Bromotryptophans and their incorporation in cyclic and bicyclic privileged peptides

While revisiting biologically active natural peptides, the importance of the tryptophan residue became clear. In this article, the incorporation of this amino acid, brominated at different positions of the indole ring, into cyclic peptides was successfully achieved. These products demonstrated improved properties in terms of passive diffusion, permeability across membranes, biostability in human serum and cytotoxicity. Moreover, these brominated tryptophans at positions 5, 6, or 7 proved to be compatible as building blocks to prepare bicyclic stapled peptides by performing on‐resin Suzuki‐Miyaura cross‐coupling reactions.


| I N TR ODU C TI ON
The relevance of the amino acid tryptophan is widely known in the field of natural products, as this particular building block represents an important biosynthetic precursor for some bioactive compounds such as alkaloids. [1] Additionally, a common feature of membrane-spanning proteins is their preference for aromatic amino acids, namely tyrosine or tryptophan. [2,3] Aromatic, as well as charged, amino acids may act as an anchor for these proteins while they also participate in interfacial interactions. [4] The indole ring of tryptophan can display p-cation interactions, which are known to be of high importance in biological systems and are of increasing interest in medicinal chemistry. These interactions are present in 65% of the protein interfaces [5] and are considered to be real driving forces in biological processes. Stability and folding of proteins [6] along with specific drug-receptor interactions [7] have been related to p-cation interactions.
The presence of tryptophan in peptides and proteins is crucial for their biological activity despite their low natural abundance (<1% of amino acids). [8] At the beginning of the 21st century, novel substitution patterns within tryptophan were discovered, thus providing structures that are not directly accessible for ribosomes, including posttranslationally modified analogues. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) comprise constrained peptides with improved pharmacological properties such as higher stability, functionality and target recognition. [9] Those biologically active peptides are formed through a rich variety of post-translational modification chemistries, some of them involving tryptophan derivatization.
Position 3 of tryptophan can, for example, be isoprenylated in bacteria with a farnesyl moiety or geranyl group, yielding a tricyclic proline-like core in the latter case. [10] Another unusual modification is produced in bacteria with metalloenzymes able to crosslink Lys and Trp side chains, to form macrocyclic peptides. [11] Within RiPPs, lanthipeptides, for instance, constitute an abundant family, some of which possess antimicrobial activities. Of note, a unique 5-ClTrp moiety, inserted through biocatalysis, is present in a clinical candidate with antimicrobial bioactivity. Here, the chlorine substituent is responsible for its increased. [12] Tryptophan's scarce presence in proteins might be the ideal opportunity for selective derivatization, although it can also suffer from a poor accessibility when aiming at conjugation. For this latter strategy, tryptophan can be introduced by recombinant engineering. Trp can be abolished in a living organism via a surrogate noncanonical amino acid named L-b-(thieno [3,2-b]pyrrolyl)alanine ( [3,2]Tpa). [13] This modification was later implemented in bioactive peptides by genetic code engineering. [14] The specific reactivity of this amino acid is an important challenge to overcome when addressing protein bioconjugation. Trp side chain labeling can be reached using malonaldehydes in harsh reaction conditions or by means of metallocarbenoids. [15] A relevant example was described by Hansen et al., [16] performing chemo-and regioselective ethynylation of Trp-containing proteins. In addition to the outstanding selectivity obtained, the alkyne substituent can serve as a handle to undergo click chemistry or Pd-catalyzed Sonogashira reactions. Owing to the great interest in tryptophan bioconjugation and the raised concerns of the current methods, novel strategies continuously appear. Interestingly, a transition metal-free tryptophan-selective bioconjugation of proteins has recently been described. [17] Natural cyclizations involving the Trp side chain mainly take place through 5-membered ring formation. For example, the Savige-Fontana tryptathionylation, gives way to a rigid bicyclic peptide crosslink utilizing Trp and Cys side chains. [18] Iodine-mediated Cys deprotection and formation of the Trp-Cys thioether bridge provided an analogous result. [19] Nevertheless, we developed an alternative strategy which involves conjugation and cyclization at the 6-membered ring. Given the broad interest in Trp-based chemistry, new synthetic strategies have been applied for the derivatization of the indole ring of tryptophan.
Among all possible derivatization products, the halotryptophan motif is especially relevant due to its presence in natural bioactive compounds. [20,21] Introduction of halogens at different positions of the indole ring of tryptophan opens a gateway to new peptides with interesting properties. Moreover, access to these building blocks is realized by biotransformation of readily available starting materials. [22] The potential fine-tuning of some properties such as the membrane permeability, biostability in human serum or fluorescence is highly significant in the future application of these building blocks in peptides. In addition to halogens, other interesting groups such as a cyano, azido, or hydroxyl function are also quite relevant, and all of them can be obtained from the same precursor, namely a boronated Trp. [23] Introduction of some Trp analogues in catalytic proteins has proved to be advantageous. [24] In this context, Trp has, for example, demonstrated its involvement in the catalysis of ligninolytic peroxidases. [25] Of relevance to this work, halogenated peptides, as well as borylated ones, can also be used as synthetic precursors for Suzuki-Miyaura reactions. Taking advantage of the previously established methodology for on-resin Suzuki-Miyaura cross-coupling reactions, [22,26,27] bicyclic tryptophan-stapled peptides can be prepared as privileged peptide scaffolds involved in membrane transport.         The halotryptophans were prepared according to a reported biotransformation method [22] and obtained as HCl salts. Briefly, the indole substrate (2 mmol) and serine (1.25 equiv., 2.5 mmol) were suspended in 100 mL buffer (100 mM potassium phosphate, adjusted to pH 7.8 using 10 M potassium hydroxide). The cell lysate (3 mL), obtained as described in Smith et al. [22] was transferred to dialysis tubing and gently placed in the reaction mixture. The reaction was incubated at 378C while shaking at 180 rpm for 48 h. The cellulose tubing was removed and washed with water. Unreacted indole was recovered by extraction with ethyl acetate (2 3 50 mL) and the aqueous phase was concentrated under reduced pressure to $50 mL. The crude halotryptophans were purified by reverse-phase (RP) column chromatography with 97:3 and 0.5:99.5 methanol:water used as mobile phases. The reverse-phase column was packed in methanol and conditioned by passing 50 mL of 20, 40, 60, 80, and 100% water in methanol through the column sequentially, never allowing the column to run dry. A further 250 mL water was passed through the column, after which the concentrated aqueous phase was loaded onto the column. About 500 mL water was passed through the column to remove salts, serine, and pyridoxal phosphate (PLP). Halotryptophans were eluted by passing 250 mL methanol through the column, following which the solvent was removed under vacuum. The sample was then treated twice with 20 mL 0.1 M hydrochloric acid to convert the tryptophan to the hydrochloride salt, removing the solvent under reduced pressure. The salt was then dissolved in 50-100 mL water and lyophilized to obtain the purified L-halotryptophan as a colorless, pink, yellow, or tan powder.
Sodium azide (1.2 equiv.), previously dissolved in H 2 O (2 mL), was added to this solution. This mixture was vigorously stirred at room temperature for 3 h. The amino acid, dissolved in dioxane (16 mL) and 2% aq. NaHCO 3 (16 mL), was then introduced dropwise. The mixture was allowed to react for 24 h at room temperature. The pH was kept at 9-10 and readjusted when needed. Completion of the reaction was monitored by thin layer chromatography (TLC).
The crude product was separated in H 2 O/methyl-tert-butyl ether (MTBE) 1:1 (100 mL), and the aqueous phase was extracted with MTBE (2 3 50 mL 21 ). The pH was adjusted to 4 with aqueous HCl 12 N. The products precipitated as yellow pale solids which were finally filtered, washed, resuspended in H 2 O and lyophilized.  In the manuscript, we follow for brevity the nomenclature described in Spengler et al. [29] The peptide was prepared using the standard methodology of García-Pindado et al. [26] pNZ-(5Br)Trp-OH and Fmoc-(5Br)Trp-OH were used as the second and fourth amino acid, respectively. The synthesis was performed on solid-phase using Fmoc/tBu chemistry.

| Head-to-tail cyclization in solution
Miyaura borylation was carried out on-resin at the tripeptide level to form the boronic acid of the tryptophan derivative. Once the pentapeptide was obtained, the on-resin Suzuki reaction yielded the desired stapled peptide, bridged between the two tryptophan moieties. UPLC

| Cyclo(Lys-(6&)Trp-Lys-(6&)Trp-D-Pro)
The peptide was prepared using the standard methodology of García-Pindado et al. [26] pNZ-(6Br)Trp-OH and Fmoc-(6Br)Trp-OH were used as the second and fourth amino acid, respectively. The synthesis was performed on solid-phase using Fmoc/tBu chemistry. Miyaura borylation was carried out on-resin at the tripeptide level to form the boronic acid of the tryptophan derivative. Once the pentapeptide was obtained, on-resin Suzuki reaction yielded the desired stapled peptide between the two tryptophans. The peptide was prepared using the standard methodology of García-Pindado et al. [26] pNZ-(7Br)Trp-OH and Fmoc-(7Br)Trp-OH were used as the second and fourth amino acids, respectively. The synthesis was performed on solid-phase using Fmoc/tBu chemistry. Miyaura borylation was carried out on-resin at the tripeptide level to form the boronic acid of the tryptophan derivative. Once the pentapeptide was obtained, on-resin Suzuki reaction yielded the desired stapled peptide between the two tryptophans.

| Cytotoxicity, XTT assay
Nearly 5000 HeLa cells were seeded in 96-well plates 24 h before starting the assay. After this period, cells with peptides at 200 mM and 500 mM in DMEM (1 mg mL 21 glucose) supplemented with 10% serum were incubated for 24 h. Then, medium with peptides was removed and the XTT reagent was added to a final concentration of 0.5 mg mL 21 . After a 3-h incubation, absorbance was measured at 475 nm.
The same measure was performed after 6-h incubation. Cell viability was calculated by dividing the absorbance of wells treated with a given peptide by the absorbance of the untreated wells. The 6-h incubation values were used for the calculations, since an absorbance higher than 2 is required to reach the plateau zone of fluorescence. Measurements were performed in triplicate. As a negative control, cells were incubated with medium and as positive control 1% of DMSO was introduced.

| RE SUL TS A ND D I SCUSSION
Encouraged by the notorious presence of halogenated tryptophans in natural products, [9,10] we aimed to study the effects of introducing this particular motif in a small size peptide sequence, containing five amino acids.

| Design of the peptides
Within the current study, the core sequence was selected based on positive results obtained with a previously studied "Phe analogue," i.e., H-Lys-Phe-Lys-Phe-D-Pro-OH. We selected a pentapeptide scaffold to carry out the cyclization since this number of amino acids yields macrocycles with some rigidity, after amide bond formation between the Nand C-termini. D-Proline was selected as C-terminal amino acid (i.e., anchorage point to the resin) to favor cyclization in solution by inducing a turn conformation. [30] Lysine incorporation provided good results in terms of passive diffusion permeability and poor membrane retention, as well as non-cytotoxicity in HeLa cells. [25] In this study, the phenyalanines were substituted by tryptophans that were suitably decorated with bromine, to yield head-to-tail cyclized and brominated peptides.
Herein, the incorporation of Trp could positively influence peptide conformation and stability. [31] Moreover, the biaryl staple between two phenyalanines was extensively studied in our group and could be successfully replaced with a staple involving positions 5-5, 6-6, or 7-7 of two tryptophans. It is also worth mentioning that Trp-containing peptides with antimicrobial, and hence possibly membrane interfering, properties were reported. [22,32] Head-to-tail cyclic pentapeptides displaying two tryptophans, brominated at the same position (5,5; 6,6, or 7,7 of the indole ring) were synthesized using standard Fmoc/tBu solid-phase peptide synthesis.
We decided to introduce two identically substituted bromotryptophans to evaluate the potential effect of the indole substitution. The selected core sequence was exactly the same for all the peptides, that is, H-Lys- Bicyclic peptides have demonstrated to be privileged scaffolds that display improved pharmacological properties and prominent activities toward relevant biological targets. [33] Using our previously developed methodology for the preparation of bicyclic biarylcontaining pentapeptides, [11] we decided to prepare bicyclic peptides stapled between two tryptophans, at positions 5-5, 6-6, and 7-7 of the indole rings, taking advantage of the Suzuki-Miyaura reaction ( Figures 1 and 2).
Briefly, the bicyclic peptides were prepared using a modified Fmoc/tBu strategy and applying Wang resin as solid support. At the tripeptide level, on-resin borylation was carried out using Trt as the temporary protecting group and pNZ as the "permanent" one. Once the tryptophan was borylated through a Miyaura borylation, Trt was removed and the peptide was elongated on the solid support. The pentapeptide was then subjected to an on-resin Suzuki-Miyaura reaction, having Boc as temporary protecting group and pNZ as the more permanent orthogonal one. Cleavage of the stapled precursor was carried out providing the peptide with side chain protecting groups on both lysines. After performing a pre-purification of the peptide and subsequent head-to-tail cyclization, removal of pNZ was performed in solution yielding the desired stapled bicyclic peptide ( Figure 3).
Furthermore, this new construct presents an interesting motif (i.e., two stapled tryptophans) that could be used when trying to target surface recognition, as was demonstrated for the biaryl feature, [11,12] due to the possible p-cation interactions with the tryptophan staple.
Once the compounds were prepared, we aimed to evaluate their properties in terms of passive diffusion permeability, resistance towards proteolytic degradation and cytotoxicity.

| Passive diffusion permeability
Owing to the special interest in peptide-based CNS drugs, these compounds were studied by PAMPA (Parallel Artificial Membrane Permeability Assay). The assay allows unveiling the potential passive diffusion permeability of these compounds across a mimic of the blood-brain barrier (BBB). We tested the three bromine-containing cyclic peptides and the bicyclic ones together with the two controls, obtaining the following results (Table 1, Figure 4). Propranolol was used as positive control of the assay.
Cyclization of the linear peptide gave way to an enhanced permeability, also when performed in conjunction with the introduction of bromine at any of the studied positions of the indole ring of tryptophan.
FIG URE 1 Cyclic pentapeptides with (5,5), (6,6), and (7,7) bromotryptophans, named monocyclic 5Br, monocyclic 6Br, and monocyclic 7Br, respectively P e represents the permeability of the investigated compounds across the artificial BBB, while T stands for the percentage of transport of the peptides in the assay. These parameters allow to understand whether the tested compound show a relevant permeability through passive diffusion. An effective permeability higher than 1Á3 10 26 cm s 21 correlates with a good transport prediction in vivo. [34] Upon comparison of the four head-to-tail cyclic peptides we observed an enhanced permeability when incorporating 5Br-Trp and 6Br-Trp, while in case of 7Br-Trp the permeability was slightly lower.
These results demonstrated that halogens can be incorporated in a peptide and, in selected cases, this improves passive diffusion permeability. This tendency was previously reported by Malakoutikhak et al. [35] Nonetheless, it is worth noting the significant differences in the transport depending on the halogen position at the tryptophans, thus implying subtle changes in the properties of the brominecontaining cyclic peptides.
The bicyclic peptides did show a different behavior in terms of permeability. While bicyclic 5,5 and 6,6 displayed a slightly lower value of permeability, as compared to the monocyclic versions, bicyclic 7,7 demonstrated to have the highest BBB permeability. Therefore, we could not correlate the incorporation of the stapled constraint to an increased transport. The differences can arise from the constraint of the structures between the aromatic residues involved in the stapling.
Regarding membrane retention, we did not observe a significant value for any of the head-to-tail cyclic versions. On the contrary, the linear control was mostly retained on the membrane. This result was not surprising since tryptophans as well as N-and C-termini are described to interact with membranes. Therefore, cyclization is suggested to be a tool to avoid high membrane retention due to the absence of these termini. However, this strategy could lead to lower transport of the final compounds owing to the loss of both polar groups.

| Cell viability assay
We were especially concerned by the potential cytotoxicity of these peptides due to the presence of two bromines. Therefore, an XTT

| Protease resistance in human serum
As previously mentioned, head-to-tail cyclization is a well-known strategy used to improve peptide biostability in human serum. After having prepared these compounds, it became interesting to determine which effect the incorporation of bromine shows in terms of protease resistance. Therefore, the peptides were incubated in human serum for 24 h and the different time-point aliquots were analyzed by HPLC to determine half-life times. We observed that all of the head-to-tail cyclized peptides, including the cyclic control, were stable for more than 3 h in human serum. Even though the incorporation of bromine could not be linked to increased serum stability, the insertion of bromotryptophan amino acids did not lead to a more rapid degradation of the cyclic peptide analogues. Here, the linear peptide was used as positive control of the assay, displaying a half-life close to 1 h.
Earlier studies of our group [26] demonstrated that the incorporation of an extra cyclization increases considerably the biostability in human serum. The published results demonstrated outstanding values of protease resistance, which can be quite beneficial. Nonetheless,   owing to the minimum amount of the bicyclic peptides obtained, these were not subjected to stability assays.

| C ONC LUSI ON S
This piece of work illustrates how the incorporation of bromotryptophan amino acids in cyclic peptides enables to improve passive diffusion permeability devoid of cytotoxicity, and presents an adequate resistance toward protease degradation. Not only can these compounds be of interest for biological purposes, but also the bromotryptophan motif can be used to prepare stapled bicyclic peptides, known as privileged scaffolds for recognition of protein surfaces.