(S)-N-Acetyl-2'-fluorophenylalanine ((S)-85)
A-Acetyl-2'-fluorophenylalanine
(85) (0.110 g, 0.488 x 10
*3mol) was dissolved in
0.05 mol dm
*3pH 7.0 phosphate buffer (10 cm3), and the pH was adjusted to 7.0 with
ammonia solution. Acylase I (10 x 10
*3g, 0.12 x 10
*6mol) was added and the mixture
was stirred at 298 K for
6days. The reaction mixture was treated with charcoal at 333 K
for 10 min, filtered and the filtrate was acidified to ca. pH 1 with 2 mol dm
3hydrochloric
acid and was extracted with ethyl acetate ( 4 x 1 0 cm3). The solvent was removed in
vacuo and the residue was taken up in water (2.5 cm3). Triethylamine (0.3 cm3) and
acetic anhydride (0.3 cm3) were added and the mixture was stirred at room temperature
for 3 h. The mixture was acidified to ca. pH 1 with 2 mol dm
*3hydrochloric acid and
was extracted with ethyl acetate ( 4x10 cm3). The combined organic extracts were dried
over magnesium sulfate and the solvent was removed in vacuo. The residue was purified
by preparative thin layer chromatography (ethanol : methanol : water : acetic acid,
1 9 : 3 : 2 : 1) and recrystallised from ethyl acetate / hexane to give the title compound as a
colourless solid (28.1 x 10
*3g, 25%). m.p. 164-166 °C (lit
.,1581 68-170 °C);
NMR
Experimental
(300 MHz, d6-DMSO) 51.71 (s, 3H, -COCH3), 2.69 (dd, J = 9, 14 Hz, 1H, ß-H ), 3.17 (dd, J = 6, 14 Hz, 1H, ß-H), 4.1 (m, 1H, cc-H), 7-7.30 (m, 4H, Ar-H), 8.24 (d, J = 8 Hz, 1H, NH), 12.75 (br s, 1H, C 0 2H).
A sample (ca. 2 x 10 3 g) of the product was added to thionyl chloride (0.01 cm 3) pretreated methanol (1 cm3) and the solution was stirred for 2 h at room temperature. The solvent was removed in vacuo and the residue was analysed by GC on a Chirasil-Val capillary column (0.3 x 1 0 3 m x 25 m). GC (180 °C): tR = 5.50 min
A -A cetyltyrosine (92)
Tyrosine (42) (1.45 g, 8.00 x 10 3 mol) was dissolved in water (2.5 cm 3) and 2 mol dm 3 sodium hydroxide solution (0.65 cm3) and the solution cooled in ice. Acetic anhydride (2.0 cm 3) and 2 mol dm 3 sodium hydroxide solution (20 cm 3) were each added in 8 equal portions with cooling and shaking over 20 min and the resultant mixture was allowed to sit at room temperature for 40 min. To this was added 6 mol d m '3 sulfuric acid (8.4 cm3) and the resultant solution was cooled at 277 K for 16 h. The solvent was removed in vacuo and the residue triturated with acetone. The residue was taken up in ethyl acetate (50 cm 3) and washed with water (50 cm 3), which was subsequently washed with ethyl acetate (2 x 50 cm3). The combined organic layers were dried over magnesium sulfate and the solvent was removed in vacuo. The residue was recrystallised from ethyl acetate / hexane to give the title compound as colourless crystals (1.14 g, 64%). m.p. 121-122 °C (lit.,187 94-95 °C for the hydrate); *H NMR (300 MHz, d 6-DM SO) 51.78 (s, 3H, -COCH3), 2.73 (dd, J = 9, 14 Hz, 1H, ß-H ), 2.85 (dd, J = 6, 14 Hz, 1H, ß-H), 4.3 (m, 1H, a-H ), 6.64 (d, J = 8 Hz, 2H, Ar-H), 6.97 (d, 7 = 8 Hz, 2H, Ar-H), 8.27 (d, J = 8 Hz, 1H, NH).
A sample (ca. 10 x 1 0 3 g) o f the product was added to thionyl chloride (0.05 cm 3) pretreated methanol (5 cm3) and the solution was stirred for 2 h at room temperature. The solvent was removed in vacuo and the residue was analysed by GC on a Chirasil-Val capillary column (0.3 x 10~3 m x 25 m). GC (140 °C): tR = 13.0, 13.6 min
(S)-A -A cetyltyrosine ((S )-9 2 )
A suspension of triethylamine (14 cm3) in water (50 cm3) was prepared. (S)-Tyrosine ((S)-42) (1.51 g, 8.33 x 10'3 mol) was taken up in water (2.5 cm 3) and triethylamine suspension (4.25 cm 3). Acetic anhydride (2.0 cm 3) and triethylam ine suspension (20 cm3) were each added in 8 equal portions with cooling and shaking over 20 min and
the reaction mixture was allowed to sit at room temperature for 40 min. To this was added 6 mol dm '3 sulfuric acid (8.4 cm3) and the resultant solution was cooled at 277 K for 16 h. The solvent was removed in vacuo to give an orange oil, which was partitioned between water and ethyl acetate (3 x 50 cm3). The combined organic layers were dried over magnesium sulfate and the solvent was removed in vacuo to give a yellow solid. This was recrystallised from ethyl acetate / hexane to give the title compound as a colourless powder (0.989 g, 54%). m.p. 151-152 °C (lit.,164 153-154 °C); lH NMR (300 MHz, dö-DMSO) 81.77 (s, 3H, -COCH3), 2.70 (dd, J = 9, 14 Hz, 1H, ß-H), 2.90 (dd, J = 5, 14 Hz, 1H, ß-H), 4.3 (m, 1H, a-H ), 6.64 (d, J = 9 Hz, 2H, Ar-H), 7.00 (d, J = 9 Hz, 2H, Ar-H), 8.10 (d, J = 8 Hz, 1H, NH), 9.19 (s, 1H, Ar-OH)
A sample (c a. 20 x 10'3 g) of the product was added to thionyl chloride (0.1 cm 3) pretreated methanol (10 cm3) and the solution was stirred for 2 h at room temperature. The solvent was removed in vacuo and the residue was analysed by GC on a Chirasil-Val capillary column (0.3 x 10'3 m x 25 m). GC (140 °C): tR = 13.6 min
A^-Acetyl-2'-hydroxyphenylalanine (93)
2'-H ydroxyphenylalanine (65) (0.228 g, 1.26 x 10'3 mol) was dissolved in water (0.38 cm 3) and 2 mol dm '3 sodium hydroxide solution (0.65 cm 3) and this solution cooled in ice. Acetic anhydride (0.30 cm3) and 2 mol dm 3 sodium hydroxide solution (3.0 cm3) were each added in 6 equal portions with cooling and shaking over 20 min and the resultant mixture was allowed to sit at room temperature for 40 min. To this was added 6 mol dm '3 sulfuric acid (1.3 cm3) and the resultant solution was cooled at 277 K for 16 h. The solvent was removed in vacuo and the residue was triturated with acetone. The residue was taken up in ethyl acetate (10 cm3) and washed with water (10 cm 3), which was subsequently washed with ethyl acetate ( 2 x 1 0 cm3). The combined organic layers were dried over magnesium sulfate and the solvent was removed in vacuo. The residue was recrystallised from ethyl acetate / hexane to give the title compound as a hygroscopic colourless powder (0.201 g, 72%). m.p. 142-144 °C; ]H NMR (300 MHz, d 6-DMSO) 51.92 (s, 3H, -COCH3), 3.09 (m, 2H, ß-H), 4.76 (m, 1H, a -H ), 7.1-7.2 (m, 4H, Ar-H), 8.54 (d, J = 8 Hz, 1H, NH); 13C NMR (75.5 MHz, d6-DMSO) 522.5 (ß-C), 32.1 (-COCH3), 52.0 (a-C ), 114.9, 118.8, 123.9, 127.8, 130.9 (aromatic C), 155.5 (arom atic C-OH), 169.4 (-C O C H 3), 173.8 ( - C 0 2H); v max(N ujol, c m '1) 3000-3500, 1705, 1638, 1506, 1458, 1376, 1250, 758; m/z (El) 223 (M+, 18%), 205 (19), 164 (19), 146 (100), 130 (49), 118 (83), 112 (60), 91 (26), 77 (39); Found: C, 56.9; H, 6.1; N, 5.7. Calc, for C n H 13N 05.0.5H 20: C, 56.9; H, 6.1; N, 6.0%.
Experimental
General procedure for the treatment of regioisomeric pairs of substituted A -acetylp h en ylalan in es with acylase I, follow ed by isolation of the phenylalanines produced
Samples o f racemic ortho- and para-substituted A-acetylphenylalanines (ca. 20 x 10'3 g of each) were dissolved in 0.05 mol dm '3 pH 7.0 phosphate buffer (10 cm3) and the pH checked. Acylase I (ca. 6 x 1 0 3 g, 70 x 10'9 mol) was added and the mixtures were stirred at 298 K for 2 h, acidified to ca. pH 6 with 2 mol dm '3 hydrochloric acid and heated with charcoal at 333 K for 10 min. After cooling to room temperature, the reaction mixtures were filtered, the filtrates were acidified to ca. pH 6 with 2 mol dm 3 hydrochloric acid and were extracted with ethyl acetate (4 x 20 cm3). The aqueous layers were taken and the solvents were removed in vacuo. The residues were taken up in water (5 cm3), triethylamine (0.2 cm 3) and acetic anhydride (0.2 cm 3) were added and the solutions were stirred at room temperature for 3 h. The solutions were acidified to ca. pH 1 with 2 mol dm '3 hydrochloric acid and were extracted with ethyl acetate ( 4 x 1 0 cm 3). In each case, the combined organic layers were dried over magnesium sulfate and the solvent was removed in vacuo. Where analysis of the ]H NM R spectrum of this residue indicated a single regioisomeric product, the residue was recrystallised from ethyl acetate / hexane. In all cases, a small sample (ca. 10'3 g) of the residue or product was dissolved in thionyl chloride (0.01 cm3) pretreated methanol (1.0 cm3) and the reaction mixture was stirred for 2 h at room temperature. The solvent was removed in vacuo and the residue was analysed by GC on a Chirasil-Val capillary column (0.3 x 10'3 m x 25 m).
N-Acetyl-4'-fluorophenylalanine (84) and N-acetyl-2'-fluorophenylalanine (85) Starting Materials: A-Acetyl-4'-fluorophenylalanine (84) 18.8 x 10'3 g;
7V-Acetyl-2'-fluorophenylalanine (85) 19.2 x 10-3 g
Products: (S)-Af-Acetyl-4'-fluorophenylalanine ((S)-84) and (S)-./V-Acetyl-2'-fluorophenylalanine ((S)-85)
Ratio: 2 : 1 (based on !H NMR spectrum and GC.) Yield: 8.4 x 10'3 g (58%, 29%); *H NMR (300 MHz, d6-DMSO) 81.76 (s, 3H, 0-COCH3), 1.79 (s, 6H, p -C O C H 3), 2.8 (m, 3H, p-ß-H and o-ß-H), 3.04 (dd. J = 5, 14 Hz, 2H, p-ß-H ), 3.14 (dd, 7 = 5 , 1 4 Hz, 1H, o -ß -H ), 4.5 (m, 3H, p - a - H and o -a -H ), 7-7.40 (m, 12H, p -A r-H and o-Ar-H), 8.18 (d, 7 = 8 Hz, 2H, p-NH), 8.22 (d, 7 = 8 Hz, 1H, o-NH).
Methyl Ester: GC (160 °C): tR = 9.20, 9.60 min with integrations of 1 and 2 respectively. (These correspond to the methyl esters o f the substituted phenylalanines (S)-84 and (S)-85 as determined by injection of standards.)