Enabling Cysteine‐Free Native Chemical Ligation at Challenging Junctions with a Ligation Auxiliary Capable of Base Catalysis

Abstract Ligation auxiliaries are used in chemical protein synthesis to extend the scope of native chemical ligation (NCL) beyond cysteine. However, auxiliary‐mediated ligations at sterically demanding junctions have been difficult. Often the thioester intermediate formed in the thiol exchange step of NCL accumulates because the subsequent S→N acyl transfer is extremely slow. Here we introduce the 2‐mercapto‐2‐(pyridin‐2‐yl)ethyl (MPyE) group as the first auxiliary designed to aid the ligation reaction by catalysis. Notably, the MPyE auxiliary provides useful rates even for junctions containing proline or a β‐branched amino acid. Quantum chemical calculations suggest that the pyridine nitrogen acts as an intramolecular base in a rate‐determining proton transfer step. The auxiliary is prepared in two steps and conveniently introduced by reductive alkylation. Auxiliary cleavage is induced upon treatment with TCEP/morpholine in presence of a MnII complex as radical starter. The synthesis of a de novo designed 99mer peptide and an 80 aa long MUC1 peptide demonstrates the usefulness of the MPyE auxiliary.

1 General Information

Materials and instruments
Commercially available compounds were used without further purification. Dry solvents were taken from a MBraun Solvent Purification System SPS 800. Purification of compounds by flash chromatography was done on silica gel (0.060-0.2 mm, 60 Å) from Acros Organics using technical grade solvents. TLC silica gel plates 60 F254 from Merck were used for thin-layer chromatography. NMR-spectra were recorded on a Bruker Avance II 500 MHz Spectrometer and referenced to the residual protonated solvent signal. Elemental analysis was carried out on a HEKAtech Eurovector3000.
High-resolution ESI-MS spectra were recorded on an Agilent 6220 TOF Accurate Mass coupled to an Agilent 1200 LC (Agilent Technologies, USA) and measured at 35 °C between 100 -2000 m/z. An Accucore RP-MS (30 x 2.1 mm; 2.6 µm particle size) was used as stationary phase at a flow of 0.8 mL/min and the following gradient (A = water, B = acetonitrile): 95 % A + 5 % B for 0.2 min, then 95 % A + 5 % B to 1 % A + 99 % B to 1.1 min, then 1 % A + 99 % B to 2.5 min.

S1
S2 23  Diisobutylaluminium hydride (12.3 mL, 1.0 M in toluene, 3.0 eq.) was added carefully over the vessel wall over the course of 1 h. The reaction mixture was stirred for an additional 15 min at -94 °C. Subsequently, a mixture of DCM:MeOH (1 mL, 1:1 v/v) was added over the vessel wall over the course of 30 min at -94 °C. The reaction mixture was stirred at room temperature for 10 min and a solution of potassium sodium tartrate (20 mL saturated) was added. The resulting mixture was stirred at room temperature for 1 h and then extracted with DCM (3x). The combined organic layers were dried over MgSO4, filtered and concentrated. The residue was purified by recrystallization (MeOH) to afford the enol 25 (1.12 g, 3.4 mmol, 82 %) as bright yellow crystals.

Storage of Auxiliary Precursor
From the available NMR data, it can be concluded that the auxiliary precursor 25 is exclusively present in its enol form. The compound is stable and did not decompose over a period of 7 month (see figure S5).

Synthesis of peptides 4.1 Fmoc-strategy
Loading of Tentagel Rink Amide Resin: The resin (~0.18 µmol/mg) was transferred into a syringe equipped with a filter frit and swollen in DMF (10 min).
The Fmoc-group was removed by treatment of the resin with a solution of 20 % piperidine in DMF (2x 5 min) and the initial loading estimated by quantification of the piperidine-fulvene adduct (λ = 301 nm, ε = 7800 M -1 cm -1 ).

Selenoester Formation after SPPS on 2-Chlorotrityl Resin:
After final Fmoc-deprotection the resin was treated with a solution of Boc-anhydride (50 eq.) and DIPEA (10 eq.) in DMF (0.4 M) for 1 h to protect the N-terminal amino group. The peptides were cleaved from the 2-chlorotrityl resin by treatment with 30 vol.% HFIP in DCM (2 mL/10 µmol peptide) for 2 h and the cleavage solution was collected by filtration and transferred into a round bottom flask. The resin was washed with DCM (2x 1 mL) and the combined filtrates were concentrated in vacuo. The resulting residue was dissolved in anhydrous DMF (300 µl/10 µmol peptide) and cooled to 0 °C. Diphenyldiselenide (30 eq., c = 1 M) in anhydrous DMF was added to the solution followed by nBu3P (30 eq.). The reaction was allowed to warm to room temperature and proceed for 3 h, after which time the solvent was removed in vacuo. The crude material was cooled to 0 °C and the protecting groups removed via treatment with TFA:TIS:H2O (95:2.5:2.5 v/v/v; 3 mL/10 µmol peptide). After 1 h the cleavage cocktail was reduced under a stream of Argon. Et2O (~8-10-fold volume) was added to the remaining solution which was subsequently cooled (in dry ice ~30 min) and centrifuged (4000 rpm, 15 min, 4 °C) and the ether decanted.
Afterwards the precipitate was suspended a second time in ether, centrifuged, decanted, and the pellet dissolved in H2O:ACN:TFA (1:1:0.001 v/v/v) and purified by preparative HPLC as indicated. [2]
-Final cleavage: The resin was washed with DCM and dried under vacuum. Then a mixture of TFA:TFMSA:mCresol (16:3:1 v/v/v; 3 mL/10 µmol peptide) was added to the resin. After 2 h the cleavage cocktail was collected by filtration, the resin washed with TFA (3x 0.5 mL) and the combined filtrates were concentrated (~ 1 mL).
Gravimetric determination: Concentrations of peptides bearing no Tyr-or Trp-residue were determined by weighing of the lyophilized peptide and dissolving it afterwards in a certain volume. Basic amino acid residues (Arg, Lys, His) and free N-terminus were assumed to be present in their corresponding TFA salts and considered for the MW of the weighted peptide .

LYRAA-S(CH2)2(CO)-Gly (28A)
The synthesis was carried out on a MBHA resin (~0.67 µmol/mg) in a 25 µM scale following the manual Boc-strategy protocol for the synthesis of peptide thioester (see 4.2). After cleavage from the resin and peptide precipitation, the crude peptide thioester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide thioester 28A was isolated as a white solid after lyophilization (see figure S10),

LYRAL-S(CH2)2(CO)-Gly (28L)
The synthesis was carried out on a MBHA resin (~0.67 µmol/mg) in a 25 µM scale following the manual Boc-strategy protocol for the synthesis of peptide thioester (see 4.2). After cleavage from the resin and peptide precipitation, the crude peptide thioester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide thioester 28L was isolated as a white solid after lyophilization (see figure S11),

LYRAP-S(CH2)2(CO)-Gly (28P)
The synthesis was carried out on a MBHA resin (~0.67 µmol/mg) in a 10 µM scale following the manual Boc-strategy protocol for the synthesis of peptide thioester (see 4.2). After cleavage from the resin and peptide precipitation, the crude peptide thioester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide thioester 28P was isolated as a white solid after lyophilization (see figure S12), which was dissolved in H2O/ACN/TFA (1:1:0.001 v/v/v) for a spectroscopic determination of the synthesis yield.

LYRAA-SePh (31A)
The synthesis was carried out on a polystyrol 2-chlorotrityl resin (~1.02 µmol/mg) in a 22 µM scale. After the initial loading step the synthesis followed the automated Fmoc-strategy protocol with subsequent selenoester formation (see 4.1). After removal of all protecting groups and peptide precipitation, the crude peptide selenoester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide selenoester 31A was isolated as a white solid after lyophilization (see figure S13), which was dissolved in

LYRAL-SePh (31L)
The synthesis was carried out on a polystyrol 2-chlorotrityl resin (~1.02 µmol/mg) in a 20 µM scale. After the initial loading step the synthesis followed the automated Fmoc-strategy protocol with subsequent selenoester formation (see 4.1). After removal of all protecting groups and peptide precipitation, the crude peptide selenoester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide selenoester 31L was isolated as a white solid after lyophilization (see figure S14), which was dissolved in

LYRAP-SePh (31P)
The synthesis was carried out on a polystyrol 2-chlorotrityl resin (~1.02 µmol/mg) in a 15 µM scale. After the initial loading step the synthesis followed the automated Fmoc-strategy protocol with subsequent selenoester formation (see 4.1). After removal of all protecting groups and peptide precipitation, the crude peptide selenoester was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired peptide selenoester 31P was isolated as a white solid after lyophilization (see figure S15), which was dissolved in  6 Introduction of the Auxiliary
Afterwards the ether phase was decanted. The remaining peptide was dissolved in H2O:ACN:TFA (1:1:0.001 v/v/v) and purified by preparative HPLC as indicated.

MPyE-GRAEYSGLG (27G)
Synthesis was achieved by automated solid phase peptide synthesis following the protocol described in 4.1. After removal of the last Fmoc-group (loading 20.0 µmol) the auxiliary was introduced by reductive alkylation for 2 h (see 6.1). Complete conversion was verified by UPLC-MS analysis of test cleavage reaction (see figure S16). After cleavage from the resin and peptide precipitation, the crude peptide was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired auxiliary peptide 27G was isolated as a white solid after lyophilization, which was dissolved in H2O/ACN/TFA (1:

MPyE-RRAEYSGLG (27R)
Synthesis was achieved by automated solid phase peptide synthesis following the protocol described in 4.1. After removal of the last Fmoc-group (loading 20.0 µmol) the auxiliary was introduced by reductive alkylation for 18 h (see 6.1). Complete conversion was verified by UPLC-MS analysis of test cleavage reaction (see figure S18). After cleavage from the resin and peptide precipitation, the crude peptide was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired auxiliary peptide 27R was isolated as a white solid after lyophilization, which was dissolved in H2O/ACN/TFA (1:

MPyE-VRAEYSGLG (27V)
Synthesis was achieved by automated solid phase peptide synthesis following the protocol described in 4.1. After removal of the last Fmoc-group (loading 20.8 µmol) the auxiliary was introduced by reductive alkylation for 18 h (see 6.1). Complete conversion was verified by UPLC-MS analysis of test cleavage reaction (see figure S19). After cleavage from the resin and peptide precipitation, the crude peptide was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired auxiliary peptide 27V was isolated as a white solid after lyophilization, which was dissolved in H2O/ACN/TFA (1:

MPyE-LRAEYSGLG (27L)
Synthesis was achieved by automated solid phase peptide synthesis following the protocol described in 4.1. After removal of the last Fmoc-group (loading 21.0 µmol) the auxiliary was introduced by reductive alkylation for 18 h (see 6.1). Complete conversion was verified by UPLC-MS analysis of test cleavage reaction (see figure S20). After cleavage from the resin and peptide precipitation, the crude peptide was purified by preparative HPLC using a

Synthesis of Ligation Product AG (29AG)
500 nmol of MPyE-peptide 27G and 1 µmol of peptide thioester 28A were dissolved in 100 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S23).
The crude ligation product was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired ligation product 29AG was isolated as a white solid after lyophilization, which was dissolved in

Synthesis of Ligation Product LG (29LG)
1 µmol of MPyE-peptide 27G and 2 µmol of peptide thioester 28L were dissolved in 200 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S24). After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired ligation product 29LG was isolated as a white solid after lyophilization, which was dissolved in

Synthesis of Ligation Product AN (29AN)
2 µmol of MPyE-peptide 27N and 3 µmol of peptide thioester 28A were dissolved in 400 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S25). The reaction was quenched by addition of 20 µL of 51 % aqueous hydrazine solution and subsequently 20 µL of 1 M TCEP solution. After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient

Synthesis of Ligation Product LN (29LN)
2 µmol of MPyE-peptide 27N and 3 µmol of peptide thioester 28L were dissolved in 400 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S26). The reaction was quenched by addition of 20 µL of 51 % aqueous hydrazine solution and subsequently 20 µL of 1 M TCEP solution. After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient

Synthesis of Ligation Product AR (29AR)
2 µmol nmol of MPyE-peptide 27R and 3 µmol of peptide thioester 28A were dissolved in 400 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S27).

Synthesis of Ligation Product LR (29LR)
2 µmol of MPyE-peptide 27R and 3 µmol of peptide thioester 28L were dissolved in 400 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S28). The reaction was quenched by addition of 20 µL of 51 % aqueous hydrazine solution and subsequently 20 µL of 1 M TCEP solution. After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient

Synthesis of Ligation Product AV (29AV)
2 µmol of MPyE-peptide 27V and 3 µmol of peptide thioester 28A were dissolved in 400 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S29). The reaction was quenched by addition of 20 µL of 51 % aqueous hydrazine solution and subsequently 20 µL of 1 M TCEP solution. After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient

Synthesis of Ligation Product AL (29AL)
1 µmol of MPyE-peptide 27L and 1.5 µmol of peptide thioester 28A were dissolved in 200 µL ligation buffer (see 7.1) and allowed to shake under argon atmosphere until completion of the ligation reaction (see figure S30). The reaction was quenched by addition of 10 µL of 51 % aqueous hydrazine solution and subsequently 10 µL of 1 M TCEP solution. After quenching, the crude ligation product was purified by preparative HPLC using a linear gradient  [x] thioester formed by N→S rearrangement. [w] 27N with cleaved auxiliary. *the two auxiliary stereoisomers elute as a double peak.

Comparison between Proline Seleno-and Proline Thioester in Ligation with a MPyE-Peptide
The MPyE-peptide 27R was reacted with peptide thioester following the general procedure 7.1 and with the peptide selenoester 31P following the general procedure 8.1. We assume that the mechanism of cleavage is comparable to that of the MPE auxiliary (see figure S35).

Removal of Auxiliary from Ligation Product AG (37AG)
To 315 nmol of ligated peptide 29AG, 315 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 3 h and the crude ligation product was purified by preparative HPLC rinsing initially for 10 minutes at 15 % B and subsequently using a linear gradient from 15-30 % B in 30 min. The desired auxiliary removal product 37AG was isolated as a white solid after lyophilization (see figure S36), which was dissolved in

Removal of Auxiliary from Ligation Product LG (37LG)
To 255 nmol of ligated peptide 29LG, 255 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 3 h and the crude ligation product was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired auxiliary removal product 37LG was isolated as a white solid after lyophilization (see

Removal of Auxiliary from Ligation Product AN (37AN)
To 175 nmol of ligated peptide 29AN, 175 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 24 h and the crude ligation product was purified by preparative HPLC rinsing initially for 10 minutes at 15 % B and subsequently using a linear gradient from 15-30 % B in 30 min. The desired auxiliary removal product 37AN was isolated as a white solid after lyophilization (see figure S38), which was dissolved in H2O/ACN/TFA

Removal of Auxiliary from Ligation Product AR (37AR)
To 315 nmol of ligated peptide 29AR, 315 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 24 h and the crude ligation product was purified by preparative HPLC rinsing initially for 10 minutes at 15 % B and subsequently using a linear gradient from 15-30 % B in 30 min. The desired auxiliary removal product 37AR was isolated as a white solid after lyophilization (see figure S39), which was dissolved in  [y] nonpeptidic material.

Removal of Auxiliary from Ligation Product AV (37AV)
To 510 nmol of ligated peptide 29AV, 510 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 24 h and the crude ligation product was purified by preparative HPLC rinsing initially for 10 minutes at 15 % B and subsequently using a linear gradient from 15-30 % B in 30 min. The desired auxiliary removal product 37AV was isolated as a white solid after lyophilization (see figure S40), which was dissolved in

Removal of Auxiliary from Ligation Product LN (37LN)
To 190

Removal of Auxiliary from Ligation Product LR (37LR)
To 190 nmol of ligated peptide 29LR, 190 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 24 h and the crude ligation product was purified by preparative HPLC using a linear gradient from 3-30 % B in 40 min. The desired auxiliary removal product 37LR was isolated as a white solid after lyophilization (see

Removal of Auxiliary from Ligation Product AL (37AL)
To 420 nmol of ligated peptide 29AL, 420 µL auxiliary cleavage mixture was added (see 10.1). The mixture was shaken for 24 h and the crude ligation product was purified by preparative HPLC rinsing initially for 10 minutes at 15 % B and subsequently using a linear gradient from 15-30 % B in 30 min. The desired auxiliary removal product 37AL was isolated as a white solid after lyophilization (see figure S43), which was dissolved in

Product Formed by Removal Mixture
Incubation of the removal cocktail over a period of 24 h leads to the formation of a non-peptidic product with a m/z of 475. This not yet identified side product forms regardless of the presence of peptide (see figure S44) and has not been observed to be detrimental for removal reaction. During HPLC purification, overlap with the product peaks of polar peptides (37AN, 37AG, 37AV, 37AR) could be avoided by rinsing with 15 % ACN before applying the gradient for purification.

MUC1 (39-80) MPyE-Peptide (43)
The peptide was synthesized on a rink-amide tentagel resin via automated Fmoc-synthesis (see 4.1, loading 11.7 µmol). After removal of the N-terminal Fmoc-group the auxiliary was introduced by reductive alkylation of 49 for 24 h (see general procedure, 6.1). Following 30 h of cleavage from the resin with TFA:TIS:H2O (96:2:2 v/v/v) and peptide precipitation, the crude peptide was purified by preparative HPLC using a linear gradient from 3-20 % B in 50 min. The desired auxiliary peptide 43 (5.87 mg, 1.2 µmol, 11 % (based on 4100.51 as 7 x TFAsalt)) was isolated as a white solid after lyophilization (see figure S50).   14 Scope and Limitations of the MPyE Auxiliary

Ligation at difficult ligation sites
Reaction at LL and LV ligation sites proceeded slowly. For the LV junction, approximately half of the starting material was converted to the corresponding ligation product after 24 h and a large portion of thioester 28L was hydrolyzed (see figure S54). For these ligation sites, we therefor recommend using the peptide thioester in larger excess. Alternatively, the selenoester method may be applied.

Decomposition of MPyE-Peptide in Presence of Oxidizing Agents
We noticed a rapid decomposition of MPyE-peptides, when brought into contact with peroxide impurities (from Et2O) or DMSO. We therefore recommend avoiding the use of any oxidizing agents.