Supplemental protocols are provided below including synthetic details for each of the

Transcription

1 Supplemental Section Supplemental protocols are provided below including synthetic details for each of the compounds that are reported in the manuscript. Analytical characterization of the compounds can be found following the methods section. In addition, two supplemental figures and legends can be found at the end of this section. General methods. Unless otherwise noted, all resin and reagents were purchased from commercial suppliers and used without further purification. All solvents used were of PLC grade. All water-sensitive reactions were performed in anhydrous solvents and under a positive pressure of argon. Reactions were analyzed by thin-layer chromatography on Whatman 0.25 mm silica plates with fluorescent indicator. Flash chromatography was carried out with EMD 2-0 mesh silica gel. Reverse-phase PLC was conducted on a C 18 column using the ÄKTA explorer 0 (Amersham Pharmacia Biotech). LCMS data were acquired using an API 1EX LC/MS system (Applied Biosystems). igh-resolution MS analyses were performed by Stanford Proteomics and Integrative Research Facility using a Bruker Autoflex MALDI TF/TF mass spectrometer. General solid phase peptide synthesis method. The synthetic protocols for solid phase synthesis of peptide AMKs are a modification of the previously reported methods 1,2. alomethyl ketones (2a-f) and their solid support bound derivatives via carbazate linker (5a-e) were synthesized with modification to the procedure as described below. Unless otherwise noted, reactions were conducted in 12-mL polypropylene cartridges (Applied

2 Separations, Allentown, PA) with 3-way nylon stopcocks (BioRad Laboratories, ercules, CA). The cartridges were connected to a port vacuum manifold (Waters, Milford, MA) that was used to drain solvent and reagents from the cartridge. The resin was gently shaken on a rotating shaker during solid-phase reactions. General method for synthesis of halomethyl ketone derivatives of -α-fmocprotected amino acids (2a-f). A 0.2 M solution of the corresponding -α-fmoc amino acid (1a-e, 5 mmol) in anhydrous TF was stirred in an ice/ acetone bath at - C. To this solution, -methylmorpholine (6.25 mmol, 1.25 equiv) and isobutylchloroformate (5.75 mmol, 1.15 equiv) were sequentially added. Immediately after the addition of the latter compound, a white precipitate formed. The reaction mixture was maintained at - C for 25min. Diazomethane was generated in situ using the procedure described in the Aldrich Technical Bulletin (AL-1). Ethereal diazomethane ( mmol) was transferred to the stirred solution of the mixed anhydride at 0 C. The reaction mixture was warmed to room temperature over the course of 3 hours. To obtain the corresponding chloromethyl ketones (2a-e), 15 ml of a 1:1 solution of concentrated hydrochloric acid and glacial acetic acid was added dropwise to the reaction mixture at 0 C. Immediately after the evolution of nitrogen gas stopped, the reaction mixture was diluted with ethyl acetate and transferred to a separatory funnel. The reaction mixture was washed sequentially with water, brine solution, and saturated aqueous ac 3. The organic layer was dried over MgS 4. The solvent was removed under reduced pressure. Alternatively, the bromethyl ketone (2f) was obtained by dropwise addition of ml of a 1:2 solution of hydrogen bromide ( wt. % solution in acetic acid) and water to the

3 reaction mixture at 0 C. Workup was carried out as described for the chloromethyl ketone synthesis. Chloromethyl ketones 2a (glycine), 2c (leucine), 2d (lysine), 2e (aspartic acid) were obtained as a white solid (quantitative yield) and the bromomethyl ketone 2f (aspartic acid) was obtained as a yellow oil (quantitative yield), and used without any purification. Crude chloromethyl ketone 2b (arginine) was purified by column chromatography (-% ethyl acetate in hexane) to obtain a white solid (3.13 mmol, 62 %). Synthesis of carbazate linker on aminomethylpolystyrene resin. Aminomethylpolystyrene resin (1.1 mmol/g) was dried in vacuo overnight in a 12-mL polypropylene cartridge. The resin was presolvated with DMF for min and another min with C 2 Cl 2. A 1 M solution of, -Carbonyldiimidazole (6 equiv) in C 2 Cl 2 was added to the resin, and the resin was shaken at room temperature for 3 h. The reagent was drained and the resin was washed with C 2 Cl 2 followed by DMF. A M solution of hydrazine ( equiv) in DMF was added to the resin, and the resin was shaken at room temperature for 1 h. The resin was washed with DMF followed by C 2 Cl 2, dried in vacuo, and stored at -4 C. Loading of chloromethyl ketone derivatives and synthesis of 2,6- dimethylbenzoyloxymethyl ketone derivatives (5a-e). A 0.5 M solution of the chloromethyl ketone derivative of the corresponding -a-fmoc-l-amino acid (2a-e) in DMF was added to the resin. The cartridge was tightly sealed and shaken at C for various time periods depending on the chloromethyl ketone. The glycine CMK (2a) was

4 incubated for min; all others (2b-e) were incubated for 3 h. After the reaction the solution was removed, and the resin was washed with DMF. Formation of the AMK on resin was performed using KF as reported for solution phase synthesis of AMKs 3. This method allowed the use of a reduced amount of the carboxylic acid. Specifically a 0.5 M solution of 2,6-dimethylbenzoic acid (5 equiv) and potassium fluoride ( equiv) in DMF were added to the resin. The resin was shaken at room temperature overnight. After the solution was removed, the resin was washed with DMF followed by C 2 Cl 2, and dried in vacuo. The resin load was estimated by UV absorption of free Fmoc. Synthesis of 2,6-dimethylbenzoyloxymethyl ketone derivative of -a-fmoc-laspartic acid on Rink resin (5f). A 0.2 M solution of bromomethyl ketone derivative of -α-fmoc-l-aspartic acid-β-tert-butyl ester (2f) in DMF was stirred at 0 C, and potassium fluoride (3 equiv) was added as a solid. After 1min stirring at 0 C, 2,6- dimethylbenzoic acid (1.2 equiv) was added as a solid, the reaction mixture was warmed to room temperature. After overnight stirring, the reaction mixture was diluted with ethyl acetate, and transferred to a separatory funnel. The reaction mixture was worked up sequentially with water, brine solution, and saturated aqueous ac 3. The organic layer was dried over MgS 4. The solvent was removed under reduced pressure. The product was purified by flash chromatography (~17% ethyl acetate in hexane) yielding a yellow oil (98% yield). A 0.2 M solution of the product 2,6-Dimethylbenzoyloxymethyl ketone derivative of -α-fmoc-l-aspartic Acid-β-tert-butyl ester (3f) was dissolved in 25% v/v TFA/ C 2 Cl 2 and allowed to stand for min with occasional shaking. The reaction mixture

5 was diluted with C 2 Cl 2. The cleavage solution was removed by coevaporation with toluene. The product was further dried in vacuo.the crude product (4f; 96% yield) was used without further purification. Rink resin (0.75 mmol/ g) was presolvated by shaking in DMF for 1 h. The Fmoc-protecting group on the resin was removed with % piperidine/ DMF for 15min. The resin was washed with DMF followed by C 2 Cl 2. A 0.5 M solution of 2,6- dimetylbenzoyloxymethyl ketone derivative of -α-fmoc-l-aspartic acid (4f, 1.25 equiv) and BT (1.25 equiv) was added to the resin followed by DIC (1.25 equiv). After shaking for 2.5 h, the resin was washed with DMF, yielding the loaded resin (5f). Resin load was determined by UV absorption of free Fmoc. ptimization of base deprotection of peptide AMKs. Before solid phase peptide synthesis could be carried out for extended peptides a survey of optimal bases for deprotection of the Fmoc group was performed to identify conditions that allowed Fmoc removal without displacement of the AMK group. Eighteen aliquots of -α-fmoc-lleucine 2,6-dimethylbenzoyloxymethyl ketone loaded resin (5c, ~1 mg, ~3.7 x -4 mmol) were solvated with DMF for min. DMF solutions of each of the bases were added to each well, and the reactions were shaken for min. The resins were washed with DMF followed by C 2 Cl 2. Acetic anhydride ( equiv) and DIEA (15 equiv) in 2 µl DMF were added to each well to acylate the deprotected free amine. The reactions were shaken for 15 min and the resin washed with DMF followed by C 2 Cl 2. The reaction block was placed under vacuum for ~15 min. 0 µl of cleavage cocktail (95%

6 TFA, 5% 2 ) was added to the resin. After 1 h the cleavage mixture were collected, diluted in methanol, and analyzed by direct infusion ion-spray mass spectrometry. Solid phase peptide synthesis on aminomethylpolystyrene. -Fmoc-protected 2,6- dimethylbenzoyloxymethyl ketone derivatives linked to aminomethylpolystyrene or Rink resin (5a-f) were presolvated in DMF for min. -terminal Fmoc group was removed by treatment with a 5% diethylamine solution in DMF for 15min followed by another 15 min treatment with fresh solution. The resin was washed with DMF followed by C 2 Cl 2. A 0.2 M solution of -Fmoc-protected amino acid (3 equiv) (Z-protected amino acid for 8, 9 a-c), BT (3 equiv) in DMF and DIC (3 equiv) were sequentially added to the resin. The resin was shaken at room temperature for 2 h, and washed with DMF followed by C 2 Cl 2. For each subsequent step of the solid phase peptide synthesis, the same deprotection and coupling reactions were followed. Deprotection and coupling reactions were monitored by the ninhydrin test for primary amine. Capping of the -terminal amine for compound (7a-f, a-b) was achieved by shaking the resin with a 0.5 M solution of acetic anhydride ( equiv) and DIEA (15 equiv) in DMF. After shaking at room temperature for 15min, the resin was washed with DMF followed by C 2 Cl 2, and dried in vacuo. General method of cleavage from aminomethylpolystyrene resin. The 3-way nylon stopcocks were replaced with TFA-resistant polypropylene needle valve (Waters). A solution of 95% TFA/ 5% 2 was added to the resin. After standing at room temperature for 1.5 h, the cleavage mixture was collected, and the resin was washed with

7 fresh cleavage solution. The combined mixture was precipitated in cold ether at - C for 2 h. The precipitated peptide was collected by centrifugation at 3,000 rpm at - C for 15 min. The pellet was dried by positive flow of argon, dissolved in a minimal amount of DMS. The product was purified on a C 18 reverse phase PLC (Waters, Delta-Pak) using a linear gradient of 0-0% water-acetonitrile. Fractions containing product were pooled, then lyophilized to dryness. The identity of the product was confirmed by mass spectrometry. The general method of cleavage from Rink resin. The same procedure as for aminomethylpolystyrene resin was followed except that a solution of % TFA/ 2.5% triisopropylsilane in C 2 Cl 2 was added to the resin, and the reaction time was shortened to 15 min. Characterization of compounds. All final compounds used for biological studies were purified by PLC and characterized by high-resolution mass spectrometry (RMS) using a Bruker Autoflex TF/TF mass spectrometer. Data files for these studies can be found in the Supplementary Data section. verall yields for the complete synthesis and RMS data are listed below. Compound 7a. 5.0% yield. [Ma] + calcd for C a S, ; found, Compound 7b. 2.1% yield. RMS (m/z): [M] + calcd for C S, ; found, Compound 7c. 11% yield. RMS (m/z): [Ma] + calcd for C a S, ; found, Compound 7d. 1.5% yield. RMS (m/z):

8 [M] + calcd for C S, ; found Compound 7e. 8.4% yield. RMS (m/z): [M] + calcd for C S, ; found, Compound 7f. 8% yield. RMS (m/z): [M] + calcd for C S, ; found, Compound % yield. RMS (m/z): [Ma] + calcd for C a 7, ; found, Compound 9a..8% yield. RMS (m/z): [M] + calcd for C , ; found, Compound 9b. 3.6% yield. RMS (m/z): [M] + calcd for C , ; found, Compound 9c. 14.6% yield. RMS (m/z): [M] + calcd for C , ; found, Compound a. 13.2% yield. RMS (m/z): [M] + calcd for C , , found, Compound b. 12.5% yield. RMS (m/z): [M] + calcd for C , ; found, Coupound % yield. RMS (m/z): [M]+ calcd for C S, 11.50, found, Radiolabeling of inhibitors. A 1.5 ml microcentrifuge tube was coated with 0 mg of ID-GE (Pierce, Rockford, IL). Probe (62.5 µl of a 0.2 mm solution in phosphate buffer p 7.4) was added to the tube. a 125 I (1 mci, ml) was added to the tube and incubation continued for min. Labeled inhibitor was purified by application to a Sep- Pak (Waters, Milford, MA) column containing a C 18 stationary phase. After sample application, the column was washed with 25 ml of phosphate buffer p 7.5. Labeled inhibitor was eluted using 0% acetonitrile. Fractions of 1mL were collected and samples with the largest number of counts were pooled and used without further purification.

9 Small scale radiolabeling of probes. A 1.5mL microcentrifuge tube was coated with µg of ID-GE. Probe (6.5 µl of a 0.2 mm solution in phosphate buffer p 7.4) was added to the tube. a 125 I (0.1 mci, 1 µl) was added to the tube and incubation continued for min. Labeled inhibitor was transferred to a fresh tube containing 0 µl of 0% acetonitrile and used without further purification. Labeling of gingipains in crude cell lysates. Pelleted cells from P. gingivalis grown in batch culture for 24 h (early stationary phase) were washed three times with mm Bis- Tris, 1 mm acl, 0.02% a 3, p 6.8 containing 1.5 mm 4,4 -dithiopyridine disulfide, followed by sonication at 1,0 z for min. Unbroken cells and large debris were removed by centrifugation (27,000 x g, min, 4 o C) and the resulting supernatant diluted into reaction buffer (mm Tris, mm DTT, 5mm MgCl 2, p 7.6) to a final concentration of 1 mg/ml. Total crude lysates ( µg) were labeled with 125 I-labeled P1 AMK probes (1X 6 total counts per minute) for min at RT and labeled proteases visualized by SDS-PAGE followed by autoradiography. Supplemental References 1. Wood, W. J., uang, L. & Ellman, J. A. Synthesis of a diverse library of mechanism-based cysteine protease inhibitors. J Comb Chem 5, 869- (03). 2. Lee, A., uang, L. & Ellman, J. A. General solid-phase method for the preparation of mechanism-based cysteine protease inhibitors. Journal of the American Chemical Society 121, (19).

10 3. Mujica, T. M. & Jung, G. A novel approach to the solid phase synthesis of (acyloxy)methyl ketones. Synlett 12, (19).

11 Compound 7a S e e6 5.5e6 5.0e6 Intensity, CPS 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e e

12 2 Compound 7b S 7.5e6 7.0e e6 6.0e6 Intensity, CPS 5.5e6 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e e e

13 Compound 7c S e e6 5.5e6 5.0e6 Intensity, CPS 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e e e

14 2 Compound 7d S e e6 Intensity, CPS 2.5e e6 1.5e e

15 Compound 7e S e e6 9.00e5 cps Intensity, 8.00e5 7.00e5 6.00e5 5.00e e5 3.00e5 2.00e5 1.00e

16 2 Compound 7f S e e6 1.2e cps Intensity, 8.0e5 6.0e e5 2.0e

17 Compound e6 5.1 cps Intensity, 6.0e6 5.5e6 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 5.0e

18 Compound 9a e6 6.0e cps 5.0e6 Intensity, 4.0e6 3.0e e

19 2 Compound 9b cps Intensity, 7.0e6 6.5e6 6.0e6 5.5e6 5.0e6 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 5.0e

20 1.52 Compound 9c e e6 cps Intensity, 5.0e6 4.0e6 3.0e6 2.0e

21 Compound a e e6 5.0e6 cps Intensity, 4.5e6 4.0e6 3.5e6 3.0e6 2.5e e6 1.5e e

22 2 Compound b e Intensity, cps 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 5.0e

23 S Compound e6 6.0e6 cps Intensity, 5.0e6 4.0e6 3.0e e