Amphiphilic Iodine(III) Reagents for the Lipophilization of Peptides in Water

Abstract We report the functionalization of cysteine residues with lipophilic alkynes bearing a silyl group or an alkyl chain using amphiphilic ethynylbenziodoxolone reagents (EBXs). The reactions were carried out in buffer (pH 6 to 9), without organic co‐solvent or removal of oxygen, either at 37 °C or room temperature. The transformation led to a significant increase of peptide lipophilicity and worked for aromatic thiols, homocysteine, cysteine, and peptides containing 4 to 18 amino acids. His6‐Cys‐Ubiquitin was also alkynylated under physiological conditions. Under acidic conditions, the thioalkynes were converted into thioesters, which could be cleaved in the presence of hydroxylamine.


Preparation of amphiphilic reagents
Preparation of (5a) Following a reported procedure, 1 2-amino-5-sulfobenzoic acid (4.34 g, 20.0 mmol, 1.0 equiv.) was suspended in a 10% aqueous hydrochloric acid solution (100 mL) and cooled to 0 °C. A cooled solution of sodium nitrite (NaNO2, 3.45 g, 50.0 mmol, 2.5 equiv.) in water (18 mL) was slowly added over a period of 45 minutes. After an additional 30 minutes stirring at this temperature, a cooled solution of potassium iodide (KI, 19.9 g, 120 mmol, 6.0 equiv.) in water (75 mL) was slowly added over a period of 1 hour at 0 °C. The resulting dark solution was allowed to warm to room temperature and stirred for 16 hours. Then, the reaction was slowly quenched by small portions of sodium bisulfite (around 14 g) until the solution persistently turned as a light-yellow 2 suspension. The resulting suspension was filtered, washed with acetone (3 x 100 mL) and dichloromethane (50 mL) to afford a yellow pale solid. The collected solid was then recrystallized from water and washed with cold water (2 x 50 mL), acetone (2 x 50 mL) and dichloromethane (2 x 50 mL) to yield pure 5a (3.71 g, 10.1 mmol, 51% yield) as a pale-yellow solid. Preparation of TIPS-EBX-SO3M (4a) Trimethylsilyl trifluoromethanesulfonate (TfOTMS, 2.55 mL, 14.1 mmol, 3.0 equiv.) was added dropwise to a stirred suspension of potassium 2-iodosyl-5-sulfobenzoate (1.80 g, 4.71 mmol, 1.0 equiv.) in dichloroethane (157 mL) at 40 °C.
After 2 h stirring at this temperature, triisopropyl((trimethylsilyl)ethynyl)silane (2.64 g, 10.4 mmol, 2.2 equiv.) was slowly added to the solution. The reaction mixture was stirred for another 18 h and pyridine (2.29 mL, 28.3 mmol, 6.0 equiv.) was added. After 2 additional hours stirring, the mixture was diluted with dichloromethane (200 mL), washed with a 0.5 N aqueous sodium bicarbonate solution (150 mL) and a 0.5 N aqueous hydrochloric acid solution (150 mL). The organic layer was dried over magnesium sulfate, filtered and the volatiles were removed in vacuo. The crude orange oil was purified by column chromatography (SiO2, Dichloromethane:Methanol gradient from 9:1 to 8:2) to yield pure K/Na-5-sulfonate TIPS-

Optimization of reaction conditions
Experimental procedure for optimization of 4a A solution of glutathione (6, GSH) in non-degassed 10 mM Tris buffer pH 7.4 was prepared. Then, a solution of 4a in nondegassed 10 mM Tris buffer pH 7.4 was added to the solution of glutathione 6 in a 1.5 mL vial. The mixture was then stirred at room temperature under "open-flask" conditions. After 6 hours, the labeling furnished a remarkable 47% yield of the alkynylated glutathione 6a (Table S1, Entry 1). Notably, we did not observe any formation of VBX derivatives. A similar procedure adopted for the other entries of the optimization, Table S1, entry 2-20.

S17
Experimental procedure for optimization of 4b A 1.5 mL vial was charged with glutathione (6, 0.50 mg, 1.6 µmol) and 4b (1.1 mg, 1.9 µmol) in 163 µL 10 mM Tris pH 7.4 with small magnetic stirring bar, and the reaction mixture was stirred at rt. After 2 h, a 25 µL aliquot of the reaction mixture was diluted with 25 µL acetonitrile to make a clear solution. Then the solution was submitted to HPLC. According to HPLC-MS chromatogram, 25% alkynylated product (6b), 7% VBX (6c) and 50% disulfide were observed (Table S2, entry 1). There was a large difference in integral ration observed in HPLC-UV and HPLC-MS chromatograms due to difference in absorption of products 6b and 6c, therefore the yields reported in the table S2 and manuscript are based on HPLC-MS chromatogram, which is more accurate. A similar experimental procedure was followed for the other entries of Table S2, entries 2-12. a Relative ratio of alkynylated (6b), VBX (6c) and disulfide based on HPLC-MS chromatogram. Equivalent of 4b calculated based on K salt. Yields in parentheses refers to relative ratio of 6b and 6c based on HPLC-UV chromatogram. b By-product observed.

Reaction of TIPS-EBX (1) and glutathione (6).
A 1.5 mL vial was charged with glutathione (6, 1.0 mg, 3.2 µmol) and 1 (2.3 mg, 5.3 µmol) in 326 µL 10 mM Tris pH 7.4 with small magnetic stirring bar, and the reaction mixture was stirred at 37 °C for 5 h. After 5 h, a 20 µL aliquot of the reaction mixture was diluted with 30 µL acetonitrile to make a clear solution. Then the solution was submitted to HPLC.

HPLC-MS chromatogram of the reaction mixture
Reaction of 4a and glutathione (6). The same procedure was followed as for the reaction of TIPS-EBX (1) and glutathione (6).
HPLC-MS chromatogram of the reaction mixture GSH disulfide 6a S19 Reaction of 4b and glutathione (6). The same procedure was followed as for the reaction of TIPS-EBX (1) and glutathione (6).

HPLC-MS chromatogram of the reaction mixture
Reaction of 1 and 6 in biphasic solvents (buffer :DCM) The same procedure was followed as for the reaction of TIPS-EBX (1) and glutathione (6)

Calibration 6c
Calibration of 6a was achieved through the preparation of several samples of different concentrations and their analysis on RP HPLC. In order to obtain average curve each analysis was performed 3 times. The following linear regression was obtained: y = 1204x + 9.8942, and R = 0.9967, where axis X is the concentration in millimolar (mM) of 6c and Y the absorbance area of the peak at 214 nm. was then added and the resulting mixture was stirred vigorously at 37 °C for 6 h. The buffer was used directly from freshly prepared bottle without degassing. Although the reaction solution was not clear due to the hydrophobic nature of naphthalene-2-thiol, no effect was observed on the rate of the reaction. After 6 h, the reaction mixture was diluted with 10 mL DCM and the organic layer was extracted using separating funnel. The organic layer dried over Na2SO4.The solvent was evaporated and the crude residue was purified by column chromatography on Biotage (Büchi flashpure cartridge 12 g, Pentane) to afford pure 8a as low melting solid, yield 68% (14.5 mg, 42.5 µmol). Reaction procedure for alkynylation of homocysteine 9 using 4a

Synthesis of thioesters in one-pot
Reaction procedure for preparation of silylthio ester of Ac-Ala-Cys-Gly-Phe-NH2 11aa in one-pot A 1.5 mL vial was charged with 11 (0.50 mg, 1.1 µmol, 1.0 equiv.) and 4a (1.0 mg, 1.7 µmol, 1.5 equiv.) in 114 µL of 10 mM Tris buffer pH 7.4 with small magnetic stirring bar. The reaction mixture was stirred at 37 °C for 6 h. The buffer was used directly from a freshly prepared solution without degassing. After 6 h, an aliquot of 10 µL of the reaction mixture was diluted with 30 µL acetonitrile:water (1:1) to give a clear solution. The solution was submitted to HPLC. The HPLC revealed the complete consumption of 11. TFA (50 µL, 0.65 mmol) was added and the reaction mixture was allowed to stir at rt for additional 2 h. After 2 h, 74% 11aa (retention time = 12.5) and 17% disulfide (retention time = 6.3) were observed by HPLC-