Synthetic hyperacetylation of nucleosomal histones

We report combinations of a DMAP-based catalyst and phenyl acetate with optimal electron density as a new chemical system for high-yield, selective synthetic acetylation of histone lysine residues. The utility of this chemical system as a unique biologic tool is demonstrated by applying it to Xenopus laevis sperm chromatin.


Supporting figures
. (a) Schematic for synthetic histone acetylation by an chemical catalyst system.

Experimental procedures
General NMR spectra were recorded on JEOL JNM-ECX500 spectrometer, operating at 500 MHz for 1 H NMR and 124.51 MHz for 13 C NMR. Chemical shifts were reported in ppm on the  scale relative to residual CHCl3 ( = 7.24 for 1 H NMR and  = 77.0 for 13 C NMR) or CHD2OD ( = 3.31 for 1 H NMR and  = 49.0 for 13 C NMR) as an internal reference, respectively. Analytical HPLC was conducted by using a JASCO HPLC system equipped with a UV-2075 spectrometer, PU-2080 pumps, a DG-2080-54 degasser, and an MX-2080-32 mixer or Shimadzu HPLC system equipped with an SPD-20A spectrometer, LC-20AD pumps, and a DGU-20A3R degasser. Preparative HPLC was conducted by using a JASCO HPLC system equipped with a UV-2075 spectrometer, PU-2086 pumps, a DG-2080-53 degasser, an MX-2080-32 mixer or Shimadzu HPLC system equipped with an SPD-20A spectrometer, and LC-6AD pumps. ESI-MS spectra were measured on Bruker micrOTOF II spectrometer (for HRMS). MALDI/TOF-MS was obtained with a Shimadzu Biotech Axima ToF 2 spectrometer.
LC-MS/MS analyses were conducted with an AB Sciex Triple TOF 4600 equipped with an Eksigent ekspert microLC 200.

Materials
All protected α-amino acids were purchased from Watanabe Chemical Industries, Ltd.

16DMAP (2)
16DMAP was synthesized on a solid phase in 0.200 mmol scale using Rink-Amide-AM resin.
Fmoc-Lys(Mtt)-OH (0.600 mmol, Mtt: 4-Methyltrityl) was sequentially coupled using a DIC-HOBt method (0.600 mmol each) for 60 min at room temperature after removal of each Fmoc group with 20% piperidine-DMF for 10 min to obtain pentadeca-Lys(Mtt) on resin. The Mtt groups were removed by treatment with TFA and TIPS in CH2Cl2  mmol) were added. The mixture was stirred at room temperature for 1.5 h. Volatiles were removed under reduced pressure and the residue was taken up in CH2Cl2, and organic layer was washed with 10% CuSO4 aq and brine, dried over Na2SO4, filtered, and concentrated to afford crude PAc-gly, which was purified with silica gel column chromatography (Ac-OEt/MeOH = 6/4) to afford PAc-gly (6, 122.9 mg, 0.366 mmol, y. 93%) as colorless oil.

Antibody
Mouse monoclonal antibody against PCNA PC-10 was purchased from Santa Crutz Biotechnology and used for immunoblotting. Mouse monoclonal MCM7 antibody ab2360 was purchased from Abcam. Rabbit polyclonal MCM2 antibody ab4461 was purchased from Abcam.
Rabbit polyclonal histone H3 antibody ab1791 was purchased from Abcam. Mouse monoclonal nuclear pore complex proteins antibody ab24609 was purchased from Abcam. Cdc45 antibody was generous gift (Shintomi K, RIKEN).
Rabbit polyclonal antibody against X. laevis ELYS was raised against the peptide CRGRRGRVITSDDLRE by SCRUM Inc.

Xenopus egg extracts
Preparation of interphase egg extracts was performed as described previously 10  Underlay diluted extracts with 1.5 mL CPB containing 30% sucrose and centrifuged at 15,000 g for 10 min at 4 °C using a swing-bucket rotor. The pellets were resuspended in Laemmli sample buffer. The DNA replication efficiency was determined as described previously 11 .

Computational general information
All the calculations were carried out with Gaussian 16 (Revision B.01) program package 12 .
Unless otherwise noted, geometry optimization and vibrational analysis were carried out at M06-2X 13 with 6-31+G** basis set 14