2.1. Materials
Acetylthiocholine iodide (ATC), DTNB [5,5´-dithiobis(2-nitro-benzoic acid)], ethopropazine, and bovine serum albumin (BSA) were acquired from Sigma (St. Louis, MO, USA). Disodium hydrogen phosphate (monobasic) and sodium dihydrogen phosphate (dibasic) were acquired from Neon Comercial LTDA (Brasil). All other chemicals used were of methodical grade.
Melting points (mp) were determined by using the MQAPF-301 apparatus. 1H and 13C NMR spectra (nuclear magnetic spectra) were documented on a Bruker Dpx-400 spectrometer operative at the 400.13 MHZ for 1H and 100.62 MHZ for 13C, using tetramethylsilane (TMS) as the interior standard and CDCl3 as solvent. On pre-coated TLC plates the thin-layer chromatography (TLC) was accomplished (Merck, silica gel 60 F-254). One or more of the following methods were used for the detection of spots: UV (254 nm), o-toluidine, ninhydrin (0.1% in ethanol), and dragendorff’s reagent. the compound’s purification chromatographic was carried out by means of a column packed with silica gel 60 (230–400 mesh) acquired from Merck Co. in system solvents selected by Rf separation in TLC.
All amino acids and substances used, except threo-β-Phenylserine acquired conferring to Shaw and Fox (Kenneth et al., 1953), possess L-configuration, and were commercially available by Aldrich/Fluka, and were utilized without further purification
2.2. Synthesis of dipeptide N-Cbz-L-Leu-threo-D, L-Pheser-OH (3)
To a suspension of threo-D, L-β-Phenylserine methyl ester hydrochloride (2) (10.1 g, 75 mmol ), (HOBt) (10.14 g, 75 mmol ), N-methyl morpholine (8.24 mL, 75 mmol) and N-Cbz-L-Leu-OH (1) (Figure.1) (19.6 g, 75 mmol) (crude from the previous step) in dry THF (50 ml) at 0°C, followed by addition of DCCI (16.5 g, 80 mmol) stirring at the same temperature for 3 h, the reaction was allowed to warm at room temperature and stirred for additional 20 hrs. Then the mixture of reaction was filtered to remove the DCCU and under the reduced pressure the solvent was removed. The resulting yellow residue was diluted in CHCl3 (100 mL) and splashed with H2O (40 mL and 20 mL), with sat. Aqueous NaHCO3 (2 x 30 mL), solution aqueous 10% citric acid (2 x 30 mL), water (20 mL), with sat, aqueous NaHCO3 (30 mL) and finally brine (30 mL). The joined layers of organic were dehydrated over MgSO4. Filtration and concentration afforded the dipeptide methyl ester that was hydrolyzed in the solution of 25 mL of MeOH and 1M NaOH cooled at 0°C, by flash chromatography the resultant crude oil was cleansed to afford compounds N-Cbz-L-Leu-threo-D, L-Pheser-OH (3) in a total product of 70% as a yellow oil, after purification in flash silica gel column chromatography.
1H NMR (CDCl3): δ 7.22–7.19 (br s, 10H), 6.16 (br d, 2H), 5.73 (br d, H), 4.70 (br dd, 1H), 4.94 (br s, 2H), 4.30 (br dd, 1H), 1.49 (m, 1H), 1.16 (m, 2H), 0.76 (br d, 3H), 0.70 (br d, 3H).13C NMR (CDCl3): δ 177.52 (177.11) (C-9), 173.50 (C-1), 156.82 (156.50) (C-16), 140.72 (C-4), 136.03 (C-18),128.18 (C-7), 127.76 (C-6), 127.67 (C-20), 127.62 (C-21), 125.78 (C-5), 125.76 (C-19), 73.13 (72.97) (C-3), 66.72 (C-17), 59.05 (C-10), 53.41 (C-2), 41.38 (C-11), 24.38 (C-12), 22.54 (C-14), 21.39 (C-13).
2.3. Synthesis of tri-peptides Z-L-Leu-threo-L- Pheser-L-Phe-OMe (4) and Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5)
The tripeptide Z-L-Leu-threo- Pheser-L-Phe-OMe production was carried out through our well described procedure (Mostardeiro et al., 2013). 2.36 g (5.53 mmol) of the di-peptide N-Cbz-L-Leu-threo-D, L-Pheser-OH (3) (Figure.1) in 5 mL THF was coupled with L-Phe-OMe (0.91g, 5.53 mmol) by DCC-method according to Bodanski to give the tri-peptide 78% as a diastereomeric mixture (Bodanszky, 1984). The separation of the diastereomeric mixture was achieved by recrystallization in ethylene ether-petrol ether, yield Z-L-Leu-threo-L-Pheser-L-Phe-OMe (48%) and Z-L-Leu-threo-D-Pheser-L-Phe-OMe (30%), as white solids.
Z-L-Leu-threo-L-Pheser-L-Phe-OMe (4) mp 139.8-141.8 ºC: 1H NMR (CDCl3): δ 7.17–7.34 (br m, 15H), 7.09 (d, 1H, J16 − 10= 8.0 Hz), 7.00 (d, 1H, J8 − 2= 8.3 Hz), 5.29 (d, 1H, J11 − 10= 3.0 Hz), 5.12 (br d, 1H), 5.10, 5.03 (d, 2H, J25 − 25’= 12.3 Hz), 4.77(dd, 1H,J2 − 3=6.8 Hz, J2 − 3`= 4.4 Hz, J2 − 8= 8.2 Hz), 4.70 (dd, 1H, J10 − 16= 8.0 Hz, J10 − 11= 3.0 Hz), 4.11 (m, 1H), 3.72 (s, 3H), 3.10, 3.03 (dd, 2H, J3 − 2=6.8 Hz, J3 − 2= 4.4 Hz), 1.50 (m, 1H), 1.41, 1.28 (m, 2H), 0.85 (d, 3H, J21 − 20= 6.4 Hz), 0.85 (d, 3H, J22 − 20= 6.4 Hz)(Bodanszky, 1984) C NMR (CDCl3): δ 172.78 (C-17), 171.33 (C-1) 169.77 (C-9), 156.25 (C-24), 139.03 (C-12), 135.96 (C-4), 135.79 (C-26), 129.19 (C-5), 128.54 (C-27), 128.52 (C-14), 128.50 (C-29), 128.24 (C-6), 128.00 (C-28), 127.71 (C-15), 127.11 (C-7), 125.86 (C-13), 71.98 (C-11), 67.21 (C-25), 57.76 (C-10), 53.71 (C-2), 53.57 (C-30), 53.32 (C-18), 40.99 (C-19), 37.65 (C-3), 24.54 (C-20), 22.79 (C-21), 21.17 (C-22). FAB-M+ (m/z): 591
Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5): amorphous powder, 1H NMR (CDCl3): δ 7.48 (d, 1H, J8-2 = 7.56 Hz), 7.10–7.25 (br m, 15H), 6.98 (d, 1H, J16-10 = 7.9 Hz), 5.42 (br d, 1H), 5.31 (d, 1H, J23-17 = 7.3 Hz), 5.04, 4.94 (d, 2H, J25-25’= 12.1 Hz), 4.50 (dd, 1H, J2-3 = 4.8 Hz, J2-3`= 7.6 Hz, J2-8 = 7.9 Hz), 4.75 (dd, 1H, J10-16 = 7.9 Hz), 3.99 (m, 1H), 3.61 (s, 3H), 3.08, 3.00 (dd, 2H, J3-2 = 6.0 Hz, J3-2 = 7.6 Hz, J3-3 = 14.0 Hz), 1.32 (m, 1H), 1.36, 1.30 (m, 2H), 0.72 (d, 3H, J21-20 = 6.4 Hz), 0.72 (d, 3H, J22-20 = 6.4 Hz). 13C NMR (CDCl3): δ 173.50 (C-17), 171.00 (C-1), 171.00 (C-9), 139.40 (C-12), 136.78 (C-24), 135.60 (C-4),134.95 (C-26), 129.12 (C-5), 128.42 (C-27), 127.92 (C-14), 127.64 (C-29), 127.41 (C-6), 126.99 (C-15), 126.65 (C-28), 125.96 (C-13), 125.57 (C-7), 71.80 (C-11), 65.32 (C-25), 58.71 (C-10), 57.17 (C-2), 53.00 (C-18), 52.13 (C-30), 40.76 (C-19), 36.92 (C-3), 24.19 (C-20), 22.20 (C-21), 21.35 (C-22). FAB-M+ (m/z): 591
Scheme 2: tri-peptides Z-L-Leu-threo-L- Pheser-L-Phe-OMe (4) and Z-L-Leu-threo-D-Pheser-L-Phe-OMe (5) synthesis