Materials and Methods. Protocol for Fmoc- based solid- phase peptide synthesis

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1 Materials and Methods All commercially available materials (Aldrich, TCI, ovabiochem, Fluka ) were used without further purification. All solvents were reagent grade or PLC grade (Fisher ). Anhydrous TF, C 2 Cl 2, toluene, and benzene were obtained from a dry solvent system (passed through column of alumina) and used without further drying. All reactions were performed under an atmosphere of pre- purified dry nitrogen. MR spectra ( 1 and 13 C) were recorded on a Bruker Advance II 600 Mz or Bruker Advance DRX- 500 Mz, referenced to residual solvent. Low- resolution mass spectral analyses were performed with a Waters Micromass ZQ mass spectrometer. Analytical thin layer chromatography (TLC) was performed on E. Merck silica gel 60 F254 plates and flash column chromatography was performed on E. Merck silica gel 60. Yields refer to chromatographically pure compounds. The following compounds were purchased from the designated supplier: TCEP Cl (Thermo Scientific Pierce ), ATU (Genscript, Piscataway, ew Jersey), VA- 044 (2,2'- Azobis[2- (2- imidazolin- 2- yl)propane]dihydrochloride; Wako Pure Chemical Industries ), thiophenol (Aldrich ), ethyl 3- mercaptopropionate (TCI ), 2- nitroveratraldehyde (Aldrich ), trifluoroacetic acid (Aldrich ), 1,2- ethanedithiol (Fluka ), anisole (Aldrich ), thioanisole (Aldrich ). Protocol for Fmoc- based solid- phase peptide synthesis Automated peptide synthesis was performed on an Applied Biosystems Pioneer continuous- flow peptide synthesizer. Peptides were synthesized under standard automated Fmoc protocols, with 4.0 equivalents of each amino acid and 3.9 equivalents of ATU (1- [Bis(dimethylamino)methylene]- 1-1,2,3- triazolo[4,5- b]pyridinium 3- oxid hexafluorophosphate) as coupling reagent. The deblock mixture was a mixture of 96:2:2 DMF/piperidine/DBU (v/v/v). The following Boc- and Fmoc- protected amino acids and pseudoproline dipeptides from ovabiochem were utilized: Fmoc- Ala-, Fmoc- Arg(Pbf)-, Fmoc- Asn(Trt)-, Fmoc- Asp(tBu)-, Fmoc- Glu(tBu)-, Fmoc- Gln(Trt)-, Fmoc- Gly-, Fmoc- is(trt)-, Fmoc- Ile-, Fmoc- Leu-, Fmoc- Lys(Boc)-, Fmoc- Met-, Fmoc- Phe-, Fmoc- Pro-, Fmoc- Ser(tBu)-, Fmoc- Thr(tBu)-, Fmoc- Val-, Fmoc- Cys(Acm)-, Fmoc- Tyr(tBu)-, Boc- Cys(Trt)-, Boc- Ser(tBu)-, Boc- Glu-, Boc- Leu-, Boc- Tyr(tBu)-, Boc- Asn(Trt)-, Boc- Arg(Mtr)-, and Fmoc- Asp(Mpe)- and Fmoc- Ile- Ser(ψMe,MePro)- when specifically indicated. The following resins for peptide synthesis were acquired from ovabiochem : Fmoc- Arg(Pbf)- ovasyn TGT, Fmoc- Glu(tBu)- ovasyn TGT, Fmoc- Asp(tBu)- ovasyn TGT, Fmoc- Lys(Boc)- ovasyn TGT, Fmoc- Tyr(tBu)- ovasyn TGT, Fmoc- Gly- ovasyn TGT.

2 Protocol for the cleavage of fully protected peptyl acids from resin Solid- phase peptide synthesis was performed on a 0.10 mmol scale, and the peptide resin was transferred with C 2 Cl 2 from the synthesis column into a 15 ml fritted glass column with a stopcock. The resin cleavage was performed with 3 ml of freshly prepared C 2 Cl 2 /acetic acid/trifluoroethanol solution (3:1:1 v/v), with mild stirring for 2 hours. The cleavage solution was then drained into a 20 ml glass vial; this cleavage wash was repeated for four iterations. The combined protected peptide solutions were concentrated in vacuo with a water bath temperature of 24 C to a volume of 3 4 ml to remove the C 2 Cl 2. The protected peptide was precipitated upon the addition of 8 ml of distilled water to result in a thick, milky mixture; this was lyophilized for 48 hours and azeotroped with 8 ml of toluene prior to further use. Protocol for coupling of the aminothioesters with the protected peptidyl acids The protected peptidyl acid was massed into a 20 ml glass vial with stir bar. The protected acid was dissolved with 49:1 C 2 Cl 2 :trifluoroethanol (v/v), and cooled in a 0 C ice bath with stirring. To this solution was added - (3- dimethylaminopropyl)- ʹ - ethylcarbodiimide (EDC, 2.44 equivalents), followed immediately by 3- hydroxy- 4- oxo- 1,2,3- triazine (Bt, 2.44 equivalents). This solution was allowed to stir at 0 C for 5 minutes, before the addition of the aminothioester as a hydrochloride salt (2.91 equivalents). The coupling was monitored by thin layer chromatography (glass backed silica plates, 9:1 C 2 Cl 2 :Me (v/v) as eluent) or ran for no more than two hours. Upon completion, the ice bath was removed, and the reaction was concentrated down to a viscous oil under a stream of nitrogen. To the crude protected peptidyl thioester was added 90:5:3:2 TFA:thioanisole:1,2- ethanedithiol:anisole cleavage cocktail (v/v), at a volume of 1 ml per 20 mg of protected peptide. This solution was allowed to stir vigorously for 90 minutes, upon which it was transferred to a 50- ml polypropylene conical tube, and concentrated down to a viscous oil under a stream of nitrogen. The crude, deprotected peptide thioester was precipitated with the addition of 40 ml of cold diethyl ether, centrifuged to a pellet, and the ether layer decanted. The crude peptide thioester was dissolved with 1:1 2 :AcC with 0.05% TFA, lyophilized, redissolved with 1:1 2 :AcC with 0.05% TFA, filtered through a 0.45 μm PTFE filter, and lyophilized to provide crude peptide for PLC purification. UPLC and LC- MS analysis and PLC purification of deprotected peptides All separations involved a mobile phase of 0.05% TFA (v/v) in water (solvent A)/0.04% TFA in acetonitrile (solvent B). Reaction progress was monitored on a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) equipped with photodiode detector and single quadrupole mass detector, with Waters BE300 C4 1.7μm 2.1 x 100 mm column at a flow rate of 0.3 ml/min. Analytical LC- MS analyses for purity were performed using a Waters 2695 Separations Module and a Waters 2996 Photodiode Array Detector equipped with a Varian Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min. Preparative separations were performed using a Waters igh Performance Liquid

3 Chromatography (PLC) system equipped with a Waters 2545 binary gradient module and Waters 2996 Photodiode array detector using Agilent Dynamax reverse phase 250 x 21.4 mm Microsorb C8 or Microsorb C4 column at a flow rate of 16.0 ml/min. Preparation of ligation, dethiylation, and Thz- deprotection buffers ative chemical ligation buffer was prepared by massing guanidine hydrochloride (1.146 g, 12 mmol), disodium phosphate (56.6 mg, 400 μmol), 4- mercaptophenylacetic acid (67.3 mg, 400 μmol), and tris(2- carboxyethyl)phosphine hydrochloride (TCEP, 40 μmol) into a 15- ml polypropylene conical tube, and the volume was brought to 1.9 ml with distilled water. The p of the buffer was adjusted to 7.2 with 5.0 M sodium hydroxide (145 μl), and the buffer was degassed with argon bubbling under sonication for 30 minutes. Kinetic ligation buffer was prepared by massing guanidine hydrochloride (1.146 g, 12 mmol), disodium phosphate (56.6 mg, 400 μmol), and tris(2- carboxyethyl)phosphine hydrochloride (TCEP, 40 μmol) into a 15- ml polypropylene conical tube, and the volume was brought to 2.0 ml with distilled water. The p of the buffer was adjusted to 7.2 with 5.0 M sodium hydroxide (30 μl), and the buffer was degassed with argon bubbling under sonication for 30 minutes. Thiazolidine deprotection buffer was prepared by massing guanidine hydrochloride (1.146 g, 12 mmol), disodium phosphate (56.6 mg, 400 μmol), tris(2- carboxyethyl)phosphine hydrochloride (TCEP, 40 μmol), and methoxylamine hydrochloride (100. mg, 1.2 mmol) into a 15- ml polypropylene conical tube and the volume was brought to 1.9 ml with distilled water. The p of the buffer was adjusted to 4.0 with 2.0 M hydrogen chloride (120 μl), and the buffer was degassed with argon bubbling under sonication for 30 minutes. Dethiylation buffer was prepared by massing guanidine hydrochloride (1.146 g, 12 mmol) and disodium phosphate (56.6 mg, 400 μmol) into a 15- ml polypropylene conical tube and the volume was brought to 2.0 ml with distilled water. The p of the buffer was taken (7.4), and the buffer was degassed with argon bubbling under sonication for 30 minutes.

4 Synthesis of amino acid building blocks Boc- Leu- SPh. A stirring solution of Boc- Leu- (0.500 g, 2.0 mmol, 1.0 equiv) in dry dichloromethane (15 ml) was cooled in a 0 C ice bath. To this solution was added - (3- dimethylaminopropyl)- ʹ - ethylcarbodiimide (EDC, 355 μl, 2.0 mmol, 1.0 equiv), followed immediately by 1- hydroxy- 7- azabenzotriazole (At, 409 mg, 3.0 mmol, 1.5 equiv). This solution was allowed to stir at 0 C for 5 minutes, before the addition of thiophenol (1.026 ml, 10 mmol, 5 equiv). This solution was allowed to warm to room temperature and stirred for 24 hours. The reaction was then concentrated in vacuo, and the residue was purified using silica gel chromatography (4:1 hexane:ethyl acetate eluent) to afford Boc- Leu- SPh thioester (351 mg, 54%).!!!" : +6.9 (c 1.75, C 2 Cl 2 ); 1 MR (CDCl 3, 600 Mz) δ (m, 5), 4.94 (d, J = 8.3 z, 1), 4.49 (td, J = 9.5, 3.9 z, 1), (m, 2), (m, 1), 1.49 (s, 9), 0.97 (d, J = 6.5 z, 3), 0.96 (d, J = 6.2 z, 3); 13 C MR (CDCl 3, 150 Mz) δ 200.1, 155.3, 134.7, 129.3, 129.2, 127.6, 80.3, 59.2, 41.6, 28.4, 24.9, 23.1, 21.6; IR (film) υ max 2958, 1688, 1508, 1366, 1248, 1162, 1064, 1022 cm 1 ; MS (ESI- MS) calcd for C S [M+] + m/z 324.2, found 323.9; calcd for C Sa [M+a] + m/z 346.1, found Leu- SPh Cl. To a round- bottom flask containing a magnetic stir bar and Boc- Leu- SPh (351 mg, 1.09 mmol, 1.0 equiv) was added 4.0 M hydrogen chloride in dioxane (3.0 ml, 12 mmol, 11 equiv) in one portion. The solution was stirred vigorously, monitored by TLC (4:1 hexanes:ethyl acetate eluent), and was observed to be complete in 30 minutes. The reaction was concentrated in vacuo and used without further purification, providing Leu- SPh Cl as a white solid (282 mg, quantitative).!!!" : (c 1.15, C 2 Cl 2 ); 1 MR (CD 3 D, 500 Mz) δ (m, 5), (m, 1), (m, 2), (m, 1), 1.0 (d, J = 6.3 z, 3), 0.96 (d, J = 6.3 z, 3); 13 C MR (CDCl 3, 125 Mz) δ 196.6, 135.9, 131.5, 130.8, 126.6, 58.7, 41.9, 25.8, 23.1, 22.2; IR (film) υ max 2866, 1702, 1584, 1504, 1368, 1158, 984, 947 cm 1 ; MS (ESI- MS) calcd for C Sa [M+a] + m/z 224.1, found Boc- Tyr(tBu)- SC 2 C 2 C 2 Et. A stirring solution of Boc- Tyr(tBu)- (1.00 g, 2.96 mmol, 1.0 equiv) in dry dichloromethane (25 ml) was cooled in a 0 C ice bath. To this solution was added - (3- dimethylaminopropyl)- ʹ - ethylcarbodiimide (EDC, ml, 2.96 mmol, 1.0 equiv), followed immediately by 1- hydroxy- 7- azabenzotriazole (At, 1.21 g, 8.89 mmol, 3.0 equiv). This solution was allowed to stir at 0 C for 5 minutes, before the addition of ethyl 3- mercaptopropionate (3.18 ml, 23.7 mmol, 8 equiv). This solution was allowed to warm to room temperature and stirred for 24 hours. The

5 reaction was then concentrated in vacuo and the residue purified by silica gel chromatography (4:1 hexane:ethyl acetate eluent) to afford Boc- Tyr(tBu)- SC 2 C 2 C 2 Et thioester (1.016 g, 76%).!!!" : (c 0.54, C 2 Cl 2 ); 1 MR (CDCl 3, 500 Mz) δ 7.03 (d, J = 8.4 z, 2), 6.91 (d, J = 8.4 z, 2), 4.87 (d, J = 8.6 z, ), 4.57 (q, J = 7.1 z, 1), 4.14 (q, J = 7.2 z, 2), 3.09 (t, J = 7.0 z, 2), (m, 1), 2.96 (dd, J = 14.1, 7.5 z, 1), 2.57 (t, J = 7.0 z, 2), 1.39 (s, 9), 1.32 (s, 9), 1.25 (t, J = 7.1 z, 3); 13 C MR (CDCl 3, 125 Mz) δ 201.0, 171.6, 154.9, 154.4, 130.3, 129.7, 124.2, 80.3, 78.4, 61.0, 60.8, 37.7, 34.1, 28.8, 28.2, 23.9, 14.2; IR (film) υ max 2978, 1720, 1687, 1507, 1366, 1239, 1160, 899 cm 1 ; MS (ESI- MS) calcd for C Sa [M+a] + m/z 476.2, found Tyr- SC 2 C 2 C 2 Et Cl. To a round- bottom flask containing a magnetic stir bar and Boc- Tyr(tBu)- SC 2 C 2 C 2 Et (1.016 g, 2.24 mmol, 1.0 equiv) was added 4.0 M hydrogen chloride in dioxane (19.7 ml, 78.8 mmol, 17.6 equiv) in one portion. The solution was stirred vigorously, monitored by TLC (4:1 hexanes:ethyl acetate eluent), and was observed to be complete in 2 hours. The reaction was concentrated in vacuo and used without further purification, providing Tyr- SC 2 C 2 C 2 Et Cl as a white solid (0.748 g, quantitative).!!!" : (c 0.17, 20% Ac/C 2 Cl 2 v/v); 1 MR (CD 3 D, 500 Mz) δ 7.00 (d, J = 8.5 z, 2), 6.69 (d, J = 8.5 z, 2), 4.31 (t, J = 7.0 z, 1), 4.06 (q, J = 7.1 z, 2), 3.15 (dd, J = 13.7, 6.9 z, 1), (m, 2), 3.00 (dd, J = 14.3, 7.0 z, 1), (m, 2), 1.17 (t, J = 7.2 z, 3); 13 C MR (CD 3 D, 125 Mz) δ 197.5, 173.0, 158.6, 131.9, 125.3, 117.0, 62.1, 61.6, 38.1, 34.9, 25.4, 14.6; IR (film) υ max 2980, 1732, 1689, 1518, 1374, 1250, 1158, 1000 cm 1 ; MS (ESI- MS) calcd for C S [M+] + m/z 298.1, found ThzSLeu- TMSE. To a round- bottom flask containing a magnetic stir bar and Boc- AcSLeu- TMSE (259 mg, mmol, 1.0 equiv) was added methanol (10 ml). It was then cooled in a 0 C ice bath and degassed by argon bubbling for an hour. To this solution was added a degassed 1.0 M sodium hydroxide solution (1.6 ml, 1.60 mmol, 2.5 equiv), which stirred at 0 C. The reaction was observed as complete by TLC in 15 minutes, at which point it was quenched with 1.0 M hydrogen chloride (1.91 ml, 1.91 mmol, 3.0 equiv), diluted with 30 ml water, and extracted with ethyl acetate (20 ml, twice). The combined organic layers were washed with saturated sodium chloride (20 ml), dried over magnesium sulfate, and concentrated in vacuo to yield the crude Boc- SLeu- TMSE intermediate. To this intermediate was added 9:1 C 2 Cl 2 :TFA (10 ml), which then stirred at room temperature. The reaction was observed as complete by TLC in 2 hours, at which point the volatiles were removed and the reaction mixture was concentrated in vacuo to dryness to provide the crude SLeu- TMSE trifluoroacetate salt. This intermediate was dissolved in methanol (5 ml) with stirring, and an aqueous solution of 37% formaldehyde was added (71.3 μl, mmol, 1.5 equiv). The reaction was monitored by TLC and observed to be complete in 24 hours. The reaction was neutralized with the addition of saturated sodium bicarbonate (0.50 ml), diluted with water (20 ml), and extracted with ethyl acetate (15 ml,

6 three times). The combined organic phases were dried over magnesium sulfate and concentrated in vacuo, and the residue was purified using silica gel chromatography (gradient 39:1 to 19:1 hexane:ethyl acetate eluent) to afford ThzSLeu- TMSE (95.6 mg, 54%).!!!" : 19.0 (c 0.72, C 2 Cl 2 ); 1 MR (CDCl 3, 600 Mz) δ 4.30 (d, J = 9.8 z, 1), 4.26 (dd, J = 9.5, 7.4 z, 2), 4.08 (d, J = 9.9 z, 1), 3.65 (d, J = 5.9 z, 1), 3.52 (t, J = 5.9 z, 1), 1.96 (octet, J = 6.5 z, 1), (m, 2), 1.02 (d, J = 7.0 z, 3), 1.01 (d, J = 6.8 z, 3), 0.05 (s, 9); 13 C MR (CDCl 3, 150 Mz) δ 173.4, 69.3, 65.6, 64.1, 55.3, 33.6, 24.1, 20.2, 18.9, 0.0; IR (film) υ max 2956, 1734, 1251, 1169, 1044, 935, 859, 837 cm 1 ; MS (ESI- MS) calcd for C SSi [M+] + m/z 276.1, found Boc- ThzSLeu- TMSE. To a dry round- bottom flask fitted with a reflux condenser and magnetic stir bar was added ThzSLeu- TMSE (145.2 mg, mmol, 1.0 equiv) and dry dichloromethane (6 ml). To this solution was added di- tert- butyl dicarbonate (460 mg, 2.11 mmol, 4.0 equiv) and 4- dimethylaminopyridine (32.2 mg, mmol, 0.5 equiv), and the reaction was gently refluxed in an oil bath. The reaction progress was monitored by TLC (4:1 hexanes:ethyl acetate eluent, permanganate stain). Additional di- tert- butyl dicarbonate and DMAP equivalents were added after 24 hours, if necessary. Upon completion, the volatiles were removed in vacuo, and the desired product was purified using silica gel chromatography (gradient 39:1 to 19:1 hexane:ethyl acetate eluent) to afford Boc- ThzSLeu- TMSE (102.6 mg, 52%) along with recovered ThzSLeu- TMSE (48.9 mg, 34%). The isolated compound appeared to exhibit rotomers by 1 MR. In order to confirm that the pairs of signals were due to rotomers of the Boc group and not an undesired diastereomer, the Boc group was removed with TFA to result in a compound whose 1 spectra had a single set of peaks.!!!" : 11.2 (c 1.33, C 2 Cl 2 ); 1 MR (CDCl 3, 500 Mz) δ 4.78 (d, J = 1.5 z, 0.5 αc), 4.66 (d, J =8.4 z, 0.5 SC 2 ), 4.56 (d, J =8.0 z, 0.5 SC 2 ), 4.55 (s, 0.5 αc), 4.43 (d, J =8.4 z, 0.5 SC 2 ), 4.39 (d, J =8.1 z, 0.5 SC 2 ), 4.23 (q, J =6.8 z, 2 C 2 C 2 ), 3.44 (t, J =7.0 z, βc), (m, 1 ɣc), 1.47 (s, 4.5 C(C 3 ) 3 ), 1.44 (s, 4.5 C(C 3 ) 3 ), (m, 8 C(C 3 ) 2 + C 2 Si(C 3 ) 3 ), 0.05 (s, 4.5 Si(C 3 ) 3 ), 0.03 (s, 4.5 Si(C 3 ) 3 ); 13 C MR (CDCl 3, 125 Mz) δ [C 2 C 2 ], [C 2 C 2 ], 153.4[(C)], [(C)], 81.1 [C(C 3 ) 3 ], 81.0 [C(C 3 ) 3 ], 64.8 [αc], 64.7 [αc], 64.0 [C 2 C 2 ], 58.0 [βc], 56.6 [βc], 48.3 [SC 2 ], 47.7 [SC 2 ], 33.0 [ɣc], 32.6 [ɣc], 28.3 [C(C 3 ) 3 ], 21.0 [C(C 3 ) 2 ], 20.8 [C(C 3 ) 2 ], 19.2 [C 2 Si(C 3 ) 3 ], 17.4[C(C 3 ) 2 ], 17.3 [C(C 3 ) 2 ], [Si(C 3 ) 3 ] as a mixture of rotomers; IR (film) υ max 2958, 1748, 1705, 1389, 1367, 1250, 1157, 836 cm 1 ; MS (ESI- MS) calcd for C SSi [M+] + m/z 376.2, found Boc- ThzSLeu-. To a dry round- bottom flask containing a magnetic stir bar and Boc- ThzSLeu- TMSE (102.6 mg, mmol, 1.0 equiv) was added dry tetrahydrofuran (6 ml), and the flask was cooled in a 0 C ice bath with stirring. A 1.0 M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.683 ml, mmol, 2.5 equiv) was added dropwise over 30 seconds. The reaction was observed as complete by TLC (4:1 hexanes:ethyl acetate eluent) in 15 minutes. The solution was quenched by acidification with a 2.0 M solution of hydrogen chloride (0.683 ml, 1.37 mmol, 5 equiv), then diluted

7 with water (30 ml), and extracted with dichloromethane (20 ml, twice). The combined organic solutions were dried over magnesium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (gradient 9:1:0.05 to 5:5:0.05 hexanes:ethyl acetate:acetic acid eluent) to yield Boc- ThzSLeu- (55.6 mg, 74%).!!!" : 14.1 (c 2.06, C 2 Cl 2 ); 1 MR (CDCl 3, 600 Mz) δ (brs, 1h), 4.81 (s, 0.5), 4.65 (d, J = 8.4 z, 0.5), 4.60 (s, 0.5), 4.57 (d, J = 8.2 z, 0.5), 4.44 (d, J = 8.4 z, 0.5), 4.38 (d, J = 8.2 z, 0.5), 3.51 (t, J = 5.6 z, 1), 1.93 (q, J = 6.3 z, 1), 1.47 (s, 4.5), 1.45 (s, 4.5), 1.03 (d, J = 6.5 z, 3), 1.00 (d, J = 6.5 z, 3); 13 C MR (CDCl 3, 150 Mz) δ 177.0, 176.2, 153.7, 153.2, 81.6, 64.4, 57.8, 56.4, 48.3, 47.8, 32.9, 32.6, 28.3, 28.2, 20.9, 20.8, 19.2, 19.2; IR (film) υ max 3097, 2970, 2933, 1702, 1675, 1389, 1367, 1156 cm 1 ; MS (ESI- MS) calcd for C S [M+a] + m/z 276.1, found Boc- Arg(Mtr)- SPh. A stirring solution of Boc- Arg(Mtr)- (0.881 g, 1.8 mmol, 1.0 equiv) in dry dichloromethane (10 ml) was cooled in a 0 C ice bath. To this solution was added - (3- dimethylaminopropyl)- ʹ - ethylcarbodiimide (EDC, 320 μl, 1.8 mmol, 1.0 equiv), followed immediately by 1- hydroxy- 7- azabenzotriazole (At, 246 mg, 1.8 mmol, 1.0 equiv). This solution was allowed to stir at 0 C for 5 minutes, before the addition of thiophenol (925 μl, 9.0 mmol, 5 equiv). This solution was allowed to warm to room temperature and stirred for 24 hours. The reaction was then concentrated in vacuo, and the crude viscous oil purified using silica gel chromatography (4:1 hexane:ethyl acetate eluent) to afford Boc- Arg(Mtr)- SPh thioester (96.6 mg, 9%).!!!" : 18.1 (c 4.86, C 2 Cl 2 ); 1 MR (CDCl 3, 500 Mz) δ (m, 5), 6.50 (s, 1), 6.30 (brs, 1), 5.53 (d, J = 8.2 z, 1), 4.29 (brs, 1), 3.77 (s, 3), 3.15 (brs, 2), 2.67 (s, 3), 2.60 (s, 3), 2.10 (s, 3), 1.81 (brs, 1), (m, 2h), (m, 1), 1.45 (s, 9); 13 C MR (CDCl 3, 125 Mz) δ 200.1, 158.5, 156.4, 155.7, 138.5, 136.5, 134.7, 133.4, 129.4, 129.2, 127.3, 124.9, 111.8, 80.5, 77.4, 55.4, 40.7, 29.7, 28.4, 25.5, 24.3, 18.5, 12.0; IR (film) υ max 3334, 1699, 1546, 1247, 1158, 1104, 1020, 805 cm 1 ; MS (ESI- MS) calcd for C S 2 [M+] + m/z 579.2, found 578.9; calcd for C S 2 a [M+a] + m/z 601.2, found Arg(Mtr)- SPh Cl. To a round- bottom flask containing a magnetic stir bar and Boc- Arg(Mtr)- SPh (96.6 mg, mmol, 1.0 equiv) was added dry dichloromethane (3 ml). To this solution was added 4.0 M hydrogen chloride in dioxane (1 ml, 4.0 mmol, 24.0 equiv) in one portion. The solution was stirred vigorously, monitored by TLC (4:1 hexanes:ethyl acetate eluent), and was observed to be complete in 90 minutes. The reaction was concentrated in vacuo to form a viscous oil, which was lyophilized from

8 water (5 ml) and used without further purification, providing Arg(Mtr)- SPh Cl as a white powder (92.0 mg, quantitative).!!!" : (c 1.26, C 2 Cl 2 ); 1 MR (CD 3 D, 600 Mz) δ (m, 5), 6.68 (brs, 1), 4.34 (brs, 1), 3.72 (brs, 3), 3.29 (brs, 2), 2.60 (s, 3), 2.51 (s, 3), 2.00 (brs, 4), 1.91 (brs, 1), 1.65 (brs, 2); 13 C MR (CDCl 3, 150 Mz) δ 195.9, 162.2, 154.3, 141.4, 140.8, 136.0, 131.5, 130.8, 128.8, 127.1, 126.5, 113.8, 68.2, 59.8, 56.4, 42.6, 29.6, 24.8, 24.5, 18.6, 12.3; IR (film) υ max 2931, 1696, 1276, 1176, 1151, 1119, 1017, 843 cm 1 ; MS (ESI- MS) calcd for C S 2 [M+] + m/z 479.2, found Boc- Glu(DMB)-. To a dry round- bottom containing a magnetic stir bar and covered with aluminum foil was added crude Boc- glutamic anhydride (400 mg, 1.4 mmol, 1.0 equiv) and dry dichloromethane (14 ml), which was then cooled in a 0 C ice bath. To this solution was added 4- dimethylaminopyridine (85.3 mg, 0.70 mmol, 0.5 equiv) and 4,5- dimethoxy- 2- nitrobenzyl alcohol (446 mg, 2.1 mmol, 1.5 equiv). Reaction progress was monitored by UPLC (C18 column, 10% to 95% gradient) and TLC (9:1 C 2 Cl 2 : C 3 eluent). When complete, the reaction was concentrated in vacuo and purified using silica gel chromatography (gradient 1:1 hexanes:ethyl acetate to 100% ethyl acetate) to yield Boc- Glu(DMB)- (409 mg, 66%).!!!" : +3.6 (c 1.12, C 2 Cl 2 ); 1 MR (CDCl 3, 500 Mz) δ 10.0 (brs, 1), 7.68 (s, 1), 6.99 (s, 1), 5.48 (d, J = 2.4 z, 2), 5.24 (d, J = 7.7 z, 1), 4.35 (brs, 0.6), 4.25 (brs, 0.4), 3.97 (s, 3), 3.93 (s, 3), (m, 2), (m, 1), (m, 1), 1.4 (s, 9); 13 C MR (CDCl 3, 125 Mz) δ 176.1, 172.3, 155.7, 153.5, 148.3, 139.9, 126.8, 110.5, 108.2, 80.5, 63.5, 56.5, 56.4, 52.8, 30.3, 28.2, 27.5; IR (film) υ max 3388, 2936, 1733, 1689, 1522, 1277, 1165, 1066 cm 1 ; MS (ESI- MS) calcd for C [M+] + m/z 443.2, found Boc- Glu(DMB)- SC 2 C 2 C 2 Et. A stirring solution of Boc- Glu(DMB)- (409 mg, 0.92 mmol, 1.0 equiv) in dry dichloromethane (10 ml) was cooled in a 0 C ice bath. To this solution was added - (3- dimethylaminopropyl)- ʹ - ethylcarbodiimide (EDC, 164 μl, 0.92 mmol, 1.0 equiv), followed immediately by 1- hydroxy- 7- azabenzotriazole (At, 126 mg, 0.92 mmol, 1.0 equiv). This solution was allowed to stir at 0 C for 5 minutes, before the addition of ethyl 3- mercaptopropionate (372 μl, 2.77 mmol, 3 equiv). This solution was allowed to warm to room temperature and stirred for 24 hours. The reaction was then concentrated in vacuo and the residue purified using silica gel chromatography (gradient 4:1 hexane:ethyl acetate to 100% ethyl acetate eluent) to afford Boc- Glu(DMB)- SC 2 C 2 C 2 Et thioester (286 mg, 55%).

9 !!!" : (c 0.93, C 2 Cl 2 ); 1 MR (CDCl 3, 500 Mz) δ 7.71 (s, 1h), 7.00 (s, 1), 5.53 (d, J = 15 z, 1), 5.49 (d, J = 15 z, 1), 5.13 (d, J = 8.5 z, 1), 4.38 (brs, 1), 4.14 (q, J = 7.1 z, 2), 3.99 (s, 3), 3.95 (s, 3), 3.11 (t, J = 7.0 z, 1), 2.59 (t, J = 7.0 z, 2), 2.53 (q, J = 7.1 z, 2), 2.27 (brs, 1), (m, 1), 1.42 (s, 9), 1.24 (t, J = 7.1 z, 3); 13 C MR (CDCl 3, 125 Mz) δ 200.6, 172.1, 171.5, 155.2, 153.5, 148.3, 140.0, 126.7, 110.6, 108.3, 80.6, 63.6, 60.9, 59.9, 56.5, 56.4, 34.1, 30.3, 28.3, 27.7, 23.9, 14.1; IR (film) υ max 2961, 1693, 1522, 1366, 1330, 1278, 1160, 1066 cm 1 ; MS (ESI- MS) calcd for C Sa [M+a] + m/z 581.2, found Glu(DMB)- SC 2 C 2 C 2 Et Cl. To a round- bottom flask containing a magnetic stir bar and Boc- Glu(DMB)- SC 2 C 2 C 2 Et (286 g, mmol, 1.0 equiv) was added 4.0 M hydrogen chloride in dioxane (5.0 ml, 20. mmol, 39 equiv) in one portion. The solution was stirred vigorously, monitored by TLC (9:1 C 2 Cl 2 :C 3 eluent), and was observed to be complete in 1 hour. The reaction was concentrated in vacuo and used without further purification, providing Glu(DMB)- SC2C2C2Et Cl as a slightly yellow solid (253 mg, quantitative).!!!" : (c 0.35, C 2 Cl 2 ); 1 MR (CD 3 D, 500 Mz) δ 7.63 (s, 1), 7.08 (s, 1), 5.41 (d, J = 13.9, 1), 5.37 (d, J = 13.9, 1), 4.28 (t, J = 6.4 z, 1), 4.05 (q, J = 7.1 z, 2), 3.90 (s, 3), 3.84 (s, 3), 3.18 (octet, J = 7.0 z, 2), (m, 4), 2.24 (dq, J = 13.9, 7.1 z, 1), 2.14 (dq, J = 14.6, 7.1 z, 1), 1.16 (t, J = 7.2 z, 3); 13 C MR (CD 3 D, 125 Mz) δ 197.3, 172.9, 172.9, 155.1, 150.1, 141.7, 127.3, 112.9, 109.5, 64.9, 62.0, 59.5, 57.1, 56.9, 34.7, 30.0, 27.8, 25.5, 14.5; IR (film) υ max 2937, 1732, 1684, 1523, 1276, 1222, 1176, 1067 cm 1 ; MS (ESI- MS) calcd for C S [M+] + m/z 459.1, found

10 Preparation of peptide fragments Ser1 Leu22 SMQEEDTFRELRIFLRVTRL SPh 2 S S Boc tbu SMe Trt tbu tbu tbu tbu tbu Trt 2 tbu Trt S S 1) EDC, Bt, Leu-SPh Cl 49:1 DCM:TFE, 0 C 2) 90:5:3:2 TFA:thioanisole:EDT:anisole SMe 2 Chemical Formula: C S SPh Fragment 2 was synthesized following the general SPPS protocol outlined above on Fmoc- Arg(Pbf)- ovasyn TGT resin, terminating in the coupling of Boc- Ser(tBu)-. The protected peptide was cleaved from the resin following the general protocol described above. The protected peptidyl acid was coupled with Leu- SPh Cl following the general protocol for thioester installation and then deprotected as described. Purification of the crude peptide was done on a 250 x 21.4 mm Microsorb C8 column with a gradient of 35 50% over 30 minutes; the desired product eluted at 17 minutes and was isolated in 24% yield. LC- MS and ESI- MS analysis of pure isolated fragment 2: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (m/z) for C S 2 = , observed as by average of charged ions.

11 Fragment 3 was synthesized following the general SPPS protocol outlined above on Fmoc- Lys(Boc)- ovasyn TGT resin, terminating in the coupling of Boc- Cys(Trt)-. The protected peptide was cleaved from the resin following the general protocol described above. The protected peptidyl acid was coupled with Tyr- SC 2 C 2 C 2 Et Cl following the general protocol for thioester installation and then deprotected as described. Purification of the crude peptide was done on a 250 x 21.4 mm Microsorb C4 column with a gradient of 25 40% over 30 minutes; the desired product eluted at 21 minutes and was isolated in 35% yield. LC- MS and ESI- MS analysis of pure isolated fragment 3: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (m/z) for C S 3 = , observed as by average of charged ions.

12 Fragment 11 was synthesized following the general SPPS protocol outlined above on Fmoc- Asp(tBu)- ovasyn TGT resin, terminating in the coupling of Boc- ThzSLeu-. The protected peptide was cleaved from the resin following the general protocol described above. The protected peptidyl acid was coupled with Arg(Mtr)- SPh Cl following the general protocol for thioester installation and then deprotected as described. Purification of the crude peptide was done on a 250 x 21.4 mm Microsorb C8 column with a gradient of 25 40% over 30 minutes; the desired product eluted at 27 minutes and was isolated in 29% yield. LC- MS and ESI- MS analysis of pure isolated fragment 11: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (m/z) for C S 3 = , observed as by average of charged ions.

13 Fragment 12 was synthesized following the general SPPS protocol outlined above on Fmoc- Glu(tBu)- ovasyn TGT resin, terminating in the coupling of Boc- MeSSLeu-. The protected peptide was cleaved from the resin following the general protocol described above. The protected peptidyl acid was coupled with Glu(DMB)- SC 2 C 2 C 2 Et Cl following the general protocol for thioester installation and then deprotected as described. Purification of the crude peptide was done on a 250 x 21.4 mm Microsorb C8 column with a gradient of 25 50% over 30 minutes; the desired product eluted at 19 minutes and was isolated in 21% yield. LC- MS and ESI- MS analysis of pure isolated fragment 12: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (m/z) for C S 4 = , observed as by average of charged ions.

14 Fragment 13 was synthesized following the general SPPS protocol outlined above on Fmoc- Arg(Pbf)- ovasyn TGT resin, terminating in the coupling of Boc- MeSSLeu-. To the protected peptide on resin was added 90:5:3:2 TFA:thioanisole:1,2- ethanedithiol:anisole cleavage cocktail (v/v), at a volume of 1 ml per 20 mg of protected peptide. This solution was allowed to stir vigorously for 90 minutes, upon which it was filtered through a 20- μm polypropylene frit, transferred to a 50- ml polypropylene conical tube, and concentrated down to a viscous oil under a stream of nitrogen. The crude, deprotected peptide thioester was precipitated with the addition of 40 ml of cold diethyl ether and centrifuged to a pellet and the ether layer was decanted. The crude peptide thioester was dissolved with 1:1 2 :AcC with 0.05% TFA, lyophilized, redissolved with 1:1 2 :AcC with 0.05% TFA, filtered through a μm PTFE filter, and lyophilized to provide crude peptide for PLC purification. LC- MS and ESI- MS analysis of pure isolated fragment 13: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (m/z) for C S 3 = , observed as by average of charged ions.

15 Assembly of ATAD2 Bromodomain Peptide fragments 2 (17.63 mg, 6.11 μmol, 1.5 equiv) and 3 (21.00 mg, 4.08 μmol, 1.0 equiv) were massed into a 2- ml Eppendorf tube containing a magnetic stir bar. The atmosphere was flushed with argon, and 815 μl of freshly prepared kinetic ligation buffer added to the Eppendorf tube to result in a 5 mm concentration reaction. The atmosphere flushed with argon before capping. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed as complete at 2 hours. To the reaction mixture was added 8 μl of ethyl 3- mercaptopropionate to cleave the adduct 5 to 4 (see manuscript text). After an additional hour of stirring, the reaction was quenched with the addition of 1 ml of 20% acetonitrile in water with 0.05% TFA (v/v), and purified by PLC (250 x 21.4 mm Microsorb C8 column, 15 60% gradient, eluted at 22 min) to result in pure fragment 4 (17.64 mg, 55%). LC- MS and ESI- MS analysis of pure isolated fragment 4: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (average of isotopes) for C S 4 = , observed as by average of charged ions.

16 Peptide fragments 11 (5.87 mg, 1.64 μmol, 1.2 equiv) and 12 (3.76 mg, 1.36 μmol, 1.0 equiv) were massed into a 2- ml Eppendorf tube containing a magnetic stir bar. The atmosphere was flushed with argon, and 273 μl of freshly prepared kinetic ligation buffer added to the Eppendorf tube to result in a 5 mm concentration reaction. The atmosphere flushed with argon before capping. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed complete at hours. Upon completion, fragment 13 (4.36 mg, 2.05 μl, 1.5 equiv) was massed into a new Eppendorf tube, the atmosphere was argon flushed, and 273 μl of freshly prepared native chemical ligation buffer with MPAA was added. The solution of 13 was vortexed and centrifuged, before transferring 13 to the kinetic ligation reaction via pipette to result in a 2.5 mm concentration ligation. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed as complete in 4 6 hours. Upon completion, the reaction was quenched with the addition of 1 ml of 20% acetonitrile in water with 0.05% TFA (v/v) and purified by PLC (250 x 21.4 mm Microsorb C8 column, 35 50% gradient, eluted at 22 min) to result in pure fragment 14 (3.51 mg, 33%). LC- MS and ESI- MS analysis of pure isolated fragment 14: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (average of isotopes) for C S 4 = , observed as by average of charged ions.

17 Peptide fragment 14 (8.0 mg, 0.98 μmol, 1.0 equiv) was lyophilized into a 20- ml glass vial. A magnetic stir bar was added, and the peptide was solubilized in argon degassed 1:1 water:acetonitrile with 0.05% TFA (v/v) (3.9 ml, to yield 0.25M concentration). The vial was flushed with argon. A Spectroline EF- 240C 4W UV lamp with longwave 365- nm bulb was held 2 5 cm from the glass, and the system was enclosed loosely in aluminum foil. The glass vial was irradiated with stirring. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed as complete at 4 6 hours. Upon completion of the deprotection, the solution was lyophilized directly. Crude UPLC traces for formation of fragment SI- 1: BE300 C4 1.7μm 2.1 x 100 mm column at a flow rate of 0.3 ml/min, 20 50% gradient. 1 hour hours +8 Calculated mass (average of isotopes) for C S 4 = , observed as by average of charged ions.

18 Crude peptide fragment SI- 1 (8.00, 0.98 μmol, 1.0 equiv) was carried forward from the previous reaction and lyophilized into a 2- ml Eppendorf tube. A magnetic stir bar was added, the atmosphere was flushed with argon, 980 μl of freshly prepared thiazolidine deprotection buffer was added to the Eppendorf tube to result in a 1 mm concentration reaction, and the atmosphere was flushed with argon again before capping. The Eppendorf tube was submerged halfway into a 37 C water bath. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed as complete at 4 hours. Upon completion, the reaction was quenched with the addition of 1 ml of water with 0.05% TFA (v/v) and desalted using a Waters Sep- Pak tc2 Plus Short Cartridge (400 mg Sorbent per Cartridge, µm particle size). The desalting procedure considered of running a gradient of 1 ml each of the following solutions of acetonitrile in water with 0.05% TFA (v/v): 0%, 8%, 16%, 24%, 32%, 40%, 48%, 56%, 64%, 72%, 80%, 88%. The crude, desalted peptide eluted with 48 56% acetonitrile in water. The crude peptide was lyophilized and carried forward without further purification. Crude UPLC traces for formation of fragment SI- 2: BE300 C4 1.7μm 2.1 x 100 mm column at a flow rate of 0.3 ml/min, 20 50% gradient. 1 hour hours +8 6 hours Calculated mass (average of isotopes) for C S 4 = , observed as by average of charged ions.

19 Crude peptide fragments SI- 2 (2.08 mg, μmol, 1.0 equiv) and 4 (2.07 mg, μmol, 1.0 equiv) were lyophilized into a 2- ml Eppendorf tube containing a magnetic stir bar. The atmosphere was flushed with argon, μl of freshly prepared ligation buffer added to the Eppendorf tube to result in a 2.0 mm concentration reaction, and the atmosphere was flushed with argon before capping. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient) and was observed as complete at 6 hours. Freshly prepared dethiylation buffer was added (1.0 ml), transferred to an Amicon Ultra- 4 Centrifugal Filter Unit with a 10k MW cutoff, the atmosphere flushed with argon, and centrifuged to a volume of 200 μl. The buffer exchange centrifugation was repeated two additional times with dethiylation buffer (1.0 ml), and the remaining solution was transferred with additional dethiylation buffer to an Eppendorf tube with a magnetic stir bar to a total solution volume of 1 ml. To the solution was added tert- butyl thiol (30 μl), 0.10 M VA- 044 (30 μl), and 0.50 M TCEP p 7.0 (Bond- Breaker, 250 μl). The resulting solution was allowed to stir in a 37 C water bath. The reaction progress was monitored by UPLC every 12 hours (C4 column, 20 50% gradient), and additional 0.5M TCEP p 7.0 solution (20 μl) and 0.10M VA- 044 (10 μl) were added as necessary. The reaction was typically complete in hours. Upon completion, the reaction was quenched with the addition of 1 ml of water with 0.05% TFA (v/v) and desalted using a Waters Sep- Pak tc2 Plus Short Cartridge (400 mg Sorbent per Cartridge, µm particle size). The desalting procedure considered of running a gradient of 1 ml each of the following solutions of acetonitrile in water with 0.05% TFA (v/v): 0%, 8%, 16%, 24%, 32%, 40%, 48%, 56%, 64%, 72%, 80%, 88%. The crude, desalted peptide eluted with 48 56% acetonitrile in water. The crude peptide was lyophilized and carried forward without further purification.

20 Crude UPLC traces for formation of fragment 15: BE300 C4 1.7μm 2.1 x 100 mm column at a flow rate of 0.3 ml/min, 20 50% gradient Calculated mass (average of isotopes) for C S 9 = , observed as by average of charged ions. Crude UPLC traces for dethiylation to form fragment SI- 3: BE300 C4 1.7μm 2.1 x 100 mm column at a flow rate of 0.3 ml/min, 20 50% gradient Calculated mass (average of isotopes) for C S 5 = , observed as by average of charged ions.

21 Crude peptide fragment SI- 3 (0.13 μmol, 1.0 equiv) was lyophilized into a 2- ml Eppendorf tube containing a magnetic stir bar. The atmosphere was flushed with argon, and 0.26 μl of freshly argon degassed 7:3 acetic acid:water (v/v) was added to the Eppendorf tube to result in a 0.50 mm concentration reaction. The atmosphere was flushed with argon before capping. Silver acetate (6.5 mg, 39 μmol, 300 equiv) was added slowly to the rapidly stirring solution over 1 minute. Dithiothreitol (DTT) buffer was prepared (1.146 g guanidine hydrochloride, 56.6 mg disodium phosphate, and 41.6 mg DTT) and freshly degassed. The reaction progress was monitored by UPLC (C4 column, 20 50% gradient; 5 μl reaction mixed with 25 μl DTT buffer, centrifuged and decanted) and was observed as complete at 2 hours. An Eppendorf tube was prepared with a magnetic stirbar and DTT buffer (0.52 ml). The reaction mixture was added in 10- μl aliquots by pipette to the DTT buffer with rapid stirring; a fine white precipitate appeared. The cloudy solution was centrifuged, and the supernatant was decanted. DTT buffer (0.26 ml) was added to the pellet for an additional extraction and stirred rapidly for 30 minutes before centrifuging and decanting the supernatant. The combined supernatant solutions were purified by PLC (250 x 21.4 mm Microsorb C4 column, 35 48% gradient, eluted at 22 min) to result in pure bromodomain 1 (0.46 mg, 23%). LC- MS and ESI- MS analysis of pure isolated fragment 1: Microsorb 300-5, C4 250 x 2.0 mm column at a flow rate of 0.2 ml/min, 15 60% gradient for 30 minutes Calculated mass (average of isotopes) for C S 5 = , observed as by average of charged ions.

22 1 and 13 C MR spectra for new compounds S Boc Boc- Leu- SPh

23 S Cl 3 Leu- SPh Cl

24 tbu S Boc C 2 Et Boc- Tyr(tBu)- SC 2 C 2 C 2 Et

25 S Cl 3 C 2 Et Tyr- SC 2 C 2 C 2 Et Cl

26 S ThzSLeu- TMSE TMS

27 S Boc Boc- ThzSLeu- TMSE TMS

28 S Boc C 2 Boc- ThzSLeu-

29 S Me S Boc Boc- Arg(Mtr)- SPh

30 S Me S Cl 3 Arg(Mtr)- SPh Cl

31 2 Me Me Boc C 2 Boc- Glu(DMB)-

32 2 Me Me S Boc C 2 Et Boc- Glu(DMB)- SC 2 C 2 C 2 Et

33 2 Me Me S Cl 3 C 2 Et Glu(DMB)- SC 2 C 2 C 2 Et Cl