胺類藥物之新光學衍生劑 AFGIT 之開發及分割應用
Development new chiral derivatizing agent,AFGIT for amine drugs
中文摘要 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-glucopyranosyl isothiocyanate)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-glucunopyranosyl isothiocyanate]，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。 英文摘要
GITC(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate) is one GITCof the most widely applicable chiral
derivatizing agent to resolve racemic amines . GITC
containing isothiocyanate active moiety can react with those compounds contain amine functional group. Good resolution of the diastereomeric thiourea derivatives is generally achieved by
HPLC via UV detection .Due to weak chromophore , GITC is not sensitive enough to be detected in low
concentration . 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] was
developed previously in our laboratory . Now we like to report here the synthesis of another new fluorescent chiral
derivatizing agent，AFGIT [6-
(9'-aminofluorenyl)-2,3,4-tri-O-acetyl-b-D-glucunopyranosyl isothiocyanate] . Glucuronolactone is served as a starting
material for the preparation of AFGIT . Glucuronolactone was reacted with NaOH in 50% CH3OH aqueous to produce sodium glucuronate，and then was acetylated in the presence of p-toluenesulfonic acid . Incoporation of 9-aminofluorene to tetra-O-acetyl glucuronic acid as an amide linkage and following reaction with HBr affored the acetobromoglucuronic acid，subsequent reaction with
AgSCN yielded the fluorescent chiral derivatizing agent，AFGIT . Derivatization was performed by reaction of AFGIT with primary or secondary amines in CH3CN for one hour .
However amino acids were reacted in 50% aqueous CH3CN catalyzed by triethylamine for one hour .
These diastereoisomeric thioureas was separated on a reversed HPLC 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 amino
adrenergic b-blockers，and CH3CN : CH3OH : 0.1% H3PO4 aqueous = 40 : 20 : 40 (v/v/v) for
phenethylamines were applied . Twelve racemic amino acids (a =1.04~1.71) , four adrenergic b-blockers (a>1.1)，and four phenethylamines (a=1.05~1.15) were resolved successfully. Comparison of the detection sensitivity of DL-alanine derivatives of AFGIT and GITC, the detection sensitivity of AFGIT derivatives were at least 18 times more
stronger then those of GITC derivatives . In conclusion，the new chiral derivatizing agent，AFGIT，is not only useful
to resolve the racemic amine drugs or amino acids but also to enhance their detection sensitivity by the developed methods.
