LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Peak Scientific nitrogen generator – The Proteasome Inhibitor Reduces Mortality of Septic Mice along with Improved Bacterial Clearance

LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Peak Scientific nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless otherwise noted. Flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin layer chromatography (TLC) was performed on Agela Technologies glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless otherwise noted. Chemical names were generated and select chemical properties were calculated using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova or ACD/NMR Processor Academic Edition (http://www.acdlabs.com) using the JOC report format. High-resolution mass spectra (HRMS) were obtained at the University of Wisconsin-Milwaukee Mass Spectrometry Laboratory with a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Peak Scientific nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) as a white solid that was carried onto the next reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already containing crude material from Part 1 (496 mg crude material, max. yield is 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd,.The material was then dry loaded using silica onto a 10 g silica gel column and purified with flash chromatography (IPA/DCM, 0 to 20%) to give 4 (86 mg) as a white solid in 50% yield. synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with negative allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless otherwise noted. Flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin layer chromatography (TLC) was performed on Agela Technologies glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless otherwise noted. Chemical names were generated and select chemical properties were calculated using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova or ACD/NMR Processor Academic Edition (http://www.acdlabs.com) using the JOC report format. High-resolution mass spectra (HRMS) were obtained at the University of Wisconsin-Milwaukee Mass Spectrometry Laboratory with a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Peak Scientific nitrogen JAK1-IN-7 generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) as a white solid that was carried onto the next reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already comprising crude material from Part 1 (496 mg crude material, max. yield is definitely 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd, = 5.0, 5.9 Hz, 1H), 8.22 (d, = 7.9 Hz, 1H), 7.86 (d, = 7.6 Hz, 2 H), 7.68C7.53 (m, 2 H), 7.52C7.43 (br s., 1H), 7.43C7.07 (m, 11= 5.6 Hz, 4 H), 4.15 (br s, 3 H), 3.05C2.85 (overlapping br s, 3 H), 2.73 (app t, = 12.0 Hz, 1 H), 1.86C1.73 (m, 1 H), 1.73C1.58 (m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-= 171.8, 171.7, 156.4, 156.1, 150.8 (dd, = 45.2, 13 Hz), 147.6 (dd, = 45.8, 12.1 Hz), 144.4, 144.3, 141.35, 141.33, 139.8, 136.6, 128.9, 128.2, 127.7, 127.6, 127.4, 126.7, 125.9, 120.7, 118.9, 118.6, 117.6,.A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LC-MS to confirm reaction completion. also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar functions as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with bad allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless normally indicated, NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless normally noted. Adobe flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin coating chromatography (TLC) was performed on Agela Systems glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless normally noted. Chemical titles were generated and select chemical properties were determined using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova or ACD/NMR Processor Academic Release (http://www.acdlabs.com) using the JOC statement file format. High-resolution mass spectra (HRMS) were obtained in the University or college of Wisconsin-Milwaukee Mass Spectrometry Laboratory having a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Maximum Scientific nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 CR6 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) like a white solid that was carried onto the next reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already comprising crude material from Part 1 (496 mg crude material, max. yield is definitely 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd, = 5.0, 5.9 Hz, 1H), 8.22 (d, = 7.9 Hz, 1H), 7.86 (d, = 7.6 Hz, 2 H), 7.68C7.53 (m, 2 H), 7.52C7.43 (br s., 1H), 7.43C7.07 (m, 11= 5.6 Hz, 4 H), 4.15 (br s, 3 H), 3.05C2.85 (overlapping br s, 3 H), 2.73 (app t, = 12.0 Hz, 1 H), 1.86C1.73 (m, 1 H), 1.73C1.58 (m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-= 171.8, 171.7, 156.4, 156.1, 150.8 (dd, = 45.2, 13 Hz), 147.6 (dd, = 45.8, 12.1 Hz), 144.4, 144.3, 141.35, 141.33, 139.8, 136.6, 128.9, 128.2,.The pH was adjusted to 7.4 if necessary by adding aq. was used to confirm that vorapaxar functions mainly because an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found out to inhibit PAR1 reversibly, in a manner consistent with bad allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless normally indicated, NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless normally noted. Adobe flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin coating chromatography (TLC) was performed on Agela Systems glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless normally noted. Chemical titles were generated and select chemical properties were determined using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova or ACD/NMR Processor Academic Release (http://www.acdlabs.com) using the JOC statement file format. High-resolution mass spectra (HRMS) were obtained in the University or college of Wisconsin-Milwaukee Mass Spectrometry Laboratory having a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Maximum Scientific nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) as a white solid that was carried onto the next reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already made up of crude material from Part 1 (496 mg crude material, max. yield is usually 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd, = 5.0, 5.9 Hz, 1H), 8.22 (d, = 7.9 Hz, 1H), 7.86 (d, = 7.6 Hz, 2 H), 7.68C7.53 (m, 2 H), 7.52C7.43 (br s., 1H), 7.43C7.07 (m, 11= 5.6 Hz, 4 H), 4.15 (br s, 3 H), 3.05C2.85 (overlapping br s, 3 H), 2.73 (app t, = 12.0 Hz, 1 H), 1.86C1.73 (m, 1 H), 1.73C1.58 (m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-= 171.8, 171.7, 156.4, 156.1, 150.8 (dd, = 45.2, 13 Hz), 147.6 (dd, = 45.8, 12.1 Hz), 144.4, 144.3, 141.35, 141.33, 139.8,.Sodium triacetoxyborohydride (8.32 g, 39.2 mmol) was added in 3 portions at 5 min. The assay is also suitable for study of PAR2 ligands; a peptide antagonist reported by Fairlie was synthesized and found to inhibit PAR2 in a manner consistent with reports using epithelial cells. The assay was used to confirm that vorapaxar acts as an irreversible antagonist of PAR1 in endothelium, and parmodulin 2 (ML161) and the related parmodulin RR-90 were found to inhibit PAR1 reversibly, in a manner consistent with unfavorable allosteric modulation. 0), or CDCl3 (13C 77.16), (CD3)2CO (1H 2.05, 13C 29.84), d6-DMSO (1H 2.50, 13C 39.5), or CD3OD (1H 3.31, 13C 49.00). NMR data are reported as follows: chemical shifts, multiplicity (obs = obscured, app = apparent, br = broad, s = singlet, d = doublet, t JAK1-IN-7 = triplet, q = quartet, sxt = sextet, m = multiplet, comp = complex overlapping signals); coupling constant(s) in Hz; integration. Unless otherwise indicated, NMR data were collected at 25 C. Filtration was performed by vacuum using VWR Grade 413 filter paper, unless otherwise noted. Flash chromatography was performed using Biotage SNAP cartridges filled with 40C60 m silica gel on Biotage Isolera automated chromatography systems with photodiode array UV detectors. Analytical thin layer chromatography (TLC) was performed on Agela Technologies glass plates with 0.25 mm silica gel and F254 indicator. Visualization was accomplished with UV light (254 nm) and KMnO4 stain, unless otherwise noted. Chemical names were generated and select chemical properties were calculated using either ChemAxon Marvin suite (https://www.chemaxon.com) or ChemDraw Professional 15.1. NMR data were processed using either MestreNova or ACD/NMR Processor Academic Edition (http://www.acdlabs.com) using the JOC report format. High-resolution mass spectra (HRMS) were obtained at the University of Wisconsin-Milwaukee Mass Spectrometry Laboratory with a Shimadzu LCMS-IT-TOF with ESI and APCI ionization. 4.2. LC/MS characterization methods Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on a Shimadzu LCMS-2020 with autosampler, photodiode array detector, and single-quadrupole MS with ESI and APCI dual ionization using a Peak Scientific nitrogen generator. Method A 1C5 L of sample in MeCN or MeOH UV absorbance at 210 or 254 nm measured by UV absorbance at 210 or 254 nm 0.1C1.9 mL (2 mL sample loop) of sample in DMSO = 530.05 (M+H+); 1H NMR (300 MHz, CDCl3) = 7.76 (d, = 7.6 Hz, 2H), 7.61C7.49 (overlapping signals, 3H), 7.43C7.22 (m, 9H), 5.96 (d, = 6.7 Hz, 1H), 5.11 (br. m., 1H), 4.50C4.13 (overlapping m, 6H), 3.48C3.32 (br s., 1H), 3.04C2.90 (br m., 1H), 1.97C1.71 (overlapping signals, 2H), 1.45C1.37 (m, 9H); 13C NMR (75 MHz, CDCl3) = 171.2, 157.2, 156.3, 143.9, 141.5, 138.0, 128.8, 127.9, 127.8, 127.6, 127.3, 125.3, 120.2, 80.1, 67.2, 52.2, 47.3, 43.8, 37.0, 34.9, 28.6. 4.4.2. tert-butyl ((S)-3-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl) amino)-3-(3,4-difluorophenyl)propanamido)-4-(benzylamino)-4-oxobutyl) carbamate (3) Part 1: Fmoc removal Intermediate 2 (550 mg, 1.03 mmol) was added to a round bottomed flask with stir bar and sealed under nitrogen, then MeCN (25 mL), DCM (10 mL), and piperidine (0.220 mL, 2.22 mmol) were added. The reaction was stirred for 2 h. A sample aliquot was taken from the reaction, concentrated under reduced pressure, dissolved in a minimal amount of HPLC grade MeCN, JAK1-IN-7 and analyzed with LCMS to confirm reaction completion. The reaction was concentrated under reduced pressure and then re-dissolved in (CHCl3/EtOAc, 1: 1, 75 mL), washed with H2O (2 20 mL) and brine (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure to give crude material (496 mg) as a white solid that was carried onto the next reaction without further purification. Part 2: Coupling To a round bottomed flask with stir bar already made up of crude material from Part 1 (496 mg crude material, max. yield is usually 319 mg) was added anhydrous MeCN (30 mL) and anhydrous DCM (15 mL). (= 713.15 (M+H+), = 757.35 (formic acid adduct); 1H NMR (300 MHz, DMSO = 8.43 (dd, = 5.0, 5.9 Hz, 1H), 8.22 (d, = 7.9 Hz, 1H), 7.86 (d, = 7.6 Hz, 2 H), 7.68C7.53 (m, 2 H), 7.52C7.43 (br s., 1H), 7.43C7.07 (m, 11= 5.6 Hz, 4 H), 4.15 (br s, 3 H), 3.05C2.85 (overlapping br s, 3 H), 2.73 (app t, = 12.0 JAK1-IN-7 Hz, 1 H), 1.86C1.73 (m, 1 H), 1.73C1.58 (m, 1H), 1.34 (s, 9 H); 13C NMR (75 MHz, DMSO-= 171.8, 171.7, 156.4, 156.1, 150.8 (dd, = 45.2, 13 Hz), 147.6 (dd, = 45.8, 12.1 Hz), 144.4, 144.3,.

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