胺類藥物之新光學衍生劑 AFGIT 之開發及分割應用

1. 胺類藥物之新光學衍生劑AFGIT之開發及分割應用胺類藥物之新光學衍生劑AFGIT之開發及分割應用 • 目前已有多種光學分割衍生劑被用於分割混旋性胺類化合物，其中以GITC (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate)最炳`用，即利用其結構中之isothiocyanate與具有amine官能基之混旋性 化合物形成thioureas，這些衍生物通常可於HPLC中得到很好的分離 度，並可利用最普遍的UV偵測器檢測，但其缺點是本身僅有微弱的發 色團，在低濃度的檢品中，常因其靈敏度不夠而無法偵測到。為改進此 缺點，本實驗室曾合成一種具有螢光的光學衍生劑CGIT [6-O-(4'-me-thylcoumarin-7'-methoxycarbonyl)-2,3,4-tri-O-acetyl-α-D-glucopyranosisothiocyanate]，且經實驗可與一級或二級的混旋性胺類或胺基酸反應後 進行光學分割，其靈敏度比GITC衍生物強10~36倍。現本研究擬合成 另一種螢光性的光學衍生劑AFGIT [6-(9'-aminofluorenyl)-2,3,4-tri-O- acetyl--D-glucunopyranosylisothiocyanate]，即利用葡萄糖醛酸內酯 (glucuronolactone)作起始物，其在50%甲醇溶液中經NaOH開環形成 sodium glucuronate，在p-toluenesulfonic acid催化下進行乙醯化反應後形 成1,2,3,4-tetra-O-acetyl--D-glucuronic acid，然後與具螢光性的9-aminofluorene接合，再與HBr反應生成acetobromoglucuronic acid衍生物，再與silver isothiocyanate反應後，便可合成AFGIT . AFGIT可與一級及二級混旋性胺類化合物於室溫下在氰甲烷中反應一小時生成衍生物，而胺基酸可在50%的氰甲烷水溶液及三乙胺的鹼性條件下於室溫反應一小時生成衍生物。AFGIT與多種混旋性的一級及二級胺類或胺基酸反應，生成diastereoisomeric的thiourea衍生物，這些衍生物可利用HPLC逆相層析管分離，用螢光偵測器檢測(激發波長：270 nm，發散波長：310 nm)，以55~70%的甲醇-0.1%磷酸水溶液可分離胺基酸，60%的氰甲烷-0.1%磷酸水溶液可分離adrenergic β-blockers，氰甲烷：甲醇：0.1%磷酸水溶液(40 : 20 : 40)可分離phenethylamines，均可得到 很好的分離度(α)，如十二種混旋胺基酸之AFGIT衍生物的分離度α= 1.04~1.71，四種混旋adrenergic β-blockers之AFGIT衍生物的分離度 也均在1.1以上，以及四種混旋phenethylamines之AFGIT衍生物，包 括國內濫用的甲基安非他命，分離度α在1.05~1.15之間，且利用DL- alanine分別與AFGIT及GITC反應成衍生物後，測得AFGIT-L-alanine 之靈敏度比GITC高約十八倍。結論，本研究所開發之新光學衍生劑 AFGIT能有效的分離混旋性胺類藥物或胺基酸，並且增加它們的偵測 靈敏度。

2. Development new chiral derivatizing agent,AFGIT for amine drugs • GITC(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate)is one GITCof the most widely applicable chiralderivatizing agent to resolve racemic amines . GITCcontaining isothiocyanate active moiety can react with thosecompounds contain amine functional group. Good resolution of thediastereomeric thiourea derivatives is generally achieved byHPLC via UV detection .Due to weak chromophore , GITCis not sensitive enough to be detected in lowconcentration . In order to improve the detection sensitivity,fluorescent chiral derivatizing agent，CGIT [6-O-(4'-methylcoumarin-7'- methoxycarbonyl)-2,3,4-tri-O-acetyl-a-D-glucopyranosyl isothiocyanate] wasdeveloped previously in our laboratory . Now we like to reporthere the synthesis of another new fluorescent chiralderivatizing agent，AFGIT [6-(9'-aminofluorenyl)-2,3,4-tri-O-acetyl-b-D-glucunopyranosylisothiocyanate] . Glucuronolactone is served as a startingmaterial for the preparation of AFGIT . Glucuronolactonewas reacted with NaOH in 50% CH3OH aqueous toproduce sodium glucuronate，and then was acetylatedin the presence of p-toluenesulfonic acid . Incoporation of9-aminofluorene to tetra-O-acetyl glucuronic acid as anamide linkage and following reaction with HBr afforedthe acetobromoglucuronic acid，subsequent reaction withAgSCN yielded the fluorescent chiral derivatizing agent，AFGIT .Derivatization was performed by reaction of AFGIT with primaryor secondary amines in CH3CN for one hour .However amino acids were reacted in 50% aqueousCH3CN catalyzed by triethylamine for one hour .These diastereoisomeric thioureas was separated on a reversedHPLC column (C18) with a fluorescence detector(λex. : 270 nm ,λem. : 310 nm). Mobile phase of 55~70%CH3OH in 0.1% H3PO4 solution for racemic aminoacids，60% CH3CN in 0.1% H3PO4 aqueous (v/v) for racemicadrenergic b-blockers，and CH3CN : CH3OH : 0.1%H3PO4 aqueous = 40 : 20 : 40 (v/v/v) forphenethylamines were applied . Twelve racemic amino acids (a=1.04~1.71) , four adrenergic b-blockers (a>1.1)，and fourphenethylamines (a=1.05~1.15) were resolved successfully.Comparison of the detection sensitivity of DL-alaninederivatives of AFGIT and GITC, the detection sensitivityof AFGIT derivatives were at least 18 times morestronger then those of GITC derivatives . In conclusion，the newchiral derivatizing agent，AFGIT，is not only usefulto resolve the racemic amine drugs or amino acidsbut also to enhance their detection sensitivity by thedeveloped methods.