Uses
Building block for the synthesis of phosphothreonine containing peptides. T.Wakamiya et al., Chem. Lett. , 1994, 1099 T.Vorherr and W.Bannwarth, Bioorg. Med. Chem. Lett 5, 2661 (1995)
Synthesis
The general procedure for the synthesis of (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxybutanoic acid from (2S,3R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(((benzyloxy)(hydroxy)phosphono)oxy)butanoic acid with benzyl alcohol was as follows:
1. Raw material pretreatment: Dry the amino-protected amino acid (hydrated form) to a moisture content of <0.5% by weight. Weigh the dried amino-protected amino acids (1.0 equiv.) into a reaction flask. Anhydrous THF (4 volumes, EMD OmniSolv HPLC grade, >99.99%) was added and the total volume was labeled outside the flask. The mixture was heated to 70 °C and approximately 2 volumes of THF were removed by distillation at 700 mbar. the mixture was replenished to the original labeled volume with dry THF and this distillation and dilution process was repeated twice. The final water content of the amino acid solution was confirmed to be <0.20% by weight by Karl-Fischer titration.
2. Dichlorophosphite preparation: THF (8 volumes) was added to the flask under nitrogen protection and cooled to 0°C. PCl3 (1.30 equiv, Aldrich, Reagent Plus grade, 99%) was added slowly, keeping the reaction temperature at 0-5 °C. Pyridine (1 equiv, Aldrich, anhydrous, 99+%) was optionally added to maintain the reaction temperature at -5 to 5 °C. Benzyl alcohol (1.50 equiv, Aldrich, ACS grade, 99+%) was then added to maintain the temperature at -5 to 5 °C. The reaction mixture was stirred at 0-5 °C for 5 min and complete consumption of PCl3 was confirmed by 31P NMR. 2,6-Dimethylpyridine (3.00 equiv, Aldrich, >99%) was added and the reaction temperature was maintained at -5 to 5 °C to form a thick slurry.
3. Phosphite Intermediate Preparation: 2,6-dimethylpyridine (1.0 eq.) was added to a dry aminoprotected amino acid solution. This solution was slowly added to the mixture from Step 1, keeping the reaction temperature at -5 to 5°C. Rinse the flask with additional THF (1 volume) to ensure complete transfer. HPLC analysis (Method 1) was performed at 10-min intervals for 5 min after the start of the reaction until the ratio of APG-AA to the intermediate phosphite was stabilized.
4. Hydrolysis: After confirming that the reaction was complete, H2O (3.6 vol) was added slowly, keeping the reaction temperature at no more than 10°C to form a two-phase solution.
5. Oxidation: Add NaBr (2.32 eq., Aldrich, ACS grade, 99+%) and aqueous NaBrO3 (20 wt. %, 0.48 eq., Riedel-de-Haen, puriss grade, 98%) to the two-phase mixture, keeping the reaction at 0-5 °C. After completion of oxidation, the reaction was allowed to warm up slowly to room temperature, avoiding exceeding 30°C. The content of phosphite intermediates was confirmed to be <1.5% by HPLC. Finally, the reaction was terminated by a one-time addition of Na2S2O5 aqueous solution (10 wt%, 1 volume, Sigma-Aldrich, ACS grade, 97+%).
Product characterization: 1H NMR, 13C NMR, 31P NMR and HRMS data were structurally consistent with the target compound with a specific rotation of +5.99°.
