Histidine-specific bioconjugation via visible-light-promoted thioacetal activation

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.

Scheme S1. Examples of metal-approaches for His modification on protein. (a) Intracellular reactions promoted by bis(histidine) miniproteins stapled using Palladium(II) complexes [1] . (b) Histidine-directed arylation/alkenylation of backbone N−H bonds mediated by Copper(II) [2] .  [3] . (b) Histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation [4] . (c) peptide modification via radical-mediated chemoselective C−H alkylation of histidine using C4alkyl-1,4-dihydropyridine (DHP) reagents under visible-light-promoted conditions [5] . (d) Siteselective protein conjugation at histidine using a bis-alkylation reagent, PEG(10kDa)-monosulfone [6] .  Pummerer reaction is a well-studied organic transformation [7] and generally requires stoichiometric amounts of strong electrophilic activators including acid anhydride, trimethyloxonium, dimethylthiosulfonium fluoroborate (DMTSF), and TMSCl (Scheme S3a). Recently, thioacetals and intermediates of aldehyde-thiol condensation were well demonstrated as thionium precursor to undergo connective Pummerer-type reaction by previous literatures, where polar organic solvents, such as TFE, and catalytical Lewis acids, such as BF 3 OEt 2 , Cu(OTf) 2 and phosphate, were required (Scheme S3b). [8] Therefore, we initialed our investigation by the reaction between thioacetal and aromatic amino acids to screen a biocompatible method for Pummerer-type modification of proteins. All of the aromatic amino acids (His, Trp, Tyr and Phenylalanine, Phe) were reacted with thioacetal 1a under a polar condition (TFE solvent) with irradiation of Blue LED (10 W). The reaction of derivatives of Trp and His gave products with 89% and 71% yield (Scheme S4a and Scheme S4b), and evidences of NMR spectra validated their alkylation positions are C 2 for Trp and N 3 for His, respectively. In contrast, derivatives of Phe didn't observe any product under this condition, and trace of product of Tyr and 1a was detected in a S6 more polar solvent 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (Scheme S4). These results proved the feasibility of the modification of aromatic amino acid by Pummerer-type reaction and encouraged us to develop more biocompatible conditions. In order to ensure the dissolution of thioacetal at a 20 mM concentration, MeCN/H 2 O mixed solvent was investigated. In MeCN/20% H 2 O solvent, the reaction between Boc-His-OMe and 1a exhibited a very inefficient manner. Hence, we then shifted our focus to photoredox-catalysts.

General procedure
All chemical reagents are commercially available without purification. The reactions were monitored by TLC (silica gel-G). Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker 300 MHz, 400 MHz or 500 MHz spectrometer using trimethylsilane (TMS) as internal standard under ambient temperature (20 °C). High-Resolution Mass Spectrometry (HRMS) were measured on Orbitrap Exploris 480.

Chemicals and Reagents
All chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Leuproprelin (peptide 4) was purchased from Sangon Biotech; Angiotensin II (peptide 5) and Melanotan I (peptide 6) were purchased from Wuxi Asiapeptide Biotechnology Co. Ltd.. The procedures for the preparation of peptide 7 and 8 were illustrated in Section 4.1. Bovine Serum Albumin (BSA) was purchased from Sangon Biotech. Myoglobin (MB) and Carbonic Anhydrase (CA) were purchased from Sigma-Aldrich. TAMRA-azide (named as "TAMRA-N 3 " in this study), Biotin-dadpsazide (named as "DADPS biotin-N 3 " in this study) and PC biotin-PEG 3 -azide (named as "PC biotin-N 3 " in this study) was purchased from BIOCONE (Chengdu, CHINA). Sequencing-grade trypsin and NeutrAvidin™ agarose were purchased from Thermo Scientific. All solutions were made with ultrapure Milli-Q water (Millipore, Bedford, MA). BSA, MB and CA were dissolved in PBS buffer, pH 7.4. The peptide and probes were dissolved in DMSO to a stock concentration of 100 mM.

Cell Culture
MCF-7 cells were maintained at 37 ˚C in a humidified atmosphere containing 5% carbon dioxide using Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM insulin and 10% fetal bovine serum (FBS). We separated cells once they grew to 80-90% confluence in a 1:2 ratio. After 12~14 hours, subculture would achieve to approximately 80% confluence on 100 mm polystyrene tissue culture plates. The cells should be in log phase growth and healthy. On ice, we discard old medium and wash cells twice with ice-cold PBS. Pre-cooled microcentrifuge tube resuspended pellet in 1 ml cold DPBS buffer. Cells were sonicated until pellet is disturbed then centrifuged at x12,000 rpm 30 min. The final supernatant protein was transferred to a new tube and quantified at 562 nm using BCA (Thermo Scientific).

Gel fluorescence analysis of probe-labelled proteins
For probe labelling assay, BSA or proteins extracted from MCF7 cells were incubated with probes at indicated concentration at 37 ˚C for 2 h with blue light. A freshly pre-mixed click chemistry reaction cocktail was then added (50 μM TAMRA-N 3 , 100 μM TBTA, 1 mM TCEP, and 1 mM CuSO4) to the mixture above for another 1 h at RT. After reaction, the labelled proteins were precipitated by pre-chilled acetone (-20 ˚C) to remove excessive reagents. Precipitated proteins were subsequently collected by centrifugation (13000 rpm x 10 min at 4 ˚C) and dissolved in PBS containing 1.2% SDS and then boiled using 4× SDS-PAGE loading buffer at 95 ˚C for 10 min. The samples were analyzed by 12% SDS-PAGE gels (polyacrylamide gel electrophoresis) and imaged by Bio-Rad ChemiDoc MP. Then the gels were then stained with Coomassie staining (CBB) and scanned. For competition assay, proteins were treated with IAA or sulfo-NHS acetate at indicated concentration as above processes.

Histidine-specific profiling using mass spectrometry
We had 3 groups of proteins named as "DMSO", "10 μM TA8" and "80 μM TA-8". Then biotin was ligated to the probe-labelled proteins in as groups with click chemistry reaction. After reaction, the labelled proteins were precipitated by pre-chilled acetone (-20 ˚C) to remove excessive reagents. Precipitated proteins were subsequently collected by centrifugation (13000 rpm x 10 min at 4 ˚C) and dissolved in PBS containing 1.2% SDS then diluted to 0.2% SDS. Upon incubated with NeutrAvidin™ agarose beads for 3 h at 29 ˚C to capture the biotin-labelled proteins, the beads were washed with cold PBS (twice) and cold water (twice). The captured proteins on beads were reduced by DTT, treated by IAA, and digested at 37 ˚C overnight by trypsin into peptides. The supernatant was collected by centrifugation (1,000 g, 1 min) and combined with 2 X 50 μl water after washing beads, stored at -80 ˚C then acidified to a final concentration of 2% (v/v) formic acid and desalted for LC-MS/MS analysis.

LC-MS/MS Analysis
After dry in the speed VAC, obtained samples were loaded onto a Thermo analytical column (75 μm i.d. × 25 cm) C18 column with an Easy-nLC 1200 chromatography pump coupled with Orbitrap Exploris TM 480. For each analysis, we reconstituted peptides in 15 μl 0.1% FA and loaded 4 μl onto the column for running. Peptides in each running were separated on a 120 min (

Data Processing
Spectral data was searched against the Uniprot/Swiss-prot protein database using PD 2.4 and filtered to 1% FDR (false discovery rate) at the protein level. Default parameters used following exceptions: a minimum of 1 unique peptide was required for quantitation; peptide matching between runs was included and peptides containing oxidation (O), N-terminal acetylation (protein N-term), and carbamidomethyl (C) as variable modifications; only tryptic peptides with two missed cleavage sites were allowed; fragment mass tolerance was set to 0.02 Da for MS/MS fragment ions; mini and max peptide lengths were 6 and 144. GO enrichment was performed for cellular components of labelled proteins, and Uniprot accession numbers of identified reactivehistidine-containing proteins were subjected to LIMMA R package analysis. To a colorless transparent glass vial charged with an appropriate magnetic stir bar was added thioacetal 1 (108 mg, 0.4 mmol, 4 equiv.), Boc-His-OMe 2 (26.9 mg, 0.1 mmol) and Rose Bengal (10.2 mg, 0.01 mmol 10 mol%), and 20 mL of MeCN/H 2 O=4/1 was added as solvent. The vial was then sealed and placed on a magnetic stirrer about 2 cm away from a 10 W blue LED lamp for 1h. After reaction completion, the organic solvents were removed on a rotary evaporator, 10 mL of H 2 O was added and the crud mixture was extracted with CH 2 Cl 2 (3×10 mL). The organic phase was washed with saturated NaCl solution (2×5 mL) and dried over anhydrous Na 2 SO 4 . The crud product was purified by flash column chromatography using eluent solution Hexane/EtOAc (
Synthesis of S17 was followed General Procedure B using pentanal and propane-1-thiol as substrates.

General information
All chemical reagents are commercially available without purification. High-Resolution Mass Spectrometry (HRMS) and MS/MS Spectrometry were measured on a Q_Exactive_Focus. General procedure for SPPS: Peptide 7 and 8 were synthesized on Rink Amide MBHA resin (peptide 7) or Fmoc-Wang resin (peptide 8) by Fmoc solid-phase synthesis (SPPS). Rink-amide resin was pre-swelled with DCM for 30min, filtered, the Fmoc (9-fluorenylmethyloxycarbonyl) group was removed with 50% (vol/vol) morpholine for 30min*2; the resin was sequentially washed with DCM and DMF for three times. Fmoc-protected amino acids (2.0 equiv.) and HATU (2.0 equiv.) were dissolved in DMF, followed by DIPEA (3.0 equiv.). The mixture was pre-activated for 1min and added to the resin for 1 h with N 2 bubbling, repeated once. The resin was washed sequentially with DCM, DMF for three times, then dried under a stream of nitrogen for next step. For cleavage of resin, the final resin was treated with TFA/TIS/water (95:2.5:2.5) at room temperature for 3 h and concentrated under a stream of nitrogen. The crude peptides were precipitated and washed with cold hexane/diethyl ether (1:2, v/v) at 4°C, redissolved in 50% acetonitrile in water. Crude peptides were purified by preparative HPLC.
Acetamidation of peptide 8: A solution of un-protected peptide 8 (5.0 mg, 2 mM, PBS buffer 7.4 with 50% MeCN) was added iodoacetamide (IAM, 20 mM), and the resulted mixture was shaken in an incubator shaker (37 o C) for 1 hours. The crude reaction mixture was directly purified by preparative HPLC after filtration. 4.0 mg (78%) white powder was obtained as target product.

General procedure C for the reaction between peptide and thioacetal
A 10 mM MeCN/H 2 O (1/1) stock solution of RB and a 200 mM MeCN stock solution of thioacetal was made up. These stock solutions were stored at room temperature away from light. To a 2 mL vial was added 100 uL solution of specific peptide (1 mM, in MeCN/H 2 O=4/1) solvent and 2 μL RB (10 mM), 10 μL specific thioacetal (200 mM) stock solution. The vial was then caped and equipped with magnetic bar. The reaction was set in a reaction chamber equipped with magnetic stirrer, 450 nm LED lamp (10 W) and exhaust fan to maintain the reaction temperature at about 30 o C for 1 hour, as shown in the following figure. The resulting solution was then analyzed with an internal standard (dibenzyl sulfoxide, 0.2 mM) via HPLC-MS after filtration. The distillates of target products were collected and further analyzed by HRMS and MS/MS spectrometry. Desired distillates of preparative LC were identified by MS and lyophilized to obtain target products, and analyzed by NMR. Liquid nitrogen was used to quickly freeze distillates and lyophilize them as quickly as possible using a lyophilizer.
LC-MS yields were estimated by UV absorption at 220 nm of the peak corresponding to the thioacetal adducted product versus the internal standard (dibenzyl sulfoxide): % yield = (A p /A st ) × k. A p is the peak area of thioacetal-adducted products; A st is the peak area of the internal standard; k is the quantity coefficient between specific peptide and standard. The quantity coefficients were measured by the correlation of a gradient concentration. The isolated yield of 4a was obtained by a 10 mg level reaction, and isolated by a preparative LC. Note that the lyophilization of desired distillates need to be quick due to the potential hydrolyzation of thioacetal adducted products in weak acidic solution.

General method for LC/MS analysis
Analytical LC-MS were performed on a Shimadzu LC-MS 8030 system equipped with Kromasil 100-5-C18 column (4.6 × 250 mm, 5 µm; room temperature). Water (containing 0.1% TFA, A phase) and pure CH 3 CN (B phase) were used as solvents in linear gradient mixtures at a flow rate of 1 mL·min -1 .

General method for preparative LC
Preparative LC were performed on a Shimadzu LC-6AD system equipped with Shimadzu Shim-pack GIST C18 column (20 × 250 mm, 5 μm; room temperature). Water (containing 0.1% TFA) and pure CH 3 CN were used as solvents in linear gradient mixtures at a flow rate of 8 mL·min -1 . Due to the potential hydrolyzation of thioacetal adducted products in weak acidic solution (Figure S6), we tried a mobile phase of pure water to separate the products, and found that 0.1% TFA in water is essential. Thus, we tried quick freezing samples using liquid nitrogen and lyophilizing.

General method for MS/MS analysis
The fragmentation of modally modified peptides was investigated in positive electrospray ionization mode, loaded onto a Thermo Q Exactive Focus Orbitrap LC-MS/MS system. The protonated molecule was generated by spraying a 0.5 ng/μl solution in 20:80 water:methanol + 0.1% formic acid (FA) with a flow rate of 0.28 mL/min. Parameters are as follows in Full MS/ data dependent-MS2 TopN mode: mass analyzer over m/z range of 145−2175 with a mass resolution of 70,000 (at m/z=200) in a data-dependent mode. MS/MS spectra were obtained using collision energy values at 25% normalized activation energy with a HCD (High Energy Collision Dissociation) mode. The reaction was followed General Procedure C using peptide 4 (100 uL 1 mM solution, sequence: NH-PyrHWSYLLR-NHEt) and thioacetal 1a, 1c, 1d and 1e. A stock solution of peptide 4 (1mM) was made up by 2 mL solvent (MeCN/H 2 O = 4/1) and 2.4 mg peptide 4.
Before the reaction, a quantity relationship of matter between peptide 4 and the internal standard (dibenzyl sulfoxide) was established by a gradient concentration. The volume of the reaction mixture was adjusted to 500 uL with MeCN/H 2 O (1/1), and then 2 uL dibenzyl sulfoxide stock solution (50 mM in MeCN/H 2 O = 1/1) was added. The resulted solution was filtrated and analyzed with LC-MS.
A 10 mg level reaction between 1a and peptide 4 was followed the similar procedure. 10.0 mg of peptide 4 was dissolved in the solvent (5 mL MeCN/H 2 O = 4/1) in a 20 mL vial. 45 mg thioacetal 1a and 1.1 mg RB was added to the mixture. The vial was then caped and equipped with magnetic bar. The reaction was set in a reaction chamber equipped with magnetic stirrer, 450 nm LED lamp (10 W) and exhaust fan to maintain the reaction temperature at about 30 o C for 1 hour. The resulting solution was then purified via a preparative LC after filtration. (10-80% B phase over 20 min, 8 mL·min -1 flow rate, 00.1% TFA, λ = 220 nm, Shimadzu Shim-pack GIST C18 20 × 250 mm, 5 μm column ). Desired distillates of preparative LC were identified by MS and lyophilized to obtain target products, and analyzed by NMR. Liquid nitrogen was used to quickly freeze distillates and lyophilize them as quickly as possible using a lyophilizer. 6.8 mg (59%) white powder was obtained as target product.
Before the reaction, a quantity relationship of matter between peptide 5 and the internal standard (dibenzyl sulfoxide) was established by a gradient concentration. The volume of the reaction mixture was adjusted to 500 uL with MeCN/H 2 O (1/1), and then 2 uL dibenzyl sulfoxide stock solution (50 mM in MeCN/H 2 O = 1/1) was added. The resulted solution was filtrated and analyzed with LC-MS.
Before the reaction, a quantity relationship of matter between peptide 6 and the internal standard (dibenzyl sulfoxide) was established by a gradient concentration. The volume of the reaction mixture was adjusted to 500 uL with MeCN/H 2 O (1/1), and then 2 uL dibenzyl sulfoxide stock solution (50 mM in MeCN/H 2 O = 1/1) was added. The resulted solution was filtrated and analyzed with LC-MS.
Before the reaction, a quantity relationship of matter between peptide 7 and the internal standard (dibenzyl sulfoxide) was established by a gradient concentration. The volume of the reaction mixture was adjusted to 500 uL with MeCN/H 2 O (1/1), and then 2 uL dibenzyl sulfoxide stock solution (50 mM in MeCN/H 2 O = 1/1) was added. The resulted solution was filtrated and analyzed with LC-MS.
Before the reaction, a quantity relationship of matter between peptide 7 and the internal standard (dibenzyl sulfoxide) was established by a gradient concentration. The volume of the reaction mixture was adjusted to 500 uL with MeCN/H 2 O (1/1), and then 2 uL dibenzyl sulfoxide stock solution (50 mM in MeCN/H 2 O = 1/1) was added. The resulted solution was filtrated and analyzed with LC-MS.
Characterization data of 8 HPLC traces of the quantity relationship of matter, crud reaction and purified 8. (10-80% B phase over 25 min, 1 mL·min -1 flow rate, 0.1% TFA, λ = 220 nm, Kromasil 100-5-C18 4.6 × 250 mm, 5 µm column). The green circle, blue triangle and orange square refer to the starting peptide, internal standard and target product. The red asterisks indicate oxidative products with both oxidized Met and modified His.