Synthesis And Application Of N-Tosyl-L-alanyloxyindole

In the commercial products for leukocytes detection,pyrrole esters or indoxyl esters are usually used as the chemical reaction substrates.There are only few pyrrole esters vendors in China,and the price is very expensive.Meanwhile no domestic vendors of indoxyl esters can be found.So it is particularly important to synthesize these two esters for the efficient detection of leukocytes.According to the literature,the synthesis route of pyrrole ester was studied and optimized.Pyrrole ester was synthesized from traps-cinnamic acid by epoxidation,nucleophilic substitution,cyclization,hydrolysis and esterification.The pyrrole ester we prepared is a pink powder(white powder in literature),with a 94.89%purity,which caused false positive results.So the pyrrole ester was purified further by the decolorization in activated carbon and recrystallization in toluene,in which a white powder was obtained with a 99.08%purity.Also the false positive results were eliminated efficiently.As an important intermediate compound for the synthesis of N-Tosyl-L-alanyloxyindole,3-hydroxyindole was synthesized from o-chlorobenzoic acid by Ullmann reaction,acetic anhydride protection,cyclization,decarboxylation,isomerization,acetyl protection and hydrolysis.3-Hydroxyindole is very sensitive to the air,and further purification is difficult.In our research,the diacetate of 3-hydroxyindole was purified by column chromatography and recrystallization in AcOEt/PE,then the hydrolysis gave 3-Hydroxyindole with a 95.79%purity.Also the reaction conditions for synthesis of N-Tosyl-L-alanyloxyindole were optimized by testing the protonic acid,Lewis base and different solvents.We obtained the highest yield when the reaction ratio of N-Tosyl-L-alanyl chloride to indoxyl was 1:1,in the presence of trifluoroacetic acid,pyridine,anhydrous magnesium sulfate,with THF as the solvent.The optimal yield is 71.4%with a 99.09%purity,and the cost of the route was also reduced.The molecular weight and purity of product and some intermediate compounds were confirmed by LC-MS and HPLC.In addition,the structures of the products and intermediate compounds were confirmed by 1H NMR and 13C NMR.Finally,we prepared leukocyte test strips which used the purchased pyrrole ester,selfmade pyrrole ester and indoxyl ester respectively.By the comparison of accuracy,sensitivity and reaction time performance to Siemens leukocyte test strips,N-Tosyl-L-alanyloxyindole is the best chemical reaction substrate.