Protecting group free radical C–H trifluoromethylation of peptides

Two radical-based approaches enable the efficient trifluoromethylation of aromatic sidechains in fully unprotected peptides under mild, biocompatible conditions.


I. General Procedures
Unless otherwise noted, all reactions were conducted under inert atmosphere (N 2 ). Reactions were monitored using a Waters Acquity UPLC system with UV detection at 220, 254 or 280 nm and a low resolution electrospray ionization mode (ESI) mass spectrometer. Yields were obtained through 19 F-NMR using α,α,α-trifluorotoluene (Aldrich) as an internal standard.
Gradients for each amino acid product are detailed in the experimental procedures along with LCMS traces of final products. Semi-preparative reverse phase HPLC of peptides (5-9, 16-17) was accomplished using a Gilson HPLC equipped with a Waters SunFire Prep C18 stationary phase. Semi-preparative reverse-phase HPLC of the modified peptides (10-14, S10-S23) was accomplished using a Charged Surface Hybrid (CSH) stationary phase (Waters) and H2O:acetonitrile with 0.16% TFA gradient mobile phase with the gradients for each peptide detailed in the experimental procedures.

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High resolution mass spectrometry/mass spectrometry measurements: For compounds 11A , 12A S14-17, S20-23: For compounds 10A, S10, S11, S12, S13, S18, 13A, 13B, S19: LC/MS/MS experiments were performed using a Thermo LTQ™-Orbitrap mass spec and a Water's Acquity™ UPLC ® system. The high resolution LC/MS spectra were acquired in FTMS mode using an Orbitrap instrument at a resolution of 30,000 (at m/z 400). The electrospray ionization needle was held at 4.5 kV, and a nitrogen sheath gas and a nitrogen auxiliary gas were used to stabilize the spray. The heated capillary was set at 275 °C. All MS/MS experiments were conducted in the linear ion trap. Helium was introduced into the ion trap to improve the trapping efficiency and also to serve as the collision gas for CID. The operational pressure after introducing helium was ∼2 × 10 −5 Pa in the linear ion trap.
LC gradient conditions are described as below: The gradient started at 5% B and was heldfor 0.5 min, before increasing to 99% B in 9.5 min at a flow rate of 300 μL/min (column temperature

II. Materials
Unless otherwise noted all materials were obtained from commercial suppliers and used without further purification. Anhydrous organic solvents were purchased from Sigma Aldrich in SureSeal TM bottles. Concentrated acetic acid was purchased from Sigma Aldrich and degassed before further use. All N-acyl amino acid amides, deltorphin I (10), angiotensin I (11), angiotensin II (12), β-casomorphin (13), splenopentin (14), and dermorphin (15)  General Procedure. The substrate (1 equiv) and zinc trifluoromethanesulfinate (3 equiv) were added to a 4 mL vial with a septum cap and brought into an N 2 -filled glove box. Degassed 10% acetic acid in distilled water (final concentration 0.2 M) was added into the vial. To this solution was added TBHP (5 equiv) in a dropwise manner, and this mixture was allowed to stand at 23 ºC for 16 h. The reactions were monitored at 220 nm and 280 nm by LC/MS. Solution yields were determined using 19 F-NMR spectroscopy with α,α,α-trifluorotoluene as an internal standard. The mixture was then diluted with acetonitrile, transferred to a separate flask, and the solvents were removed by lyophilization. The resulting solid was reconstituted in DMSO and purified by semi-preparative reverse phase HPLC. Fractions were collected and solvents were removed by lyophilization. F 3 C + General Procedure: A substrate (0.05-0.2 mmol, 1 equiv), sodium trifluoromethanesulfinate (20 equiv) and [Ir(dFCF 3 ppy) 2 (dtbpy)](PF 6 ) 4 (0.15 equiv) were added to a 4 mL vial equipped with a stir bar and sealed with a septum cap and brought into a N 2 -filled glove box. A 1:1 mixture of degassed 10% acetic acid in distilled water:acetonitrile (final concentration 0.1 M solution with respect to tyrosine) was added into the vial. The vial was removed from the N 2 -filled glove box, the reaction was irradiated with the MSD photoreactor 1 , and the mixture was allowed to stand at ambient temperature for 16 h. The reactions were monitored at either 254 or 280 nm by LC-MS.

IV. Additional Optimization and Mechanistic
NMR yields were determined using 19 F-NMR with α,α,α-trifluorotoluene as an internal standard.
The mixture was diluted with 1:1 acetonitrile:water and the sample was lyophilized to remove all solvents. The resultant solid was reconstituted in DMSO and purified by semi-preparative reverse phase HPLC. The isolated peaks were characterized by HRMS-LC/MS/MS, and by 1 H, 19 F, and 13 C-NMR ( 13 C-NMR are available for compounds isolated in sufficient quantity).   S-11

Stability of Met Residue under Trifluoromethylation Conditions (Condition X & Y)
Condition X: Under these conditions, methionine in NH 2 -Met-Tyr-OH was oxidized to the corresponding sulfoxide in the presence of stoichiometric TBHP. In addition, trifluoromethylation occurred as expected and afforded the yields shown below. X.

Synthesis of Trifluoromethylated Products and Characterizations a.
Simple Aromatic Amino Acids (2)      and zinc trifluoromethanesulfinate (199 mg, 0.6 mmol, 3 equiv) was added a solution of acetic acid (200 µL) and water (1800 µL). This mixture was transferred into a N 2 -filled glove box. To this solution was added a 70% solution of TBHP in water (137 µL, 1.00 mmol, 5 equiv) in a dropwise manner. The vial was sealed and the mixture was allowed to stand at 23 ºC for 16 h.

Mixture of CF 3 [Y5]-dermorphin, CF 3 [Y1]-dermorphin and CF 3 [F3]-dermorphin
S-46      Briefly, the frozen cells were plated in 96-well (40,000 cells/well) clear tissue culture plates and allowed to recover. Test molecules at the appropriate concentrations were added and the cells were incubated for 8 min at 37 °C. The media was aspirated and chilled, and MSD cell lysis buffer was added as per MSD kit instructions. The cells were lysed on ice for 40 min and the lysate then mixed for 10 minutes at room temperature. The lysate was transferred to the MSD kit pIR detection plates. The remainder of the assay was carried out following the MSD kit recommended protocol. S-52