in methanol and the mixture was refluxed overnight, concentrated under reduced pressure and purified by silicagel column chromatography giving the title compound as a white solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-DMSO): δ=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 (s, 9H). {[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine (10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic layers were concentrated under reduced pressure, and the crude product was purified by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J = 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J = 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). ([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ= 10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 3H). ([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 3H). {((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 (m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). ([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 (quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 (s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 3H).  ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide (MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). {(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 (m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz).  Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with control dmso (upper) or MW6bio (lower). ∆mobs = 810 Dalton. ∆m for covalent modification of the enzyme with expulsion of the p-methoxybenzoate is 801.  ______________ [1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 

Steven H. L. Verhelst

Martin D. Witte

Shirin Arastu‐Kapur

Marko Fonovic

Matthew Bogyo

Crossref

Novel Aza Peptide Inhibitors and Active‐Site Probes of Papain‐Family Cysteine Proteases

University of GroningenNovel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine ProteasesVerhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo,MatthewPublished in:ChemBioChemDOI:10.1002/cbic.200600001IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza PeptideInhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.https://doi.org/10.1002/cbic.200600001CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 24-12-2025               Supporting Information  © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006    © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006       Supporting Information  for  Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family Cysteine Proteases  Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  Marko Fonovic, and Matthew Bogyo*   Synthetic Procedures Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. After LCMS analysis had proved complete conversion, the reaction was quenced with methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA (10 equiv) in DMF were added followed by shaking overnight. Acylation was performed on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-trated under reduced pressure and purified by HPLC.  N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in methanol and the mixture was refluxed overnight, concentrated under reduced pressure and purified by silicagel column chromatography giving the title compound as a white solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-DMSO): δ=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 (s, 9H). {[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine (10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic layers were concentrated under reduced pressure, and the crude product was purified by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J = 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J = 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). ([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ= 10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 3H). ([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 3H). {((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 (m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). ([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 (quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 (s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 3H).  ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide (MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). {(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 (m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz).  Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with control dmso (upper) or MW6bio (lower). ∆mobs = 810 Dalton. ∆m for covalent modification of the enzyme with expulsion of the p-methoxybenzoate is 801.  ______________ [1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 

   University of GroningenNovel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine ProteasesVerhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo,MatthewPublished in:ChemBioChemDOI:10.1002/cbic.200600001IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza PeptideInhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.https://doi.org/10.1002/cbic.200600001CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 27-04-2023               Supporting Information  © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006    © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006       Supporting Information  for  Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family Cysteine Proteases  Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  Marko Fonovic, and Matthew Bogyo*   Synthetic Procedures Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. After LCMS analysis had proved complete conversion, the reaction was quenced with methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA (10 equiv) in DMF were added followed by shaking overnight. Acylation was performed on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-trated under reduced pressure and purified by HPLC.  N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in methanol and the mixture was refluxed overnight, concentrated under reduced pressure and purified by silicagel column chromatography giving the title compound as a white solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-DMSO): δ=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 (s, 9H). {[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine (10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic layers were concentrated under reduced pressure, and the crude product was purified by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J = 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J = 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). ([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ= 10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 3H). ([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 3H). {((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 (m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). ([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 (quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 (s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 3H).  ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide (MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). {(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 (m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz).  Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with control dmso (upper) or MW6bio (lower). ∆mobs = 810 Dalton. ∆m for covalent modification of the enzyme with expulsion of the p-methoxybenzoate is 801.  ______________ [1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 

NARCIS 

   University of GroningenNovel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine ProteasesVerhelst, Steven H.L.; Witte, Martin D.; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo,MatthewPublished in:ChemBioChemDOI:10.1002/cbic.200600001IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2006Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza PeptideInhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.https://doi.org/10.1002/cbic.200600001CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022               Supporting Information  © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006    © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006       Supporting Information  for  Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family Cysteine Proteases  Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  Marko Fonovic, and Matthew Bogyo*   Synthetic Procedures Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. After LCMS analysis had proved complete conversion, the reaction was quenced with methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA (10 equiv) in DMF were added followed by shaking overnight. Acylation was performed on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-trated under reduced pressure and purified by HPLC.  N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in methanol and the mixture was refluxed overnight, concentrated under reduced pressure and purified by silicagel column chromatography giving the title compound as a white solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-DMSO): δ=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 (s, 9H). {[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine (10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic layers were concentrated under reduced pressure, and the crude product was purified by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J = 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J = 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). ([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ= 10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 3H). ([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 3H). {((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J = 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 (m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). ([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 (quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 (d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 (s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 3H).  ([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide (MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). {(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): δ=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 (m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz).  Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with control dmso (upper) or MW6bio (lower). ∆mobs = 810 Dalton. ∆m for covalent modification of the enzyme with expulsion of the p-methoxybenzoate is 801.  ______________ [1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 

University of Groningen Research Database

  
 University of Groningen
Novel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases
Verhelst, Steven H.L.; Witte, Martin; Arastu-Kapur, Shirin; Fonovic, Marko; Bogyo, Matthew
Published in:
ChemBioChem
DOI:
10.1002/cbic.200600001
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2006
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Verhelst, S. H. L., Witte, M. D., Arastu-Kapur, S., Fonovic, M., & Bogyo, M. (2006). Novel Aza Peptide
Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases. ChemBioChem, 7(6), 943-950.
DOI: 10.1002/cbic.200600001
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
  
 
 
 
 
 
 
 
 
 
 
 
 
 
Supporting Information 
 
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006 
 
 
 
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2006 
 
 
 
 
 
 
Supporting Information 
 
for 
 
Novel Aza Peptide Inhibitors and Active-Site Probes of Papain Family 
Cysteine Proteases 
 
Steven H. L. Verhelst, Martin D. Witte, Shirin Arastu-Kapur,  
Marko Fonovic, and Matthew Bogyo* 
 
 
Synthetic Procedures 
Solid phase synthesis of (aza)glycine O-acylhydroxamates: Fmoc-hydroxylamine 
was loaded onto 2-chlorotritylresin as described.[1] Next, the protecting group was depro-
tected with 20 % piperidine in DMF. For azaglycine probes, the resin was reacted for 3 h 
with carbonyldiimidazole (6 equiv) in dichloromethane. The resin was washed and a 60-
fold excess of hydrazine in DMF was added. After 1 h, resin was washed with DMF and 
elongated by solid phase peptide chemistry. For the glycine probes, Fmoc-glycine was 
coupled to the deprotected hydroxylamine resin, and further elongated by solid-phase 
peptide chemistry. Amino -termini were capped with Ac2O (10 equiv) and DIEA (10 
equiv) in DMF (15 min). Peptides were cleaved with 1% TFA in DCM (5 x 2 min), and 
dried by coevaporation with toluene. The crude peptide was dissolved in THF, cooled in 
ice, and subsequently pyridine (2 equiv) and p-anisoylchloride (1.9 equiv) were added. 
After LCMS analysis had proved complete conversion, the reaction was quenced with 
methanol and evaporated to dryness. Crude inhibitors were deprotected with TFA/H2O/ 
TIS 95:2.5:2.5 for 1h, concentrated under reduced pressure and purified by HPLC. 
Solid phase synthesis of O-acylhydroxyureas: Peptides were synthesized on Rink 
amide resin by standard protocols, using Fmoc-amino acids (3 equiv), DIC (3 equiv) and 
HOBt (3 equiv) for elongation. After final Fmoc-deprotection, the resin was reacted at 
4 °C with carbonyldiimidazole (6 equiv) in DMF. After 5 min. the reactants were drained 
and the resin was washed with DMF. Subsequently, hydroxylamine (10 equiv) and DIEA 
(10 equiv) in DMF were added followed by shaking overnight. Acylation was performed 
on resin by treatment with p-anisoylchloride (1.9 equiv) and pyridine (2 equiv) in THF. 
Products were cleaved from the resin with with TFA/H2O/TIS 95:2.5:2.5 (1 h), concen-
trated under reduced pressure and purified by HPLC.  
N-Acetyl-(O-tert-butyl)tyrosylphenylalanine hydrazide (2): N-acetyl-(O-tert-butyl)-
tyrosyl-phenylalanine methyl ester (1) was synthesized by coupling Fmoc-Tyr( t-Bu)-OH 
and phenylalanine methyl ester under influence of DIC/HOBt. Crude product was treated 
with 20 % piperidine, concentrated under reduced pressure and acetylated using an ex-
cess of acetic anhydride and DIEA. Crude 1 and hydrazine (60 equiv), were dissolved in 
methanol and the mixture was refluxed overnight, concentrated under reduced pressure 
and purified by silicagel column chromatography giving the title compound as a white 
solid in 53% yield over 4 steps. ESI-MS: m/z 441.4 [M+H]+. 1H NMR (500 MHz, [D6]-
DMSO): d=9.13 (s, 1H), 8.06 (d, 1H, J = 8.3 Hz), 7.98 (d, 1H, J = 8.4 Hz), 7.28-7.17 (m, 
5H), 7.08 (d, 2H, J = 8.4 Hz), 6.83 (d, 2H, J = 8.4 Hz), 4.48-4.40 (m, 2H, 4.24 (br s, 2H), 
2.94 (dd, 1H, J = 5.3 Hz, J = 13.8 Hz), 2.86 (dd, 1H, J = 4.3 Hz, J = 13.7 Hz), 2.80 (dd, 
1H, J = 8.8 Hz, J = 13.7 Hz), 2.60 (dd, 1H, J = 10.0 Hz, J = 13.9 Hz), 1,71 (s, 3H), 1.25 
(s, 9H). 
{[N-Acetyl-(O-tert-butyl)tyrosyl]phenylalanyl}azaglycine hydroxyamide (4): Car-
bonyldiimidazole (6.0 equiv) was added to a soution of compound 2 (1.0 equiv) in DMF, 
and the reaction mixture was stirred for 4 h at room temperature. Next, hydroxylamine 
(10 equiv) and DIEA (10 equiv) were added and the resulting mixture was stirred for 1 h 
at 60 °C. Diluted KHSO4 was added, and product was extracted with DCM (2x). Organic 
layers were concentrated under reduced pressure, and the crude product was purified 
by HPLC, yielding the title compound in 44%. ESI-MS: m/z 500.4 [M+H]+. 1H NMR (500 
MHz, [D6]DMSO): d=9.77 (s, 1H), 8.78 (s, 1H), 8.71 (s, 1H), 8.54 (s, 1H), 8.05 (d, 1H, J 
= 8.1 Hz), 7.98-7.94 (m, 1H), 7.29-7.13 (m, 5H), 7.08 (d, 2H, J = 8.0 Hz), 6.83 (d, 2H, J 
= 7.9 Hz), 4.62-4.56 (m, 1H), 4.47-4.41 (m, 1H), 3.10-3.06 (m, 1H), 2.87-2.79 (m, 2H), 
2.59-2.54 (m, 1H), 1.70 (s, 3H), 1.25 (s, 9H). 
([N-Acetyl-tyrosyl]phenylalanyl)azaglycine p-methoxybenzoyloxyamide (MW4): P-
anisoyl chloride (1.9 equiv) and pyridine (2.0 equiv) were added to a stirred solution of 
compound 4 in THF, cooled to 4 °C. After 2 h, the reaction was quenched by addition of 
methanol. The mixture was concentrated under reduced pressure, treated with TFA/TIS/ 
H2O 95:2.5:2.5 for 1 h and again concentrated. HPLC purification gave the title com-
pound in 21 % yield. ESI-MS: m/z 578.4 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): d= 
10.26 (s, 1H), 9.93 (s, 1H), 9.14 (br s, 2H), 8.08 (d, 1H, J = 8.3 Hz), 8.00 (d, 2H, J = 8.8 
Hz), 7.93 (d, 1H, J = 8.5 Hz), 7.28-7.18 (m, 5H), 7.10 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J 
= 8.4 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.60-4.55 (m, 1H), 4.41-4.36 (m, 1H), 3.86 (s, 3H), 
3.09 (dd, 1H, J = 3.4 Hz, J = 13.9 Hz), 2.85-2.78 (m, 2H), 2.53-2.48 (m, 1H), 1.70 (s, 
3H). 
([N-Acetyl-tyrosyl]phenylalanyl)glycine p-methoxybenzoyloxyamide (MW2): The 
title compound was isolated as a white solid in 12% yield. ESI-MS: m/z 577.3 [M+H]+. 1H 
NMR (500 MHz, [D6]DMSO): d=9.16 (br s, 1H), 8.38-8.34 (m, 1H), 8.05 (d, 1H, J = 8.6 
Hz), 8.01-7.96 (m, 3H), 7.27-7.18 (m, 6H), 7.11 (d, 2H, J = 8.5 Hz), 6.97 (d, 2H, J = 8.3 
Hz), 6.61 (d, 2H, J = 8.1 Hz), 4,58-4.52 (m, 1H), 4.40-4.35 (m, 1H), 3.90-3.68 (m, 5H), 
3.07 (dd, 1H, J = 4.1 Hz, J = 13.9 Hz), 2.84-2.78 (m, 2H), 2.56-2.50 (m, 1H), 1.71 (s, 
3H). 
{((N-[Methoxybenzoyloxyaminocarbonyl]leucyl)tyrosyl)-6-aminohexanoyl}lysyl-
amide (MW6): The title compoud was isolated as a white solid in 52% yield. ESI-MS: 
m/z 728.7 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): d=9.23-9.10 (br s, 1H), 7.97 (d, 2H, J 
= 8.2 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.85-7.79 (m, 2H), 7.71 (br s, 2H), 7.34 (s, 1H), 7.12-
7.07 (m, 3H), 7.03-6.94 (m, 3H), 6.63 (d, 2H, J = 8.4 Hz), 4.38-4.32 (m, 1H), 4.18-4.11 
(m, 2H), 3.86 (s, 3H), 3.05-2.98 (m, 1H), 2.95-2.89 (m, 1H), 2.83 (dd, 1H, J = 5.8 Hz, J = 
13.9 Hz), 2.76-2.67 (m, 3H), 2.10 (t, 2H, J = 7.4 Hz), 1.67-1.61 (m, 1H), 1.52-1.24 (m, 
14H), 1.18-1.12 (m, 2H), 0.85-0.76 (m, 6H). 
([N-Acetyl-tyrosyl]leucyl)azaglycine p-methoxybenzoyloxyamide (MW20): The title 
compound was isolated as a white solid in 10 % yield. ESI-MS: m/z 544.3 [M+H]+. 1H 
NMR (500 MHz, [D6]DMSO): d=10.20 (s, 1H), 9.80 (s, 1H), 9.15 (br s, 1H), 9.08 (br s, 
1H), 8.04 (d, 1H, J = 8.2 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.19 (s, 1H), 7.12-7.08 (m, 2H), 
7.02 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz), 4.46-4.40 (m, 1H), 4.38-4.33 (m, 1H), 
3.85 (s, 3H), 2.85 (dd, 1H, J = 3.3 Hz, J = 14.2 Hz), 2.64-2.51 (m, 3H), 1.74 (s, 3H), 1.62 
(quint, 1H, J = 6.5 Hz), 0.89 (d, 3H, J = 6.5 Hz), 0.85 (d, 3H, J = 6.5 Hz). 
([N-Acetyl-tyrosyl]-4-methylphenylalanyl)azaglycine p-methoxybenzoyloxyamide 
(MW21): The title compound was isolated as a white solid in 12% yield. ESI-MS: m/z  
592.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): d=10.23 (s, 1H), 9.91 (s, 1H), 9.14 (br s, 
1H), 9.12 (br s, 1H), 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 2H, J = 8.9 Hz), 7.93 (d, 1H, J = 
8.2 Hz), 7.15 (d, 2H, J = 8.1 Hz),  7.10 (d, 2H, J = 9.0 Hz), 7.05 (d, 2H, J = 7.9 Hz), 6.96 
(d, 2H, J = 8.4 Hz), 6.61 (d, 2H, J = 8.6 Hz), 4.55-4.50 (m, 1H), 4.40-4.35 (m, 1H), 3.86 
(s, 3H), 3.05-3.01 (m, 1H), 2.83-2.41 (m, 2H), 2.54-2.50 (m, 1H), 2.25 (s, 3H), 1.70 (s, 
3H).  
([N-Acetyl-tyrosyl]-4-methylphenylalanyl)glycine p-methoxybenzoyloxyamide 
(MW22): The title compound was isolated as a white solid in 43% yield. ESI-MS: m/z  
591.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): d=11.9 (s, 1H), 9.15 (br s, 1H), 8.35 (s, 
1H), 8.03-7.96 (m, 4H), 7,15-7.10 (d, 4H, J = 8.5 Hz), 7.05 (d, 2H, J = 7.6 Hz), 6.97 (d, 
2H, J = 8.1 Hz), 6.61 (d, 2H, J = 8.3 Hz), 4.54-4.51 (m, 1H), 4.40-4.35 (m, 1H), 3.04-
2.00 (m, 1H), 2.84-2,50 (m, 4H), 2.24 (s, 3H), 1.72 (s, 3H). 
{(N-[Methoxybenzoyloxyaminocarbonyl]-4-methylphenylalanyl)alanyl}tyrosyl-
amide (MW23): The title compound was isolated as a white solid in 42% yield. ESI-MS: 
m/z 606.5 [M+H]+. 1H NMR (500 MHz, [D6]DMSO): d=9.83 (s, 1H), 9.16 (s, 1H), 8.20 (d, 
1H, J = 7.1 Hz), 7.94 (d, 2H, J = 9.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 7.32 (s, 1H), 7.11-6.96 
(m, 9H), 6.63 (d, 2H, J = 8.5 Hz), 4.38-4.30 (m, 2H), 4.26-4.22 (m, 1H), 3.86 (s, 3H), 
2.94-2.86 (m, 2H), 2.78-2.70 (m, 2H), 2.23 (s, 3H), 1.19 (d, 3H, J = 7.0 Hz). 
 Figure S1. Parts of the MALDI-TOF spectrum of recombinant cathepsin L after incubation with 
control dmso (upper) or MW6bio (lower). Dmobs = 810 Dalton. Dm for covalent modification of the 
enzyme with expulsion of the p-methoxybenzoate is 801. 
 
______________ 
[1] S. L. Mellor, C. McGuire, W. C. Chan, Tetrahedron Lett. 1997, 38, 3311- 3314. 


Novel Aza Peptide Inhibitors and Active-Site Probes of Papain-Family Cysteine Proteases

Abstract

Similar works

Full text

Available Versions

ARTS repository - University of Groningen

University of Groningen

Crossref

University of Groningen

ARTS repository - University of Groningen

University of Groningen

NARCIS

ARTS repository - University of Groningen

University of Groningen Research Database