Peptide aromatic interactions modulated by fluorinated residues: Synthesis, structure and biological activity of Somatostatin analogs containing 3-(3′,5′difluorophenyl)-alanine

Somatostatin is a 14-residue peptide hormone that regulates the endocrine system by binding to five G-protein-coupled receptors (SSTR1–5). We have designed six new Somatostatin analogs with L-3-(3′,5′-difluorophenyl)-alanine (Dfp) as a substitute of Phe and studied the effect of an electron-poor aromatic ring in the network of aromatic interactions present in Somatostatin. Replacement of each of the Phe residues (positions 6, 7 and 11) by Dfp and use of a D-Trp8 yielded peptides whose main conformations could be characterized in aqueous solution by NMR. Receptor binding studies revealed that the analog with Dfp at position 7 displayed a remarkable affinity to SSTR2 and SSTR3. Analogs with Dfp at positions 6 or 11 displayed a π-π interaction with the Phe present at 11 or 6, respectively. Interestingly, these analogs, particularly [D-Trp8,L-Dfp11]-SRIF, showed high selectivity towards SSTR2, with a higher value than that of Octreotide and a similar one to that of native Somatostatin.


General methods and instrumentation for non-natural amino acids synthesis:
All reactions were carried out under nitrogen atmosphere unless otherwise specified. When dry solvents were necessary (dichloromethane, diethyl ether and tetrahydrofuran), the Innovative Technology Inc. Puresolv purification system Solvent Purification System (SPS) were used.
Other dry solvents were purchased from Sigma-Aldrich and used without further purification.
All experiments were monitored by analytical thin layer chromatography (TLC) performed on silica gel TLC-aluminum sheets (Merck 60 F254). Chromatographic purifications were carried out using a CombiflashR (Teledyne Isco) automated chromatography system unless otherwise stated. Silica gel RediHepR columns were used. The elution was carried out using hexanes/EtOAc gradients.
NMR spectra of small molecules (non peptidic) were recorded at room temperature on a Varian Mercury 400. 1H NMR and 13C NMR spectra were referenced to residual solvent peaks. 19F NMR spectra were referenced by the spectrometer without external reference.
Signal multiplicities in the 13C spectra have been assigned by DEPT (Direct Enhancement by Polarization Transfer) and HSQC (Hetero-nuclear Single Quantum Correlation) experiments and are described as C (quaternary), CH (tertiary), CH2 (secondary) and CH3 (primary). The following abbreviations were used to define the multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The coupling constants (J) are measured in hertz (Hz).
Melting points were measured using DSC 822e Mettler-Toledo apparatus. HRMS experiments were carried out in The Mass Spectrometry Core Facility located in the Institute for Research in Biomedicine of the using NanoESI techniques. CHNS elemental analyses have all been determined by the Unitat de Tècniques Separitives i Síntesi de Pèptids (The Separation Techniques and Peptide Synthesis Unit) located at the Barcelona Science Park. All IR spectrums have been obtained using a Thermo Nicolet Nexus FT-IR Fourier transform spectrometer. The samples were prepared by either dissolution in solvent and subsequent formation of a film on a NaCl disc by evaporation of the prepared solution or by the preparation of a KBr disc.
Optical rotations were measured at room temperature (25°C) using a Jasco P-2000 iRM-800 polarimeter. A cell with a length of 1 dm and a volume of 1 mL has been used. The concentration is expressed in the form g/100 mL. A sodium lamp with a wavelength of 589 nm has been employed.
The crude was allowed to cool to room temperature and a stirrable paste was formed. Then, it was cooled to 0 C and cold water (60 mL) was added. The mixture was stirred 10 min.
Filtration, washing the collected solid with cold water (3x15 mL) and drying at 50 C in vacuo provided 14.1 g (90% yield) of the titled compound (1) as a light brown powder, which was used in the next reaction without further purification.   (Z)-4-(3,5-Difluorobenzylidene)-2-methyloxazol-5(4H)-one (1) (10 g, 45 mmol, 1 eq) was dissolved in anhydrous MeOH (133 mL) in a two-neck 250-mL round-bottom flask fitted with a mechanical stirrer and a condenser, and under N2 atmosphere. MeONa (3.8 g, 49 mmol, 1.1 eq) was added dropwise, and the reaction mixture was stirred for 2 h at 70 ºC. The crude was allowed to cool to room temperature and the solvent was evaporated under reduced pressure.
The crude mixture was redissolved, neutralized with HCl and extracted with AcOEt. The combined extracts were washed with brine solution and dried over MgSO4. After filtration of MgSO4, the solvent was removed under vacuum and the brown colored residue was purified by silica gel flash chromatography to give pure 6.9 g (27 mmol, 65 % yield) of the titled product (2) as a white solid.

N-Fmoc-L-3-(3,5-difluorophenyl)-alanine, (5)
The L-3-(3,5-difluorophenyl)-alanine hydrochloride (5) (6.2 g, 26.2 mmol) was suspended in 66 mL of an aqueous solution of Na2CO3 (7.6 g, 72 mmol). The mixture was cooled to 0 ºC and a solution of FmocOSu (12.1 g, 36 mmol) in acetone (66 mL) was slowly added. The resulting mixture was allowed to warm to room temperature and stirred for 20h. Then, 20mL of water was added and the reaction mixture was extracted with ethyl acetate. The organic layer was back extracted with water, and the combined aqueous layers were washed with AcOEt, acidified to a pH of 1 with aqueous HCl and extracted with AcOEt.
The resulting solution was kept in a flask protected from light.

NMR and Computational methods:
All NMR data was processed with NMRPipe. 3 Cara 4 was used to assign the spectra. Distance restraints derived from fully assigned peaks in NOESY experiments were used for structure calculation. The structures were calculated with the programs CNS 5 and StructCalc. 6