Trifluoroacetic acid → p-Toluenesulfonic acid

After 9.96 g of 1-13C-L-leucine (Masstrace) was dissolved in 75 ml of 1N NaOH, a solution of di-t-butyl dicarbonate (Boc2O) (18.0 g) in acetone (50 ml) was added. Then, 5.21 ml of triethylamine was added dropwise thereto and stirred at room temperature. After one hour, a solution of Boc2O (9.8 g) in acetone (40 ml) was added and stirred overnight at room temperature. After acetone was distilled out under reduced pressure, 500 ml of ethyl acetate was added, precipitated with 6N HCl, washed with water and dried over anhydrous sodium sulfate. After the desicating agent was filtered out, 10 ml of cyclohexylamine (CHA) was added. After washing with 1N HCl, Boc-(13C-Leu) was extracted with saturated sodium hydrogencarbonate solution and the aqueous layer was acidified with 6N HCl, extracted with ethyl acetate, washed with water and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residual oily product was dissolved in hexane and 1.2 ml of water was added to crystallize to yield Boc-(13C-Leu)-OH.H2O. Ten (10) g of Boc-(13C-Leu)-OH.H2O was dissolved in ethanol (50 ml)-water (20 ml) and 6.52 g of cesium carbonate dissolved in 20 ml of distilled water was added. After concentration under reduced pressure, ethanol and toluene were added and the remaining water was azeotropically removed out to yield a gel product, which was suspended in 300 ml of dimethylformamide (DMF). To the suspension, 5.2 ml of benzyl bromide was dropwise added at room temperature under stirring. After stirring at room temperature for 45 minutes, the solvent was distilled out under reduced pressure. Ethyl acetate was added and washed with water. After drying over anhydrous sodium sulfate, ethyl acetate was distilled out under reduced pressure to yield an oily product. To the product, 59 ml of TFA was added and stirred at room temperature for 40 minutes. After adding 8.37 g of p-toluenesulfonic acid monohydrate, TFA was distilled out under reduced pressure. Diisopropyl ether was added to the residue to crystallize to yield TosOH.H-(13C-Leu)-OBzl. After dissolving 7.89 g of TosOH.H-(13C-Leu)-OBzl, 5.57 g of t-Boc-L-phenylalanine (Boc-Phe) and 2.97 g of HOBt in 80 ml of DMF, 4.03 ml of water soluble carbodiimide (WSCD: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) was added dropwise while being ice-cooled and stirred at room temperature for 3 hours. Ethyl acetate and ice water were added and separated into two layers. The organic layer was washed with saturated sodium hydrogen-carbonate, water, 0.1N HCl and water and dried over anhydrous sodium sulfate. After distilling out ethyl acetate under reduced pressure, hexane was added to crystallize to yield Boc-Phe-(13C-Leu)-OBzl. The Boc-Phe-(13C-Leu)-OBzl (5.2 g) was dissolved in 10 ml of acetic acid and 500 mg of 5% palladium carbon (Pd—C) was added. While stirring at room temperature, hydrogen gas was blown thereinto for 1 hour. After the catalyst was filtered out, the mother liquid was concentrated under reduced pressure to about 10 ml. While being ice-cooled, 7.23 ml of 4.6N HCl/dioxane was added and stirred for 10 minutes followed by further stirring at room temperature for 1 hour. After concentration under reduced pressure, ether was added to solidify. The resulting solid was filtered out, washed with ether and dissolved in 100 ml of distilled water. The insolubles were filtered off with a membrane filter and lyophilized. This product was purified in RP-HPLC (YMC-ODS (10 μm), 30 mm×250 mm, 20 ml/min, aq. CH3CN (0.05% HCl)) (5%–5%–20%, 0–15–75 minutes) and the main fraction was lyophilized to yield 2.84 g of Phe-(13C-Leu).

1. additionwas added
2. concentrationAfter concentration under reduced pressure, ethanol and toluene
3. additionwere added
4. customthe remaining water was azeotropically removed out
5. customto yield a gel product, which
6. stirringAfter stirring at room temperature for 45 minutes
7. distillationthe solvent was distilled out under reduced pressure
8. additionEthyl acetate was added
9. washwashed with water
10. dry with materialAfter drying over anhydrous sodium sulfate, ethyl acetate
11. distillationwas distilled out under reduced pressure
12. customto yield an oily product
13. stirringstirred at room temperature for 40 minutes
14. distillationwas distilled out under reduced pressure
15. additionDiisopropyl ether was added to the residue
16. customto crystallize

ORD record

ord-e75a431165a7465f8647d12efe10e50c

Source dataset

uspto-grants-2007_06

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07232559B1