Solid-Phase Synthesis of Biaryl Cyclic Peptides Containing a Histidine-Phenylalanine Linkage

The feasibility of the solid-phase intramolecular 4(5)-arylation of a histidine residue to obtain biaryl cyclic peptides bearing a His-Phe linkage was established. The synthetic strategy involved the preparation of a linear peptidyl resin incorporating a 5-bromohistidine and a 4-boronophenylalanine, and its cyclization through the formation of the biaryl bond between the imidazole of histidine and the phenyl group of phenylalanine via a microwave-assisted Suzuki–Miyaura cross-coupling. This methodology was applied to the preparation of biaryl cyclic peptides consisting of a 3- or 5-residue ring, incorporating the His residue at the N- or the C-terminus and bearing a Leu-Leu spacer or a –NH2 group at the C-terminus. In the case of the 3-residue ring peptides, the position of the His did not influence the macrocyclization. In contrast, to obtain the 5-member ring biaryl cyclic peptides, the His residue should be located at the N-terminus. It was also observed that the Leu-Leu spacer is crucial for the intramolecular arylation. These results suggest that this approach could be useful for the preparation of a diversity of synthetic and natural biaryl cyclic peptides bearing a His-Phe linkage.


Introduction
Unsymmetrical biaryl systems are present in many naturally occurring cyclic peptides that show a variety of important biological activities including antimicrobial or cytotoxic (Feliu and Planas 2005). In recent years, much attention has been turned to the incorporation of biaryl amino acids into biologically active peptides (Haug et al. 2007;Le Quement et al. 2011;Ng-Choi et al. 2014;Mendive-Tapia et al. 2015;García-Pintado et al. 2017). It has been reported that the presence of the biaryl motif restricts the conformational flexibility of peptides, enhances their proteolytic stability, increases their selectivity, and improves their bioavailability (Willemse et al. 2017). In particular, 5-arylhistidines are present in cytotoxic and antifungal marine peptides and it has been observed that the imidazole ring is crucial for their activity (Bewley et al. 1996). Moreover, we have shown that the incorporation of a 5-arylhistidine in a linear antimicrobial peptide does not influence its antimicrobial activity and provides peptides with low hemolysis. This low cytotoxicity has been attributed to the presence of the imidazole ring of histidine (Ng-Choi et al. 2012).
Despite the significance of biaryl motifs in drug discovery, the preparation of biaryl cyclic peptides still remains an important synthetic challenge. In fact, a scarce number of research groups has addressed the solid-phase synthesis of cyclic peptides incorporating a Phe-Phe, Phe-Tyr, Tyr-Tyr, Trp-Phe or a Trp-Tyr linkage (Afonso et al. 2011(Afonso et al. , 2012Meyer et al. 2012;Mendive-Tapia et al. 2015;García-Pintado et al. 2017;Ng-Choi et al. 2019a). In this context, we have recently developed a solid-phase strategy for the synthesis of biaryl cyclic peptides containing a His-Tyr bond (Ng-Choi et al. 2019b). A linear peptidyl resin incorporating a 5-bromohistidine and a 3-boronotyrosine was used as precursor and the cyclization was performed through a microwave-assisted Suzuki-Miyaura cross-coupling. This approach provided access to 3-or 5-member biaryl cyclic peptides with a His at the N-or at the C-terminus, and was extended to the preparation of analogues of the northern and southern hemispheres of aciculitins. To the best of our knowledge, this is the first report on the solid-phase cyclization of peptides by arylation of a His residue. In fact, it has been described that the derivatization of the position 4(5) of the imidazole ring of a His is troublesome.
Herein, we were interested in studying the feasibility of the above strategy to the solid-phase synthesis of biaryl cyclic peptides incorporating a His-Phe linkage, which to the best of our knowledge has not yet been reported. In particular, we planned to obtain such biaryl cyclic peptides by formation of a biaryl bond between the position 4(5) of the imidazole ring of His and the position 4 of the benzene ring of Phe. Accordingly, the synthesis of the biaryl cyclic peptides depicted in Figs. 1 and 2, consisting of a 3-or 5-residue ring and bearing the His at the N-or C-terminus was investigated.

General Methods
Manual peptide synthesis was performed in polypropylene syringes (2 or 5 mL) fitted with a polyethylene porous disk. Solvents and soluble reagents were removed by suction. Most chemicals were purchased from commercial suppliers Sigma-Aldrich (Madrid, Spain), Iris Biotech GmbH (Marktredwitz, Germany), Scharlab (Sentmenat, Spain) or Panreac (Castellar del Vallès, Spain), and used without further purification.
Peptides were analyzed under standard analytical HPLC conditions with a Dionex liquid chromatography instrument composed of an UV/Vis Dionex UVD170U detector, a P680 Dionex bomb, an ASI-100 Dionex automatic injector, and CHROMELEON 6.60 software. Detection was performed at 220 nm. Solvent A was 0.1% aq TFA and solvent B was 0.1% TFA in CH 3 CN. Conditions A: Analysis was carried out with a Kromasil 100 C 18 (4.6 mm × 40 mm, 3.5 μm) column with a 2-100% B over 7 min at a flow rate of 1 mL/min. Conditions B: Analysis was carried out with a Kromasil 100 C 18 (4.6 mm × 250 mm, 5 μm) column with a 2-100% B over 28 min at a flow rate of 1 mL/min. Conditions C: Analysis was carried out with a Kromasil 100 C 18 (4.6 mm × 250 mm, 5 μm) column with a 2-25% B over 3 min followed by a 25-35% B over 30 min and a 35-100% B over 1 min at a flow rate of 1 mL/min. Peptides were also analyzed with an Agilent Technologies LC 1200 series liquid chromatography instrument at 220 nm. Solvent A was 0.1% aq TFA and solvent B was 0.1% TFA in CH 3 CN. Conditions D: Analysis was carried out with a Kromasil 100 C 18 (4.6 mm × 250 mm, 5 μm) column with a 2-15% B over 1 min followed by a 15% B over 1 min, 15-60% B over 26 min, 60% over 1 min, and a 60-100% B over 1 min at a flow rate of 1 mL/min. ESI-MS analyses were performed with an Esquire 6000 ESI ion Trap LC/MS (Bruker Daltonics) instrument equipped with an electrospray ion source. The instrument was operated in the positive ESI(+) ion mode. Samples Fig. 1 Structure of the biaryl cyclic peptides incorporating a His at the N-terminus (5 μL) were introduced into the mass spectrometer ion source directly through an HPLC autosampler. The mobile phase (80:20 CH 3 CN/H 2 O at a flow rate of 100 μL/min) was delivered by a 1200 Series HPLC pump (Agilent). Nitrogen was employed as both the drying and nebulising gas. HRMS were recorded on a Bruker MicroTof-QIITM instrument using ESI ionization source at Serveis Tècnics of the University of Girona. Samples were introduced into the mass spectrometer ion source by direct infusion using a syringe pump and were externally calibrated using sodium formate. The instrument was operated in the positive ion mode.
Microwave-assisted reactions were performed with a single mode Discover S-Class labstation microwave (CEM) (0-300 W). The time, temperature, and power were controlled with the Synergy software. The temperature was monitored through an infrared sensor in the floor of the cavity.
Peptide purifications were performed on a CombiFlash Rf200 automated flash chromatography system using RediSep Rf Gold reversed-phase C 18 column packed with high performance C 18 derivatized silica. 1 H and 13 C NMR spectra were measured with a Bruker 300 or 400 MHz NMR spectrometer. Chemical shifts were reported as δ values (ppm) directly referenced to the solvent signal.
Upon completion of the peptide sequence, the N-terminal Fmoc group was removed and the trityl group was introduced using TrCl (10 equiv.) and DIEA (10 equiv.) in DMF at room temperature for 4 h. Then, the resulting resin was washed with DMF (6 × 1 min) and CH 2 Cl 2 (3 × 1 min), and air-dried. The completion of this reaction was monitored by the Kaiser test (1970). An aliquot of the resulting peptidyl resin was cleaved with TFA/H 2 O/TIS (95:2.5:2.5) for 2 h at room temperature. Following TFA evaporation and diethyl ether extraction, the corresponding crude peptide was dissolved in H 2 O/CH 3 CN (1:1), lyophilized, analysed by HPLC, and characterized by mass spectrometry.

Solid-Phase Synthesis of Cyclic Biaryl Peptides Containing a Histidine Residue at the N-terminus
We planned to synthesize the biaryl cyclic peptides containing a His at the N-terminus depicted in Fig. 1. They consist of a 3-or 5-amino acid ring and incorporate an amide group or a Leu-Leu spacer at the C-terminus. The use of 2-(trimethylsilyl)ethoxymethyl (SEM) or methyl as imidazole protecting group was evaluated.
The strategy for the solid-phase synthesis of these biaryl cyclic peptides involved as key steps: (i) the preparation of a linear peptidyl resin incorporating a 4-boronophenylalanine at the C-terminus and a 5-bromohistidine at the N-terminus, and (ii) the cyclization of this linear peptidyl resin via a microwave-assisted Suzuki-Miyaura cross-coupling (Scheme 1). The synthesis of the linear precursor was envisaged through preparation of the corresponding N-terminal trityl protected 4-iodophenylalanine peptidyl resin, solidphase Miyaura borylation, trityl group removal and coupling of a conveniently protected 5-bromohistidine.
First, the synthesis of the biaryl cyclic peptide BPC758 was investigated (Scheme 1). According to the above strategy, the peptidyl resins 1 were required as linear precursors. Thus, we synthesized the N-terminal trityl protected 4-iodophenylalanine peptidyl resin 2 starting from a Fmoc-Rink-MBHA resin following a standard 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy. Elongation of the peptide sequence was performed through sequential Fmoc group removal and coupling steps. The Fmoc group was removed using piperidine/DMF ( Oxyma and N,N-diisopropylethylamine (DIEA) in DMF. After assembling the Fmoc-protected peptide sequence, the Fmoc group was then replaced by a trityl group to overcome the instability of Fmoc under the basic Miyaura borylation conditions. Thus, after treatment with piperidine/DMF (3:7) the resin was subjected to trityl chloride (TrCl) and DIEA. An aliquot of the resulting resin 2 was treated with a mixture of trifluoroacetic acid (TFA)/ triisopropylsilane (TIS)/H 2 O (95:2.5:2.5) for 2 h, providing the corresponding iodopeptide in 94% purity, which was also characterized by mass spectrometry.
Next, we attempted the synthesis of the biaryl cyclic peptide BPC750, analog to BPC758 but without a Leu-Leu spacer at the C-terminus (Scheme 2). For this purpose, peptidyl resins 7 bearing the 4-boronophenylalanine residue bound to the Rink linker were prepared. The intramolecular Suzuki-Miyaura cross-coupling using the above conditions yielded the expected biaryl cyclic peptide BPC750 in 14% HPLC purity together with the linear peptides H-His-Lys-Lys-Leu-Phe-NH 2 , H-His-Lys-Lys-Leu-Tyr-NH 2 , H-His-Lys-Lys-Leu-Phe(4-B(OH) 2 )-NH 2 resulting from protodeborylation, debromination and/or oxidation of the linear precursor 7. The lower purity observed for these biaryl cyclic peptide in the crude reaction mixture compared to that of their analogue containing a Leu-Leu spacer could be attributed to the steric hindrance posed by the resin.
To investigate if this approach could also be applied to the formation of 3-member ring biaryl cyclic peptides, resins 8 and 9 were synthesized, which differ on the presence of a Leu-Leu spacer at the C-terminus (Scheme 3). Cyclization of resins 8 and 9 afforded the biaryl cyclic peptides BPC760 and BPC752 in 33% and 13% HPLC purity, respectively, together with linear peptides resulting from protodeborylation, debromination and/or oxidation of the corresponding precursors 8 and 9. BPC760 and BPC752 were purified by column chromatography (> 99% and 91% purity, respectively) and were characterized by mass spectrometry. Therefore, similar results were obtained for the preparation of biaryl cyclic peptides incorporating 3 or 5 amino acids in their ring and it was observed that the presence of a Leu-Leu spacer at the C-terminus favoured the cyclization.

Solid-Phase Synthesis of Cyclic Biaryl Peptides Containing a Histidine Residue at the C-terminus
The feasibility of synthesizing 5-arylhistidine-containing cyclic peptides in which the His residue is located at the C-terminus was investigated (Fig. 2). In particular, taking into account the previous results, we studied the synthesis of biaryl cyclic peptides containing a 3-or 5-residue ring, and incorporating an amide group or a Leu-Leu spacer at the C-terminus.
Towards this aim, a similar strategy to that described for biaryl cyclic peptides bearing a His at the N-terminus was planned. In this case, it would involve the preparation of a linear peptidyl resin incorporating a 4-boronophenylalanine at the N-terminus and a 5-bromohistidine at the C-terminus, which it would be then cyclized by means of a microwaveassisted intramolecular Suzuki-Miyaura cross-coupling (Scheme 4).
This approach was first applied to the synthesis of the 3-member ring biaryl cyclic peptide BPC780 which incorporates a Leu-Leu spacer at the C-terminus. The synthesis of the required linear peptidyl resins 10 started from a Fmoc-Rink-MBHA resin and followed a Fmoc/tBu strategy. The two Fmoc-Leu-OH residues were incorporated by sequential Fmoc removal and coupling steps. The Fmoc group was cleaved with piperidine/DMF (3:7) and couplings were performed with DIPCDI and Oxyma in DMF. Next, after Fmoc removal, the regioisomeric mixture of Boc-His(5-Br,1-SEM)-OH and Boc-His(5-Br,3-SEM)-OH (Cerezo et al. 2008) was coupled using COMU, Oxyma and DIEA in DMF. The Boc group of the resulting 5-bromohistidine peptidyl resins was selectively removed with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in the presence of 2,6-lutidine (Zhang et al. 1998). Peptide elongation was performed by coupling of the corresponding Fmoc-Leu-OH and of Boc-Phe(4-BPin)-OH (Ng-Choi et al. 2019a) as the N-terminal residue. An aliquot of the resulting resins 10 was cleaved with TFA/TIS/H 2 O (95:2.5:2.5) for 3 h yielding the corresponding 4-boronophenylalanyl peptide H-Phe(4-B(OH) 2 )-Leu-His(5-Br)-Leu-Leu-NH 2 in 69% purity. Formation of the boronic acids took place during HPLC analysis which was confirmed by mass spectrometry.
With the boronopeptidyl resins 10 in hand, the intramolecular Suzuki-Miyaura arylation was carried out using Pd 2 (dba) 3 , P(o-tolyl) 3 , and KF under microwave irradiation at 140 °C for 30 min, which were the best conditions for the formation of the biaryl bond in cyclic peptides with a His at the N-terminus (Scheme 4). After acidolytic cleavage, the crude reaction mixture was analyzed by HPLC and mass spectrometry. Results showed that the intramolecular arylation of resins 10 did provide the biaryl cyclic peptide BPC780 in 30% HPLC purity. Purification through column chromatography rendered BPC780 in > 99% purity and was characterized by mass spectrometry.
This methodology was applied to the synthesis of BPC774, BPC776 and BPC778 (Fig. 2). The 3-member ring biaryl cyclic peptide BPC776 without the Leu-Leu spacer was obtained in lower purity (10%) than its analog BPC780. This result is in accordance to that obtained for biaryl cyclic peptides with a His at the N-terminus. Unexpectedly, in the case of the 5-member ring cyclic peptides BPC778 and BPC774, differing on the presence of a Leu-Leu spacer, the cyclization of the corresponding linear peptidyl resins was not successful. Mass spectrometry analysis of the crude reaction mixtures showed only traces of the expected biaryl cyclic peptide BPC778, whereas BPC774 was not observed.
Taking all these results together, it can be concluded that the synthesis of biaryl cyclic peptides bearing a His-Phe linkage with a His at the C-terminus is more difficult than that of analogues with a His at the N-terminus, specially in the case of the 5-member ring peptides. Moreover, this synthesis is favoured when a Leu-Leu spacer is present. In a previous study focused on the preparation of biaryl cyclic peptides containing a His-Tyr bond a different behavior was observed (Ng-Choi et al. 2019b). In this case, best results were obtained for those derivatives incorporating a His at the C-terminus and the presence of the Leu-Leu spacer was not required .

Conclusions
This paper reports the development of a strategy for the solidphase synthesis of biaryl cyclic peptides containing a His-Phe linkage. It has been evaluated the position of the His in the sequence and the presence of a spacer at the C-terminus. It was observed that the intramolecular Suzuki-Miyaura crosscoupling was favoured when a Leu-Leu spacer is located at the C-terminus. Moreover, in the case of the biaryl cyclic peptides consisting of a 3-residue ring, similar results were obtained irrespective of the His position. In contrast, the formation of the 5-member biaryl cyclic peptides was only achieved when the His was at the N-terminus.
This methodology takes full advantage of the solid-phase synthesis because in the case of the biaryl cyclic peptides incorporating a His at the N-terminus, apart from macrocyclization, borylation is also performed on the solid support. This protocol avoids the synthesis and purification of the amino acid boronic ester in solution and at the same time it facilitates the removal of the palladium catalyst used in the Suzuki-Miyaura reaction. Therefore, this approach could be useful for the preparation of cyclic peptides containing a biaryl linkage between a His and a Phe in a flexible manner under mild conditions.