One-pot Native Chemical Ligation of Peptide Hydrazides Enables Total Synthesis of Modified Histones. Supporting Information

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1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2014 One-pot Native Chemical Ligation of Peptide Hydrazides Enables Total Synthesis of Modified Histones Jiabin Li, a Yuanyuan Li, a Qiaoqiao He, a,b Yiming Li, a,b* Haitao Li a and Lei Liu a* a Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry; MOE Key Laboratory of Protein Sciences, Center for Structural Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing , China, lliu@mail.tsinghua.edu.cn b School of Medical Engineering, Hefei University of Technology, Hefei, Anhui , China, lym2007@mail.ustc.edu.cn These authors contributed equally to this work Supporting Information Table of Contents 1. General Information...S Materials 1.2 General procedures for SPPS 1.3 HPLC 1.4 Mass spectrometry 1.5 Solutions used in the ligation reaction 2. Experimental Section S Preparation of hydrazine resin 2.2 Synthesis of H3K4me3 by using N-to-C sequential ligations 2.3 Tests for the use of Dobz-peptides 2.4 Synthesis of H3K4me3 by using H 2 O 2 -controlled one-pot strategy 2.5 Synthesis of H4K16ac by using H 2 O 2 -controlled one-pot strategy 2.6 Synthesis of H3K36me3, H4K12ac, H4K5acK8acK12acK16ac 2.7 Histones octamer reconstitution 2.8 Isotope envelope of H3K4me3 and H4k16ac on different charge states S1

2 1. General Information...S Materials The resins, amino acid derivatives, coupling reagents and cleavage reagents were purchased from GL Biochem (Shanghai) Ltd. Nova-PEG Wang Resin was purchased from Novabiochem. DMF, DCM, Et 2 O, MeOH, THF, Ac 2 O, NH 2 NH 2 (85%) and other common reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. 4-(4,4,5,5 Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl methanol and 4-nitrophenyl chloroformate were purchased from Alfa Aesar. Tris(2-carboxyethyl)phosphine (TCEP), DL-dithiothreitol (DTT) and 4-mercapto-benzeneacetic acid (MPAA) were purchased from Sigma-Aldrich. All reagents and solvents were purified when necessary General procedure of Fmoc SPPS The desired resin was swelled in DMF/DCM = 1:1 for about 2 h. The SPPS procedure was performed on an automated peptide synthesizer (C.S. Bio, Co. CS136XT). The amino acid residues were coupled by 4 equiv of protected amino acid (AA), 8 equiv of DIEA and 3.8 equiv of HCTU to the initial loading of resin. For peptide hydrazides, the first amino acid was double coupled for 1h by 4 equiv of AA, 16 equiv of DIEA and 3.8 equiv of HCTU. The coupling was performed once (60 min) or twice (10 min + 60 min) and the Fmoc was deprotected with 20% piperidine/dmf (5 min + 10 min). Then, the finished peptide was cleaved from the resin with a mixture of 87.5% TFA, 5% water, 5% phenol and 2.5% TIPS. After 2.5 h, the resin was removed by filtration and washed with neat TFA. The combined solution was concentrated under N 2 flowing. Then, the crude peptide was obtained by precipitating in cold Et 2 O and centrifugation for three times. Finally, the crude peptide was dissolved in H 2 O/CH 3 CN and purified by preparative HPLC HPLC Analytical HPLC was run on a SHIMADZU (Prominence LC-20AT) instrument using an analytical column (Grace Vydac Protein C18, mm or Grace Vydac Protein C4, mm, 5 um particle size, flow rate 1.0 ml/min, rt). Semi-preparative HPLC was run on a SHIMADZU (Prominence LC-20AT) instrument using a semi-preparative column (Grace Vydac Protein C18, mm, 10 um particle size, flow rate 8.0 ml/min, rt) used for purification of crude peptides or a semi-preparative column (Grace Vydac Protein C18, mm, 10 um particle size, flow rate 4.0 ml/min, rt). Solvent A was 0.1 % TFA in acetonitrile and solvent B was 0.1 % TFA in water Mass spectrometry and NMR spectrometry Peptides were confirmed by ESI-MS (Agilent 6210 Time of Flight Mass Spectrometer), MALDI-TOF (ABI 4800 plus MALDI-TOF-TOF mass spectrometer), MS-MS analysis (Thermo Scientific Q Exactive LC-MS/MS) and isotope envelope (Waters Xevo G2). 1 H and 13 C NMR spectra of small molecular were recorded on an S2

3 Oxford 400 MHz spectrometer in CDCl Solutions used in ligation reaction Ligation buffer: 6.0 M Gn HCl, 0.2 M Na 2 HPO 4 in water; Acidic ligation buffer: ph = 3.0; NaNO 2 solution: 0.2 mm in neat water; MPAA solution: 0.2 mm in ligation buffer; TCEP solution: 57 mg TCEP HCl in 1mL of ligation buffer (ph = 6.5). 2. Experimental Section... S Preparation of hydrazine resin Scheme S1. Preparation of hydrazine resin 2 g 2-Chlorotrityl chloride resin was swelled in DMF (12.5mL) for 15min and cooled to 0 o C. A mixture of DIEA (1.334 ml) and hydrazine hydrate (0.4 g, 85%) in DMF (4 ml) was added slowly. Then, the suspension was stirred at room temperature. After 2 h, 0.8 ml MeOH was added and stirred for additional 15 min. Finally, the resin was washed with DMF, H 2 O, DMF, MeOH, Et 2 O and dried in vacuo for 2 h. 2.2 Synthesis of H3K4me3 by using N-to-C sequential ligations S3

4 Scheme S2. N-to-C sequential strategy for the synthesis of H3K4me3 Histone H3 contains 135 amino acid residues. There is only one Cys at the site 110, which is not suitable for the ligation. The sequence was divided into three segments and desulfurization would be performed later. Thus we chose Val 45 -Ala 46 and Gly 90 -Ala 91 as the ligation sites. In order to obtain the native sequence, Cys 110 was protected by Acm which would be removed after desulfurization. For the three segments, an N-to-C sequential strategy was performed. First, 2 was ligated to 1 and generated the 4. Next the ligation between 4 and 3 was performed. Then desulfurization and Acm removal were carried out, generating the native H3K4me3. Synthesis of H3K4me3[Ala 1 -Val 46 ]-NHNH 2 (1): The synthesis was performed on the automated peptide synthesizer using hydrazine resin (400 mg, 0.25 mmol). The first amino acid was double coupled for 1 h by 4 equiv of AA, 16 equiv of DIEA and 3.8 equiv of HCTU. The Fmoc-Lys(me3)-OH was coupled manually by 2 equiv of AA, 4 equiv of DIEA, 2 equiv of HOAt and 1.9 equiv of HATU to the initial loading of resin and checked by ninhydrin test. After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of 5-27 % solvent A over 38 min. Yield: 30.0 %. Figure S1. A): HPLC analysis (λ = 214 nm) of the crude and purified 1. Gradient: 5-30 % solvent A over 38 min. B): ESI-MS analysis of the product. ESI-MS: Observed ± 0.7 Da, Calcd Da. (average isotopes). Synthesis of H3K4me3[Cys 47 -Gly 90 ]-NHNH 2 (2): The synthesis was performed on the automated peptide synthesizer using hydrazine resin (400 mg, 0.25 mmol). All the amino acids were doubly coupled. After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of % solvent A over 38 min. Yield: 14.5 %. S4

5 Figure S2. A): HPLC analysis (λ = 214 nm) of the crude and purified 2. Gradient: 20-55% solvent A over 38 min. B): ESI-MS analysis of the product. ESI-MS: Observed ± 0.6 Da, Calcd Da. (average isotopes). Synthesis of H3K4m3[Cys 91 -Ala 135 ] (3): The synthesis was performed on the automated peptide synthesizer using Nova-PEG Wang Resin. The first coupling (4 equiv of amino acid, 16 equiv of DIEA, 4 equiv of HOBt, 0.1 equiv of DMAP and 3.8 equiv of HBTU) was performed overnight. Then the resin was capped with reagent (Acetic anhydride: DIEA: DMF = 1 : 1 : 8 ). After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of % solvent A over 38min. Yield: 12.5 %. Figure S3. A): HPLC analysis (λ = 214 nm) of the crude and purified 3. Gradient: % solvent A over 38 min. B): ESI-MS of the product. ESI-MS: Observed ± 0.3 Da, Calcd Da. (average isotopes). Ligation of 1 with 2: H3K4me3[Ala 1 -Val 46 ]-NHNH 2 (1) (23 mg, 4.6 µmol, 1.5 equiv to 2) was dissolved in 1.0 ml of acidic ligation buffer and cooled to -12 o C. Then 150 µl of NaNO 2 solution was added. After stirring at -12 o C for 20 min, 1.5 ml of MPAA solution was added. Next, the ph of the mixture was slowly adjusted to S5

6 6.8 with NaOH (2 M), and H3K4me3[Cys 47 -Gly 90 ]-NHNH 2 (2) (17 mg, 3.2 µmol) was added. The ligation reaction was stirred at room temperature and monitored by analytical HPLC using a gradient of 5-55 % solvent A over 38 min. Due to the ligation site was Val and the HPLC retention time of ligation product was the same as 2. The reaction was conducted for hours. After completion, the ligation solution was reduced by TCEP solution and isolated by HPLC. The isolation yield was 41% approximately and afforded 13.4 mg (1.3 µmol) ligation product H3K4me3[Ala 1 - Cys 47 -Gly 90 ]-NHNH 2 (4). Figure S4. A): analytical HPLC traces (λ = 214 nm) of ligation between 1 and 2 at 0 min (a), 24 h (b) and purified product (c); B): ESI-MS analysis of the ligation product. Observed ± 1.4 Da, Calcd Da. (average isotopes). Note: the retention times of H3K4me3[Cys 47 -Gly 90 ]-NHNH 2 and H3K4me3[Ala 1 -Cys 47 -Gly 90 ] -NHNH 2 were almost identical in the reverse-phase HPLC. The purification of the ligation product was very difficult and microheterogeneity was shown clearly in mass spectra. Ligation of 4 with 3: H3K4me3[Ala 1 -Cys 47 -Gly 90 ]-NHNH 2 (4) (13.4 mg, 1.3 µmol) was dissolved in 0.5 ml of acidic ligation buffer. 42 µl of NaNO 2 solution was added under -12 o C and kept for 20 min. Then, the mixture was treated by 420 µl of MPAA solution and its ph was slowly adjusted to 6.5 with 2.0 M NaOH solution. At the same time, H3K4me3[Cys 91 -Ala 135 ] (3) (13.4 mg, 2.6 µmol) was added. The reaction was monitored by analytical HPLC using a gradient of % solvent A over 38 min. The ligation product was isolated by semi-preparative HPLC after reduction by TCEP solution. The isolation yield was 33% approximately and afforded 6.7 mg (0.44 µmol) product H3K4me3[Ala 1 -Cys 47 -Cys 91 -Ala 135 ]. S6

7 Figure S5. A): analytical HPLC traces (λ = 214 nm) of ligation between 4 and 3 at 0 min (a), 2 h (b) and purified product (c). B): ESI-MS analysis of the ligation product. Observed ± 2.1 Da, Calcd Da. (average isotopes). Note: Unreacted H3K4me3[Cys 47 -Gly 90 ]-NHNH 2 in H3K4me3[Ala 1 - Cys 47 -Gly 90 ]-NHNH 2 contributed to the complexity of the second ligation. The mass spectra in Figure S5, S6, S7 show evidence of microheterogeneity. Therefore we had to give up the N-to-C sequential ligations. Desulfurization: 6.7 mg (0.44 µmol) H3K4me3[Ala 1 -Cys 47 -Cys 91 -Ala 135 ] was dissolved in 1 ml solution of 6.0 M Gn HCl, 0.2 M Na 2 HPO 4 and 1.0 M TCEP (ph = 6.9). To the above solution, 60 µl t BuSH and 600 µl VA-044 solution (0.1 M in water) was added. The final ph of the solution was adjusted to 6.9 and kept at 37 o C with stirring overnight. After isolation, H3K4me3[Ala 1 -Ala 47 -Ala 91 -Ala 135 ] was obtained, giving 5.2 mg of product with a yield of 77%. S7

8 Figure S6. Analytical HPLC trace (λ = 214 nm) and ESI-MS analysis of the product after desulfurization. Observed ± 1.4 Da, Calcd Da. (average isotopes). Acm removal: 5.2 mg (0.34 µmol) H3K4me3[Ala 1 -Ala 47 -Ala 91 -Ala 135 ] was dissolved in 1.1 ml solution of 1:1 AcOH/H 2 O. To the above mixture, 2.0 mg AgOAc was added and stirred at room temperature overnight. After completion, 1.1 ml solution of 1.0 M DTT in 6.0 M Gn HCl was added and stirred for another 20 min. After centrifugation, the product was isolated by HPLC, giving 5.0 mg native H3K4me3 with a yield of 85%, and the overall isolated yeild was 10.3%. Figure S7. HPLC (λ = 214 nm) and ESI-MS analysis of the final product H3K4me3. ESI-MS: Observed ± 0.7 Da, Calcd Da. (average isotopes). 2.3 Tests for the use of Dobz-peptides Synthesis of pdobz-cys(trt)-oh S8

9 Scheme S3. The synthesis of pdobz-cys(trt)-oh Compound 1: 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl methanol (1.17 g, 5.0 mmol) was dissolved in dry THF (10.0 ml). Triethylamine (1.40 ml, 10.0 mmol) and 4-nitrophenyl chloroformate (1.12 g, 5.5 mmol) were added. The mixture was stirred at room temperature for 1 h and evaporated to dryness under vacuo. The residue was dissolved in ethyl acetate (100 ml). The mixture was washed with 1 M HCl, saturated sodium carbonate, brine and dried over Na 2 SO 4.The crude product was further purified by column chromatography over silica (ethyl acetate/petroleum ether = 1 : 20) to give compound 1. Yield: 1.64 g (82%). 1 H-NMR (400 MHz, CDCl 3 ): δ 8.27 (d, 2H), 7.85 (d, 2H), 7.44 (d, 2H), 7.38 (d, 2H), 5.31 (s, 2H), 1.35 (s, 12H). 13 C-NMR (400 MHz, CDCl 3 ): δ , , , , , , , , 83.98, 70.76, pdobz-cys(trt)-oh: Compound 1 (1.64 g, 4.1 mmol) was dissolved in dry DMF (83 ml). H 2 N-Cys(Trt)-OH (1.90 g, 5.2 mmol) was added, followed by triethylamine (1.73 ml, 12.4 mmol). The reaction mixture was stirred at room temperature overnight and then evaporated to dryness. The residue was dissolved in ethyl acetate (100 ml). The mixture was washed with water, brine and dried over Na 2 SO 4. The crude product was further purified by column chromatography over silica (ethyl acetate/petroleum = 1: 4). Finally, pdobz-cys(trt)-oh was obtained as light yellow solids (1.75g, 2.8 mmol, 68 %). HRMS, Calcd for C 36 H 38 BNO 6 S [M-H] - : Observed: H-NMR (400 MHz, CDCl 3 ): δ 7.80 (d, 2H), (m, 17H), (m, 3H), (m, 1H), 1.35 (s, 12H); 13 C-NMR (400 MHz, CDCl 3 ): δ , , , , , , , , , 83.93, 67.18, 67.04, 52.84, 29.73, Scheme S4. Synthesis of Dobz-CHMYCRWK-NHNH 2 (5) Synthesis of Dobz-CHMYCRWK-NHNH2 (5): The synthesis was performed manually using hydrazine resin. The final amino acid was coupled by 2 equiv of pdobz-cys(trt)-oh, 2 equiv of HOAt, 1.9 equiv of HATU and 4 equiv of DIEA and checked by ninhydrin test. After cleavage, the crude peptide was purified on semi-preparative column. ESI-MS: Observed ± 0.7 Da, ± 0.7 Da S9

10 (M-18), Calcd Da, Da (M-18) (Average isotopes). Note: M-18 and M-36 peaks corresponded to the loss of one or two water molecules between Dobz and amino acid side chains via internal boronate ester formation. Such internal boronate ester formation was known in other boronic acid-containing systems [1]. In water such internal boronate ester formation reaction should be reversible. Indeed, the dehydration peaks disappeared after deprotection of Dobz in our experiments. Therefore, the dehydration process observed in mass spectra was not harmful to the peptides. Scheme S5. Conversion of 5 to a thioester and its ligation with Cys Ligation of 5 with Cys: Dobz-CHMYCRWK-NHNH 2 (5) (1.6 mg, 1.2 µmol) was dissolved in 0.2 ml of ligation buffer (ph = 3.0) and cooled to -12 o C. 40 µl of NaNO 2 was added and kept for 20 min. Then, the reaction mixture was treated by 400 µl of MPAA solution and Cys (0.3 mg, 2.5 µmol). The ph was adjusted to 6.5 with 2.0 M NaOH. The reaction was monitored by analytical HPLC using a gradient of % solvent A over 30 min. S10

11 Figure S8. Conversion of 5 to a thioester and its ligation with Cys. A): analytical HPLC traces (λ = 214 nm) of 5; B): ligation of 5 with Cys at 0 min. C) ligation of 5 with Cys at 60 min. D): ESI-MS analysis of 5. Observed ± 0.3 Da, ± 0.3 Da (M-18) [1], Calcd Da, Da (M-18) [1]. (average isotopes). E): ESI-MS analysis of 7. Observed ± 0.3 Da (M-18) [1], Calcd (average isotopes). Note: two side products (thiolactone and lactam of Lys at C-terminal, observed: ± 0.1 Da ) were observed. Ligation of 8 with 9: Dobz-CRKSGGA-NHNH 2 (8) (5.0 mg, 5.15 µmol) was dissolved in 0.2 ml of acidic ligation buffer and cooled to -12 o C. 150 µl of NaNO 2 solution was added and kept for 20 min. Then, the ph of reaction mixture was adjusted to 6.5 after 1.5 ml of MPAA solution and H 2 N-CPRQL-OH (9) (3.83 mg, 5.15 µmol) was added. The reaction was monitored by analytical HPLC using a gradient of 0-25 % solvent A over 30 min. S11

12 Figure S9. A) ligation of 8 with 9. B) analytical HPLC traces (λ = 214 nm) of 8 and reaction at 10 min, 3 h. C) ESI-MS analysis of 8: Observed ± 0.2 Da. Cacld Da. 9: Observed ± 0.3 Da. Cacld Da. 10: Observed ± 0.3 Da. Cacld Da. Peaks of M-18 and M-36 were observed [1] (average isotopes). Scheme S6. Dobz removal by H 2 O 2 Dobz removal: Dobz-CHMYCRWK-NHNH 2 (5) (1.6 mg, 1.2 µmol) was dissolved in 0.3 ml solution of ligation buffer (ph = 6.8) and MPAA solution (390 µl, 78 µmol) was added. Then the reaction mixture was treated by H 2 O 2 (1 M in water). After 10 min, the reaction was monitored by analytical HPLC using a gradient of 5-50% solvent A over 30 min. S12

13 Figure S10. A): analytic HPLC traces (λ = 214 nm) of Dobz removal by H 2 O 2 in model peptide. B): ESI-MS analysis of 5. C): ESI-MS analysis of 11. Observed ± 0.2 Da, Calcd Da. D): ESI-MS analysis of 12. Observed ± 0.3 Da, Calcd Da. (average isotopes). 2.4 Synthesis of H3K4me3 by using H 2 O 2 -controlled one-pot strategy S13

14 Scheme S7. Synthesis of H3K4me3 by using H 2 O 2 -controlled one-pot strategy Synthesis of Dobz-H3K4me3[Cys 47 -Gly 90 ]-NHNH 2 (13): The synthesis was performed on hydrazine resin (400 mg, 0.25 mmol) using the same procedure as 2. The last amino acid was coupled by 2 equiv of pdobz-cys(trt)-oh, 2 equiv of HOAt, 1.9 equiv of HATU and 4 equiv of DIEA and checked by ninhydrin test. After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of % solvent A over 38min. Finally, 88.8 mg of pure product was obtained with a yield of 6.5 %. S14

15 Figure S11. A): HPLC analysis (λ = 214 nm) of the crude and purified 13. Gradient: 25-55% solvent A over 38 min. B): ESI-MS analysis of 13. Observed ± 0.5 Da, Calcd Da (average isotopes). Peaks of M-18 and M-36 were observed [1]. C), D), E): the detail of the ESI-MS. Synthesis of H3K4me3 by using one-pot strategy: Dobz-H3K4me3[Cys 47 -Gly 90 ] -NHNH 2 (13) (20.8 mg, 3.8 µmol) was dissolved in 0.4 ml of acidic ligation buffer (6.0 M Gn HCl, 0.2 M Na 2 HPO 4, ph = 3.0). The mixture was treated by NaNO 2 solution (113 µl) at -12 o C and kept for 20 min ml of MPAA solution was added, followed by H3K4me3[Cys 91 -Ala 135 ] (3) (19.5mg, 3.8 µmol) and its ph was slowly adjusted to 6.5 with 2.0 M NaOH. After about 2 h, the reaction mixture was treated by 1 M H 2 O 2 (115 µl) and H3K4me3[Ala 1 -Val 46 ]-NHNH 2 (1) (37 mg, 7.6 µmol) was activated at the same time in another tube. After 10 min, the activated 1 was added to the reaction mixture and its ph was slowly adjusted to 6.8 with 2.0 M NaOH. Then the reaction was kept at room temperature for h. The procedure was monitored by analytical HPLC using a gradient of 20-55% solvent A over 38 min. The ligation product was reduced by TCEP solution and purified by HPLC. The isolation yield was 21% affording 12.0 mg H3K4me3[Ala 1 -Cys 47 -Cys 91 -Ala 135 ]. S15

16 Figure S12. A) analytic HPLC traces (λ = 214 nm) of One-Pot synthesis of H3K4me3. a): ligation of 13 with 3 at 0 min. b): ligation of 13 with 3 at 90 min. c): analytic HPLC traces of Dobz removal. d): analytic HPLC traces of the second ligation at 45 h. * corresponds to ligation product of 1 and unreacted 3; # was an unidentified peak with high absorption at 254 nm (possibly quinonemethides as mass spectra showed messy peaks in low MW). B) ESI-MS analysis of 14. Observed: ± 0.2 Da. Calcd: Da. C) ESI-MS analysis of 15. Observed: ± 1.4 Da. Calcd: Da. D) HPLC and ESI-MS analysis of the purified ligation S16

17 product. ESI-MS: Observed ± 2.3 Da, Calcd Da. (average isotopes). Desulfurization and Acm removal: The same procedure as above was used. From 12.0 mg of H3K4me3[Ala 1 -Cys 47 -Cys 91 -Ala 135 ], 8.3 mg of final product was obtained with a yield of 70 %. Figure S13. HPLC (A) (λ = 214 nm) and ESI-MS (B) analysis of the product after desulfurization. Observed: ± 2.8 Da. Calcd: Da (average isotopes). S17

18 Figure S14. A) HPLC analysis (λ = 214 nm) of the product (H3K4me3) after Acm removal. B) ESI-MS analysis. Observed: ± 1.6 Da. Calcd: Da. (average isotopes). C) MALDI-TOF analysis. Observed: Da. The enlarged spectrum showed that no peak was observed at the expected mass of oxidized side product. D) The deconvoluted ESI-MS. S18

19 Figure S15. Isotope envelope of the final product (native H3K4me3). A) Observed and calculated isotope envelopes of different charge states (19H + and 13H + ). B) Observed isotope envelope (doing statistics from m/z peaks in table S1) and Calculated isotope envelope of final product and oxidized side product. S19

20 Figure S16. MS-MS analysis of the synthetic H3K4me3 (and below). A) The m/z of observed fragments was marked through the sequence of H3. B) MS-MS analysis of the underlined fragment in H3 sequence. Details of MS-MS analysis Fragment: TDLRFQSAAIGALQEASEAYLVGLFEDTNLcAIHAK, amidomethyl ( Da) (Observed , Calcd ) C31-Carb- Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #14627 RT: ITMS, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₁₆²+-h₂o, y₂₄³+-h₂o, y₂₄³+-nh₃, y₁₆²+-nh₃ y₂₂³+-nh₃, y₇ y₉ b₁₁+-nh₃ b₁₀ b₃₄³+, y₃₄³ b₁₃ y₁₂ y₁₃ y₁₄ y₁₅ y₁₆ y₃₄² y₁₇ m/z Fragment: RVTIMPKDIQLARRIRGERA (Observed , Calcd ) S20

21 Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #8356 RT: ITMS, z=+4, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₄+, b₁₁³ y₁₂³ b₁₉⁴ y₁₉³ b₁₈³ b₇ b₈ m/z Fragment: STELLIRKLPFQRLVREIAQDFK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #13457 RT: ITMS, CID@35.00, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₁₈³ b₂₁³ y₁₅²+-h₂o, y₁₅²+-nh₃ b₂₁²+-nh₃ m/z Fragment: AARKSAPSTGGVK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #3589 RT: ITMS, CID@35.00, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da 30 Intensity [counts] (10^3) b₆²+-nh₃ y₃ y₄ [M+2H]²+-NH₃-H₂O y₁₁²+-nh₃ y₅ y₈ b₈+-h₂o y₉ y₁₀ b₁₁ b₁₂ m/z Fragment: STELLIRK (Observed , Calcd ) S21

22 Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #5988 RT: ITMS, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^6) b₂+-h₂o b₂ b₃+-h₂o y₆²+-h₂o, y₆²+-nh₃ y₃ y₄+-nh₃ y₄ b₅+ y₅ y₆ b₇+ y₅+-nh₃ m/z Fragment: LPFQRLVREIAQDFK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #12062 RT: ITMS, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₁₃² [M+2H]²+-H₂O, [M+2H]²+-NH₃ m/z Fragment: DIQLARRIRGERA (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #10651 RT: ITMS, CID@35.00, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₁₂²+-h₂o, y₁₂²+-nh₃ y₁₁²+-h₂o, b₁₁²+, y₁₁²+-nh₃ b₁₂² m/z Fragment: APRKQLATK (Observed , Calcd ) S22

23 Extracted from: D:\Liu-lei\MS131354\MS H3_ raw #3546 RT: ITMS, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₈²+-h₂o, b₈²+-nh₃ [M+2H]²+-H₂O, [M+2H]²+-NH₃ b₆ b₇ b₈ m/z 2.5 Synthesis of H4K16ac by using H 2 O 2 -controlled one-pot strategy Scheme S8. Synthesis of H3K16ac by using H 2 O 2 -controlled one-pot strategy According to the sequence of histone H4, it contains 102 amino acid residues but no Cys residue. The sequence was divided into three segments and desulfurization would be performed later. Thus we chose Leu 37 - Ala 38 and His 75 -Ala 76 as the ligation sites. S23

24 Synthesis of H4K16ac[Ser 1 -Leu 37 ]-NHNH 2 (16): The synthesis was performed on the automated peptide synthesizer using hydrazine resin (400 mg, 0.25 mmol). The Fmoc-Lys(Ac)-OH was coupled manually by 2 equiv of AA, 4 equiv of DIEA, 2 equiv of HOAt and 1.9 equiv of HATU to the initial loading of resin and checked by ninhydrin test. After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of 5-50 % solvent over 38 min. Yeild: mg (27.5 %). Figure S17. A) HPLC analysis (λ = 214 nm) of the crude and purified 16. Gradient: 5-50% solvent A over 38 min. B) ESI-MS analysis of the product 16. ESI-MS: Observed ± 0.1 Da, Calcd Da. (average isotopes). Synthesis of Dobz-H4K16ac[Cys 38 -His 75 ]-NHNH 2 (17): The synthesis was performed on hydrazine resin (400mg, 0.25 mmol). All the amino acid residues were double coupled. After cleavage, the crude peptide was purified on semi-preparative C18 column using a gradient of 30-70% solvent over 38min. Finally, 34.8 mg of pure product was obtained with a yield of 3.0%. Figure S18. A) HPLC analysis (λ = 214 nm) of the crude and purified 17. Gradient: 30-70% solvent A over 38 min. B: ESI-MS analysis of 17. Observed ± 0.3 Da, Calcd Da. The peaks of M-18 and M-36 were observed. [1] (average isotopes). Synthesis of H4K16ac[Cys76-Gly 102 ] (18): The synthesis was performed on the S24

25 automated peptide synthesizer using Nova-PEG Wang Resin (0.25 mmol). The first amino acid was coupled in the presence of 4 equiv of amino acid, 4 equiv of HOBt, 3.8 equiv of HBTU, 0.1 equiv of DMAP and 16 equiv of DIEA. After coupling for 12 h, the resin was capped with reagent (acetic anhydride: DIEA: DMF = 1 : 1 : 8 ). Then, all the amino acid residues were coupled normally. Finally, mg of pure product was obtained with a yield of 19.2 %. Figure S19. A) HPLC analysis (λ = 214 nm) of the crude and purified 18. Gradient: % solvent A over 38 min. B) ESI-MS analysis of 18. ESI-MS: Observed ± 0.7 Da, Calcd Da. (average isotopes). Synthesis of H4K16ac by using one-pot strategy: Dobz-H4K16ac[Cys 38 -His 75 ] -NHNH 2 (17) (16.2 mg, 3.5 µmol) was dissolved in 0.4 ml of ligation buffer (6.0 M Gn HCl, 0.2 M Na 2 HPO 4, ph = 3.04) and cooled to -12 o C. 125 µl of NaNO 2 was added and kept for 20 min. The mixture was treated by 1185 µl of MPAA solution, followed by 18 (10.7 mg, 3.5 µmol) and its ph was slowly adjusted to 6.5 with 0.5 M NaOH. After 120 min, the reaction mixture was treated by 106 µl of 1 M H 2 O 2. At the same time, 16 (28.3 mg, 7.0 µmol) was activated. After 20 min, activated 16 was added to the reaction mixture and its ph was slowly adjusted to 6.8 with 2.0 M NaOH. The procedure was monitored by analytical HPLC using a gradient of 10-70% solvent A over 38 min. After stirred at room temperature for 24 h, the reaction mixture was reduced by TCEP solution and isolated by HPLC. Finally, 11.0 mg of ligation product was obtained, giving an isolated yield of 27.4 %. S25

26 Figure S20. A): analytic HPLC traces (λ = 214 nm) of One-Pot synthesis of H4K16ac. a) Ligation of 17 with 18 at 0 min. b) Ligation of 17 with 18 at 120 min. c)the second ligation at 0 min (Dobz removal). d) The second ligation at 24 h. # was unidentified peak with high absorption at 254 nm (quinonemethides as mass spectra showed messy peaks in low MW).B) ESI-MS analysis of 20. Observed: ± 0.3 Da, Calcd: Da (average isotopes). C), D): HPLC and ESI-MS analysis of the purified ligation product. ESI-MS: Observed ± 1.1 Da, Calcd Da (average isotopes). S26

27 Desulfurization: The same procedure as above was used. After purification, 7.3 mg of final product was obtained with a yield of 66.7 %. Figure S21. HPLC (A) (λ = 214 nm) and ESI-MS (B) analysis of the native H4K16ac. ESI-MS: Observed ± 0.7 Da, Calcd Da. (average isotopes). C) MALDI-TOF analysis. Observed: Da. The enlarged spectrum showed that no peak was observed at the expected mass of oxidized side product. D) The deconvoluted ESI-MS. S27

28 Figure S22. Isotope envelope of native H4K16ac. A) Observed and calculated isotope envelopes of different charge states (15H + and 11H + ). B) Observed isotope envelope (doing statistics from m/z peaks in table S2) and Calculated isotope envelope of final S28

29 product and oxidized side product. Figure S23. MS-MS analysis of the synthetic H4K16ac. A) The m/z of observed fragments was marked through the sequence of H4. B) MS-MS analysis of the underlined fragment. Details of MS-MS analysis: Fragment: VFLENVIRDAVTYTEHAKR (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #12681 RT: ITMS, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₂+, y₄²+-nh₃, y₆³ y₉²+-h₂o, b₉²+, y₉²+-nh₃ b₆ y₇+-h₂o, y₁₅²+, y₇+-nh₃ y₁₆² y₈ y₁₀+-h₂o, b₁₀ b₁₁ m/z Fragment: VLRDNIQGITKPAIR (Observed , Calcd ) S29

30 Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #6849 RT: ITMS, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₃+-nh₃ y₇² y₄ y₈² y₉²+-nh₃, b₄ y₅ b₅ y₆ b₆ b₇ y₈ b₈ b₉ b₁₀ m/z Fragment: RKTVTAMDVVYALK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #9362 RT: ITMS, CID@35.00, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₆²+-h₂o, b₆²+-nh₃ b₇²+, y₇²+-h₂o b₈² b₉² y₄ b₅ y₅ b₆ y₆ b₇ y₇ b₈ y₈ y₉ m/z Fragment: KTVTAMDVVYALK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #10723 RT: ITMS, CID@35.00, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₃ b₇² b₅+-h₂o b₅ y₄ y₅ [M+2H]²+-H₂O, [M+2H]²+-NH₃ b₈ y₈ y₉ y₁₀ y₁₁ y₁₂ m/z Fragment: TVTAMDVVYALK (Observed , Calcd ) S30

31 Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #11582 RT: ITMS, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₆³+-h₂o, b₂ y₄² y₅² y₆² b₇² b₈² y₄ y₁₀² y₅ b₆ y₆ b₇ b₈ m/z Fragment: ISGLIYEETRGVLK (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #9978 RT: ITMS, CID@35.00, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) b₃ b₄ y₁₀² y₅+, b₁₀²+-h₂o [M+2H]²+-H₂O, [M+2H]²+-NH₃ y₆ y₁₃² y₈ y₉+-nh₃ y₉ b₁₂ y₁₀ b₁₁ b₁₃ m/z Fragment: VLRDNIQGITKPAIRR (Observed , Calcd ) Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #6392 RT: ITMS, CID@35.00, z=+3, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₅² b₈²+-h₂o, b₁₂³+-h₂o y₁₂³ b₄ y₁₀² y₅+-nh₃ y₁₃²+-h₂o, y₁₃²+-nh₃ y₁₂² y₁₃² b₁₅²+, b₇ b₈ b₉ b₁₀ b₁₁ m/z Fragment: TVTAMDVVYALKR (Observed , Calcd ) S31

32 Extracted from: D:\Liu-lei\MS131354\MS H4_ raw #10841 RT: ITMS, z=+2, Mono m/z= Da, MH+= Da, Match Tol.=0.8 Da Intensity [counts] (10^3) y₂ b₄+-h₂o y₇²+-nh₃, y₃ y₄ b₁₁²+, y₁₀² y₅+-nh₃, y₁₁² y₆ y₇ y₈ y₉ y₁₀ y₁₁ m/z 2.6 Synthesis of H3K36me3, H4K12ac, H4K5acK8acK12acK16ac Figure S24. HPLC (A) (λ = 214 nm) and ESI-MS (B) analysis of the native H3K36me3. ESI-MS: Observed ± 1.6 Da, Calcd Da (average isotopes). Figure S25. HPLC (A) (λ = 214 nm) and ESI-MS (B) analysis of the native H4K12ac. ESI-MS: Observed ± 1.7 Da, Calcd Da (average isotopes). S32

33 Figure S26. HPLC (A) (λ = 214 nm) and ESI-MS (B) analysis of the native H4K5acK8acK12acK16ac. ESI-MS: Observed ± 0.6 Da, Calcd Da (average isotopes). 2.7 Histones octamer reconstitution Scheme S9. Histones octamer reconstitution Histones H2A and H2B dimer purification S33

34 Histones H2A and H2B dimer was purified using a non-denaturing method according to the previous method with minor modifications. Briefly, histones H2A and H2B genes were assembled into the prsfduet-1 vector as a polycistronic plasmid, with a hexahistidine-sumo tag-ulp1 cleavage site (His6-SUMO tag) preceding the N-terminus of H2A. The resulting plasmid encoding H2A and H2B histones was transformed into BL21(DE3) cells and plated on to an LB agar plate containing kanamycin (50 μg/ml). The plate was incubated overnight (~16 h) at 37 C. For a 1 liter scale preparation, one colony was inoculated into 50 ml LB media containing kanamycin (50 μg/ml) and was shaken at 250 rpm for 4-5 h at 37 C, which was then amplified to 1 liter of the same media and was grown for another 3-4 h at 37 C. When the OD600 reached ~0.4, histone coexpression was induced by adding 0.4 mm isopropyl β-d-1-thiogalactopyranoside (IPTG) for h at 25 C. Cells were then harvested by centrifugation at 4,500 x g for 10 min at 4 C and were processed immediately or stored at -80 C for future purification. Cell pellets were re-suspended in lysis buffer (20 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1 mm phenylmethanesulfonylfluoride and 1mM dithiothreitol). Re-suspended cells were lysed by EmulsiFlex-C3 high pressure homogenizer (Avestin) and clarified by centrifugation at 14,000 g at 4 C for 1 h. The supernatant was collected and imidazole stock solution was added to adjust the concentration to 30 mm. The clarified lysate was then loaded onto a 5 ml HisTrap FF column (GE Healthcare) pre-equilibrated in the Ni-buffer A (20 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM dithiothreitol) followed by a 10 column volumes (CV) wash with Ni-buffer A containing 30 mm imidazole and then another 10 CV wash with buffer B (20 mm Tris-HCl ph 8.0, 4.0 M sodium chloride, 1mM dithiothreitol). The bound proteins were eluted by increasing the imidazole concentration from 30 mm to 500 mm linearly over 23.5 CV. Each faction was analyzed by 15% SDS-PAGE: 10 μl incubated at 98 C for 10 min and centrifuged at 14,000 g for 10 min, after which all supernatant was loaded on the gel. Electrophoresis was run at 180 V for 50 min. Ulp1 digestion S34

35 Ulp1digestion was carried out by adding purified His6-Ulp1 in 25:1 mass ratio and incubating the samples at 4 C for 3 h. The digestion was confirmed by SDS-PAGE. Size exclusion chromatography Ulp1-digested histones were loaded onto a 5 ml HisTrap FF column (GE Healthcare) again to remove his-ulp1 protease and undigested his-h2a/b. The flowthrough containing native H2A/B was then concentrated to reduce sample volume to ~0.5 ml with ultrafiltration using Amicon YM50 membrane (Millipore, MWCO 30 kda) at 4 C. The concentrated sample was then injected onto a Superdex /300GL column (GE Healthcare) pre-equilibrated in 20 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM dithiothreitol. The histone H2A/B dimer peak was eluted at an elution volume of 14.9 ml. The peak fractions were pooled and concentrated up to 8 mg/ml, aliquoted and flash-frozen in the presence of 50% glycerol for long-term storage. Histones H3 with K4me3 and H4 with K16ac tetramer reconstitution Chemically synthesized histones H3 with K4me3 and H4 with K16ac powder were firstly dissolved in unfolding buffer (6M Guanidine hydrochloride, 20 mm Tris-HCl ph 8.0, 5mM dithiothreitol) and left at room temperature for 1-2 h before four rounds of dialysis against refolding buffer (10 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM Na-EDTA, 5mM dithiothreitol) at 4 C for in total of about h. Then the refolded H3-H4 sample was injected onto a Superdex /300GL column (GE Healthcare) pre-equilibrated in 20 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM dithiothreitol. The histone H3-H4 tetramer peak was eluted at an elution volume of 13.6 ml. The peak fractions were pooled and concentrated up to 8 mg/ml, aliquoted and flash-frozen in the presence of 50% glycerol for long-term storage. S35

36 Figure S27: Histone tetramer formation of synthetic H3K4me3 and H4K16ac. Blue curve: gel filtration profile of histone H3-H4 tetramer eluted over a Superdex /300 column. The peak of H3-H4 tetramer was eluted at 13.6 ml. Left to the peak: SDS-PAGE of the H3-H4 tetramer peak stained by commassie brilliant blue. Octamer reconstitution H2A-H2B dimer and H3-H4 tetramer were mixed at molar ratio of ~2.5:1 and then dialyzed against refolding buffer (10 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM Na-EDTA, 5mM dithiothreitol) for four rounds at 4 C for in total of about h at 4 C. Then the resultant product were injected onto a Superdex /300GL column (GE Healthcare) pre-equilibrated in 20 mm Tris-HCl ph 8.0, 2.0 M sodium chloride, 1mM dithiothreitol. The histone octamer peak was eluted at an elution volume of 12.8 ml. The peak fractions were pooled and concentrated up to 8 mg/ml, aliquoted and flash-frozen in the presence of 50% glycerol for long-term storage. Western blotting Sandwiched nitrocellulose membrane-gel-filter paper was transferred in transfer buffer (25mM Tris-base, 192mM glycine, 20% methanol) at 4 C under 80V for 90 mins. The membrane was blocked using 5% non-fat milk at room temperature for 1 h. Rabbit anti-h3k4me3 (Millipore) and H4K16ac (Cell signalling) primary antibody S36

37 were diluted by 5000 fold using 5% non-fat milk, mixed and were then added onto the membrane for incubation overnight at 4 C by rotation. Next day, the membrane was washed using wash buffer (TBST) at least three times, 10 mins per time before the anti-rabbit IgG secondary antibody was added for another 1 h at room temperature. Then the membrane was washed using wash buffer (TBST, 50 mm Tris 7.6, 150 mm NaCl, 0.05% Tween 20) at least three times, 10 mins per time before the freshly prepared HRP (horseradish peroxidase) chemiluminescent (TIANGEN) was added, followed by exposure and analyzed using a BioRad ChemDoc +XRS system (BioRad). Figure S28. Synthetic H3K4me3 and H4K16ac can form histone octamer, which were purified over a Superdex 200 (10/300) column after extensive dialysis. a) The chromatogram shows that histone octamer was eluted at 12.8 ml. It was well separated from excess H2A-H2B dimer (eluted at 15.2 ml). b) The SDS PAGE gel (left) and Western blotting (right) show that the reconstituted histone octamer from synthetic H3K4me3 and H4K16ac was intact and stoichiometric (Com. Blue: commassie brilliant blue; Western: Western blotting). S37

38 2.8 Isotope envelope of H3K4me3 and H4k16ac on different charge states Table S1. Isotope envelope of H3K4me H 13H 14H 15H + + observed calculated observed calculated observed calculated observed calculated m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int S38

39 16H + 17H + 18H + + observed calculated observed calculated observed calculated observed calculated m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. 19H H+ 21H+ 22H+ 23H+ observed calculated observed calculated observed calculated observed calculated m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int. m/z Int S39

40 19H + Table S2. Isotope envelope of H4K16ac + 18H 17H + + Observed Calculated Observed Calculated Observed Calculated Observed Calculated m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. 16H H + 14H + 13H + + Observed Calculated Observed Calculated Observed Calculated Observed Calculated m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. m/z int. 12H S40