SYNTHESIS OF COMPOUND 1
Method
In a 300 ml round-bottom flask, dissolve D-threoninol ,4-(phenylazo)benzoic acid ,DCC and HOBt in 50 ml DMF.Stir the reaction mixture at room temperature (25 ºC) for 24 h.
Check the reaction by TLC with 25:1 (vol/vol) ethyl acetate/methanol.
Remove the precipitated by filtration and then remove the solvent with a rotary vacuum evaporator at 60 ºC to obtain an oily product.
Dissolve the oily product in ethyl acetate, and wash the solution with saturated aqueous solution of NaHCO3and then of NaCl through extraction in a separatory funnel.Add Na2SO4to the ethyl acetate solution to dry.Remove Na2SO4by filtration and rotary evaporator at 60 ºC to obtain a crude oily product.
Extraction solution was purified by flash column chromatography with the same eluent.
Check the purity of compound 1 by TLC with the same eluent. Measure 1H-NMR for further purity check.
SYNTHESIS OF COMPOUND 2
Method
Dissolve compound 1 and 4-dimethylaminopyridine in 40 ml dry pyridine in a 100 ml round-bottom flask and keep the solution under dry nitrogen (using a balloon full of dry nitrogen gas) on an ice bath (approximately 0–5 ºC).
Dissolve DMT-Cl in 10 ml dry CH2Cl2 in a 50 ml round-bottom flask under nitrogen and add to the above pyridine solution slowly under dry nitrogen on ice bath with stirring. Remove the reaction flask from the ice bath and continue stirring at room temperature for 24 h.
Check the reaction by TLC with 50:50:3 (vol/vol/vol) ethyl acetate/hexane/triethylamine.
Remove the solvent (pyridine and CH2Cl2) using a rotary evaporator at 35-60 ºC to obtain a crude oily product.
Dissolve the oily product in ethyl acetate, and wash the solution with saturated aqueous solution of NaHCO3 and then of NaCl through extraction in a separatory funnel.Add Na2SO4 to the ethyl acetate solution to dry.Remove Na2SO4by filtration and rotary evaporator at 35-60 ºC to obtain a crude oily product.
Extraction solution was purified by flash column chromatography with the same eluent.
Check the purity of compound 2 by TLC with the same eluent Measure 1H-NMR for further purity check.
SYNTHESIS OF COMPOUND 3
Method
Evaporate 1H-tetrazole with dry AN (1–2 ml) in an oven-dried 20 ml round-bottom flask twice to remove as much water as possible.Then add 5 ml dry AN. The evaporation can be carried out by removing the AN under vacuum, where the temperature is kept room temperature.
Evaporate compound 2 with dry AN (2–3 ml) in a 50 ml round-bottom flask twice to remove as much water as possible. Then add 10 ml dry AN. The evaporation can be carried out by removing the AN, where the temperature is kept below room temperature.
Add 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite to the above AN solution of compound 2 at room temperature under dry nitrogen and cool the reaction mixture in an ice bath (approximately 0–5 ºC). Add 1H-tetrazole solution dropwise to the reaction mixture under dry nitrogen and stir for approximately 1 h at room temperature.
Check the progress of the reaction by TLC with 40:60:3 (vol/vol/vol) ethyl acetate/hexane/triethylamine When the reaction is more than 85% complete, remove the solvent (AN) using a rotary evaporator below 35 ºC.
Dissolve the oily product in ethyl acetate, and wash the solution with saturated aqueous solution of NaHCO3 and then of NaCl through extraction in a separatory funnel.Add Na2SO4 to the ethyl acetate solution to dry.Remove Na2SO4 by filtration and rotary evaporator at 35 ºC to obtain a crude oily product.
Extraction solution was purified by flash column chromatography with the same eluent.
Remove the solvent using a rotary evaporator.
Dry compound 3 completely overnight. Measure TLC as a further purity check.
