Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji–Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.


Reversibility of the Prenylation
. Time dependence of the n/i ratio during the Pd-catalyzed prenylation of 1-octanethiol.

Kinetics of the Pd-Catalyzed Prenylation
To ensure catalyst integrity during reaction monitoring the reactions were carried out in a glovebox.

Preparation of stock solutions: (under oxygen-free conditions)
In a flame-dried and argon-flushed 10 mL Schlenk flask, equipped with a Teflon-coated magnetic stirring bar, Pd(dba)2 (1.4 mg, 2.4 µmol) and BIPHEPHOS (1.9 mg, 2.4 µmol) were suspended in 2.0 mL anhydrous CH3CN, stirred in a pre-heated oil bath at 60 °C for 30 min to obtain a bright yellow solution and cooled to rt. To this solution, Rc (0.67 mg, 2.4 µmol) was added (= stock A).

Reaction: (under oxygen-free conditions)
In a flame-dried and argon-flushed 10 mL Schlenk flask, equipped with a Teflon-coated magnetic stirring bar,

General Information
If reactions were performed under inert conditions, e.g. exclusion of water, oxygen or both, all experiments were carried out using established Schlenk techniques or inside a Glovebox (MBraun UNIlab pro). Herein solvents were dried and/or degassed with common methods and afterwards stored under inert gas atmosphere (argon or nitrogen) over molecular sieves. In some cases, when explicitly mentioned, dry solvents were received from the mentioned suppliers. In general, when high vacuum (in vacuo) was stated in experimental procedures, typically a vacuum of 10 -2 -10 -3 mbar was applied. Degassing of solvents or reaction mixtures was performed by bubbling argon from a balloon via cannula through the solvent or the reaction mixture during ultrasonication for about 20 min.
All reagents were added in a counterstream of inert gas to keep the inert atmosphere. All reactions were stirred with Teflon-coated magnetic stirring bars.
Molecular sieves (Sigma-Aldrich, beads with 8-12 mesh) were activated in a round-bottom flask with a gas inlet adapter by heating them carefully in a heating mantle at level 1 at least for 24 h under high vacuum until complete dryness was obtained. These activated molecular sieves were stored at rt under argon atmosphere.
Temperatures were measured externally if not otherwise stated. When working at a temperature of 0 °C, an icewater bath served as the cooling medium. Lower temperatures were achieved by using an acetone/dry ice cooling bath. Reactions, which were carried out at higher temperatures than rt, were heated in a silicon oil bath on a heating plate (RCT basic IKAMAG® safety control, 0-1500 rpm) equipped with an external temperature controller.

Chemicals
All commercially available chemicals and solvents were purchased from Acros Organics, Alfa Aesar, Fisher, Fluka, Honeywell, Merck, Roth, Sigma-Aldrich, TCI, VWR and used without further purification, unless otherwise stated.
Protected Fmoc-amino acids, resins and coupling reagents were purchased from Novabiochem and Iris. The medium and buffers for protein expression and purification were prepared with substances from Roth, Sigma-Aldrich and PanReac AppliChem.
Acetonitrile: Anhydrous acetonitrile was purchased from Alfa Aesar. It was transferred into an amber 1 L Schlenk bottle and stored over activated 3 Å MS under argon atmosphere.
Dichloromethane: Anhydrous dichloromethane was produced by pre-drying EtOH stabilized dichloromethane over P4O10 and afterwards heating it under reflux over CaH2 for 24 h under argon atmosphere. It was distilled into an amber 1 L Schlenk bottle over activated 4 Å MS and under argon atmosphere. N,Dimethylformamide was purchased in extra dry quality from Alfa Aesar. It was transferred into an amber 1 L Schlenk bottle and stored over activated 4 Å MS under argon atmosphere.
Methanol: Methanol was purchased from Fisher and heated under reflux over Mg and I2 for 2 h. It was distilled into an amber 1 L Schlenk bottle and stored over activated 3 Å MS under argon atmosphere.
Solvents for peptide synthesis and chromatography were of "peptide synthesis grade" or "HPLC grade".

Thin Layer Chromatography
Analytical thin layer chromatography (TLC) was carried out on Merck TLC silica gel aluminum sheets (silica gel 60,   F254, 20 x 20 cm). All separated compounds were visualized by UV light (λ = 254 nm and/or λ = 366 nm) and by the listed staining reagents followed by the development in the heat.

Flash Column Chromatography
Flash column chromatography was performed on silica gel 60 from Acros Organics with particle sizes between 35 µm and 70 µm. Depending on the problem of separation, a 30 to 100 fold excess of silica gel was used with respect to the dry amount of crude material. The dimension of the column was adjusted to the required amount of silica gel and formed a pad between 10 cm and 30 cm. In general, the silica gel was mixed with the eluent and the column was equilibrated. Subsequently, the crude material was dissolved in the eluent and loaded onto the top of the silica gel and the mobile phase was forced through the column using a rubber bulb pump. The volume of each collected fraction was adjusted between 20 % and 40 % of the silica gel volume.

Gas Chromatography
GC-MS analyses were performed on an Agilent Technologies 7890A GC system equipped with a 5975C mass selective detector (inert MSD with Triple Axis Detector system) by electron-impact ionization (EI) with a potential of E = 70 eV. Herein, the samples were separated depending on their boiling point and polarity. The desired crude materials or pure compounds were dissolved and the solutions were injected by employing the autosampler 7683B in a split mode 1/20 (inlet temperature: 280 °C; injection volume: 0.2 μL). Separations were carried out on an Agilent Technologies J&W GC HP-5MS capillary column ((5 %-phenyl)methylpolysiloxane, 30 m x 0.2 mm x 0.25 μm) with a constant helium flow rate (He 5.0 (Air Liquide), 1.085 mL•min -1 , average velocity: 41.6 cm•s -1 ). A general gradient temperature method was used: 50S: initial temperature: 50 °C for 1 min; linear increase to 300 °C (40 °C•min -1 ); hold for 5 min; 1 min post-run at 300 °C; detecting range: 50.0-550.0 amu; solvent delay: 2.60 min.

High Resolution Mass Spectrometry
High-resolution mass spectra were recorded on a Waters Micromass GCT Premier system. Ionization was realized by an electron impact source (EI ionization) at a constant potential of 70 eV. Herein, individual samples were either inserted directly (direct inlet electron impact ionization; DI-EI) or prior to this gas chromatographically separated on an Agilent 7890A system equipped with an Agilent Technologies J&W GC-column DB-5MS (length: 30 m; innerdiameter: 0.250 mm; film: 0.25 µm) at a constant helium flow. Molecule ions were analyzed by a time-of-flight (TOF) mass analyzer in the positive mode (TOF MS EI+).
Further high-resolution mass spectra were recorded using MALDI TOF on a Waters Micromass® MALDI micro MX Mass spectrometer. Dithranol (1,8-dihydroxy-9,10-dihydroanthracen-9-one) served as matrix and PEG as internal standard. Besides molecular formulas, calculated as well as determined m/z ratios of each molecule peak are denoted.
Further high-resolution mass spectra were recorded using a Bruker maXis UHR-TOF system (Qq-TOF instrument).
The samples were injected directly, ionized via electrospray ionization (ESI) and analyzed in positive mode.

Determination of Melting Points
Melting points were determined on a Mel-Temp® melting point apparatus from Electrothermal with an integrated microscopical support. They were measured in open capillary tubes with a mercury-in-glass thermometer and were not corrected.

Determination of Optical Rotation
The specific optical rotation was determined on a Perkin Elmer Polarimeter 341 with an integrated sodium vapor lamp. All samples were measured at the D-line of the sodium light (λ = 589 nm) in a 10 cm cell. Concentrations are given in g/100 mL. Each optical rotation measurement was performed five times and the mean value is reported.

Synthesis of Allylic Alcohols Ethyl (E)-6-bromohex-2-enoate (1)
The intermediate (4-bromobutanal) was prepared according to a procedure described by Brown et al. [1] In a flame-dried and argon-flushed 1 L two-necked round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, ethyl 4-bromobutyrate (3.91 g, 20.0 mmol) was dissolved in anhydrous CH2Cl2 (300 mL) and cooled to -78 °C (dry ice/acetone). Subsequently, DIBAL-H (24.0 mL, 1.0 M soln. in CH2Cl2, 24.0 mmol) was added over 10 min and the mixture was stirred at -78 °C for 1 h. Upon complete consumption of the starting material (according to TLC), the reaction mixture was quenched by the addition of 200 mL 1 M HCl and slowly warmed to rt. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and carefully concentrated under reduced pressure (700 mbar, 35 °C) to a final volume of approx. 30 mL.
Then a solution of hex-5-yn-1-ol (0.997 g, 10.2 mmol) in anhydrous CH2Cl2 (12.5 mL) was slowly added. The resulting suspension was stirred for 15 min before triethylamine (7.1 mL, 51 mmol) was added. The reaction mixture was stirred for 30 min at -78 °C after which it was allowed to warm to rt (90 min, reaction monitoring via TLC).
Then (carbethoxymethylene)triphenylphosphorane (4.24 g, 12.2 mmol) was added in one portion and the light orange suspension was stirred at rt overnight (reaction monitoring via TLC) and concentrated under reduced pressure. The crude product was adsorbed on 15 g SiO2 and purified via flash column chromatography (500 g SiO2, 19.5 x 8.0 cm, cyclohexane:EtOAc = 15:1 (v/v)) to give the desired compound as yellowish oil (1.29 g, 76 % over two steps).  7, 147.9, 122.2, 83.6, 69.1, 60.3, 31.0, 26.9, 18.0, 14.4. Analytical data is in accordance with the literature. [4] (E)-Oct-2-en-7-yn-1-ol (5) This compound was prepared similar to a procedure described by Grafton et al. [4] In an evacuated and argon-flushed 100 mL round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, ethyl (E)-oct-2-en-7-ynoate (4) (1.26 g, 7.6 mmol) was dissolved in anhydrous CH2Cl2 (20 mL) and cooled to -78 °C (dry ice/acetone). Subsequently, DIBAL-H (16 mL, 1.0 M soln. in CH2Cl2, 16 mmol) was added over 15 min and the mixture was stirred at -78 °C for 30 min. During addition the colorless solution turns brightly yellow and decolorizes afterwards again. Upon complete consumption of the starting material (according to TLC), the reaction mixture was quenched by the addition of 20 mL 1 M HCl and slowly warmed to rt. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (10 mL). The combined organic layers were washed with H2O (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give the desired compound as slightly yellowish oil (0.928 g, 98 %), which was used without further purification.

Synthesis of Allylic Carbonates General Procedure for the Synthesis of Allylic Carbonates
In a round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, methyl chloroformate was slowly added to an ice-cold solution of the corresponding allylic alcohol and pyridine in CH2Cl2. The resulting suspension was allowed to warm to rt. Upon complete consumption of the starting material (according to TLC), the reaction mixture was quenched by the addition of H2O (1/3 of solvent volume) and stirred vigorously for at least 15 min. The organic layer was separated, washed twice with 1 M HCl (1/1 of solvent volume) and once with sat. NaHCO3 (1/2 of the solvent volume), dried over Na2SO4, filtered and concentrated under reduced pressure (in case of volatile products a minimal pressure of 40 mbar at 35 °C was applied).
Allylic carbonates Ra-Rc, Ri, Rj were distilled to afford colorless products prior to use in allylation reactions, although crude products did not show any impurities according to 1 H-NMR-spectroscopy.

Ac-Cys-OMe (6)
This compound was prepared according to a procedure described by Bang et al. [9] In a flame-dried and argon-flushed 250 mL round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, N-Acetyl-L-cysteine (5.01 g, 30.7 mmol) was dissolved in anhydrous MeOH (100 mL). Then SOCl2 (2.6 mL, 36 mmol) was added over a period of 10 min and the colorless solution was stirred at rt for 90 min. Upon complete consumption of the starting material (according to TLC), the solution was concentrated under reduced pressure.
The residue was dissolved in EtOAc (80 mL

Boc-Cys-Tyr-OMe (P1)
In an evacuated and argon-flushed 10 mL round-bottom flask, equipped with a Teflon-coated magnetic stirring bar, Boc-Cys(Trt)-Tyr-OMe (7) (169 mg, 264 µmol) was dissolved in anhydrous CH2Cl2 (2.7 mL) and Et3SiH (51.0 µL, 319 µmol) was added. Subsequently, TFA (102 µL, 1.32 mmol) was added to the stirred solution at room temperature whereupon a bright yellow color appeared. After 30 min the yellow color had disappeared and complete consumption of the starting material was indicated by TLC. The reaction mixture was quenched by the addition of sat. NaHCO3 (3 mL). The aqueous phase was separated and back-extracted with CH2Cl2 (2 x 3 mL).

General Procedure for the Allylation of Small Peptides
In a flame-dried and argon-flushed 10 mL Schlenk flask, equipped with a Teflon-coated magnetic stirring bar, Pd(dba)2 and BIPHEPHOS were suspended in 1.0 mL anhydrous CH3CN and stirred in a pre-heated oil bath at 60 °C for 30 min to obtain a bright yellow solution. Then allylic carbonate and Boc-Cys-Tyr-OMe (P1) were added and the resulting mixture was stirred at 60 °C for 2 h. The yellow-tan solution was cooled to rt and concentrated under reduced pressure. The crude product was purified via flash column chromatography to afford the desired compound.

Peptide Synthesis
Peptides were synthesized by the Fmoc strategy and pre-loaded Wang and Tentagel resins were used as solid support. All equivalents are calculated based on the theoretical loading of the resin.

Manual Peptide Synthesis
All synthesis steps were performed in fritted syringes at rt on a rotator.
Before starting the synthesis, the resin was swollen in DMF for 1 h. Fmoc deprotection was performed with 20 % piperidine in DMF for 2 x 5 min and the resin was washed with DMF (3x). Fmoc amino acids (2.5 eq.) were dissolved in HBTU (0.5 M, 2.38 eq.) with DIPEA (5 eq.) and added to the resin for 2 x 30 min. After coupling, the resin was washed with DMF (3x) and at the end of the synthesis the resin was washed with DCM (3x) and dried.
For final cleavage the dry resin was treated with the cleavage solution (92.5 % TFA, 2.5 % EDT, 2.5 % TIPS, 2.5 % H2O) for 3 h. The peptide was precipitated with cold diethyl ether and after centrifugation (4000 rpm, 10 min, 4 °C) the supernatant was removed. The crude peptide was dissolved in 6 M Gdn-HCl pH 4.7 and purified by preparative HPLC.

Automated Peptide Synthesis
The following peptides were synthesized with a microwave-based synthesizer (Liberty Blue, CEM). Fmoc was removed with 20 % piperidine in DMF for 80 s at 90 °C. Amino acids were coupled in DMF, Fmoc-Xaa-OH (5 eq., 0.2 M), Oxyma (5 eq., 1 M) and DIC (5 eq., 0.5 M) for 2 min at 90 °C. For some amino acids double or triple couplings were performed (see below). Histidine was coupled 8 min at 50 °C.

UBL3 Variants
The following plasmids were ordered from eurofins and delivered in a pEX-A2 vector with ampicillin resistance.
They were cloned into a pET-28a(+) vector with a kanamycin resistance. An overnight culture of E. coli BL21 DE3 transformed with the UBL3 plasmid was incubated in 100 mL LB medium containing kanamycin (30 µg/mL) at 37 °C. It was diluted with 1 L of fresh medium (30 µg/mL kanamycin) to an OD600 of 0.2-0.3 and incubated at 37 °C again. The cells were induced on reaching an OD600 of 0.6-0.8 with 1 mM IPTG and grown at 37 °C for 6 h. After harvesting by centrifugation (6000 rpm, 30 min, 4 °C), the pellet was resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.5) and the cells were disrupted. After centrifugation (20000 rpm, 30 min, 4 °C) the supernatant was loaded on a HisTrap Ni-NTA column equilibrated with TBS buffer and it was eluted by using a linear imidazole gradient (0-300 mM imidazole in TBS). The pooled fractions were dialyzed against TBS buffer and the His6 Tag was removed by a ratio of 1:30 of TEV protease. The TEV protease was removed by a HisTrap Ni-NTA column (linear gradient, 0-300 mM imidazole in TBS). Fractions containing the protein were pooled, 20 µL 100 µM TCEP were added and the protein was dialyzed against H2O, lyophilized and stored at -20 °C. The protein was analyzed by HPLC and MS to confirm purity and molecular weight.

Hsp27 Protein
The following plasmid in pAK3038 vector was used for the expression of Hsp27:   Following the general procedure, peptide (P3) (3.8 mg, 2.

CuAAC of Hsp27-alkyne with azide-PEG3-biotin
The CuAAC was carried out in an Eppi with a reaction volume of 10 µL with final concentrations 1 mM Hsp27alkyne, 3 mM azide-PEG3-biotin, 24 mM CuSO4, 26 mM TBTA and 40 mM sodium ascorbate. 1.2 µL 200 mM CuSO4 (in water) and 2.6 µL 100 mM TBTA (in DMF) were mixed and 0.8 µL 500 mM sodium ascorbate (in water) was added. To this, the mixture of Hsp27-alkyne and azide-PEG3-biotin in DMF was added. The reaction was stirred for 10 min at rt and analysis by HPLC(-MS) showed full conversion. For this, the sample was diluted with