Supporting Information

HIV Protease-Mediate Activation of Sterically Capped Proteasome Inhibitors and Substrates

Dennis L. Buckley,† Timothy W. Corson,

‡,|| Nicholas Aberle,

‡ and Craig M. Crews*

,†,‡,§

Departments of Molecular, Cellular & Developmental Biology, Chemistry, and Pharmacology, Yale

University, New Haven, Connecticut 06511

E-mail: craig.crews@yale.edu

Contents

A. Biological Methods

- HIV Protease Digests

- 20S Proteasome Assays

- Cytotoxicity Assays

B. General Chemical Information and Methods

- General Chemistry

- Instrumentation

- Standard Peptide Synthesis Procedures

C. Synthesis and Characterization of Compounds 1-5

A. Biological Methods

HIV Protease Digests

We obtained purified HIV-1 protease from the Programme EVA Centre for AIDS Reagents and stored it in aliquots at –80°C. Standard reaction conditions consisted of 0.5 ng of this enzyme in a 20 µL reaction containing 20 mM sodium acetate pH 5.0, 200 mM NaCl, 1.0 mM EDTA, 0.5 mM DTT, and 10% glycerol (v/v). Compounds in DMSO (1 µL) were added at maximum soluble concentrations: 2 and 3 at 250 µM, 5 at 500 µM. Where indicated, egg white avidin (Sigma) or BSA (Sigma) was added to 0.72 mg/mL. Reactions were conducted at 37°C, overnight, and stopped by boiling for 10 minutes. Enzyme activity was regularly checked by cleavage of a canonical substrate, VSQNY/PIVQ (W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University) and observation of the predicted cleavage masses by MALDI.

20S Proteasome Assays

Compounds dissolved in DMSO (0.1% final volume, final concentrations 10 µM) or protease digest aliquots (0.04%–0.4% final volume, to add final compound concentrations: Figure 2b, 2 at 1 µM; Figure 2c, 3 at 0.1 µM; Figure 2d, 3 as indicated; Figure 3d, 5 at 10 µM) were mixed with chymotryptic proteasome assay buffer: Tris, EDTA, 0.035% sodium dodecyl sulfate. Each condition was set up in quadruplicate or octuplicate in Delfia yellow 96-well fluorescence assay plates (Perkin-Elmer). For epoxyketone-containing compound experiments, chymotryptic proteasome substrate Suc-LLVY-AMC (Bachem) was added at a final concentration of 10 µM. Where indicated, avidin or BSA was added to 0.72 mg/mL. Final reaction volume was 100 µL. Human 20S proteasomes (450 pM final; Boston Biochem), stored in single-use aliquots at –80°C, were added to prewarmed plates containing other reactants to start enzymatic reactions, which were conducted at 30°C. Fluorescence (355 ex / em 460) was read 99 times, 0.2 s/well, with 78 s between consecutive reads of the same well, using a Wallac Victor 2 plate reader. Data were plotted with GraphPad Prism v. 5.0c, and presented relative to fluorescence at time 0. For IC50 calculations, fluorescence readings at t = 2028 s were plotted against log[compound] and three-parameter non-linear curve fitting was performed to determine IC50.

Cytotoxicity Assays

HeLa cervical carcinoma cells were plated 7500 cells/well in white, 384-well tissue culture plates (Nunc), in DMEM supplemented with 10% FBS and penicillin-streptomycin. 1 was added in DMSO (0.4% final) to the indicated concentrations. After 72 hours, proliferation was measured using CellTiterGlo (Promega) according to the manufacturer’s instructions, with luminescence readings on a Wallac Victor 2 plate reader.

B. General Chemical Information and Methods

General Chemistry

All reactions were performed in oven-dried or flame-dried glassware fitted with rubber septa under a positive pressure of nitrogen, unless otherwise noted. Air-and moisture-sensitive liquids were transferred via syringe or cannula. THF was distilled from sodium/benzophenone. Dichloromethane was distilled from calcium hydride. Analytical thin layer chromatography (TLC) was performed using glass plates precoated with silica gel (0.25 mm). TLC plates were visualized by exposure to UV light (UV). Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents.

Instrumentation

1H and

13C spectra were recorded on Bruker Avance DPX-500 or Bruker Avance DPX- 400 NMR

spectrometers. 1H NMR spectra are represented as follows: chemical shift, multiplicity (s =

singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), integration, and coupling constant (J) in Hertz (Hz).

1H NMR chemical shifts are reported relative to CDCl3 (7.26 ppm), d6-

DMSO (2.50 ppm) and d4-MeOD (3.31 ppm). 13

C NMR was recorded relative to the central line of CDCl3 (77.16 ppm), d6-DMSO (39.52 ppm) and d4-MeOD (49.00 ppm). Mass spectra were obtained using a Perkin-Elmer API 150 EX spectrometer. MALDI-TOF analyses of purified samples were performed in a Voyager-DE- PRO 6268 (Applied Biosystems) using cyano-4-hydroxycinnamic acid matrices. HPLC was performed using a Dynamax SD200 solvent delivery system connected to a Dynamax UV-1 Absorbance Detector with a YMC-Pack ODS-AM preparative column (250 x 20 mm, 5 µm particle size, 12 nm pore size). A linear gradient of MeCN in H2O from 5% to 30% MeCN was run over 15 minutes, followed by a linear gradient of 30% to 100% MeCN for an additional 14 minutes, followed by 100% MeCN for 6 minutes.

General Solution Peptide Coupling Procedure

The TFA salt of an amine (1 eq) was dissolved in CH2Cl2 or DMF (0.2 M) and cooled to 4 °C in an ice bath, followed by the addition of the appropriate Boc-protected amino acid (1.3 eq), EDC (1.4 eq) HOBt (1.5 eq) and DIPEA (3.5 eq). The reaction was allowed to slowly warm to room temperature and stirred for 15 h, after which it was diluted with EtOAc and washed successively with aqueous solutions of 1M HCl, saturated sodium bicarbonate, water and saturated sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (0% to 20% MeOH:CH2Cl2) yielded Boc protected peptide.

General Boc Deprotection Procedure

The Boc protected amino acid was dissolved in 10% TFA in CH2Cl2 and stirred overnight, or until the reaction was deemed complete by TLC. The solution was then concentrated under reduced pressure to give the TFA salt of the peptide.

General Solid Phase Peptide Synthesis Procedure

Resin bound Fmoc protected amino acids were deprotected with 20% piperidine in DMF for 30 minutes and washed with DMF, MeOH and CH2Cl2. For steps involving the deprotection of multiple free amines, this step was repeated once before acylation to ensure completeness of reaction. The free amine beads were then suspended in a solution of the protected amino acid (3 eq per eq of free amine), PyBOP (3 eq per eq of free amine) and DIPEA (5 eq per eq of free amine) and mixed for 2 h, and then washed with DMF, MeOH and CH2Cl2. For steps involving the acylation of multiple free amines, the coupling procedure was repeated once before deprotection

to ensure completeness of reaction. These steps were repeated until the desired peptide was synthesized.

General Cleavage of Peptides from Wang Resin

The resin bound peptide was mixed with CH2Cl2 (4.9 mL), TFA (4.9 mL) and tri-isopropylsilane (0.2 mL), mixed for 2 h and then filtered. The resin was washed with DCM, which was combined with the filtrate and condensed under reduced pressure to almost dryness. Cold Et2O was then added to precipitate the desired deprotected peptide.

General Cleavage of Peptides from 2-Chlorotrityl resin

The resin bound peptide was mixed with CH2Cl2 (16 mL), trifluoroethanol (2 mL) and acetic acid (2 mL). After 2 h, the mixture was filtered. The resin was washed with DCM, trifluoroethanol and acetic acid. The washes were combined with the filtrate and condensed under reduced pressure. The crude mixture was then purified through column chromatography (10% to 15% MeOH:CH2Cl2) to give the desired protected peptide.

C. Synthesis and Characterization of Compounds 1-5

(S)-Benzyl (1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (S1)

Cbz-Leu-OH (4.68 g, 17.65 mmol, 1 eq) was dissolved in DMF (70 mL) at

room temperature and cooled to 4 °C in an ice bath. N,O-dimethylhydroxylamine hydrochloride (2.6 g, 26.5 mmol, 1.5 eq) was added, followed by EDC (5.4 g, 28.2 mmol, 1.6 eq), HOBt (4.1 g, 30 mmol, 1.7 eq) and DIEA (13.8 mL, 79 mmol, 4.5 eq). The reaction was allowed to slowly warm to room temperature and stirred for 15 h, after which it was diluted with

EtOAc and washed successively with aqueous solutions of 1M HCl, saturated sodium bicarbonate, water and saturated sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (20% to 50% EtOAc:hexanes) yielded S1 as a colorless oil (3.34 g, 62%).

1H NMR (400 MHz, CDCl3) δ

7.39–7.28 (m, 5H), 5.36 (d, J = 9.1 Hz, 1H), 5.09 (q, J = 12.3 Hz, 2H), 4.79 (d, J = 6.4 Hz, 1H), 3.79 (s, 3H), 3.20 (s, 3H), 1.71 (dd, J = 6.7, 13.3 Hz, 1H), 1.55-1.34 (m, 2H), 0.93 (dt, J = 10.4, 20.9 Hz, 6H).

13C NMR (100 MHz, CDCl3) δ 173.53, 156.38, 136.44, 128.60, 128.19, 128.10,

66.92, 61.69, 49.65, 42.09, 32.22, 24.79, 23.46, 21.62. MS (ESI) 309 (M+H+).

(S)-Benzyl (2,6-dimethyl-3-oxohept-1-en-4-yl)carbamate (S2)

(S)-benzyl (1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (S1, 3.34 g, 10.8 mmol, 1 eq) was dissolved in THF (20 mL) and cooled to 0

°C (ice plus brine). Isopropenyl magnesium bromide (0.5 M in THF; 108 mL; 54 mmol; 5 eq) was added slowly over the course of 20 minutes. The

reaction was stirred at 0°C for 4 h, after which it was poured slowly into a

saturated aqueous ammonium chloride solution at 0 °C. The THF was removed under reduced pressure and the mixture was extracted 4 times with

EtOAc. The combined organic layers were then washed with saturated sodium bicarbonate, water and saturated aqueous sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (15% EtOAc:hexanes) gave S2 as a colorless oil (2.2 g, 70%).

1H NMR (500 MHz, CDCl3) δ 7.40-7.28

(m, 5H), 6.08 (s, 1H), 5.91 (d, J = 1.3 Hz, 1H), 5.42 (d, J = 8.6 Hz, 1H), 5.14 (td, J = 3.6, 9.7 Hz, 1H), 5.09 (s, 2H), 1.90 (s, 3H), 1.77-1.69 (m, 1H), 1.50 (ddd, J = 3.6, 9.4, 13.2 Hz, 1H), 1.36 (ddd, J = 4.4, 10.1, 14.2 Hz, 1H), 1.02 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.7, 3H).

13C NMR (125 MHz,

CDCl3) δ 201.24, 156.22, 142.31, 136.48, 128.67, 128.28, 128.19, 126.51, 67.03, 53.22, 43.47, 25.06, 23.53, 21.82, 17.96. MS (ESI) 290 (M+H

+).

Benzyl ((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)carbamate (S3)

S3 was synthesized in a method adapted from Laidig et al.13

(S)-benzyl (2,6-dimethyl-3-oxohept-1-en-4-yl)carbamate (S2, 2.20 g, 7.6 mmol, 1 eq)

was dissolved in methanol (80 mL) and cooled to 0°C (ice plus brine). Cerium(III) chloride heptahydrate (3.8 g, 10 mmol, 1.3 eq) was added, followed by the portionwise addition of sodium borohydride (0.37 g, 10

mmol, 1.3 eq). The reaction was stirred at 0°C for 3 h, upon which it was deemed complete by TLC (20% EtOAc:hexanes). The reaction was

quenched by the addition of acetic acid (1.5 mL) at 0 °C for 0.5 h and then concentrated under reduced pressure. The crude mixture was dissolved in EtOAc and then washed twice with water and saturated aqueous sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure to give a colorless oil (2.23 g). The majority of the resulting

oil (2.04 g, 7 mmol, 1 eq) was then dissolved in CH2Cl2 (180 mL) and cooled to 4 °C in an ice bath. Vanadyl acetylacetonate (65 mg, 0.21 mmol, 3 mol%) and then tert-Butyl hydroperoxide (5.0-6.0 M in decane; 14 mmol; 2 eq) were then added. The reaction was allowed to warm slowly

to room temperature. After 18 h, the solution was again cooled to 4 °C in an ice bath. Dess-Martin periodinane (4.7 g, 11 mmol, 1.6 eq) was added. The reaction was allowed to warm slowly to room temperature and after 24 h, some starting material remained by TLC (20% EtOAc:hexanes).

The mixture was then cooled to 4 °C in an ice bath and more Dess-Martin periodinane (2.6 g, 6 mmol, 0.9 eq) was added. The solution was allowed to warm slowly to room temperature and after 3 h, the reaction was deemed complete by TLC (20% EtOAc:hexanes). The mixture was then filtered through celite, and concentrated to almost dryness under reduced pressure. The solution was then diluted with EtOAc and washed three times with water and once with brine. The combined aqueous layers were then back extracted once with CH2Cl2 and CHCl3. The combined organic layers was dried with Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (10% EtOAc:hexanes) gave S3 (0.73 g, 2.4 mmol, 34% over 3 steps). The spectral data matched the desired stereoisomer as reported by Laidig et al.

13

1H NMR (400 MHz, CDCl3) δ 7.43-7.28 (m, 5H), 5.12-5.02 (m, 2H), 4.46-4.33 (m, 1H), 3.28 (d, J

= 4.9, 1H), 2.90 (d, J = 4.9 Hz, 1H), 1.72 (ddd, J = 3.4, 6.5, 10.5 Hz, 1H), 1.56 – 1.35 (m, 4H), 1.30 – 1.14 (m, 1H), 1.05 – 0.83 (m, 6H).

13C NMR (100 MHz, CDCl3) δ 209.30, 156.27, 136.27,

128.69, 128.33, 128.14, 67.08, 59.14, 52.40, 51.96, 40.62, 25.18, 23.53, 21.37, 16.87. MS (ESI) 306 (M+H

+)

(S)-2-Amino-4-methyl-1-((R)-2-methyloxiran-2-yl)pentan-1-one 2,2,2-trifluoroacetate (S4)

Benzyl ((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)carbamate (S3, 174 mg, 0.57 mmol, 1 eq) was dissolved in trifluoroacetic acid (5.7 mL) and stirred at room temperature. 10 wt. % palladium on carbon (44 mg, 25 wt. %) was added and the mixture was fitted with a balloon of hydrogen (1 atm). The mixture was stirred for 80 minutes, upon which the reaction was deemed

complete by TLC (25% EtOAc:hexanes). The balloon of hydrogen was removed and the mixture was filtered through celite and concentrated under reduced pressure to yield S4 (200 mg, quant.).

1

Fmoc-Pro-Ile-Val-Phe-OH (96 mg, 0.138 mmol, 1 eq), which was synthesized according to standard solid phase peptide synthesis on Wang Resin, and S4 (47 mg, 0.164 mmol, 1.2 eq) were dissolved in DMF

(1 mL) and cooled to 4 °C in an ice bath. A separate solution of HBTU (54 mg, 0.142 mmol, 1.03 eq) and HOBt (23 mg, 0.17 mmol,

1.2 eq) in DMF (1 mL) was then added, followed by addition of DIPEA (0.20 mL, 1.15 mmol, 8.3 eq). After 3 h, the reaction mixture was warmed to room temperature and stirred for 30 minutes, after which it was diluted with EtOAc and washed successively with aqueous solutions of 1M HCl, saturated sodium bicarbonate, water and saturated sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. The crude material was triturated with cold Et2O and isolated as a beige solid (51 mg, 42%). This solid (9.6 mg, 0.011 mmol, 1 eq) was then dissolved in CH2Cl2 (0.3 mL) at room temperature. Piperidine ( 0.011 mL, 0.11 mmol, 10 eq) was added and the reaction was stirred for 2 h, after which it was deemed complete by TLC (15% methanol:CH2Cl2). Upon addition of Et2O, a white solid, 1 (4.8 mg, 71%) was isolated.

1H

NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.72 (dd, J = 7.5, 22.2 Hz, 1H), 7.42 – 7.26 (m, 2H), 7.18 (s, 5H), 5.30 (s, 1H), 4.82 (s, 1H), 4.13 (d, J = 68.6 Hz, 2H), 3.80 (s, 1H), 2.97 (d, J = 84.9 Hz, 6H), 2.58 (d, J = 8.1 Hz, 2H), 2.38 (s, 2H), 2.14 (s, 2H), 1.95 – 1.88 (m, 1H), 1.84 (s, 1H), 1.76 – 1.71 (m, 1H), 1.67 (d, J = 5.7, 1H), 1.37 (d, J = 5.5 Hz, 1H), 1.25 (s, 3H), 0.85 (t, J = 47.8 Hz, 18H). MS (ESI) 628 (M+H

+)

(S)-2-Amino-4-methyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)pentanamide 2,2,2-trifluoroacetate (S5)

Boc-Leu-OH•H2O (0.87 g, 3.5 mmol, 1 eq) and Boc2O (0.938g, 4.3 mmol, 1.25 eq) were dissolved in 1,4-dioxane (14 mL) at room temperature. Pyridine (0.27 mL, 3.4 mmol, 0.96 eq) and 7-amino-4-methylcoumarin (0.76 g, 4.3 mmol, 1.25 eq) were added and the reaction was stirred for 24 hours, after which it was diluted with EtOAc, washed with 10% KHSO4 (aq), water, saturated aqueous sodium

bicarbonate, water and saturated aqueous sodium chloride. The organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. The crude material (1.71 g) was then dissolved in CH2Cl2 (25 mL) at room temperature. TFA (25 mL) was added and the reaction mixture was stirred for 4 h, upon which it was deemed complete by TLC (EtOAc), after which it was concentrated under reduced pressure. Purification by column chromatography (0 to 20% MeOH:CH2Cl2) gave S5 (1.32 g, 93% over 2 steps) as a yellow solid.

1H NMR (500 MHz, CD3OD)

δ 7.87 (d, J = 2.0 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.50 (dd, J = 2.1, 8.7 Hz, 1H), 6.25 (d, J = 1.1 Hz, 1H), 4.08 (t, J = 7.2 Hz, 1H), 2.46 (d, J = 1.0 Hz, 3H), 1.85 – 1.74 (m, 3H), 1.04 (d, J = 6.2 Hz, 6H).

13C NMR (125 MHz, CD3OD) δ 169.71, 163.08, 162.91, 155.32, 155.16, 142.73, 126.92,

119.37, 117.68, 117.11, 117.04, 113.95, 108.22, 53.84, 41.59, 25.48, 23.27, 21.84, 18.52. MS (ESI) 289 (M+H

+).

2

Biotin-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Phe-OH (19 mg, 0.016 mmol, 1 eq), which had been synthesized on solid phase according to standard methods, and S5 (19 mg, 0.047 mmol, 3 eq)

were dissolved in DMF (0.30 mL) and cooled to 4 °C in an ice bath. To this a solution of HBTU (18 mg, 0.047 mmol, 3 eq) and HOBt (9 mg, 0.067 mmol, 4.1 eq) in DMF (0.20 mL) was added, followed by DIPEA (0.022 mL, 0.23 mmol, 14 eq). The solution was allowed to warm to room temperature slowly and after 14 h, cold Et2O was added, which led to the precipitation of 2 (17 mg, 73%) as a white solid.

1H NMR (400 MHz, DMSO-d6) δ 10.75 – 10.62 (m, 1H), 10.50 – 10.37

(m, 1H), 9.16 (s, 1H), 8.32 – 8.21 (m, 2H), 8.09 – 7.98 (m, 3H), 7.95 (s, 4H), 7.84 – 7.62 (m, 4H), 7.54 – 7.43 (m, 2H), 7.40 – 7.26 (m, 2H), 6.97 – 6.87 (m, 1H), 6.84 – 6.74 (m, 1H), 6.64 (s, 1H), 6.28 (s, 2H), 5.01 – 4.95 (m, 1H), 4.65 – 4.59 (m, 1H), 4.56 – 4.42 (m, 3H), 4.42 – 4.36 (m, 1H), 4.35 – 4.26 (m, 2H), 4.25 – 4.09 (m, 3H), 3.69 – 3.47 (m, 5H), 3.15 – 3.06 (m, 1H), 3.03 (s, 4H), 2.89 (s, 3H), 2.85 – 2.76 (m, 2H), 2.73 (s, 1H), 2.69 (s, 2H), 2.41 (s, 3H), 2.36 – 2.25 (m, 1H), 2.22 – 2.13 (m, 1H), 2.11 – 2.06 (m, 1H), 1.97 – 1.85 (m, 2H), 1.85 – 1.66 (m, 5H), 1.65 – 1.55 (m, 2H), 1.55 – 1.40 (m, 4H), 1.23 (s, 7H), 1.13 – 0.61 (m, 18H).

Compound 2

Compound 2

3

Biotin-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Phe-OH (10 mg, 0.008 mmol, 1 eq), which had been synthesized on solid phase according to standard methods, and S4 (5 mg, 0.018 mmol, 2.2 eq)

were dissolved in DMF (0.175 mL) and cooled to 4 °C in an ice bath. To this a solution of HBTU (6 mg, 0.016 mmol, 2 eq) and HOBt (5 mg, 0.037 mmol, 4.6 eq) in DMF (0.175 mL) was added, followed by DIPEA (0.008 mL, 0.083 mmol, 10.4 eq). The solution was allowed to warm to room temperature slowly and after 14 h, cold Et2O was added, which led to the precipitation of 3 (3 mg, 28%) as a white solid.

1H NMR (500 MHz, DMSO-d6) δ 9.15 (s, 2H), 8.26 (d, J = 7.5 Hz, 2H), 8.15

– 7.81 (m, 7H), 7.76 – 7.60 (m, 2H), 7.31 – 7.12 (m, 5H), 7.06 (d, J = 8.1 Hz, 2H), 6.91 (s, 1H), 6.76 (t, J = 25.5 Hz, 2H), 6.63 (d, J = 8.4 Hz, 2H), 6.43 (s, 1H), 6.37 (s, 1H), 4.98 (t, J = 5.5 Hz, 1H), 4.59 (d, J = 4.8 Hz, 1H), 4.55 – 4.43 (m, 2H), 4.34 (d, J = 40.8 Hz, 4H), 4.25 – 4.06 (m, 4H), 3.55 (dd, J = 16.4, 21.6, 5H), 3.20 – 3.08 (m, 2H), 3.08 – 2.96 (m, 5H), 2.96 – 2.87 (m, 2H), 2.87 – 2.77 (m, 2H), 2.72 (dd, J = 9.5, 22.1 Hz, 2H), 2.36 (d, J = 1.8 Hz, 1H), 2.13 (d, J = 42.1 Hz, 3H), 1.98 – 1.84 (m, 2H), 1.80 (s, 2H), 1.72 (s, 3H), 1.62 (s, 2H), 1.57 – 1.48 (m, 2H), 1.39 (s, 3H), 1.36 – 1.18 (m, 5H), 1.08 (d, J = 21.7 Hz, 2H), 0.93 – 0.64 (m, 13H).

Compound 3

Compound 3

0th Generation of 4

Boc-Lys(Boc)-Ala-Pro-Ile-Val-Phe-Leu-AMC (0.130 g, 0.113 mmol, 1eq), which was synthesized in an iterative fashion using the in solution general peptide synthesis and deprotection methods

described above, was dissolved in CH2Cl2 (4.5 mL) and cooled to 4°C in an ice bath. TFA (0.5 mL) was added and the solution was allowed to warm to room temperature slowly. After 10 h, the reaction was deemed complete by TLC (20% MeOH:CH2Cl2) and concentrated under reduced pressure to give the 0th Generation of 4 (0.145 g, quantitative yield) as a brown oil.

1H NMR

(400 MHz, CD3OD) δ 7.76 (s, 1H), 7.67 (d, J = 8.7, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.20 – 7.02 (m, 5H), 6.21 (s, 1H), 4.83 (s, 1H), 4.64 (d, J = 6.8 Hz, 2H), 4.56 (d, J = 4.5 Hz, 1H), 4.33 (d, J = 6.6 Hz, 2H), 3.91 (t, J = 6.3 Hz, 1H), 3.81 (s, 1H), 3.68 (s, 1H), 3.18 – 2.82 (m, 4H), 2.43 (s, 3H), 2.14 (s, 1H), 1.98 (s, 4H), 1.94 – 1.82 (m, 2H), 1.78 (s, 1H), 1.69 (d, J = 6.3 Hz, 5H), 1.52 (s, 3H), 1.37 (dd, J = 6.9, 21.2 Hz, 3H), 1.21 – 1.03 (m, 1H), 1.00 – 0.70 (m, 18H). MS (ESI) 945 (M+H

+).

1st Generation of 4

The 0th Generation of 4 (0.145 g, 0.124 mmol, 1 eq) was dissolved in DMF (4 mL) and cooled to

4 °C in an ice bath. Boc-Lys(Boc)-OH (0.112 g, 0.322 mmol, 2.6 eq), EDC (0.067 g, 0.347 mmol, 2.8 eq), HOBt (0.050 g, 0.372 mmol, 3 eq) and DIPEA (0.15 mL, 0.868 mmol, 7 eq) were added and the reaction mixture was allowed to warm slowly to room temperature. After 18 h, the solution was diluted with 1 M aqueous HCl and extracted 3 times with CH2Cl2. The organic layer was then washed with saturated aqueous sodium bicarbonate, which was then extracted 3 times with DCM. The combined organic layer was dried with Na2SO4, filtered and concentrated under reduced pressure. Purification by column chromatography (3% to 10% MeOH:CH2Cl2) gave a

pure white solid (0.129 g, 0.0803 mmol) which was dissolved in DCM (4.5 mL) and cooled to 4 °C in an ice bath. TFA (0.5 mL) was added and the reaction was allowed to warm slowly to room temperature. After 17 h, the reaction was deemed complete by TLC (20% MeOH:CH2Cl2) and concentrated under reduced pressure to give the 1st Generation of 4 (130 mg, 63% over 2 steps) as a yellow solid.

1H NMR (500 MHz, CD3OD) δ 7.82 (d, J = 1.4 Hz, 1H), 7.68 (d, J = 8.7

Hz, 1H), 7.55 – 7.49 (m, 1H), 7.18 (t, J = 9.1 Hz, 2H), 7.10 (dt, J = 7.2, 25.8 Hz, 3H), 6.21 (s, 1H), 4.78 – 4.72 (m, 1H), 4.60 (dt, J = 8.2, 14.8 Hz, 2H), 4.52 (dd, J = 4.5, 7.7 Hz, 1H), 4.38 (dd, J = 4.9, 9.2 Hz, 1H), 4.24 (d, J = 7.2 Hz, 1H), 3.97 (s, 2H), 3.85 (t, J = 6.6 Hz, 1H), 3.83 – 3.76 (m, 1H), 3.69 (s, 1H), 3.28 – 3.08 (m, 3H), 2.95 (dt, J = 7.7, 20.4 Hz, 5H), 2.43 (s, 3H), 2.21 – 2.14 (m, 1H), 2.11 – 2.03 (m, 1H), 2.03 – 1.94 (m, 3H), 1.90 (ddd, J = 7.4, 13.0, 19.5 Hz, 3H), 1.86 – 1.78 (m, 3H), 1.78 – 1.61 (m, 8H), 1.61 – 1.43 (m, 8H), 1.39 (t, J = 13.7 Hz, 4H), 1.34 – 1.24 (m, 1H), 1.20 – 1.10 (m, 1H), 0.99 – 0.78 (m, 18H). MS (ESI) 1201 (M+H

+).

2nd Generation of 4

The 1st Generation of 4 (130 mg, 0.078 mmol, 1 eq) was dissolved in DMF (5 mL) and cooled to

4 °C in an ice bath. Boc-Lys(Boc)-OH (0.145 g, 0.418 mmol, 5.12 eq), EDC (0.086 g, 0.418 mmol, 5.6 eq), HOBt (0.065 g, 0.482 mmol, 6 eq) and DIPEA (0.20 mL, 1.124 mmol, 14 eq) were added and the reaction mixture was allowed to warm slowly to room temperature. After 19 h, the solution was diluted with 1 M aqueous HCl and extracted 3 times with CH2Cl2. Chloroform was added in an attempt to break the intractable emulsion, which settled with time and agitation. The organic layer was washed with saturated aqueous sodium bicarbonate, which was back extracted twice with CH2Cl2. The combined organic layers were concentrated under reduced pressure and purified by column chromatography (3% to 20% MeOH:CH2Cl2) to give a colorless solid (0.1103

g, 0.044 mmol). This was suspended in CH2Cl2 (9 mL) and cooled to 4 °C in an ice bath. TFA (1 mL) was added, improving the solubility, and the mixture was allowed to warm to room temperature slowly. After 15 h, the solution was concentrated under reduced pressure to give the 2nd Generation of 4 (0.122 g, 60%) as an off white, oily solid.

1H NMR (500 MHz, CD3OD) δ

7.85 (d, J = 1.7 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 7.2 Hz, 2H), 7.18 (t, J = 7.4 Hz, 2H), 7.12 (d, J = 7.3 Hz, 1H), 6.25 (d, J = 1.1 Hz, 1H), 4.77 (d, J = 49.7 Hz, 1H), 4.61 (dd, J = 7.4, 14.7 Hz, 2H), 4.53 – 4.43 (m, 2H), 4.40 – 4.26 (m, 3H), 4.18 – 4.06 (m, 2H), 4.03 – 3.77 (m, 4H), 3.70 (s, 1H), 3.29 – 3.10 (m, 7H), 3.02 – 2.90 (m, 9H), 2.47 (d, J = 0.9 Hz, 3H), 2.21 (s, 1H), 2.12 – 1.95 (m, 5H), 1.95 – 1.80 (m, 9H), 1.80 – 1.61 (m, 16H), 1.58 – 1.44 (m, 17H), 1.43 – 1.34 (m, 7H), 1.24 – 1.14 (m, 2H), 0.98 – 0.85 (m, 18H).

2nd Generation of 4

2nd Generation of 4

3rd Generation of 4

The 2nd Generation of 4 (121 mg, 0.0438 mmol, 1 eq) was dissolved in DMF (6 mL) and cooled

to 4 °C in an ice bath. Boc-Lys(Boc)-OH (0.15 g, 0.438 mmol, 10 eq), EDC (0.08 g, 0.438 mmol, 10 eq), HOBt (0.06 g, 0.438 mmol, 10 eq) and DIPEA (0.21 mL, 1.23 mmol, 28 eq) were added and the reaction mixture was allowed to warm slowly to room temperature. After 21 h, the solution was concentrated under reduced pressure. The crude residue was redissolved in CH2Cl2 and washed with 1 M aqueous HCl, which was back extracted once with CH2Cl2 and once with chloroform. The combined organic layer was then washed with saturated aqueous sodium bicarbonate, which was back extracted once with CH2Cl2 and once with chloroform. The combined organic layers were concentrated under reduced pressure and purified by column chromatography (2% to 15% MeOH in CH2Cl2) to give a white solid (0.0891g, 0.021 mmol, 47%). This solid (0.0628 g, 0.014 mmol, 1 eq) was dissolved in CH2Cl2 (8 mL) and MeCN (1 mL) and

cooled to 4 °C in an ice bath. TFA (1 mL) was added, improving the solubility, and the mixture was allowed to warm to room temperature slowly. After 22 h, the solution was concentrated under reduced pressure to give the 3rd Generation of 4 (0.092 g, quantitative) as a white solid. 1H NMR (500 MHz, CD3OD) δ 7.87 (s, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 6.7 Hz, 2H), 7.17 (t, J = 6.8 Hz, 2H), 7.12 (d, J = 7.3 Hz, 1H), 6.26 (s, 1H), 4.61 (d, J = 7.2 Hz, 2H), 4.49 (s, 2H), 4.46 – 4.40 (m, 1H), 4.35 (d, J = 5.0 Hz, 3H), 4.29 (s, 3H), 4.20 – 4.12 (m, 1H), 3.99 (d, J = 9.1 Hz, 5H), 3.87 (dd, J = 6.9, 15.0 Hz, 5H), 3.74 – 3.66 (m, 1H), 3.27 (d, J = 5.7 Hz, 4H), 3.22 – 3.11 (m, 11H), 3.03 – 2.89 (m, 17H), 2.48 (s, 3H), 1.93 – 1.83 (m, 49H), 1.77 – 1.66 (m, 27H), 1.58 – 1.45 (m, 34H), 1.41 – 1.35 (m, 11H), 0.96 (dd, J = 6.3, 9.9, 6H), 0.92 – 0.85 (m, 12H).

3rd Generation of 4

3rd Generation of 4

1st Generation of 5

(Boc-Lys(Boc))2Lys-OMe (11.3 mg, 0.0138 mmol, 1 eq), synthesized according to standard

peptide procedures described above, was dissolved in THF (0.40 mL) and cooled to 4 °C in an ice bath. A 0.2 M aqueous solution of lithium hydroxide (0.18 mL) was added and the solution was allowed to slowly warm to room temperature. After 8 h, the solution was concentrated. The crude product (6.4 mg) was then dissolved in DMF (0.6 mL) at room temperature in a capped vial. A solution of Ser-Gln-Asn-Tyr-Pro-Ile-Val-Phe-Leu-AMC (20.6 mg, 0.014 mmol, 1.8 eq), which was synthesized according to the standard methods described above, in DMF (0.9 mL) was added, followed by EDC (1.8 mg, 0.0094 mmol, 1.2 eq), HOBt (1.3 mg, 0.0101 mmol, 1.3 eq) and

DIPEADIEA (4.1 µL, 0.0234 mmol, 3 eq). After 16 h, cold Et2O was added, and the resulting precipitate (7.1 mg) was washed with acetone and CH2Cl2. This was dissolved in CH2Cl2 (1.5 mL) and TFA (1.5 mL). After 2 hours, the solution was concentrated under reduced pressure and purified by HPLC (eluting at 24 min) to give the 1st generation of 5 (1.6 mg) as a white solid.

1st Generation of 5

2nd Generation of 5

(((Boc-Lys(Boc))2Lys)2Lys-Ser(OtBu)-Gln(NHTrt)-Asn(NHTrt)-Tyr(OtBu)-Pro-Ile-Val-Phe-OH (32 mg, 0.01 mmol, 1 eq), which was synthesized on solid phase and cleaved from 2-chlorotrityl resin using the standard procedures described above, was dissolved in DMF (2 mL) at room temperature. TFA-Leu-AMC, S5, (6 mg, 0.015 mmol, 1.5 eq) was added, followed by EDC (3.1

mg, 0.016 mmol, 1.6 eq), HOBt (2.3 mg, 0.017 mmol, 1.7 eq) and DIPEADIEA (7.8 µL, 0.045 mmol, 4.5 eq). The solution was stirred for 15 h, after which it was diluted with an aqueous solution of 1 M HCl. This was extracted 3 times with CH2Cl2 and once with EtOAc. The combined organic layer was concentrated under reduced pressure and purified by column chromatography (7.5% to 10% MeOH:CH2Cl2) to give the fully protected peptide (26.3 mg). This was dissolved in CH2Cl2 (4 mL) at room temperature. TFA (1 mL) was slowly added and the mixture was stirred for 12 h. The solution was then condensed and purified by HPLC to give the 2nd generation of 5 (4.4 mg) as a white solid.

2nd Generation of 5

3rd Generation of 5

((((Boc-Lys(Boc))2Lys)2Lys)2Lys-Ser(OtBu)-Gln(NHTrt)-Asn(NHTrt)-Tyr(OtBu)-Pro-Ile-Val-Phe-OH (0.11 g, 0.022 mmol, 1 eq) which was synthesized on solid phase and cleaved from 2-chlorotrityl resin using the standard procedures described above, was dissolved in DMF (5 mL) at room temperature. TFA-Leu-AMC, S5, (13 mg, 0.033 mmol, 1.5 eq) was added, followed by EDC

(6.7 mg, 0.035 mmol, 1.6 eq), HOBt (5 mg, 0.037 mmol, 1.7 eq) and DIPEA (17 µL, 0.099 mmol, 4.5 eq). The solution was stirred for 14 h, after which it was diluted with an aqueous solution of 1 M HCl. This was extracted three times with CH2Cl2 and once with EtOAc. The combined organic layer was concentrated under reduced pressure and purified by column chromatography (7.5% to 10% MeOH in CH2Cl2) to give the fully protected peptide (58.5 mg). This was dissolved in CH2Cl2 (4 mL) at room temperature. TFA (1 mL) was slowly added and the mixture was stirred for 12 h. The solution was then condensed and purified by HPLC (eluting at 26 min) to give the 3rd Generation of 5 (1.6mg) as a white solid.

3rd Generation of 5