Solid-phase synthesis of biaryl bicyclic peptides containing a 3-aryltyrosine or a 4-arylphenylalanine moiety

A methodology for the solid-phase synthesis of biaryl bicyclic peptides containing a Phe-Phe, a Phe-Tyr or a Tyr-Tyr motif has been devised. This approach comprises two key steps. The first one involves the cyclization of a linear peptidyl resin containing the corresponding halo- and boronoamino acids via a microwave-assisted Suzuki–Miyaura cross coupling. This step is followed by the macrolactamization of the resulting biaryl monocyclic peptidyl resin leading to the formation of the expected biaryl bicyclic peptide. This study provides the first solid-phase synthesis of this type of bicyclic compounds being amenable to prepare a diversity of synthetic or natural biaryl bicyclic peptides.

The reaction mixture was stirred at room temperature for 5 h. After this time, the organic solvents were evaporated under reduced pressure and H 2 O (100 mL) was added to the resulting residue. The resulting solution was adjusted to pH 5 by addition of glacial AcOH followed by extraction with S4 EtOAc (4 × 30 mL). The organic layers were combined, washed with brine (50 mL), and dried over anhydrous magnesium sulfate. Removal of the solvent afforded Boc-Phe(4-BPin)-OH [4] as a white solid (602 mg, 92% yield). t R = 6.43 min (98% purity). 1
After stirring for 24 h at room temperature, the reaction mixture was concentrated in vacuo. EtOAc (50 mL) was added, and the solution was washed with 1 N HCl (30 mL) and H 2 O (3 × 30 mL). The aqueous layers were combined and adjusted to pH 1 and the solution was washed with EtOAc (3 × 40 mL). All the organic layers were combined, washed with brine (30 mL) and dried over anhydrous magnesium sulfate. Removal of the solvent followed by digestion of the resulting precipitate in pentane/diethyl ether (1:1, 60 mL) for 2 h afforded Fmoc-Phe(4-I)-OH [5]

Fmoc-Glu-OpNB
Fmoc-Glu(Ot-Bu)-OpNB (1.50 g, 2.68 mmol, 1 equiv) was dissolved in TFA/CH 2 Cl 2 (1:1, 25 mL) and stirred at room temperature for 2 h. After this time, the solvent mixture was removed under vacuum. Diethyl ether was then added and evaporated under vacuum. This process was repeated three times in order to completely remove the TFA. The resulting product was dried in vacuo overnight to afford Fmoc-Glu-OpNB [

Boc-Tyr(3-B(OH) 2 ,Me)-OH
An aqueous solution of LiOH (3 mL, 4.17 mmol, 3 equiv) was added to a solution of Boc-Tyr(3-B(OH) 2 ,Me)-OMe (450 mg, 1.27 mmol, 1 equiv) in MeOH/THF (1:1, 6 mL). The reaction mixture was stirred at room temperature for 1.5 h. After this time, the organic solvents were evaporated under reduced pressure and water (25 mL) was added to the resulting residue. The solution was adjusted to pH 5-6 by addition of 1 N HCl followed by extraction with EtOAc (3 × 25 mL). The organic layers were combined, washed with brine (25 mL), and dried over anhydrous magnesium sulfate. Removal of the solvent afforded Boc-Tyr(3-B(OH) 2

H-Tyr(3-I,Me)-OMe
Boc-Tyr(3-I,Me)-OMe [7] (4.50 g, 10.34 mmol, 1 equiv) was dissolved in TFA/CH 2 Cl 2 (1:1, 30 mL) and stirred at room temperature for 2 h. After this time, the solvent mixture was removed under vacuum. Diethyl ether was then added and evaporated, this process was repeated three times to completely remove the TFA. The resulting product was dried in vacuo overnight to afford H-

H-Tyr(3-I,Me)-OH
A solution of LiOH (1.24 g, 28.91 mmol, 3 equiv) in water (17 mL) was added to a solution of H-Tyr(3-I,Me)-OMe (3.23 g, 9.64 mmol, 1 equiv) in MeOH/THF (1:1, 34 mL). The reaction mixture was stirred at room temperature for 2 h. After this time, the organic solvents were evaporated under reduced pressure and water (60 mL) was added to the resulting residue. The solution was adjusted to pH 5 by addition of glacial AcOH and the resulting precipitate was filtered, washed with cold diethyl ether, and dried in vacuo overnight, yielding H-Tyr(3-I,Me)-OH as a white solid (3 g, 96% yield). t R = 6.21 min (>99% purity). 1

Fmoc-Tyr(3-I,Me)-OH
A solution of H-Tyr(3-I,Me)-OH (3 g, 9.34 mmol, 1 equiv) in dioxane (32 mL) was adjusted to pH 7-8 by addition of aqueous 10% Na 2 CO 3 . The reaction mixture was stirred at room temperature for 30 min and Fmoc-OSu (3.31 g, 9.81 mmol, 1.05 equiv) was then added. The mixture was stirred for 24 h at room temperature and then concentrated in vacuo. EtOAc (40 mL) was added and the organic solution was washed with 1 N HCl (30 mL) and H 2 O (3 × 30 mL). The aqueous layers were combined, adjusted to pH 1 and washed with EtOAc (3 × 40 mL). All the organic layers were combined, washed with brine (30 mL) and dried with anhydrous magnesium sulfate. Removal of the solvent followed by digestion of the resulting precipitate in pentane/diethyl ether (1:1, 50 mL) for 2 h afforded a white solid, which was purified by column chromatography.

General procedure for the synthesis of octapeptidyl resins 4, 7 and 10
The octapeptidyl resins 4, 7 and 10 were synthesized manually by the solid-phase method by using standard Fmoc chemistry. MBHA resin (0.4 mmol/g) was placed in a polypropylene syringe fitted with a polyethylene filter disk, and then was swollen with CH 2 Cl 2 (1 × 20 min) and DMF (1 × 20 min), treated with piperidine/DMF (3:7, 1 × 5 min) and washed with DMF (6 × 1 min). The Fmoc-Rink amide linker (4 equiv) was coupled using DIPCDI (4 equiv) and Oxyma (4 equiv) in DMF under stirring at room temperature overnight. After this time, the resin was washed with DMF After each coupling and Fmoc group removal step, the resulting resin was washed with DMF (6 × 1 min). Once the peptidyl sequence was complete, the resin was washed with DMF (6 × 1 min),

OpNB (10)
This peptidyl resin was prepared using the procedure described above employing Fmoc-Glu-

Biaryl cyclic peptidyl resin 8
This biaryl cyclic peptidyl resin was prepared from the linear peptidyl resin Boc-Tyr

Biaryl cyclic peptidyl resin 11
This biaryl cyclic peptidyl resin was prepared from the linear peptidyl resin Boc-Tyr

General method for the synthesis of the biaryl bicyclic peptides 1, 2 and 3
The

Biaryl bicyclic peptide 1
The biaryl bicyclic peptide 1 was prepared from biaryl cyclic peptidyl resin 5 following the general procedure described above. Acidolytic cleavage of the resulting resin gave the expected

Biaryl bicyclic peptide 2
The biaryl bicyclic peptide 2 was prepared from biaryl cyclic peptidyl resin 8 following the general procedure described above.

Biaryl bicyclic peptide 3
The biaryl bicyclic peptide 3 was prepared from biaryl cyclic peptidyl resin 11 following the general procedure described above. Acidolytic cleavage of the resulting resin gave the expected biaryl bicyclic peptide 3, as confirmed by mass spectrometry.