Site‐Selective Installation of N ϵ ‐Modified Sidechains into Peptide and Protein Scaffolds via Visible‐Light‐Mediated Desulfurative C–C Bond Formation

Abstract Post‐translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site‐specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible‐light‐mediated desulfurative C(sp3)–C(sp3) bond forming reaction that enables the site‐selective installation of N ϵ ‐modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site‐selectively installing an N ϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).


Work-up:
The combined solutions were concentrated under a stream of nitrogen to < 5 mL. 40 mL of diethyl ether was added to precipitate the peptide and the suspension centrifuged. The pellet was then dissolved in water containing 0.1% TFA, filtered and purified by preparative HPLC and analyzed by LC-MS and ESI mass spectrometry.

Automated Fmoc-SPPS
Automated Fmoc-SPPS was carried out on a Biotage Initiator + Alstra microwave peptide synthesizer. General synthetic procedures for Fmocdeprotection and capping were carried out in accordance with the manufacturer's specifications. Standardized amino acid couplings were performed for 15 min at 50 o C under microwave irradiation in the presence of amino acid (0.5 M in DMF, 4 eq.), Oxyma (0.5 M in DMF, 4 eq.) and diisopropylcarbodiimide (0.5 M in DMF, 4 eq.). Peptide cleavage and work-up were carried out as described above for manual SPPS.

General conjugation protocol A
To peptide dissolved in 20% acetonitrile (ACN) in 6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7 to a concentration of 1 mM was added a solution of TCEP (0.5 M stock solution in LB pH adjusted to 7, 5 eq.), allyl compound (200 eq.) and (Ir[dF(CF3)ppy]2(dtbpy))PF6 (1 mM stock solution in ACN, 0.05 eq.). The pH of 5 the reaction mixture was checked to be 8 then the reaction mixture diluted to the final peptide concentration of 0.5 mM. The reaction vessel was then placed into blue LEDs (photochemistry set up 1), or into a PhotoRedOx Box (HepatoChem) (photochemistry set up 2); once the starting material was shown to be fully consumed by analytical HPLC, the reaction mixture was purified by semi-preparative HPLC.

General conjugation protocol B
To peptide dissolved in 20% acetonitrile (ACN) in 6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7 to a concentration of 1 mM was added a solution of TCEP (0.5 M stock solution in LB pH adjusted to 7, 10 eq.), allyl compound (200 eq.) and (Ir[dF(CF3)ppy]2(dtbpy))PF6 (1 mM stock solution in ACN, 0.1 eq.). The pH of the reaction mixture was checked to be 8 then the reaction mixture diluted to the final peptide concentration of 0.5 mM. The reaction vessel was then placed into blue LEDs (photochemistry set up 1), or into a PhotoRedOx Box (HepatoChem) (photochemistry set up 2); once the starting material was shown to be fully consumed by analytical HPLC, the reaction mixture was purified by semi-preparative HPLC

General conjugation protocol C
To peptide dissolved in 20% acetonitrile (ACN) in 6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7 to a concentration of 1 mM was added a solution of TCEP (0.5 M stock solution in LB pH adjusted to 7, 100 eq.), allyl compound (200 eq.) and (Ir[dF(CF3)ppy]2(dtbpy))PF6 (1 mM stock solution in ACN, 0.1 eq.). The pH of the reaction mixture was checked to be 8 then the reaction mixture diluted to the final peptide concentration of 0.5 mM. The reaction vessel was then placed into blue LEDs (photochemistry set up 1); once the starting material was shown to be fully consumed by analytical HPLC, the reaction mixture was purified by semi-preparative HPLC 6 Photochemistry apparatus Set up 1 A blue LED light strip wrapped around a pyrex dish, placed on top of a stirrer plate. To ensure consistency, places for up to 4 vials were marked on the plate.
The temperature was monitored and observed to reach no higher than 30 °C.

Allyltrimethylammonium chloride (17)
To trimethylamine (12.0 ml, 33% wt solution in ethanol, 92.7 mmol) was slowly added allyl chloride (1.50 ml, 18.5 mmol). The reaction was left to stir at room temperature for 2 hours then concentrated in vacuo to give a colourless solid.
The solid was then redissolved in chloroform and crystallised by slow addition of acetone. The crystals were then filtered and washed with acetone before drying under a high vacuum to give the desired product as colourless crystals (

N-allylbenzamide (22)
To N-allylamine (0.64 ml, 8.54 mmol) and triethylamine (2.97 ml, 21.3 mmol) in DCM (25 ml) at 0 o C was added benzoyl chloride (0.83 ml, 7.11 mmol) dropwise. The reaction mixture was allowed to warm to room temperature and stir for 16 hours before being diluted with DCM (25 ml) and washed with 1 M HCl (3 x 10 ml), sat. NaHCO3 (3 x 10 ml) and brine (10 ml). The organic layer was then dried over MgSO4, filtered and concentrated to product a pale-yellow oil. This was purified by column chromatography (DCM) to yield the title compound as a colourless oil, which formed colourless crystals when stored at

Installation of N  -acetyl on an isolatable scale Ac-CWHISKEY-NH2 (1) + N-allyl acetamide (2)photochemistry set up 1
The optimized conditions (protocol A) were applied to an isolatable scale reaction on Ac-CWHISKEY-NH2 (1) with N-allyl acetamide (2) using set up 1 to ascertain the overall isolated yield and reaction rate. The reaction was monitored at 15-minute intervals and was shown to reach completion after 45 minutes. At this time, the conversion of starting material to product was 55%.
Isolated of the desired product by preparative HPLC afforded peptide 3a in 51% yield.

Ac-CWHISKEY-NH2 (1) + N-allyl acetamide (2)photochemistry set up 2
Protocol A was then applied to Ac-CWHISKEY-NH2 (1) with N-allyl acetamide (2) on an analytical scale using set up 2 (PhotoRedOx Box) to ascertain the reaction rate using this specialized equipment. Aliquots were taken in oneminute intervals and analyzed by analytical HPLC. The reaction was observed to reach full consumption of the starting peptide 1 after just 2 minutes in comparable conversion to the reaction run using set up 1 (blue LEDs). To confirm the endpoint, a 2-minute irradiation was repeated, and the sample immediately injected into the HPLC. This reaction was repeated on a preparative scale to determine the isolated yield.
This reaction was repeated in degassed solvents and also in a glove box with degassed solventsno change in the ratio of product:by-products was observed.

NMR comparison of N-allyl acetamide modified Ac-CAY-NH2
and Ac-D-CAY-NH2 with chemically synthesized Ac-K(Ac)AY-NH2 Figure S19 -NMR comparison of L (5a, green spectra) and D (5b, red spectra) modified peptides.  The NMR comparison clearly shows key differences between the spectrum for the L (77) and D (78) conjugated peptides. The absence of certain peaks without peak broadening shows that racemisation of the conjugated amino acid is not occurring. This was vital as it shows that the native Lys PTM is being formed.

Modification of multiple Cys residues
Peptide 6a (3 mg, 0.002 mmol) was subjected to protocol A (set up 2) using Nallyl acetamide (2, 33 mg, 0.33 mmol). The reaction yielded a crude mixture of products, primarily consisting of an inseparable mixture of peptides 6b and 6c.
Repeating these conditions in the PhotoRedOx Box (set up 2), we observed comparable conversion to the desired product in 2 mins.

Site-selective modification of a cyclic peptide
Peptide 34

H NMR analysis of folded N  Ac-Ub
Purified N  Ac-Ub 36 was dissolved in 6 M Gdn•HCl, 0.1 M Na2HPO4, pH 7 and dialysed into 25 mM Na2PO4, 100 mM NaCl, pH 7.0 overnight. 10 vol% D2O was added to the sample which was analyzed on a Bruker 600 MHz Avance III NMR system using excitation sculpting for solvent suppression. Data were zero-filled and a 2 Hz line broadening applied prior to Fourier Transform and baseline correction. Data were referenced indirectly using the internal D2O signal that was calibrated to give 0 ppm for TMS, following the methods detailed by IUPAC.

mide (13)
ine ( 0.64 ml, 8.54 mmol) and triethylamine (2.97 ml, 21.3 mmol) ml) at 0 o C was added benzoyl chloride (0.83 ml, 7.11 mmol) e reaction mixture was allowed to warm to room temperature and urs before being diluted with DCM (25 ml) and washed with 1 M l), sat. NaHCO3 (3 x 10 ml) and brine (10 ml). The organic layer d over MgSO4, filtered and concentrated to product a pale yellow purified by column chromatography (DCM) to yield the title a colourless oil, which formed colourless crystals when stored at