Supporting Information For Structure-Based Design of Pseudopeptidic Inhibitors for SIRT1 and SIRT2

Transcription

1 Supporting Information For Structure-Based Design of Pseudopeptidic Inhibitors for SIRT1 and SIRT2 Tero Huhtiniemi,, Heikki S. Salo,*,, Tiina Suuronen, Antti Poso, Antero Salminen,, Jukka Leppänen, Elina Jarho,, and Maija Lahtela-Kakkonen School of Pharmacy and Department of Neurology, Institute of Clinical Medicine, University of Eastern Finland, P.O.Box 1627, Kuopio, Finland Department of Neurology, Kuopio University Hospital, P.O. Box 1777, Kuopio, Finland Department of Chemistry, Medicinal Chemistry, University of Gothenburg, Gothenburg, Sweden Contents Abbreviations General Manual solid phase peptide synthesis (SPPS)... 2 Elemental analysis data for compounds 8, 13, 16-24, Additional docking pose figures... 9 References *Corresponding Author. These authors contributed equally to this work.

2 Abbreviations. Boc Cbz or Z DIPEA DCM DMF EDC HCl Fmoc h H2O HOBt Lys min PE rt SPPS TBTU THF TFA tert-butyloxycarbonyl benzyloxycarbonyl N,N-Diisopropylethylamine dichloromethane dimethylformamide N-(3-Dimethylaminopropyl)-n'-ethyl-carbodiimide fluorenylmethyloxycarbonyl hour ion exchanged water 1-Hydroxybenzotriazole l-lysine minute petroleum ether room temperature solid phase peptide synthesis O-(Benzotriazol-1-yl)-N,N,N,N -tetramethyluronium tetrafluoroborate tetrahydrofuran Trifluoroacetic acid General. All reagents and solvents were commercial high purity quality. Purification of products 8, 13, 16-18, and was performed by CombiFlash column chromatography on normal phase silica (average particle size: 35 to 70 µm, mesh: 230 to 400, average pore size 60 Å) or by crystallization methods. Compounds 14, 15 and 19 were purified by preparative HPLC (Shimadzu LC-10Avp (Shimadzu, Kyoto, Japan)) on a reverse phase C18 column (Supelcogel ODP-50, 25 cm x 21.2 mm, 5µm) with a linear gradient of 5-90% solvent B (0.05%acetic acid/acetonitrile) in solvent A (0.05 acetic acid/h 2 O) in 30 min with the flow rate 10 ml/min. The peptide was detected by UV at = 215 nm. NMR spectra were recorded on a Bruker Avance 500 AV (Bruker Biospin, Switzerland) MHz for 1 H and MHz for 13 C. The chemical shifts are expressed in ppm relative to the shift of used solvent as an internal standard ( 1 H NMR: DMSO at 2.50 ppm, CH 3 OH at 3.31 ppm and CHCl 3 at 7.26 ppm. 13 C NMR: (CD 3 ) 2 SO at ppm, CD 3 OD at ppm and CDCl 3 at ppm). Positive ion mass spectra were acquired with a quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an electrospray ionization source (ESI-MS). The purity of compound 14 was determined using Agilent 1100 HPLC (Agilent Technologies Inc., Waldbronn, Karlsruhe, Germany) with diode array detection, using reversed phase column (Zorbax Eclipse XDB C 18, 4.6 x 50 mm, 1.8 µm, Agilent Technologies, Palo Alto, CA, USA), 5% Solvent B (0.05% AcOH in CH 3 CN) in solvent A (0.05% AcOH in H 2 O) for 5 min, then linear gradient 5-80% B in A in 15 min with the flow rate 1 ml/min. The purity of compound 15 was determined using Shimadzu LC-10Avp (Shimadzu, Kyoto, Japan), using a reverse phase column (Supelcogel ODP-50 C 18, 4 x 150 mm, 5 µm), with a isocratic eluent (0.05% AcOH, 2.5% MeOH, 97.45% H 2 0) in 30 min with the flow rate 1 ml/min. The purity of the rest of the compounds was determined by combustion analysis for CHN by Thermo Quest CE Instruments EA 1110 CHNS-O elemental analyzer. Peptide 14 showed 93% purity and all other compounds had purity of 95%. Manual solid phase peptide synthesis (SPPS) Wang resin (polymer-bound p-alkoxy-benzyl alcohol) was used as solid support for the peptide synthesis of compounds 5 and Solid phase synthesis was performed in a 10 ml syringe equipped with a frit. Fmoc-Ala-Wang resin (loading mmol/g) was swelled for 1 h in 3 ml of DMF. In the deprotection phase, the N -Fmoc protection group was removed with 5 ml of 20% (V/V) piperidine in DMF for 15 min and the resin was rinsed 5 times with DMF. In the coupling phase, the following N -Fmoc-amino acid or acetic acid (2-4 equiv) was preactivated (1-3 min) with the coupling reagent TBTU (2-4 equiv) and DIPEA (5-10 equiv) in 3-5 ml of DMF. This solution was added on the resin, and the syringe was shaken at rt for 60 min. Then the resin was rinsed 5 times with DMF. The cycle of Fmoc-deprotection and coupling was repeated until the desired peptide-bound resin was completed. Before cleavage from the resin, the resin was washed once with AcOH, five times with dichloromethane (DCM), and once with MeOH to remove the excess solvents and then dried under vacuum. Cleavage of Peptide-Resin as Carboxylic Acid. The dried peptide-resin was preswelled in dioxane for 15 min. The excess of dioxane was removed, and 8 ml of cool (0 C) cleavage mixture of 1M NaOH (aq)/dioxane (2 and 6 ml) was added on the peptide-resin. The reaction mixture was shaken 1 h at rt. The mixture was filtrated, filtrate was collected and the resin was washed with H 2 O / dioxane (1:3), dioxane and H 2 O. The filtrate was neutralized with 3 M HCl (aq) and the solvents were evaporated. Acetone was added on the residue, and the mixture was filtrated to remove inorganic salts. The solvent of the filtrate was evaporated under reduced pressure to yield the crude product, which was purified by column chromatography or by crystallization. Cleavage of Peptide-Resin as Methyl Ester. The dry peptide-resin was placed in a flask. Cleavage mixture DIPEA/MeOH/DMF (1:5:5, ml) was added and the reaction mixture was refluxed under argon atmosphere overnight. The mixture was filtrated, filtrate was collected and the resin was rinsed 5 times with 2

3 MeOH/DMF (1:1). Solvent was evaporated from the combined filtrates under reduced pressure to yield the crude product, which was purified by column chromatography or by crystallization. (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(benzyloxycarbonylamino)hexanoic acid (4). H- Lys(Z)-OH (2.25 g, 8.01 mmol) and NaHCO 3 (2.52 g, 30.0 mmol) were dissolved in the mixture of 350 ml H 2 O and 350 ml THF. The solution was cooled to 0 C in an ice bath, and 9-fluorenylmethyl chloroformate (2.28 g, 8.81 mmol) dissolved in dry THF was added dropwise over 10 min. The reaction mixture was stirred for 4 h. THF was evaporated and the aqueous phase was acidified with 3 M HCl (aq.) till ph 2-3. The product was extracted 3 times with EtOAc, the organic phase was dried and evaporated. The product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.05% AcOH)). The selected fractions were dissolved in DCM, washed twice with sat. NaCl solution (aq.), dried with Na 2 SO 4 and evaporated in vacuum. Yield 3.69 g (7.34 mmol, 92%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 6 H), 2.98 (m, 2 H), 3.90 (m, 1 H), 4.22 (t, 1 H), (m, 2 H), 5.00 (s, 2 H), 7.25 (t, 1 H), (m, 7 H), 7.42 (dd, 2 H), 7.62 (d, 1 H), 7.73 (d, 2 H), 7.89 (d, 2 H), (br, 1 H). (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-(benzyloxycarbonylamino)hexanamido)propanoate (5). Compound 5 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (1.80 g), compound 4 (1.09 g, 2.16 mmol), Fmoc-Ala-OH (0.67 g, 2.16 mmol) and AcOH (0.13 g, 2.16 mmol) using TBTU (0.69 g, 2.16 mmol) and DIPEA (925 µl, 5.40 mmol) in each step as coupling reagents. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded 0.37 g (0.77 mmol, 72%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.16 (d, 3 H), 1.27 (d, 3 H), (m, 2 H), 1.39 (m, 2 H), (m, 2 H), 1.82 (s, 3 H), 2.96 (m, 2 H), 3.60 (s, 3 H), (m, 3 H), 4.99 (s, 2 H), 7.20 (t, 1 H), (m, 5 H), 7.81 (d, 1 H), 8.02 (d, 1 H), 8.24 (d, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 16.79, 18.04, 22.39, 22.46, 29.14, 31.73, 40.23, 47.51, 48.19, 51.79, 51.90, 65.10, , , , , , , , , ESI- MS (m/z): [M+H] +, [M+Na] +. Anal. (C 23 H 34 N 4 O 7 ) C, H, N. (S)-2-((S)-2-Acetamidopropanamido)-6-amino-N-((S)-1-(methylamino)-1-oxopropan-2-yl)hexanamide (7). Compound 6 (0.40 g, 0.84 mmol) and g palladium on activated charcoal (puriss. 10% Pd basis) were mixed with methanol (50 ml, molecular sieves) under argon atmosphere. Continuous slow hydrogen gas bubbling was added to the mixture and the mixture was stirred overnight at room temperature under H 2 atmosphere. The mixture was filtered and the filtrate was evaporated to yield 0.29 g (0.84 mmol, 100%). 1 H NMR ((CD 3 ) 2 SO): = (m, 6 H), (m, 4 H), (m, 2 H), 1.84 (s, 3 H), (m, 3 H), 3.14 (m, 2 H), (m, 3 H). Benzyl (S)-5-((S)-2-acetamidopropanamido)-6-((S)-1-(methylamino)-1-oxopropan-2-ylamino)-6- oxohexylcarbamate (6). Compound 5 (0.40 g, 0.84 mmol) was dissolved in 8 M methylamine in ethanol and heated with microwaves at 100 C for 20 minutes. Solvents were evaporated. Yield 0.40 g (0.84 mmol, 100%) as a solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 6 H), 1.25 (m, 2 H), 1.38 (m, 2 H), (m, 2 H), 1.82 (s, 3 H), 2.56 (d, 3 H), 2.96 (m, 2 H), (m, 3 H), 4.99 (s, 2 H), 7.23 (t, 1 H), (m, 5 H), 7.73 (m, 1 H), 7.82 (d, 1 H), 7.92 (d, 1 H), 8.07 (d, 1 H). (S)-2-((S)-2-Acetamidopropanamido)-6-ethanethioamido-N-((S)-1-(methylamino)-1-oxopropan-2- yl)hexanamide (8). Compound 7 (0.050 g, 0.15 mmol) was mixed with EtOH (10 ml) and cool 10% Na 2 CO 3 (aq.) (3 ml). Ethyl dithioacetate (0.019 ml, 0.16 mmol) was added and the reaction mixture was stirred two days at room temperature. Solvents were evaporated and the residue was extracted with CH 3 CN. Purification by column chromatography (MeOH gradient 5-30% in DCM) yielded g (0.041 mmol, 28%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 6 H), 1.29 (m, 2 H), (m, 4 H), 1.83 (s, 3 H), 2.37 (s, 3 H), 2.57 (d, 3 H), 3.43 (m, 2 H), (m, 3 H), 7.73 (m, 1 H), 7.82 (d, 1 H), 7.92 (d, 1 H), 8.07 (d, 1 H), 9.93 (m, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.92, 18.29, 22.45, 22.81, 25.52, 26.81, 31.33, 32.77, 45.32, 48.08, 48.34, 52.36, , , , , ESI-MS (m/z): [M + H] +, [M + Na]+. Anal. (C 17 H 31 N 5 O 4 S 0.3hexane 0.2H 2 O) C, H, N. (R)-1-(Tert-butoxycarbonyl)piperidine-3-carboxylic acid (9). (R)-Piperidine-3-carboxylic acid (0.52 g, 4.00 mmol and Na 2 CO 3 (1.59 g, mmol) were dissolved in 1:1 THF/H 2 O (200 ml) and cooled to 0 C in an ice bath. Di-tert-butyl dicarbonate (0.96 g, 4.40 mmol) was dissolved in THF (30 ml) and added dropwise over 10 min. The reaction mixture was stirred for 2 h at 0 C. THF was evaporated and the remaining aqueous solution was made acidic with 3 M HCl (aq.), and extracted with EtOAc (3 x 50 ml). Organic phase was dried with Na 2 SO 4 and evaporated in vacuum. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded 0.88 g (3.84 mmol, 96%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.34 (m, 1 H), 1.39 (s, 9 H), 1.51 (m, 1 H), 1.61 (m, 1 H), 1.90 (m, 1 H), 2.29 (m, 1 H), 2.83 (m, 2 H), 3.68 (m, 1 H), 3.90 (br, 1 H), (s, 1 H). 3-(2-Acetoxyphenyl)propanoic acid (10). 3-(2-Hydroxyphenyl)propanoic acid (1.66 g, 10.0 mmol) was dissolved in TFA (10 ml) under argon and cooled to 0 C in an ice bath. Acetyl chloride (1.46 ml, 20.5 mmol) 3

4 was added dropwise and the reaction mixture was stirred for 0.5 h at 0 C and 1 h at rt. Solvents were evaporated, the residue was dissolved in DCM (150 ml). Organic phase was washed with 0.5 M HCl (aq.) (2 x 40 ml) and sat. NaCl (aq.) (2 x 40 ml) solutions, dried with Na 2 SO 4 and evaporated in vacuum. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded 0.90 g (4.32 mmol, 43%) as oil. 1 H NMR ((CD 3 ) 2 SO): = 2.29 (s, 3 H), 2.46 (t, 2 H), 2.71 (t, 2 H), 7.06 (d, 1 H), 7.19 (dd, 1 H), 7.25 (dd, 1 H), 7.31 (d, 1 H), (br, 1 H). (R)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phenylpropanoic acid (11). D-Phenylalanine (0.66 g, 4.00 mmol) and Na 2 CO 3 (1.59 g, mmol) were dissolved in 1:1 THF/H 2 O (200 ml) and cooled to 0 C in an ice bath. Fmoc-Cl (1.14 g, 4.40 mmol) was dissolved in THF (30 ml) and added dropwise over 10 min. The reaction mixture was stirred for 2 h at 0 C. THF was evaporated and the remaining aqueous solution was made acidic with 3 M HCl (aq.), and extracted with EtOAc (3 x 50 ml). Organic phase was dried with Na 2 SO 4 and evaporated in vacuum. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded 1.03 g (2.01 mmol, 67%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 2 H), (m, 4 H), (m, 7 H), (m, 2 H), 7.64 (dd, 2 H), 7.73 (d, 1 H), 7.88 (d, 2 H), (br, 1 H). (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-ethanethioamidohexanoic acid (12). Fmoc-Lys- OH HCl (2.20 g, 5.43 mmol) was dissolved in EtOH (30 ml). The solution was cooled to 0 C in an ice bath and 10 ml of 10% (w/v) Na 2 CO 3 (aq.) was added. The reaction mixture was allowed to warm to rt and stirred extensively. Ethyl dithioacetate (0.69 ml, 5.98 mmol) was added and the reaction mixture was stirred extensively for 3 h and placed in a fridge overnight. Solvent was evaporated, the crude product was made acidic with 3 M HCl (aq.) on ice and extracted 3 times with DCM. The organic phase was washed twice with sat. NaCl solution (aq.), dried with Na 2 SO 4, filtered and evaporated to obtain 12 as solid. Yield: 1.93 g (4.52 mmol, 83%). 1 H NMR ((CD 3 ) 2 SO): = 1.36 (m, 2 H), (m, 4 H), 2.37 (s, 3 H), 3.46 (m, 2 H), 3.93 (m, 1 H), 4.23 (t, 1 H), (m, 2 H), 7.33 (dd, 2 H), 7.42 (dd, 2 H), 7.62 (d, 1 H), 7.73 (m, 2 H), 7.89 (d, 2 H), 9.95 (m, 1 H), (br, 1 H). (S)-Methyl 2-((S)-2-((S)-2-acetamidopropanamido)-6-ethanethioamidohexanamido)propanoate (13). Compound 13 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.48 g), compound 12 (0.48 g, 1.12 mmol), Fmoc-Ala-OH (0.35 g, 1.12 mmol) and AcOH (0.067 g, 1.12 mmol) using TBTU (0.36 g, 1.11 mmol) and DIPEA (470 µl, 2.75 mmol) in each step as coupling reagents. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded g (0.13 mmol, 47%) as solid. 1 H NMR (MeOD): = 1.34 (d, 3 H), 1.39 (d, 3 H), 1.47 (m, 2 H), (m, 4 H), 1.98 (s, 3 H), 2.45 (s, 3 H), 3.59 (m, 2 H), 3.71 (s, 3 H), (m, 3 H). 13 C NMR (MeOD): = 17.28, 17.80, 22.39, 24.06, 28.27, 32.86, 33.12, 46.96, 49.46, 50.63, 52.76, 54.12, , , , , ESI-MS (m/z): [M+H] +, [M+Na] +. Anal. (C 17 H 30 N 4 O 5 S) C, H, N. (S)-2-((S)-2-((S)-2-Aminopropanamido)-6-ethanethioamidohexanamido)propanoic acid (14). Compound 14 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.30 g), compound 12 (0.31 g, 0.72 mmol) and Fmoc-Ala-OH (0.22 g, 0.72 mmol) using TBTU (0.23 g, 0.72 mmol) and DIPEA (300 µl, 1.75 mmol) in each step as coupling reagents. The product was purified with preparative HPLC on a reverse-phase C18 column (ODP50), CH 3 CN (0.05% AcOH) linear gradient (5%-90%, 30min) in H 2 O (0.05% AcOH). The selected fractions were evaporated and the product was dried in vacuum. Yield g (0.13 mmol, 72%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.25 (d, 3 H), (m, 2 H), 1.32 (d, 3 H), (m, 4 H), 2.37 (s, 3 H), 3.42 (m, 2 H), 3.83 (m, 1 H), 4.09 (m, 1 H), 4.27 (m, 1 H), 8.12 (d, 1 H), 8.48 (d, 1 H), 9.99 (m, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.50, 17.55, 22.78, 26.95, 31.87, 32.81, 45.37, 47.95, 48.18, 52.51, , , , ESI-MS (m/z): [M+H] +. HPLC: Rt min, area percent 93% at 260 nm. (S)-2-((S)-2-Amino-6-ethanethioamidohexanamido)propanoic acid (15). Compound 15 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.40 g) and compound 12 (0.31 g, 0.72 mmol) using TBTU (0.23 g, 0.72 mmol) and DIPEA (300 µl, 1.75 mmol) as coupling reagents. The peptide was cleaved from the resin with 5 ml of a mixture of TFA:H 2 O:thioanisole:phenol:ethanedithiol (82.5:5:5:5:2.5) for 90 minutes. The resin was rinsed with TFA and the excess of TFA was evaporated. Cold diethyl ether was added on the residue and the product was precipitated overnight in a freeze. The mixture was centrifuged for 5 minutes at 6000 rpm and the solvent was decanted. Cold diethyl ether was added on the precipitate and centrifugation was repeated with once. The product was purified by preparative HPLC 0.5-5% MeOH in H 2 O (0.05% AcOH in both solvents) for 30 minutes. 1 H NMR (D 2 O): = 1.44 (d, 3 H), 1.50 (m, 2 H), 1.73 (m, 2 H), 1.96 (m, 2 H), 2.51 (s, 3 H), 3.63 (t, 2 H), 4.03 (t, 1 H), 4.36 (q, 1 H). 13 C NMR (D 2 O): = 17.82, 22.71, 27.86, 31.87, 33.71, 47.11, 50.92, 54.36, , , ESI-MS (m/z): [M+H] +. HPLC: Rt min, area percent 99% at 260 nm. (S)-Methyl 2-((S)-2-acetamido-6-ethanethioamidohexanamido)propanoate (16). Compound 16 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.40 g), compound 12 (0.31 g,

5 mmol) and AcOH (55 µl, 0.96 mmol) using TBTU (0.23 g, 0.72 mmol) and DIPEA (300 µl, 1.75 mmol) in each step as coupling reagents. Cleavage as methyl ester. Purification by column chromatography (MeOH gradient 0-10% in DCM), followed by trituration with Et 2 O, yielded g (0.12 mmol, 49%) as solid. 1 H NMR (CDCl 3 ): = 1.43 (d, 3 H), 1.46 (m, 2 H), (m, 4 H), 2.01 (s, 3 H), 2.56 (s, 3 H), 3.65 (m, 2 H), 3.76 (s, 3 H), 4.52 (m, 1 H), 4.57 (m, 1 H), 6.63 (m, 1 H), 7.12 (m, 1 H), 8.21 (m, 1 H). 13 C NMR (CDCl 3 ): = 17.47, 22.30, 23.13, 26.81, 32.62, 33.71, 45.84, 48.15, 52.30, 52.52, , , , ESI-MS (m/z): [M+H] +, [M+Na] +. Anal. (C 14 H 25 N 3 O 4 S 0.3 Et 2 O) C, H, N. (S)-2-((S)-2-((S)-1-Acetylpyrrolidine-2-carboxamido)-6-ethanethioamidohexanamido)propanoic acid (17). Compound 17 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.49 g), compound 12 (0.25 g, 0.59 mmol), Fmoc-Pro-OH (0.20 g, 0.59 mmol) and AcOH (0.091 g, 0.59 mmol) using TBTU (0.19 g, 0.59 mmol) and DIPEA (251 µl, 1.47 mmol) in each step as coupling reagents. Purification by column chromatography (MeOH gradient 0-10% in DCM) yielded g (0.15 mmol, 49%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 5 H), (m, 5 H), (m, 3 H), (m, 3 H), (m, 3 H), (m, 4 H), (m, 3 H), (m, 1 H), (m, 1 H), (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 16.97, 17.05, 21.03, 22.01, 22.38, 22.50, 22.76, 22.86, 24.31, 26.77, 26.79, 29.44, 31.28, 31.58, 31.73, 32.78, 45.27, 45.36, 46.29, 47.41, 47.47, 47.65, 51.71, 51.94, 59.34, 60.19, , , , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 18 H 30 N 4 O 5 S 0.8AcOH) C, H, N. (6S,9S,12S)-9-(4-Ethanethioamidobutyl)-2,2,6,12-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan- 13-oic acid (18). Compound 18 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and Boc-Ala-OH (0.11 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.50 mmol) in each step as coupling reagents. Product was purified by column chromatography (MeOH gradient 0-10% in DCM). The selected fractions were evaporated; the residue was scrubbed with hexane to precipitate the product. Yield: g (0.13 mmol, 45%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.16 (d, 3 H), 1.26 (d, 3 H), 1.37 (s, 9 H), (m, 2 H), (m, 4 H), 2.37 (s, 3 H), 3.41 (m, 2 H), 3.96 (m, 1 H), 4.17 (m, 1 H), 4.28 (m, 1 H), 6.96 (d, 1 H), 7.69 (d, 1 H), 8.17 (d, 1 H), 9.90 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.09, 17.99, 21.04, 26.89, 28.17, 32.11, 32.79, 45.38, 47.45, 49.73, 51.75, 78.10, , , , , ESI-MS (m/z): [M+H] +. Anal. (C 17 H 30 N 4 O 5 S 0.6AcOH 0.15hexane) C, H, N. (S)-2-((S)-2-((R)-2-Amino-3-phenylpropanamido)-6-ethanethioamidohexanamido)propanoic acid (19). Compound 19 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and compound 11 (0.23 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.94 mmol) in each step as coupling reagents. The product was purified with preparative HPLC on a reverse-phase C18 column (ODP50), CH 3 CN linear gradient (3%-90%, 30min) in H 2 O. The selected fractions were evaporated and the product was dried in vacuum. Yield g (0.12 mmol, 39%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.15 (m, 2 H), 1.25 (d, 3 H), (m, 4 H), 2.37 (s, 3 H), (m, 2 H), 3.39 (m, 2 H), 3.68 (m, 1 H), 4.09 (m, 1 H), 4.25 (m, 1 H), (m, 5 H), 8.19 (m, 2 H), 9.97 (m, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.43, 22.41, 26.86, 32.07, 32.76, 45.31, 47.82, 51.95, 55.15, , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 20 H 30 N 4 O 4 S 0.5H 2 O) C, H, N. (S)-2-((S)-2-(Benzyloxycarbonylamino)-6-ethanethioamidohexanamido)propanoic acid (20). Compound 20 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g) and compound 26 (0.20 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.94 mmol) in each step as coupling reagents. The product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.05% AcOH)). The selected fractions were evaporated. The residue was dissolved in small amount of EtOH and hexane was added; white precipitate was filtered, rinsed with hexane and dried in vacuum. Yield: g (0.22 mmol, 73%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.27 (d, 3 H), 1.34 (m, 2 H), (m, 4 H), 2.37 (s, 3 H), 3.43 (m, 2 H), 4.00 (m, 1 H), 4.19 (m, 1 H), 5.02 (s, 2 H), (m, 6 H), 8.15 (d, 1 H), 9.92 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.11, 22.97, 26.89, 31.66, 32.79, 45.31, 47.38, 54.21, 65.31, , , , , , , , ESI-MS (m/z): [M+H] +, [M+Na] +. Anal. (C 19 H 27 N 3 O 5 S 0.1hexane) C, H, N. (S)-2-((S)-6-Ethanethioamido-2-(3-phenylpropanamido)hexanamido)propanoic acid (21). Compound 21 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and hydrocinnamic acid (0.090 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.94 mmol) in each step as coupling reagents. The product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.05% AcOH)). The selected fractions were evaporated; the residue was scrubbed with hexane to precipitate the product. Yield: g (0.12 mmol, 41%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.24 (m, 2 H), 1.27 (d, 3 H), (m, 4 H), 2.37 (s, 3 H), 2.45 (m, 2 H), 2.80 (m, 2 H), 3.41 (m, 2 H), 4.18 (m, 1 H), 4.29 (m, 1 H), (m, 5 H), 7.93 (d, 1 H), 8.17 (d, 1 H), 9.90 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): 5

6 = 17.03, 22.73, 26.92, 31.07, 31.93, 32.78, 36.68, 45.37, 47.35, 51.84, , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 20 H 29 N 3 O 4 S 0.1H 2 O 0.1hexane) C, H, N. (S)-2-((S)-6-Ethanethioamido-2-(3-(2-fluorophenyl)propanamido)hexanamido)propanoic acid (22). Compound 22 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and 3-(2-fluorophenyl)-propionic acid (0.090 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.94 mmol) in each step as coupling reagents. The product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.05% AcOH)). The selected fractions were evaporated; the residue was scrubbed with hexane to precipitate the product. Yield: 0.10 g (0.24 mmol, 78%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 2 H), 1.26 (d, 3 H), (m, 4 H), 2.37 (s, 3 H), 2.44 (m, 2 H), 2.82 (dd, 2 H), 3.40 (m, 2 H), 4.18 (m, 1 H), 4.28 (m, 1 H), (m, 2 H), (m, 2 H), 7.97 (d, 1 H), 8.18 (d, 1 H), 9.90 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.03, 22.74, (J CF = 2.49 Hz), 26.91, 31.90, 32.78, 35.06, 45.36, 47.36, 51.89, (d, J CF = Hz), (d, J CF = 3.32 Hz), (d, J CF = Hz), (d, J CF = 8.09 Hz), (J CF = 4.89 Hz), (J CF = Hz), , , , ESI-MS (m/z): [M+H] +. Anal. (C 20 H 28 FN 3 O 4 S 0.4AcOH 0.05hexane) C, H, N. (S)-2-((S)-6-Ethanethioamido-2-(3-(2-hydroxyphenyl)propanamido)hexanamido)propanoic acid (23). Compound 23 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and compound 10 (0.13 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.94 mmol) in each step as coupling reagents. Coupling phase with compound 10 was performed twice, because of the possible formation of chroman-2-one as a side product. Product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.1% AcOH)). The selected fractions were evaporated; the residue was scrubbed with hexane to precipitate the product. Yield: 0.90 g (0.21 mmol, 71%) as solid. 1 H NMR ((CD 3 ) 2 SO): = (m, 2 H), 1.27 (d, 3 H), (m, 4 H), 2.37 (s, 3 H), 2.39 (m, 2 H), 2.72 (dd, 2 H), 3.41 (m, 2 H), 4.18 (m, 1 H), 4.29 (m, 1 H), 6.68 (m, 1 H), 6.76 (d, 1 H), 6.98 (m, 1 H), 7.04 (d, 1 H), 7.91 (d, 1 H), 8.17 (d, 1 H), 9.28 (br, 1 H), 9.91 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.05, 22.79, 25.62, 26.92, 31.89, 32.79, 35.09, 45.36, 47.39, 51.91, , , , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 20 H 29 N 3 O 5 S 0.1H 2 O 0.7AcOH) C, H, N. (S)-2-((S)-2-((R)-1-(Tert-butoxycarbonyl)piperidine-3-carboxamido)-6- ethanethioamidohexanamido)propanoic acid (24). Compound 24 was synthesized according to SPPS procedure from Fmoc-Ala-Wang resin (0.50 g), compound 12 (0.26 g, 0.60 mmol) and (R)-1-(tertbutoxycarbonyl)piperidine-3-carboxylic acid (9) (0.14 g, 0.60 mmol) using TBTU (0.19 g, 0.60 mmol) and DIPEA (257 µl, 1.50 mmol) in each step as coupling reagents. The product was purified by column chromatography (MeOH gradient 0-10% in DCM (0.05% AcOH)). The selected fractions were evaporated; the residue was dissolved in small amount of EtOH and hexane was added; white precipitate was filtered, rinsed with hexane and dried in vacuum. Yield: g (0.12 mmol, 41%) as solid. 1 H NMR ((CD 3 ) 2 SO): = 1.26 (d, 3 H), (m, 4 H), 1.39 (s, 9 H), (m, 6 H), 2,32 (m, 1 H), 2.37 (s, 3 H), (m, 2 H), 3.43 (m, 2 H), (m, 2 H), 4.16 (m, 1 H), 4.24 (m, 1 H), 7.98 (d, 1 H), 8.09 (m, 1 H), 9.92 (m, 1 H), (br, 1 H). 13 C NMR ((CD 3 ) 2 SO): = 17.11, 21.03, 22.79, 22.84, (br), 26.88, 27.44, 28.04, 31.73, 32.78, 41.86, 45.33, 47.38, 51.79, 78.65, , , , , ESI-MS (m/z): [M+H] +, [M+Na] +. Anal. (C 22 H 38 N 4 O 6 S 0.4AcOH 0.2hexane) C, H, N. (S)-2-(Benzyloxycarbonylamino)-6-ethanethioamidohexanoic acid (26). N-(Benzyloxycarbonyl)-L-lysine (5.04 g, mmol) was dissolved in EtOH (100 ml). The solution was cooled to 0 C in an ice bath and 30 ml of 10% (w/v) Na 2 CO 3 (aq.) was added. The reaction mixture was allowed to warm to rt and stirred extensively. Ethyl dithioacetate (1.05 ml, 19.8 mmol) was added and the reaction mixture was stirred overnight at rt. Solvent was evaporated; the crude product was made acidic with 3 M HCl (aq.) on ice and extracted with DCM (3 x 100 ml). The organic phase was washed with sat. NaCl solution (aq.) (2 x 30 ml), dried with Na 2 SO 4, filtered and evaporated to obtain 26 as yellowish gum. Yield: 6.0 g (17.7 mmol, 99%). 1 H NMR ((CD 3 ) 2 SO): = 1.36 (m, 2 H), (m, 4 H), 2.37 (s, 3 H), 3.44 (m, 2 H), 3.93 (m, 1 H), 5.03 (s, 2 H), (m, 5 H), 7.53 (d, 1 H), 9.93 (m, 1 H). (S)-Benzyl 1-(cyclohexylamino)-6-ethanethioamido-1-oxohexan-2-ylcarbamate (27). Cyclohexylamine (0.060 ml, 0.88 mmol) was dissolved in 1:1 DMF/pyridine (5 ml, dried with molecular sieves) under argon. Compound 26 (0.27 g, 0.80 mmol) and TBTU (0.28 g, 0.88 mmol) dissolved in 1:1 DMF/pyridine (10 ml, dried with molecular sieves) were added dropwise and the solution was stirred 1 h at rt. Solvents were evaporated; the residue was dissolved in EtOAc (100 ml) and washed with 0.5M HCl (aq.), sat. NaCl (aq.) and sat. NaHCO 3 (aq.), dried with Na 2 SO 4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (MeOH gradient 0-5% in DCM) yielded 0.30 g (0.72 mmol, 89%) as solid. 1 H NMR (CDCl 3 ): = (m, 16 H), 2.54 (s, 3 H), (m, 3 H), 4.09 (m, 1 H), 5.11 (s, 2 H), 5.45 (d, 1 H), 5.86 (d, 1 H), (m, 5 H), 7.72 (m, 1 H). 13 C NMR (CDCl 3 ): = 22.67, 24.88, 25.54, 26.99, 6

7 32.56, 33.01, 33.12, 34.22, 45.89, 48.64, 54.51, 67.28, , , , , , , ESI-MS (m/z): [M+H] +, [M+Na] +. Anal. (C 22 H 33 N 3 O 3 S) C, H, N. (S)-Benzyl 6-ethanethioamido-1-(2-fluorophenylamino)-1-oxohexan-2-ylcarbamate (28). 2-Fluoroaniline (1.06 ml, 1.10 mmol) was dissolved in 1:1 DMF/pyridine (5 ml, dried with molecular sieves) under argon. Compound 26 (0.34 g, 1.00 mmol) and TBTU (0.35 g, 1.10 mmol) dissolved in 1:1 DMF/pyridine (10 ml, dried with molecular sieves) were added dropwise and the solution was stirred 1 h at rt. Solvents were evaporated; the residue was dissolved in EtOAc (100 ml) and washed with 0.5M HCl (aq.), sat. NaCl (aq.) and sat. NaHCO 3 (aq.), dried with Na 2 SO 4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (MeOH gradient 0-5% in DCM) yielded 0.28 g (0.65 mmol, 65%) as solid. 1 H NMR (CDCl 3 ): = 1.50 (m, 2 H), (m, 4 H), 2.53 (s, 3 H), 3.66 (m, 2 H), 4.36 (m, 1 H), 5.15 (s, 2 H), 5.43 (d, 1 H), (m, 3 H), (m, 5 H), 7.53 (br, 1 H), 8.11 (br, 1 H), 8.19 (dd, 1 H). 13 C NMR (CDCl 3 ): = 22.82, 27.23, 31.89, 34.30, 45.81, 55.42, 67.65, (d, J = Hz), , (d, J = 3.65 Hz), (d, J = 7.63 Hz), (J = Hz), , , , , (J = Hz), , , ESI-MS (m/z): [M+H] +. Anal. (C 22 H 26 FN 3 O 3 S 0.2H 2 O) C, H, N. (S)-Benzyl 6-ethanethioamido-1-(2-hydroxyphenylamino)-1-oxohexan-2-ylcarbamate (29). 2-Aminophenol (1.04 ml, 1.10 mmol) was dissolved in 1:1 DMF/pyridine (5 ml, dried with molecular sieves) under argon. Compound 26 (0.34 g, 1.00 mmol) and TBTU (0.35 g, 1.10 mmol) dissolved in 1:1 DMF/pyridine (10 ml, dried with molecular sieves) were added dropwise and the solution was stirred 1 h at rt. Solvents were evaporated; the residue was dissolved in EtOAc (100 ml) and washed with 0.5M HCl (aq.), sat. NaCl (aq.) and sat. NaHCO 3 (aq.), dried with Na 2 SO 4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (MeOH gradient 0-5% in DCM) yielded 0.22 g (0.50 mmol, 50%) as solid. 1 H NMR (CDCl 3 ): = 1.47 (m, 2 H), (m, 4 H), 2.51 (s, 3 H), 3.66 (s, 2 H), 4.41 (br, 1 H), 5.13 (s, 2 H), 5.52 (d, 1 H), 6.86 (dd, 1 H), 6.98 (d, 1 H), 7.11 (dd, 2 H), (m, 5 H), 7.52 (br, 1 H), 8.33 (s, 1 H), 8.55 (br, 1 H). 13 C NMR (CDCl 3 ): = 22.71, 27.23, 31.84, 34.31, 45.67, 55.12, 67.78, , , , , , , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 22 H 27 N 3 O 4 S) C, H, N. (S)-Benzyl 6-ethanethioamido-1-oxo-1-(2-oxo-2-phenylethylamino)hexan-2-ylcarbamate (30). Compound 26 (0.34 g, 1.00 mmol) and TBTU (0.35 g, 1.10 mmol) were dissolved in 1:1 DMF/pyridine (10 ml, dried with molecular sieves) under argon. 2-Aminoacetophenone hydrochloride (0.17 g ml, 1.00 mmol) dissolved in DMF (5 ml, dried with molecular sieves) was added dropwise and the solution was mixed 2 h at rt. Solvents were evaporated; the residue was dissolved in EtOAc (100 ml) and washed with 0.5M HCl (aq.), sat. NaCl (aq.) and sat. NaHCO 3 (aq.), dried with Na 2 SO 4, filtered and concentrated under reduced pressure. The product was purified by column chromatography (MeOH gradient 0-5% in DCM) yielded 0.37 g (0.81 mmol, 81%) as solid. 1 H NMR (CDCl 3 ): = 1.50 (m, 2 H), (m, 4 H), 2.53 (s, 3 H), 3.64 (m, 2 H), 4.35 (m, 1 H), 4.76 (m, 2 H), 5.14 (s, 2 H), 5.54 (d, 1 H), 6.95 (br, 1 H), (m, 5 H), 7.51 (dd, 2 H), 7.64 (dd, 1 H), 7.74 (br, 1 H), 7.96 (d, 2 H). 13 C NMR (CDCl 3 ): = 22.72, 27.11, 32.83, 34.21, 46.07, 46.45, 54.49, 67.37, , , , , , , , , , , , ESI-MS (m/z): [M+H] +. Anal. (C 24 H 29 N 3 O 4 S) C, H, N. 7

8 Elemental analysis data for compounds 8, 13, 16-24, Compound Formula Calculated Analyzed %C %H %N %C %H %N 8 C17H31N5O4 0.3hexane 0.2H2O C17H30N4O5S C14H25N3O4 0.3 Et2O C18H30N4O5 0.8AcOH C17H30N4O5 0.6AcOH 0.15hexane C20H30N4O4 0.5H2O C19H27N3O5 0.1hexane C20H29N3O4 0.1H2 0.1hexane C20H28FN3O4S 0.4AcOH 0.05hexa ne 23 C20H29N3O5 0.1H2 0.7AcOH C22H38N4O6 0.4AcOH 0.2hexane C22H33N3O3S C22H26FN3O3 0.2H2O C22H27N3O4S C24H29N3O4S

9 Additional docking pose figures Figure S1. The docking poses of the synthesized and tested pseudopeptide compounds docked to the SIRT3 x-ray crystal structure 3glr.pdb. The figures were prepared with MOE (version ) 1 and interactions displayed using the Ligand Interactions tool of MOE. The grid box definition differs from the one used later in the multiple protein dockings docking calculations on 3glr.pdb (Figure S1.) and hence slight variation in the docking poses can be seen between these two docking (see figure S2). 9

10 Figure S2. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses were 3glr.pdb (SIRT3). The figures were prepared with Maestro (version 9.1) 2. 10

11 Figure S3. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses were 3gls.pdb (SIRT3). The figures were prepared with Maestro (version 9.1) 2. 11

12 Figure S4. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses were 3glt.pdb (SIRT3). The figures were prepared with Maestro (version 9.1) 2. 12

13 Figure S5. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses were 3glu.pdb (SIRT3). The figures were prepared with Maestro (version 9.1) 2. 13

14 Figure S6. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 2h2d.pdb (Sir2Tm). The figures were prepared with Maestro (version 9.1) 2. 14

15 Figure S7. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 2h2f.pdb (Sir2Tm). The figures were prepared with Maestro (version 9.1) 2. 15

16 Figure S8. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 2h2g.pdb (Sir2Tm). The figures were prepared with Maestro (version 9.1) 2. 16

17 Figure S9. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 2h2h.pdb (Sir2Tm). The figures were prepared with Maestro (version 9.1) 2. 17

18 Figure S10. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 2h2i.pdb (Sir2Tm). The figures were prepared with Maestro (version 9.1) 2. 18

19 Figure S11. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was 1j8f.pdb (SIRT2). The figures were prepared with Maestro (version 9.1) 2. 19

20 Figure S12. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was a SIRT1 homology model. The figures were prepared with Maestro (version 9.1) 2. 20

21 Figure S13. The resulting poses of the docking calculations made to study the effect of the target protein structure conformation and sequence. The target structure of these poses was a SIRT2 homology model. The figures were prepared with Maestro (version 9.1) 2. 21

22 References (1) MOE (The Molecular Operating Environment), Version software available from Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910, Montreal, Canada H3A 2R7 (2) Maestro version 9.1; Schrödinger, LLC, New York, NY,