Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

These methods simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene or thio-yne click chemistry that possesses superior functional group tolerance and good yield. The main advantage of this technique is that a thioether tether provides a traceless modification site, which significantly expand the chemical space of stable peptides following peptide synthesis modification. To begin the procedure, attach a column filled with 50 milligrams of Rink Amide MBHA resin to both a benchtop vacuum manifold and a nitrogen or argon gas line via a three-way stopcock.

Add one to two milliliters of dichloromethane to the bed of resin, and swell the resin by gently agitating the mixture with a stream of inert gas bubbles for 10 minutes. Drain the resin with a rubber pipette bulb when finished. Then, add one to two milliliters of N-terminal Fmoc deprotection solution to the resin.

Gently agitate the mixture by bubbling with inert gas for 10 minutes, and then drain the resin with a rubber pipette bulb. Wash the column with one to two milliliters of DMF in the same way, followed by one to two milliliters of DCM. Repeat the deprotection sequence once.

Next, combine 0.125 millimoles of an Fmoc-protected amino acid and 50.5 milligrams of HCTU with 0.5 milliliters of DMF in a polypropylene container. Shake the mixture for a minute to dissolve the solids. Then, add 43.5 microliters of DIPEA, and shake the container for another minute to obtain a 0.25-molar activated amino acid solution.

Add this solution to the washed resin, and bubble inert gas through the mixture for one to two hours. After that, deprotect and wash the bound amino acid residue. Continue coupling amino acids to the peptide chain in this way to form the linear peptide.

Use solid-phase peptide synthesis to prepare resin supporting a linear peptide that includes a trityl-protected cysteine residue separated by three units from an mono-S-five residue. Add one to two milliliters of trityl deprotection solution to 50 milligrams of the peptide-bearing resin. Bubble nitrogen gas through the mixture for 10 minutes.

Drain the deprotection solution, and wash the resin with one-to two-milliliter portions of DCM three times. Repeat the deprotection procedure six times or until the solution no longer appears yellow to fully deprotect the cysteine. Full deprotection of the Trt group of cysteine is very important for the following photoreaction step.

Wash the resin with one-to two-milliliter portions of DMF and DCM in sequence. Then, soak the resin in one to two milliliters of methanol for two minutes to shrink the resin. Drain the methanol, and dry the resin with nitrogen gas for five minutes.

Transfer the dry resin to a 10-milliliter, round-bottom flask via weighing paper. Suspend the resin in two milliliters of nitrogen-sparged DMF. Then, add 5.6 milligrams of MMP and 38 milligrams of MAP as photoinitiators.

Place a stir bar in the flask, and cap the flask with a rubber septum. Purge the flask atmosphere with nitrogen gas three times, for a total purge time of about five minutes. Fix the reaction flask in a photoreactor equipped with UVA lamps, an inert nitrogen gas inlet, and a stir plate.

Start stirring the mixture, and close the photoreactor. Purge the photoreactor with argon for 10 minutes. Then, set the argon flow rate to one liter per minute, and irradiate the mixture with UVA light for one hour at room temperature.

Monitor the reaction with HPLC. Wash, cleave, and purify the cyclic peptide when finished. Prepare 50 milligrams of resin bearing a linear peptide that includes a gamma-substituted mono-S-five residue.

Wash the resin with DMF and DCM in sequence. Shrink the resin with methanol, and dry it with nitrogen gas. Then, in a 10-milliliter, round-bottom flask, suspend the resin in two milliliters of nitrogen-sparged DMF.

Add to the mixture 5.6 milligrams of MMP, 3.8 milligrams of MAP, and 25.7 milligrams of Fmoc-protected cysteine. Add a stir bar to the flask, seal it with a rubber septum, and purge the flask atmosphere with nitrogen gas. Then, irradiate the mixture with UVA light in an inert nitrogen gas-filled photoreactor while stirring at room temperature for one to two hours.

Monitor the reaction with LC-MS. When the photoreaction is complete, wash the bound peptide and perform the macrolactamization reaction. Afterwards, resume coupling amino acids to the cyclic peptide to form the desired structure.

Cleave and purify the peptide when finished. Prepare 50 milligrams of resin bearing a linear peptide that includes residue with a terminal alkyne separated by three units from a residue with a terminal thiol. Cleave the peptide from the resin, and precipitate it with one milliliter of cold diethyl ether.

Centrifuge the peptide mixture at 12, 000 times g for two minutes. Gently decant the supernatant, and add another milliliter of cold diethyl ether. Repeat the washing twice more, and then dry the peptide residue under vacuum.

Dissolve the residue in 50 milliliters of nitrogen-sparged DMF in a round-bottom flask. Add 3.2 milligrams of DMPA to the peptide solution, and bubble nitrogen through the reaction mixture for 10 minutes. An inert atmosphere is strictly required for effective thio-yne photoreaction in a solution phase.

Otherwise, we observed oxidation of sulfur during UV irradiation. Then, irradiate the mixture with UVA light without stirring in an inert nitrogen gas-filled photoreactor at room temperature for 30 minutes to one hour. Isolate and purify the cyclic peptide when finished.

On-resin cyclization of this peptide produced a thioether-tethered one, five cyclic peptide with an identical molecular weight to the linear peptide. The HPLC retention time of the cyclic product was two minutes shorter than that of the linear peptide. Good conversion was also observed for the on-resin cyclization of several other short peptides.

Linear peptides were screened for the ability to undergo peptide stapling via photo-induced thiol-yne hydrothiolation. The low yield of two c was attributed to a conformational preference for trapping the thiyl radical at the N-terminus during contraction to a 20-membered macrocycle. Peptides one a and one b both generated two isomers with an eight-member vinyl sulfide crosslink.

The product peptides two a-A and two a-B had distinct retention times and were produced in different ratios at different irradiation times. Proton NMR spectroscopy indicated that two a-A was the E-isomer of the vinyl sulfide and two a-B was the Z-isomer. The Z-isomer was the major product when forming peptides two d, two e, and two f, which was attributed to the conformational preference during the formation of the seven-member vinyl sulfide crosslink.

Circular dichroism spectroscopy showed a random coil for all forms of peptides two a and two b and helical conformation for peptide two d. We developed a series of chemical protocols for the construction of thioether and vinyl sulfide-tethered peptides by using photo-induced thio-ene and thio-yne click chemistry. We believe it will be helpful for the research community.