Palladium prompted on-demand cysteine chemistry for the synthesis of challenging and uniquely modified proteins.

Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome–enzyme complex.


General methods
SPPS was carried out manually in syringes, equipped with teflon filters, purchased from Torviq or by using an automated peptide synthesizer (CS336X, CSBIO). If not differently described, all reactions were carried out at room temperature. Analytical grade N,Ndimethylformamide (DMF) was purchased from Bio-Lab Ltd. Commercial reagents were used without further purification. Resins were purchased from Creosalus, protected amino acids were purchased from GL Biochem and activating reagents [(2-(1H-benzotriazol-1-yl)-

List of the protected amino acids used in peptides synthesis
Fmoc HPLC analysis was carried out on a C18 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, C18 column in gradient of 0-40% B was used to provide peptide 1 in ~50% yield.

Synthesis of peptide 2
The synthesis of AC(t-butyl)LYRAGLYRAG was carried out using Fmoc-SPPS as described in peptide 1. The peptide was cleaved from the resin as described above and lyophilized. The HPLC analysis was carried out on a C4 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, C18 column in gradient of 0-40% B was used to provide peptide 2 in ~55% yield.

Synthesis of peptide Thz-LYRAC(Acm)LYRAC(t-butyl)LYRAG
The synthesis of the peptide was carried out using Fmoc-SPPS on Rink amide resin (0.43 mmol/g, 0.1 mmol scale). was carried out using Fmoc-SPPS as described in peptide 1. The HPLC analysis was carried out on a C4 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, C18 column in gradient of 0-40% B was used to provide Thz-LYRAC(Acm)LYRAC(t-Butyl)LYRAG peptide in ~45% yield.

Synthesis of peptide 3
The synthesis of Thz-LYRAGC(Acm)LYRCA was carried out using Fmoc-SPPS as described in peptide 1. The HPLC analysis was carried out on a C4 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, C18 column in gradient of 0-40% B was used to provide peptide 3 in ~50% yield. For scavenging excess of Pd(II), 4 equiv. of resin to the Pd(II) was incubated with the reaction mixture in centrifugal filters (0.22µm) and shacked for 1 h at 37 ºC followed by separation of the resin from the reaction mixture. Similar procedure was performed in order to cap excess of the alkylating reagents: (N-(bromoacetyl)alanine-methyl ester, N-(bromoacetyl)glycine-ethyl ester and iodoacetamide) from the reaction mixture.

Inverse Sequential modification of peptide 3
2 mg of peptide 3 (1.35×10 -3 mmol, 2.7mM) was dissolved at 500 µL, 20 % CH 3 CN/H 2 O, and treated with 1 equiv. of N-(bromoacetyl)glycine-ethyl ester, which was pre-dissolved as a stock solution in MeOH with 100 equiv. of NaI and added to the reaction mixture. The reaction was monitored by HPLC-MS, which showed complete reaction within 30 min. Then, the reaction mixture was treated with 10 equiv. of PdCl 2 and incubated at 37 o C for 30 min, followed by DTT treatment to provide complete Acm deprotection to provide mono-alkylated peptide 3.
1 mg of mono-alkylated peptide 3 (0.6×10 -3 mmol, 2mM) was dissolved at 320 µL, 6 M Gn.HCl, pH 8, and treated with 1 equiv. of N-(bromoacetyl)alanine-methyl ester, which was pre-dissolved in MeOH, and the reaction mixture was incubated at 37 o C for 30 min. For the following Thz removal, 10 equiv. of [Pd(allyl)Cl] 2 and GSH (1:1) were added to the reaction and incubated at 37 o C for 45 min to observe complete Thz opening. The solution mixture was treated with 40 equiv. Cys-PEGA resin for 1 h at 37 o C and 1 equiv. of DTT. Finally, 15 equiv. of iodoacetamide at 37 o C were added to give the tri-alkylated peptide 3.

Synthesis of CSP-1-1 bearing a solubilizing tag
The synthesis was carried out using Fmoc-SPPS on Rink amide resin (0.43 mmol/g, 0.1 mmol scale). The pre-swollen resin was treated with 20% piperidine in DMF containing 0.1 mmol HOBt (3-5-3 min) in order to remove the Fmoc-protecting. Peptide synthesis was performed on an automated peptide synthesizer in presence of 4 equiv. of amino acid, HCTU and 8 equiv. DIEA. The Phacm linker was coupled manually in position 116, using 2.5 equiv. of the linker, HATU and 5 equiv. DIEA for 1.5 h. Alloc deprotection from the linker was carried out by using 0.2 equiv. tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 ) 4 ) and 20 equiv.
phenylsilane in dry DCM for 1 h. Fmoc-Arg(Pbf) was coupled to the linker using HATU as a coupling agent. This step was repeated to couple the second Arg. The third Arg of the tag was coupled as Boc-Arg(Pbf)-OH. For side chains deprotection and cleavage from the resin, the solid support was washed with DMF, MeOH, DCM and dried. Then the resin was treated with the cleavage mixture containing TFA:TIS:water (95:2.5:2.5) for 2 h. The cleavage mixture was filtered and the combined filtrate was added dropwise to a 10-fold volume of cold ether and centrifuged. The precipitated crude peptide was dissolved in 50% acetonitrile/water and lyophilized. HPLC analysis was carried out on a C4 analytical column using a gradient of 0-60% B over 30 min. For preparative HPLC, C18 column in gradient of 20-60%B was used to purify CSP-1-1 in ~35% yield.
amino acid, HCTU and 8 equiv. of DIEA. The Phacm linker was coupled manually in position 63 using 2.5 equiv. of linker, HATU and 5 equiv. DIEA for 1.5 h. Alloc deprotection from the linker and 3 Arg coupling was carried out as described above. N-MeDbz cyclization: The resin was washed with DCM and a solution of p-nitrophenyl chloroformate (120 mg, for 0.1 mmol scale) in 4 mL DCM was added and shaken for 30 min at room temperature and washed with DCM (3 × 5 mL). This step was repeated twice. Following this, the resin was treated with a solution of DIEA (0.45mL) in DMF (4 mL) and was shaken for 30 min for a complete cyclization and washed with DMF (×2). Analytical HPLC analysis was performed to ensure complete reactions. The peptides were cleaved from the resin as described above and lyophilized.

Synthesis of CSP-1-3 bearing solubilizing tag
This peptide was prepared by using the same procedure described for CSP-1-2. The HPLC analysis was carried out on a C4 analytical column using a gradient of 0-60% B over 30 min.

Circular dichroism spectroscopy of purifies full-length CSP-1:
CSP-1 was dissolved in 6 M urea solution (5% of the total volume) and then further diluted with 20 mM Tris buffer solution and pH was adjusted to 7.3 . The exact concentration of the protein solution was determined by Pierce® BCA Protein Assay Kit, Thermo scientific. With this solution circular dichroism spectrum was recorded in a Chirascan (Applied Photophysics) instrument.
of DIEA for 1.5 h. The Alloc protecting group was removed using 0.2 equiv. of (Pd[P(C 6 H 5 ) 3 ] 4 ) and 20 equiv. phenylsilane in dry DCM. Then Fmoc-Cys(t-Butyl)-OH was coupled manually. Analytical HPLC analysis was performed to ensure complete reactions.
The peptide was deprotected and cleaved from the resin as described above. The HPLC analysis was carried out on a C18 analytical column and a gradient of 0-60% B over 30 min.
of DIEA for 1.5 h. N-MeDbz cyclization was performed as described above. The peptides were cleaved from the resin as described above and lyophilized. For preparative HPLC, C18 with the same gradient was used to purify the H2A-3 in ~40% yield.
The progress of the reaction was monitored by analytical HPLC using C4 analytical column and a gradient of 0-60% buffer B over 30 min. After completion of the reaction, the product