Site‐Selective Modification of Peptides and Proteins via Interception of Free‐Radical‐Mediated Dechalcogenation

Abstract The development of site‐selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free‐radical‐mediated dechalcogenation. By exploiting phosphine‐induced homolysis of the C−Se and C−S bonds of selenocysteine and cysteine, respectively, we demonstrate the site‐selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low‐oxidation‐state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.


General additive-free selenocystine-selenoester ligation procedure
Peptide selenoesters (1.05-1.20 eq.) and peptide dimers bearing N-terminal selenocystine (0.5 eq) were dissolved separately in ligation buffer (6 M guanidine hydrochloride, 100 mM Na2HPO4, pH 7.2) to a concentration of 10.5-12 mM with respect to the peptide selenoester and 5 mM with respect to the peptide diselenide dimer. The solutions were combined to give an overall concentration of 2.5 mM with respect to the selenopeptide dimer, 5.25-6 mM with respect to the peptide selenoester and a final pH of 6.2-6.5. The reaction was monitored by RP-HPLC and once complete the DPDS was extracted with hexane. Immediately before purification by preparative RP-HPLC a small amount of TCEP was added to the ligation mixture. Fractions containing the desired ligated product were lyophilized.

General additive-free cysteine-selenoester ligation procedure
The peptide selenoester (1.05 eq.) and peptide bearing N-terminal cysteine (1.0 eq) were dissolved separately in ligation buffer (6 M guanidine hydrochloride, 100 mM Na2HPO4, pH 7.2) to a concentration of 10.5 mM and 10 mM respectively. The solutions were combined to give an overall concentration of 5 mM with respect to the peptide bearing an N-terminal cysteine and 5.25 mM with respect to the peptide selenoester, and a final pH of the reaction of 6.5-7.0. If the pH was below 6.5 the pH was carefully adjusted with 2 M NaOH, ensuring to not exceed pH 7. The reaction was monitored by RP-HPLC and once complete the DPDS was extracted with hexane. Hydroxylamine (10 eq) was added to the ligation solution and the pendant thioester hydrolysis monitored by RP-HPLC. Once hydrolyzed, TCEP was added and purification by preparative HPLC was performed. Fractions containing the desired ligated product were lyophilized.

General one-pot ligation-conjugation protocol
Peptide selenoesters (1.05-1.20 eq.) and peptide dimers bearing N-terminal selenocystine (0.5 eq) were dissolved separately in ligation buffer (6 M guanidine hydrochloride, 100 mM Na2HPO4, pH 7.2) to a concentration of 10.5- For selenopeptide fragments containing Cys the same ligation protocol was followed, using general protocol C for the conjugation:

General conjugation protocol A -Accelerated modification of Sec
To peptide dissolved in 20% DMSO in ligation buffer to a concentration of 5 mM was added a solution of TCEP (0.625 M stock solution in LB pH adjusted to 7, 50 eq.), TEMPO trap (0.1 M stock solution in DMSO, 2 eq.) and Mn(OAc)3 (0.1 M stock solution in DMSO, 4 eq.). The pH of the reaction mixture was checked to be 7-7.5 then the reaction mixture diluted to the final peptide concentration was 2.5 mM. The reaction was then heated to 50 o C and shaken for 1 hour.
Once the starting material was shown to be fully consumed by HPLC the reaction mixture was purified by semi-preparative HPLC.

General conjugation protocol B -Accelerated modification of Cys
To peptide dissolved in 20% DMSO in ligation buffer to a concentration of 2 mM was added a solution of TCEP (0.625 M stock solution in LB pH adjusted to 7, 50 eq.), TEMPO (0.1 M stock solution in DMSO, 2 eq.) and Mn(OAc)3 (0.1 M stock solution in DMSO, 5 eq.). The pH of the reaction mixture was checked to be 7-7.5 then the reaction mixture diluted to the final peptide concentration was 1.0 mM. The reaction was then heated to 50 o C and shaken for 2 hours. Once the starting material was shown to be fully consumed by HPLC the reaction mixture was purified by semi-preparative HPLC.

General conjugation protocol C -Sec-selective TEMPO conjugation in presence of Cys
To peptide dissolved in 20% DMSO in ligation buffer to a concentration of 5 mM was added a solution of TCEP (0.625 M stock solution in LB pH adjusted to 7, 50 eq.) and TEMPO (0.1 M stock solution in DMSO, 5 eq.). The pH of the reaction mixture was checked to be 7-7.5 then the reaction mixture diluted so the final peptide concentration was 2.5 mM. The reaction was then heated to 37 o C and shaken for 4 hours. Analytical RP-HPLC was used to show full consumption of starting peptide and the reaction mixture was purified by semipreparative HPLC.

General conjugation protocol D -Internal Cys residue conjugation
To peptide dissolved in 20% DMSO in ligation buffer to a concentration of 5 mM was added a solution of TCEP (0.625 M stock solution in LB pH adjusted to 7, 50 eq.), TEMPO (0.1 M stock solution in DMSO, 2 eq.) and Mn(OAc)3 (0.1 M stock solution, 4 eq.). The pH of the reaction mixture was checked to be 7-7.5 then the reaction mixture diluted to the final peptide concentration was 2.5 mM.
The reaction was then heated to 50 o C and shaken for 1 hour. Once the starting material was shown to be fully consumed by HPLC the reaction mixture was purified by semi-preparative HPLC.

General conjugation protocol E -Internal Cys residue protein conjugation
To peptide dissolved in 20% DMSO in ligation buffer to a concentration of 2 mM was added a solution of TCEP (0.625 M stock solution in LB, pH adjusted to 7, 100 eq.), TEMPO (0.1 M stock solution in DMSO, 5 eq.) and Mn(OAc)3 (0.25 M stock solution in DMSO, 20 eq.). The pH of the reaction mixture was checked to be 7-7.5 then the reaction mixture diluted to the final peptide concentration was 1.0 mM. The reaction was then heated to 50 o C and shaken for 2 hours.
Once the starting material was shown to be fully consumed by HPLC the reaction mixture was purified by semi-preparative HPLC. Table S1. Summary of the conditions used in the general protocols above. Temp/
To the crude mesylate (1.30 g, 4.54 mmol) in 35% ammonia in water (50 ml) was added ammonium acetate (4.86 g, 90.8 mmol). This was stirred at room temperature for 48 hours. The reaction mixture was saturated with NaCl and extracted with DCM (5 x 20 ml). The combined organic layers were washed with brine (10 ml), dried over MgSO4, filtered and concentrated in vacuo.
The resulting colourless oil (0.85 g, 4.10 mmol) was redissolved in dry MeOH (20 ml) and, under N2, 4-oxoTEMPO (0.63 g, 3.73 mmol) and glacial acetic acid (0.19 ml, 3.36 mmol) were added. The reaction mixture was heated to 50 o C and stirred for 2 hours. The reaction mixture was cooled to room temperature and sodium cyanoborohydride (0.70 g, 11.2 mmol) was added and the reaction mixture again heated to 50 o C and stirred under N2 for 16 hours. The reaction mixture was cooled, then poured into saturated NaHCO3 (10 ml) and the aqueous layer extracted with DCM (5 x 20 ml). The organic layers were washed with brine (20 ml), dried over MgSO4, filtered and concentrated in vacuo. The resultant orange oil was purified by column chromatography (DCM: MeOH 9:1) to give the title compound as an orange oil (1.13 g, 3.13 mmol, 84% yield).  D-Biotin-OMe (47) was synthesized following a literature procedure [1] . To methanol (10 ml) at 0 o C was added acetyl chloride (1.46 ml, 20.5 mmol)

HRMS
dropwise. This was stirred for 5 minutes before adding dropwise to a suspension of D-Biotin (1.0 g, 4.09 mmol) in methanol (10 ml) at 0 o C. The resulting solution was allowed to warm to room temperature and stirred for 1 hour. Over this time the solution became clear. The reaction mixture was concentrated in vacuo, then redissolved in 2% methanol in DCM (50 ml). This solution was extracted with saturated NaHCO3 (3 x 10 ml), water (10 ml) and brine (10 ml), then dried over MgSO4, filtered and concentrated in vacuo to give the title compound as an off-white solid (1.04 g, 4.01 mmol, 98% yield). 1

D-Biotin-ethylenediamine (48)
D-Biotin-ethylenediamine (48) was synthesized following a literature procedure [1] . To D-Biotin-OMe (47, 1.0 g, 3.87 mmol) in methanol (25 ml) was added ethylenediamine (6.5 ml, 96.8 mmol). The resulting solution was stirred at 60 o C for 16 hours. TLC was used to confirm complete consumption of the S14 starting material at this point. The reaction mixture was concentrated in vacuo, with excess ethylenediamine being co-evaporated with toluene (3 x 20 ml

D-Biotin-EDA-TEMPO (6)
To 4-oxoTEMPO (0.5 g, 2.94 mmol) and D-biotin-EDA (48, 1.01 g, 3.52 mmol) in dry methanol (5 ml) under nitrogen was added glacial acetic acid (0.15 ml, 2.65 mmol). The solution was heated to 50 o C and stirred under nitrogen for 3 hours. At this time the solution was allowed to cool to room temperature and sodium cyanoborohydride (0.20 g, 3.23 mmol) was added in a single portion.

DMTMM
DMTMM was synthesized following a literature procedure [2] . To a solution of
The resultant oil was purified by column chromatography (CHCl3: MeOH 9.5:0.5) to give the title compound as an orange solid (0.15 g, 0.336 mmol, 40%
The layers were separated, and the aqueous phase was washed using diethyl ether (50 ml). The aqueous phase was then acidified to pH 2 using 1 M HCl(aq) and extracted using ethyl acetate (3 x 50 ml). The combined organics were

S20
The combined organic layers were washed with 1 M HCl (3 x 5 ml), saturated NaHCO3 (3 x 5 ml), water (5 ml) and brine (5 ml), then dried over MgSO4, filtered and concentrated in vacuo. The resultant residue was dissolved in a 1:1 solution of DCM: TFA (10 ml) and stirred at room temperature for 30 minutes before being concentrated in vacuo. The remaining TFA was co-evaporated using toluene (

EPR analysis of TEMPO traps (3 -7)
See General Methods for details of EPR equipment and analysis parameters.

Initial optimization of the conjugation protocol
Unless otherwise specified the following method was followed: To H-UAF-OMe ( minutes. All reactions were performed in a Thermo heater/shaker set to the specified temperature and shaken at 800 rpm. Conversion to product reported was measured based on integration of the product peak from the HPLC trace compared to integration of starting material and by-product peaks.

Temperature -Attempts to increase reaction rate
Trial 34 did not require doping to reach completion in a similar time-frame relative to the 'doped' comparison (trial 32) -using a third of the amount of TCEP and TEMPO. Mn(OAc)3 showed most promising result -completion in 30 minutes

H-VDNKFNKEMWAAWEEIRNLPNLNGWQMT-SePh
Synthesized using general automated synthesizer procedure with microwave assistance on 2-chlorotrityl chloride resin (0.2 mmol calculated loading). The selenoester was formed using the general procedure in the general methods section. The crude peptide was purified by preparative RP-HPLC (20-80% B over 30 minutes) and lyophilized to produce the desired peptide (40 mg, 0.011 mmol, 6% yield).

Desulfurization of Ac-CHISKY-OH (57) to give Ac-AHISKY-OH (55)
Analytical scale reaction performed using Ac-CHISKY-OH following general conditions A without the addition of TEMPO. Reactions were monitored after being at 37 o C for 30 minutes.

NMR comparison of product from L and D Sec TEMPO conjugation
Both the L-TEMPO conjugate (2a) and D-TEMPO conjugate (2b) were synthesized following general protocol A. Briefly, H-UAF-OMe (D or L) dissolved in LB/DMSO (20%) at pH 7 to 7.5 was added TCEP (50 eq.) and TEMPO (2 eq.) followed by Mn(OAc)3 (4 eq.). The pH was checked to be 7 to 7.5 and reaction mixture shaken at 50 o C for 30 minutes, then purified by preparative RP-HPLC.            Absorbance (mAU)

H-CAF-OMe (13) TEMPO Conjugation (14) Optimization Reactions
General protocol -Reactions performed on analytical scale (0.5 ml reaction volume) in ligation buffer (pH 7 to 7.5) with 20% DMSO at 50 o C using the specified peptide concentration and reagent equivalences. The reaction was analyzed at 1 and 2 hours. All resulted in complete conversion to TEMPO trapped product, with trial 48 using the lowest equivalences of each reagent.

Cys Conjugation -Optimization
All reactions performed at 50 o C for 4 hours. Reaction mixture analyzed by RP-HPLC (10-100% B over 5 min) at 2 and 4 hours.        Absorbance (mAU)

Conjugate stability studies
The TEMPO conjugate (2a) was dissolved in H2O/MeOH 1:1 to a concentration of 12.5 mM, then diluted to a concentration of 2.5 mM with the reagent specified in the "Conditions" column of Table S15. For entries 1 and 2 a solution of 0.01 M HCl (pH 2) and 0.01 M NMM buffer (pH 9) was added to the conjugate stock respectively. The final pH of these was checked to be as specified in Table S12.
For entry 6 the peptide was dissolved in H2O: MeOH: AcOH 8:1:1 and powdered zinc was added directly to the peptide solution. The percentage degradation was based on comparing the starting material peak integration from HPLC analysis to any by-product peaks formed during the reaction.

No Zinc
The same reaction conditions were applied to starting peptide 28, without the addition of zinc as a control. The reaction mixture was shaken at 37 o C for 16 hours then analyzed by analytical RP-HPLC.

Site-selective modification of ubiquitin K48C
Ubiquitin K48C mutant protein 45 was recombinantly expressed and purified as described in Garner et al. 2011 Biochemistry [3] with SDS PAGE analysis confirming ~99% purity (not shown).

QDKEGIPPDQQRLIFAGCQLEDGRTLSDYNIQKESTLHLVLRLRGG-OH
Synthesized following general protocol E using K48C Ubiquitin mutant (45, 6.6 mg, 0.774 µmol) followed by purification using semi-preparative RP-HPLC (25-45% B over 30 minutes) and lyophilisation to produce the desired conjugated protein 46 (4.2 mg, 0.482 µmol, 62% yield).    (red); NH region extended across 6.5-9.5 ppm indicating a tertiary fold.  (black) and Ub conjugate 46 (red); signal shifts around modified residue 48 highlighted. S139 Figure S124. Space filling and ribbon models of Ub; modified residue at position 48 highlighted in green, residues that experience significant deviation in chemical shifts for NH signal highlighted in red.