Development Of Amino Functional Group-labeled Fluorescent Chiral Derivatization Reagent DBD-M-Pro

Chirality is the basic characteristic of biological systems.There are amino acid,lactic acid and other low-molecular-weight metabolites of optical isomers in organisms.Their synthesis and decomposition are able to various of diseases.For example,The synthesis and secretion of D-serine which is existed in the hippocampus of the brain are related to Alzheimer's disease.In many of methods about chiral separation,the pre-column derivatization method is one of the more effective methods.Although the operation is cumbersome,it has high sensitivity and can be separated by a cheap reversed-phase column with good repeatability.In view of this,A novel fluorescent chiral derivatization reagent which can improve the detection sensitivity of fluorescence and achieve chiral separation,easily,was developed and synthesized by ultra-high performance liquid chromatography(UPLC)-fluorescence(FL)method.The high sensitivity and high selective method for the analysis of trace amino functional chiral metabolites in the organism was developed and established.The present study focused on the chemical structure of 4-(N,N-dimethylaminosulfonyl)-7-fluorobenzofuran(DBD-F)with a fluorescent group as the precursor.(S)-2-methylproline(M-L-Pro)with the chiral center and a carboxyl group was introducedto.Synthesize the fluorescent chiral reagent DBD-trans-2-methyl-L-proline(DBD-M-Pro)with the identification of amino functional groups and the ability of chiral separation,Using ultra-high performance liquid chromatography(UPLC)with fluorescence(FL)analysis method,19 D,L-amino acids and phenethylamine was used as the amino compound model,to evaluate the chiral separation efficiency by UPLC-FL method,gradient analysis at C18 column,using 10 mmol/L ammonium acetate-0.05%of formic acid in water(pH 3),and containing 0.1%of formic acid in acetonitrile or 0.1%of formic acid in methanol as the mobile phase,column was used at a temperature of 40? and the fraction parts were sensitively detected by UPLC-fluorescence(FL,Ex:450nm,Em:560 nm).The results showed that in the 19 D,L-amino acids and phenethylamines isolated,The resoulyion of(Rs)17 amino acids and PEA were 1.59-24.11 except histidine(Rs 1.32)and serine(Rs 1.47),which was achieved complete separation.We observed that the degree of separation of amino compounds increased with the increase of absolute hydrophobicity.And D,L-Pro,D,L-Val,D,L-Trp,D,L-Phe,D,L-Leu,D,L-Lys as examples.In 30 min,Six kinds of D,L-amino acids achieved complete separation and the resolution were ranged from 2.8 to 14.03.The good linearity of the amino acids were achieved from the calibration curves(R2>0.9990)in the range of 2.5-500 pmol.The lowest detection limit(S/N=3)in the FL were 0.50-3.75 pmol.The relative standard of intra-day and inter-day about D,L-amino acids were 2.31-11.73%,1.75-11.26%.Respectively the average recovery of amino acid enantiomers were 95.99-106.97%.All meets the requirements of biological sample determination.D,L-Pro,D,L-Val,D,L-Trp,D,L-Phe,D,L-Leu,D,L-Lys in saliva of 40 healthy volunteers(15 males and 25 females)undergone the quantitative analysis.The content of D-Pro in males saliva are more than those from females saliva(p<0.05),Meanwhile,the valve of D-Lys in females saliva are significantly more than those from males saliva(p<0.001).In this study,a novel fluorescent chiral reagent,DBD-M-Pro,with the identification of amino functional groups and the ability of chiral separation was synthesized for the first time,19 kinds of chiral amino acids and phenylethylamine are used as the model of amino compounds to evaluate the chiral separation efficacy of this reagent as well as the quantitative analysis of six kings of chiral amino acids in human saliva was measured by UPLC-FL method.The reagent can achieve good chiral resolution by extending the retention time of derivatized products on reversed-phase columns.An effective fluorescent derivatization reagent for the development and establishment of high-sensitivity analysis methods about chiral amino metabolites was provided.A novel methodological strategy for metabolomics was formed.