Small peptide diversification through photoredox-catalyzed oxidative C-terminal modification

A photoredox-catalyzed oxidative decarboxylative coupling of small peptides is reported, giving access to a variety of N,O-acetals. They were used as intermediates for the addition of phenols and indoles, leading to novel peptide scaffolds and bioconjugates. Amino acids with nucleophilic side chains, such as serine, threonine, tyrosine and tryptophan, could also be used as partners to access tri- and tetrapeptide derivatives with non-natural cross-linking.


General methods
All reactions were carried out in oven dried glassware under an atmosphere of nitrogen, unless for the oxidative decarboxylation and if stated otherwise. For flash chromatography, distilled technical grade solvents were used. THF, CH3CN, toluene and CH2Cl2 were dried by passage over activated alumina under nitrogen atmosphere (H2O content < 10 ppm, Karl-Fischer titration). All chemicals were purchased from Acros, Aldrich, Fluka, VWR, TCI, Merck or Bachem and used as such unless stated otherwise. All dipeptides starting materials were commercially available and used as received. Chromatographic purification was performed as flash chromatography using Macherey-Nagel silica 40-63, 60 Å, using the solvents indicated as eluent with 0.1-0.5 bar pressure. TLC was performed on Merck silica gel 60 F254 TLC aluminum or glass plates and visualized with UV light and KMnO4 stain. 1 H-NMR spectra were recorded on a Brucker DPX-400 400 MHz spectrometer in chloroform-d, DMSO-d 6 or acetonitrile-d 3 , all signals are reported in ppm with the internal chloroform signal at 7.26 ppm, the internal DMSO signal at 2.50 ppm or the internal acetonitrile signal at 1.94 ppm as standard. The data is being reported as (s = singlet, d = doublet, t= triplet, q = quadruplet, qi = quintet, m = multiplet or unresolved, br = broad signal, app = apparent, coupling constant (s) in Hz, integration, interpretation). 13 C-NMR spectra were recorded with 1 H-decoupling on a Brucker DPX-400 100 MHz spectrometer in chloroform-d, DMSO-d 6 or acetonitrile-d 3 , all signals are reported in ppm with the internal chloroform signal at 77.0 ppm, the internal DMSO signal at 39.5 ppm or the internal acetonitrile signals at 1.32 and 118.26 ppm as standard. Infrared spectra were recorded on a JASCO FT-IR B4100 spectrophotometer with an ATR PRO410-S and a ZnSe prisma and are reported as cm-1 (w = weak, m = medium, s = strong, br = broad).
High resolution mass spectrometric measurements were performed by the mass spectrometry service of ISIC at the EPFL on a MICROMASS (ESI) Q-TOF Ultima API. MS-MS analyses were performed on a LTQ Orbitrap FTMS instrument (LTQ Orbitrap Elite FTMS, Thermo Scientific, Bremen, Germany) operated in the positive mode coupled with a robotic chip-based nano-ESI source (TriVersa Nanomate, Advion Biosciences, Ithaca, NY, U.S.A.). A standard data acquisition and instrument control system was utilized (Thermo Scientific) whereas the ion source was controlled by Chipsoft 8.3.1 software (Advion BioScience). Samples were loaded onto a 96-well plate (Eppendorf, Hamburg, Germany) within an injection volume of 5µl. The experimental conditions for the ionization voltage was +1.4kV and the gas pressure was set at 0.30 psi. The temperature of ion transfer capillary was 275 °C, tube voltages. FTMS spectra were obtained in the 80-1000 m/z range in the reduce profile mode with a resolution set to 120,000. In all spectra one microscan was acquired with a maximum injection time value of 1000ms. Typical CID experiments were carried out using Normalized collision energy values of 26-28 and 5 Da of isolation width.
Photoredox catalyzed reactions were performed in test tubes (5 and 10 mL), which were hold using a rack for test tubes placed at the center of a crystallization flask. On this flask were attached the blue LEDs (RUBAN LED 5MÈTRES -60LED/M -3528 BLEU -IP65 with Transformateur pour Ruban LED 24W/2A/12V, bought directly on RubanLED.com). The distance between the LEDs and the test tubes was approximatively 2 cm for the test tubes and 5 cm for the Schlenk flasks. Long irradiation resulted in temperature increasing up to 37°C during overnight reactions.
Tetramers peptides were synthesized by solid phase peptide synthesis using a Multipep RSi Intavis. Crude products were purified by preparative RP-HPLC on an Agilent 1260 HPLC system with a G2260A 1260 Prep ALS Autosampler, a G1361a 1260 Prep Pump, a G1365C 1260 MWD detector and a G1364B 1260 FC-PS collector, coupled with a Waters XBridge semi-preparative C18 column (19 x 150 mm, 5 µm). Water (solvent A) and water:acetonitrile 5:95 (solvent B), each containing 0.1% TFA, were used as the mobile phase at a flow rate of 20 mL.min-1. The following method was used: 100% A to 100% B in 20 minutes.
RP-HPLC-MS measurements were performed on an Agilent 1290 Infinity HPLC system with a G4226a 1290 Autosampler, a G4220A 1290 Bin Pump and a G4212A 1290 DAD detector, connected to a 6130 Quadrupole LC/MS MS, coupled with a Waters XBridge C18 column (250 x 4.6 mm, 5 µm). Water:acetonitrile 95:5 (solvent A) and water:acetonitrile 5:95 (solvent B), each containing 0.1% formic acid, were used as the mobile phase at a flow rate of 0.6 mL/min-1. The gradient was programmed as follows: 100% A to 100% B in 20 minutes then isocratic for 5 minutes. The column temperature was set up to 25 °C. Low resolution mass spectrometric measurements were acquired using the following parameters: positive electrospray electrospray ionization (ESI), temperature of drying gas = 350 °C, flow rate of drying gas = 12 L. min-1, pressure of nebulizer gas = 60 psi, capillary voltage = 2500 V and fragmentor voltage = 70 V.

Peptide synthesis
The used dipeptides were commercially available. All peptide tetramers were synthesized by solid phase peptide synthesis using a 2-chlorotrityl chloride resin (1.0-1.6 mmol/g, 100-200 mesh). The first amino acid was loaded on the resin by incubation of the Fmoc-protected monomer (3 equiv of the number of active sites on the resin), DIPEA (4 equiv) in dichloromethane for 2 h. A cycle consisted first of the deprotection, achieved by stirring for 20 min with a 20% solution of piperidine in DMF, twice. Then the resin was washed with DMF (7x). Double couplings were performed by adding the Fmoc-protected monomer (4 equiv), HBTU (4 equiv), HOBt (4 equiv), NMM (4 equiv) and stirring for 45 min. Capping was carried out at the end of each cycle, followed by a DMF wash (7x). Acetylation of the N-terminal was achieved by incubating the resin with an Acetic Anhydride/DIPEA/DMF 10/15/75 solution for 30 min, twice. Cleavage of peptides with no protecting groups on the side-chains was performed by stirring the resin in a 20% solution of HFIP in dichloromethane for 30 min. In the presence of protecting groups, a TFA/water/triisopropylsilane 95/2.5/2.5 was used instead and the stirring time increased to 2 h. The cleavage mixture was poured into cold diethyl ether and precipitated peptides were recovered. The crude peptides were purified by preparative RP-HPLC using a gradient water-95% acetonitrile in 20 min. Pure peptides were analyzed by RP-HPLC and HRMS.

4.1.
Optimization of the oxidative decarboxylation Degassed solvent was added in a 10 mL test tube containing a teflon coated stirring bar, Z-Gly-Pro (1a) (31 mg, 0.10 mmol, 1.0 equiv), R-BX (2) (0.15 mmol, 1.5 equiv), the base and the catalyst under a nitrogen atmosphere. The reaction mixture was irradiated using blue light LEDs at RT.

Procedure for HPLC yields:
The reaction was monitored by dilution of 50 L of the crude with 950 L of acetonitrile. The yield was estimated by the absorbance of product in comparison to the overall absorbance of product, unreacted starting material and side-products if any.

Procedure for isolated yields:
The crude mixture was diluted with 10 mL of sat. NaHCO3 and extracted with diethyl ether (3 x 50 mL). The combined organic layers were washed with brine (3 x 20 mL), dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by preparative TLC (DCM/ethyl acetate 7:3). [a] Ratio of integration at 214 nm by RP-HPLC, [b] green LEDs, [c] isolated yield.
Control experiments were carried out and only traces of the desired product were observed in the absence of light or catalyst.
A solution obtained by dilution of 50 L of the crude with 950 L of acetonitrile was injected into the HPLC to monitor the conversion of the starting material.
The crude mixture was diluted with 10 mL of sat. NaHCO3 and extracted with diethyl ether (3 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography DCM to DCM/ethyl acetate on triethylamine deactivated silica (5 vol% in DCM).
A solution obtained by dilution of 50 L of the crude with 950 L of acetonitrile was injected into the HPLC to monitor the conversion of the starting material.
The crude mixture was diluted with 10 mL of sat. NaHCO3 and extracted with diethyl ether (3 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography DCM to DCM/ethyl acetate on triethylamine deactivated silica (5 vol% in DCM).
A solution obtained by dilution of 50 L of the crude with 950 L of acetonitrile was injected into the HPLC to monitor the conversion of the starting material.
The phenol (0.45 mmol, 1.5 equiv) was added and the reaction mixture degassed by Ar bubbling before cooling at 0 °C. BF3.OEt2 (158 L, 0.600 mmol, 2.00 equiv) was added dropwise and the mixture stirred for 2 h at 0 °C. The crude mixture was diluted with 10 mL of sat. NaHCO3 and extracted with diethyl ether (3 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography DCM to DCM/ethyl acetate on triethylamine deactivated silica (5 vol% in DCM).
A solution obtained by dilution of 50 L of the crude with 950 L of acetonitrile was injected into the HPLC to monitor the conversion of the starting material.
The indole (0.306 mmol, 1.02 equiv) was added and TFA (23 L, 0.30 mmol, 1.0 equiv) was added dropwise and the mixture stirred for 1 h at RT.
The crude mixture was diluted with 10 mL of sat. NaHCO3 and extracted with diethyl ether (3 x 30 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, filtered and concentrated under vacuum. The crude product was purified by column chromatography DCM to DCM/ethyl acetate on triethylamine deactivated silica (5 vol% in DCM).