Rapid, traceless and facile peptide cyclization enabled by tetrazine‐thiol exchange

Cyclic peptides offer many advantages compared to their linear counterparts, including prolonged stability within the biological environment and enhanced binding affinity. Typically, peptides are cyclized by forming an amide bond, either on‐resin or in solution, through extensive use of orthogonal protecting groups or chemoselective ligation strategies, respectively. Here, we show that the chemoselective tetrazine‐thiol exchange is a powerful tool for rapid in situ cyclization of peptides without the need for additional activation reagents or extensive protecting group reshuffling. The reaction between N‐terminal sulfide‐bearing unsymmetric tetrazines and internal cysteines occurs spontaneously within a mildly acidic environment (pH 6.5) and is of traceless nature. The rapidly available unsymmetric sulfide tetrazine building blocks can be incorporated on resin using standard solid‐phase peptide synthesis protocols and are orthogonal to trifluoroacetic acid cleavage conditions. The cyclized peptides display high stability, even when incubated with a large excess of free thiols. Due to its traceless and mild nature, we expect that the tetrazine‐thiol exchange will be of high value for the in situ formation of cyclic peptide libraries, thus being applicable in drug discovery and development.

0][11][12] Naturally, cyclic peptides still benefit from the grand pool of unnatural peptide building blocks, thus allowing rapid incorporation of unnatural amino acids, such as additions of lipophilic side chains to facilitate cell permeability, which further improves their pharmacokinetic properties.For these reasons, various cyclic peptides have been FDA-and EMA-approved as biologically active species with several candidates being in the last phases of clinical trials. 7spite the frequent use of cyclic peptides, cyclization of linear peptides is often challenging and associated with extensive protecting group reshuffling and additional synthesis steps in the workflow.In general, the cyclization of peptides can be performed in four ways: head-to-tail, head-to-side chain, side chain-to-tail and side chainto-side chain. 13,14Conventional approaches include amide bond formation via head-to-tail cyclization, side chain-to-head ligation using a carboxylic acid functionality, or side chain-to-tail ligation by making use of a lysine residue. 7,13Disulfide bridges are often employed for side chain-to-side chain cyclization. 157][18][19][20][21] Native chemical ligation for the cyclization of otherwise unprotected peptides is a powerful tool for the late-stage modification of peptides. 22,23Yet, these strategies typically require additives for activation or removal of formed byproducts, thus hindering their applications for in situ drug screening and high throughput assays.Alternatively, the cyclization of unprotected peptides can be achieved using highly chemoselective reactions that proceed in a traceless manner.These reactions can be readily incorporated into standard peptide workflows.For instance, the Nitsche group demonstrated the efficient cyclization of a cyclic peptide, serving as a substrate for the Zika virus protease, through the use of oxime ligation. 24Although this example showcases the power of such transformations, the pool of available reactions that not only exhibit high chemoselectivity but are also traceless in nature, is significantly limited.
Recently, the group of Fox and our group independently reported the rapid and chemoselective exchange between methyl sulfide tetrazines and thiols, which we refer to as tetrazine-thiol exchange (TeTEx). 25,26We envisioned that TeTEx would be a powerful tool for the fully traceless cyclization of peptides in the absence of additives such as activation reagents and without extensive protecting group reshuffling.The strong water-dependency of this reaction suggests that an aqueous environment would be sufficient to trigger cyclization.Finally, we hypothesized that the dynamic nature of this reaction might enable cyclization under relatively high concentrations.In this study, we report the development of an in situ method to facilitate effortless peptide cyclization using the reaction between an N-terminal methyl sulfide tetrazine and an internal or C-terminal cysteine residue.We show that the required building blocks are readily available and fully compatible with standard SPPS protocols.We expect that TeTEx will be of high value for the in situ formation of cyclic peptide libraries, thus offering new advances in the field of peptide chemistry.
Reagents were used without further purification unless otherwise stated.Solvents were dried by passing over activated alumina columns in an MBraun MB SPS800 under a nitrogen atmosphere and stored under nitrogen.Water was demineralized using a QPOD Milli-Q system.Reactions were carried out under air unless stated otherwise.Reactions and fractions from flash column chromatography were monitored by thin layer chromatography (TLC) using glass TLC plates (Merck, TLC silica gel 60 F254) and if necessary, visualized by staining with potassium permanganate.Column chromatography was performed on VWR SiO2 Type (40-63 mesh) using a forced flow of air at 0.5-1.0bar.Mass spectra were measured on a Bruker Microflex LRF Maldi-TOF system and JEOL AccuTOF CS JMST100CS.Reversephase high-performance liquid chromatography (RP-HPLC) was carried out on an Agilent AG1120 instrument equipped with a Phenomenex PRODIGY ODS C18 column, 250 Â 4.60 mm, particle size 5 μm at 25 C and a flow of 1.0 mL/min using a linear gradient (5%-100% B in A, with A: 0.1% trifluoroacetic acid [TFA] in MilliQ and B: MeCN, 25 min).The peptides were purified using a Shimadzu LC-20 RP-HPLC equipped with a Phenomenex C18 column using 0.1% formic acid in ACN and in MilliQ as eluents and differential refractive index or UV absorbance (215 nm).
The solution was washed with sat.sodium bicarbonate solution, H 2 O and brine, and the organic layer was dried over sodium sulfate and concentrated under reduced pressure.The crude product was purified by flash column chromatography on silica gel (1:2 EtOAc/n-heptane), yielding a colourless oil (1.449 g).Yield: 98%.R f : 0.63 (1:1 EtOAc/nheptane). 1

| Methyl thiocarbohydrazide hydrogen iodide salt (2)
Compound 2 was synthesized following a protocol described in literature. 27In brief, thiocarbohydrazide (2.00 g, 18.8 mmol, 1.0 equiv.),methyl iodide (1.29 mL, 20.7 mmol, 1.1 equiv.)and ethanol (45.0 mL, 0.3 M) were added in a dry round bottom flask.After refluxing at 80 C for 3 h, the reaction mixture was cooled down to RT and heptane was added.The mixture was filtered after cooling down in the freezer (À20 C) overnight.The solid was washed with a mixture of cold ethanol/heptane (1:1, v/v) and dried under reduced pressure.

| General protocol
Peptides were synthesized by manual Fmoc-SPPS using HBTU activation procedure.Cysteine was used as trityl-protected buidling block.
Wang resin was preloaded (0.5 mmol/g) and was swollen and capped prior to SPPS.Amino acid residues were coupled by a preactivated solution of 4.1 equiv.protected amino acid (0.3 M in DMF) with 4 equiv.HBTU in the presence of DIPEA .After 1-3 h, depending on the difficulty of the coupling, the resin was washed with DMF (3Â), DCM (3Â) and DMF (3Â).All amino acids were double coupled.After the second coupling of each amino acid, the resin was capped using a mixture of DMF/DIPEA/acetic anhydride (10:2:1, v/v/v) for 30 min.
The resin was washed and piperidine (20% in DMF) was added to the alcohol to form oxetane ester 1, which was converted to the unsymmetric tetrazine 3 by using a protocol of Fox. 27tert-Butyl ester deprotection was achieved by treatment with TFA for 2 h (Figure 1a).formed cations to reattach to the peptide. 28,29This cleavage method provided a pink-coloured crude peptide.MS and HPLC analysis revealed that triethyl silane partially reduced the tetrazine residue towards the corresponding dihydro-tetrazine.Because of these findings, we also reviewed an alternative cleavage cocktail containing only H 2 O as a scavenger (8% in TFA).The characteristic deep purple colour of the cleaved peptide suggested partial degradation of sulfide tetrazine into oxygen-substituted tetrazines (Figure 2b).The HPLC and MS analysis of this peptide did not give any unambiguous results, and it cannot be excluded that degradation in the form of hydrolysis had occurred.A combination of triethyl silane and H 2 O as scavengers was further used as a cleavage cocktail.Importantly, our data indicates that the partially reduced tetrazine undergoes rapid re-oxidation in the presence of air, suggesting that reduction is not a significant concern.
We observed that cyclization did not readily occur after cleavage, likely because of the highly acidic conditions caused by remaining TFA.To trigger the cyclization, peptides P3 and P4 were incubated at 37 C within mildly acidic buffers.The acidic conditions were chosen to limit the cyclization via lysine and instead favour cyclization via TeTEx mechanism.For this study, we chose three different buffer systems with pH = 4.5, 5.5 and 6.5.In our initial attempt to achieve cyclization, we utilized the crude peptide without further purification (Table 1).The crude peptide was rapidly soluble in the different buffer systems and no organic co-solvent was required.Incubation in citric acid buffering at pH 4.5 led to little change in the composition of the crude peptide mixture with only minor amount of cyclized product being observed, suggesting that the pH is too acidic for cysteine to undergo nucleophilic aromatic substitution.The use of acetate buffer (pH = 5.5) provided better results giving a ratio of 7:3 for P3/P5.In contrast, we observed around 60% conversion to P5 when incubated in PBS (100 mM, pH = 6.5).To further improve the cyclization, we purified the linear peptide P3 via preparative HPLC prior cyclization.
Once incubated in PBS (100 mM, pH = 6.5), nearly complete conversion of P3 into P5 could be observed after 1 h of incubation (Figure 1c).Because we intended to provide a general applicable system, we employed acetonitrile as a co-solvent.In contrast, when P4 was subjected to the same conditions, only trace amounts of cyclized product were observed, which we attributed to lysine mediated cyclization.While we did not observe any formed oligomers throughout our studies, the dynamic nature of such oligomers makes them more difficult to detect, thus we cannot exclude that these are formed temporarily.
For many applications, it is beneficial to cyclize peptides headto-tail rather than head-to-side chain avoiding unnecessary synthetic overheads and branching amino acids.For that purpose, we targeted the synthesis of peptide P6, which displayed a C-terminal cysteine and N-terminal tetrazine residue.The peptide was purified and subjected to cyclization using the optimized conditions (ACN/PBS 100 mM, pH 6.5).Despite more side-reactions, the desired cyclic peptide P7 was formed as the major product (Figure 3).Throughout these and our previous studies, we observed the formation of disulfide bonds as a competing side-reaction, which can possess a challenge for this cyclization strategy. 26This is because tetrazines are prone to reduction by trialkyl phosphines; while the formed dihydrotetrazine is readily converted back to the aromatic tetrazine by simple exposure to air, the same applies to disulfide bonds.Naturally, the competing reaction strongly depends on the substrate and may vary.
Finally, we attempted the synthesis of highly hydrophobic peptide P8, a derivative of gypsophin B from Gypsophila oldhamiana. 30In case of peptide P8, we observed the reduced dihydrotetrazine as the major product after cleavage.We believe that the high hydrophobicity of  Consequently, cyclization attempts failed because the substrate was not soluble in sufficient amounts of buffered aqueous solvent mixtures.However, we believe that this example is still of value for future applications of TeTEx because it demonstrates one current limitation, namely, the required aqueous component in the solvent mixture.
The exchange between sulfide tetrazines and thiols is a reversible process.Even though we have observed in earlier work that the exchange of sterically more demanding sulfide tetrazine is significantly slower, we investigated the stability of the cyclic sulfide tetrazine-bearing peptide P5. 26 For this purpose, we incubated In conclusion, we have shown that the chemoselective exchange between methyl sulfide tetrazines and thiols (TeTEx) presents a rapid and traceless method for in situ cyclization of peptides.The reported cyclization strategy can be readily incorporated into standard SPPS protocols and eliminates the need for activation reagents or protecting group reshuffling, offering a straightforward and efficient cyclization method.This technique holds significant promise for the generation of cyclic peptide libraries in drug discovery and development, thus providing a valuable tool for accelerating the search for therapeutically active compounds.
For the cyclization studies, peptide P1 (H-AKWSGCL-OH) with several nucleophilic reactivity centres besides the required cysteine (lysine, serine, tryptophan and the C-terminus) was accessed by manual Fmoc-SPPS.To prove our hypothesis that the cyclization between N-terminal tetrazine and internal cysteine residues proceeds chemoselectively, peptide P2 was designed, which lacked the cysteine residue and displayed an alanine residue instead.The coupling of tetrazine 4 to peptides P1 and P2 was achieved by using standard SPPS protocol, namely, HBTU in the presence of DIPEA.After coupling of Tet 4 to peptides P1 and P2, the resin coloured pink suggesting successful incorporation of tetrazine to provide peptides P3 and P4, respectively.For the cleavage of tetrazine-modified peptides, we made use of a combination of H 2 O and triethyl silane as carbocation scavengers, which are commonly employed in peptide chemistry preventing F I G U R E 1 (a) Reaction scheme depicting the synthetic pathway of Tet residue 4. a: 3-Methyl-3-oxethanemethanol, EDCÁHCl, DMAP (98%); b: BF 3 ÁOEt 2 , À12 C; c: 2 in DMF, 80 C; d: (diacetoxyiodo)benzene (76% over three steps); e: trifluoroacetic acid (TFA) in DCM (26%).(b) Schematic overview of the SPPS synthesis of P1-P4 (HBTU, DIPEA in DMF), cleavage (4% H 2 O, 4% TES in TFA, 90 min) of P3-P4 and cyclization of P3 (c = 12.5 mM) in ACN/PBS (100 mM, pH 6.5, 1:4) at 37 C for 1 h yielding P5.(c) Reaction monitoring via RP-HPLC with (t 0 = 0 h and t = 1 h) and MALDI-TOF (t = 1 h) of P3 and P4, indicating the complete cyclization of P3 yielding P5 and the unresponsive P4 under cyclization conditions.F I G U R E 2 Global deprotection and cleavage using (a) 4% H 2 O, 4% TES in trifluoroacetic acid (TFA), or (b) 8% H 2 O in TFA.

3 a
peptide P8 increases the interactions with the scavenger during cleavage.Because sulfide-bearing dihydrotetrazines are oxidized upon exposure to air, we attempted cyclization of peptide P8.It turned out that peptide P8 was completely insoluble in all common solvent combinations and only showed moderate solubility in DMSO.T A B L E 1 Relative ratio of linear to cyclic peptide after 1 h of incubation of crude peptide P3 (2 mM) at 37 C as determined by HPLC.Citric acid buffer (100 mM).b Sodium acetate buffer (200 mM).c Phosphate buffer (100 mM).

F I G U R E 3
Cyclization of peptide P6 that displays a C-terminal cysteine enabling head-to-tail cyclization.Peptide P7 was obtained by incubating P6 (1 mM) in cyclization buffer (ACN/PBS [100 mM, pH 6.5, 1:4]) at 37 C for 2 h and purification by preparative HPLC.
the cyclized peptide P5 (300 μM in 1:1 H 2 O/ACN, v/v) with an excess of glutathione (2 mM) at 37 C for 2 h.Analysis by HPLC and MS indicated only minor amounts of linear peptides and decomposition products.Considering the recent work of Tallon et al. who showed that sulfide tetrazines can be employed as reversible covalent cysteine warheads, one can imagine that these cyclic sulfide tetrazine derivates are promising scaffolds for the development of selective covalent inhibitors with improved stability in the near future.25