with the riboswitch highlights new interactions possibly leading to tighter binding pocket as exemplified by the rotation of the base of guanosine 60 synthesis and purification ptpp is synthesized using conventional means briefly ortho phosphoric acid 0 5 g fluka 79622 is liquefied over flame and cooled to room temperature pyrithiamine hydrobromide 5 mg sigma p0256 is then added and the mixture is stirred for 15 min in an oil bath heated to 110 u00b0 c after cooling the reaction is quenched by adding 2 ml of water and magnesium oxide powder until the ph value reached 7 0 this step allows the removal of the unreacted inorganic phosphate for further purification using a c18 column vydac 218tp 54 or 1022 with triethylammonium solution at 100 mm ph 7 0 pyrithiamine mono di and tri phosphate can easily be separated by the hplc chromatography fractions containing the ptpp are collected and concentrated an identical procedure is used for the synthesis of otpp starting from oxythiamine chloride hydrochloride sigma o4000 tpp binding domain of the a thaliana tpp specific riboswitch is synthesized using general methods for the in vitro synthesis of rna molecules in detail the synthesis involves obtaining large quantities of dna with the sequence of the tpp binding domain preceded by a t7 promoter the dna is in vitro transcribed with t7 rna polymerase for 3.