Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis

While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol–thioester exchanges in water at mildly acidic pH and the production of peptide thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity.


Analyses
Products were characterized by analytical UPLC-MS using a System Ultimate 3000 UPLC (Thermofisher) equipped with a diode array detector, a charged aerosol detector (CAD) and a mass spectrometer (Ion trap LCQfleet). The column eluate was monitored by UV at 215 nm and CAD. The peptide masses were measured by on-line UPLC-MS (LCQ Fleet Ion Trap Mass Spectrometer, ThermoFisherScientific). Unless otherwise specified, heat temperature is set at 350 °C, spray voltage at 2.8 kV, capillary temperature at 350 °C, capillary voltage at 10 V, tube lens voltage at 75 V.
MALDI-TOF mass spectra were recorded with a Bruker Autoflex Speed using alpha cyano 4hydroxycinnaminic acid, sinapinic acid or 2,5-dihydroxybenzoic acid as matrix. The observed m/z corresponded to the monoisotopic ions, unless otherwise stated.

Purifications
Preparative reverse phase HPLC of crude peptides were performed with a preparative HPLC Waters system using the appropriate linear gradient of increasing concentration of eluent B in eluent A (flow rate of 6 mL min -1 , detection at 215 nm). Selected fractions were then combined and lyophilized.

General procedures
Peptide amides were synthesized on a NovaSyn TGR solid support (0.25 mmol/g) using standard Fmoc chemistry and an automated peptide synthesizer ( SEA off peptides were synthesized on bis(2-sulfanylethyl)aminotrityl polystyrene solid support (SEA PS) (0.16 mmol/g) or on SEA ChemMatrix ® solid support using standard Fmoc chemistry and an automated peptide synthesizer (Figure S 2). The SEA on peptide obtained after acidic cleavage was converted into the corresponding SEA off peptide by oxidation with iodine. The same intermediate provided the MPA thioester via a SEA/MPA exchange reaction. Detailed procedures to prepare these peptides are available in previous publications. 3,5,6 Note that the first amino acid was directly linked to the solid support. Coupling of the first amino acid on SEA PS and SEA ChemMatrix ® beads The first amino acid (10 equiv) was coupled to SEA PS solid support (1.0 equiv) using HATU (10 equiv)/DIEA (20 equiv) activation in DMF. The amino acid was preactivated for 2 min and then added to the beads swelled in the minimal volume of DMF. The beads were agitated at RT for 1.5 h, then washed with DMF (3 × 2 min) and drained. The absence of unreacted secondary amino groups was checked using the chloranyl colorimetric assay. A capping step was then performed using Ac2O/DIEA/DMF 10/5/85 v/v/v (2 × 5 min) and the beads were washed with DMF (3 × 2 min).

Automated peptide elongation
Peptide elongation was performed using standard Fmoc chemistry on an automated peptide synthesizer without microwaves. Couplings were performed using a 4-fold molar excess of each Fmoc L-amino acid, a 3.6-fold molar excess of HBTU and an 8-fold molar excess of DIEA. A capping step was performed with Ac2O/DIEA/DMF 10/5/85 v/v/v before Fmoc removal using piperidine/DMF 80/20 v/v.

Final peptide deprotection and cleavage
At the end of the synthesis, the beads were washed with DCM (3 × 2 min) and diethyl ether (3 × 2 min) and dried in vacuo. The crude peptide was cleaved from the solid support using TFA cleavage and deprotection cocktails, precipitated by addition of cold diethyl ether/n-heptane 1/1 v/v (20 mL per mL of TFA cocktail) and recovered by centrifugation.

Oxidation of the SEA group (SEA on → SEA off )
The SEA on peptide recovered by centrifugation was solubilized in deionized water, lyophilized and dissolved in AcOH/water 1/4 v/v. A solution of I2 in DMSO (≈ 100 mg mL -1 ) was added dropwise until complete oxidation of the SEA group (persistence of the yellow color of iodine in the reaction mixture). After 30 s of stirring, a solution of DTT in AcOH/water 1/4 v/v (≈ 100 mg mL -1 ) was added to consume the excess of I2. Then the peptide was immediately purified by HPLC.

Transthioesterification (SEA off → MPA)
The SEA off peptide recovered after purification was converted into the corresponding MPA thioester by reaction with MPA (5% in volume) at pH 4.0. A detailed procedure is given below for the synthesis of MPA thioesters.

Characterization of DYRT-SEA off peptide
The DYRT-SEA off peptide was analyzed by UPLC-MS ( Figure S 7) and MALDI-TOF mass spectrometry ( Figure S

Synthesis and characterization of MPA thioester peptides
DYRT-MPA peptide 24 was synthesized by converting the SEA off peptide into the corresponding MPA thioester using the following procedure.
The crude solid diselenide 18 was dried overnight under vaccum and then recrystallized from methanol to provide diselenide 18 as yellow crystals (462 mg, 0.788 mmol, 40 %).

Generalities
Kintek Global Kinetic Explorer Software (version 10.0.200514) was used for kinetic modelization. The model reaction used for numerical fitting corresponds to a second-order reaction in the form of A + B  C.
The standard deviation for each trace was first estimated upon fitting the experimental dataset with an analytical function (3-exponential) so as to determine an average sigma value further used for numerical data fitting. The subsequent numerical fit allowed determining the apparent second order rate constant kapp for each experiment. Fitting to a given model was achieved by nonlinear regression analysis based upon an iterative search to find a set of reaction parameters that gives a minimum ². The process was completed by careful visual examination of the fits.

Experimental procedure
The general procedure for studying the effect of the catalyst and catalyst concentration on the rate of thiol-thioester exchange is illustrated with the following conditions: 1 mM SEA-peptide, 6 M Gn·HCl, 100 mM diselenide precatalyst 17, 100 mM TCEP, 3-mercaptopropionic acid (5% v/v), pH 4.0 in 0.1 M phosphate buffer at 37 °C.
To a solution of TCEP·HCl (28.67 mg, 100 μmol, 100 mM final concentration) in 6 M Gn·HCl, 0.1 M, pH 7.2 sodium phosphate buffer (1 mL) was added the alkyldiselenide precatalyst 17 (30.2 mg, 0.1 mmol, 100 mM final concentration). To the resulting mixture was added MPA (5% v/v). Finally, the pH was adjusted to 4.0 by addition of 6 M NaOH and the SEA peptide (1.42 mg, 1.0 μmol) was dissolved in the previous solution. The reaction mixture was stirred at 37 °C under nitrogen atmosphere. The progress of the reaction was monitored by HPLC. For each point, a 2 μL aliquot was taken from the reaction mixture and quenched by addition of 50 μL of 10% aqueous AcOH. The chromatograms were processed on the basis of the absorbance signal at 215 nm to deduce the conversion ratio of the different species (no absorbance correction was applied as SEA peptides and MPA thioester were considered to have the same molecular extinction coefficient). 6.25 3.42 0.38 a General conditions are as follows: 1 mM SEA-peptide, 6 M Gn·HCl, catalyst, 100 mM TCEP, 3mercaptopropionic acid (5% v/v), pH 4.0 in 0.1 M phosphate buffer at 37 °C. Rate constants were extracted using KinteK Explorer Software TM . A standard deviation estimate for experimental measurements was obtained from fitting with a multi-exponential function. Rate constants and associated standard errors were produced by chi² minimization fitting. Standard errors were estimated based upon the covariance matrix using Kintek software. b Raw data with MPAA used as catalyst were extracted from reference 47 (J. Org. Chem. 2018, 83, 12584-12594).

Analytical approach
Data fitting based on numerical approaches (as proposed above) does not involve translating the problem into an analytical expression. Alternatively, the problem could be formulated analytically with respect to standard equations for a pseudo-first order kinetics. In this case, MPA is being considered as the reagent in large excess.

Folding of 9-GN-l and characterization of 9-GN
Characterization of folded 9-GN

Disulfide bridge pattern of 9-GN
The disulfide bridge patterns of 9-GN is given in Figure S 25.
Experimental determination of the disulfide bridge pattern was achieved by trypsin digestion of 9-GN and identification of the resulting fragments by mass spectrometry using non-reducing conditions. Fragments obtained by digestion with trypsin permitted the direct assignment of Cys69-Cys132 and Cys96-Cys107 disulfide bonds.
The HPLC fraction containing the folded protein was collected, dried under vacuum and the residue was redissolved in a solution of trypsin (5 μL, 50 ng/μL in 20 mM NH4HCO3). The digestion was carried out at 37 °C and the progress of the enzymatic reaction was monitored by MALDI-TOF mass spectrometry ( Figure S 26).