烙印分子聚合物之合成及其對胜肽分子之辨識探討

Abstract

本研究首先以L-苯丙胺酸為模板分子，將其溶解於二甲基乙醯胺和水的混合液體中，加入N’N-亞甲基雙丙烯醯胺為交聯劑、2,2’-偶氮二異腈為起始劑，將此溶液於50℃水浴下攪拌20分鐘至完全溶解，隨後加入甲基丙烯酸作為聚合單體，室溫下攪拌10分鐘後以氮氣導入預先使用3-三甲氧基矽丙胺基氯化物進行矽烷化之內徑75 μm毛細管內，在65.5 ℃下聚合13.5分鐘，待聚合完成後，依序使用二甲基甲醯胺與水的混合液(v/v 1:1)、甲醇與醋酸混合液(v/v 10:1)萃取模板分子，萃取完成後將此管柱應用於三種芳香族胺基酸的分離，其最佳化條件為偵測波長210 nm、分析電壓10 kV，磷酸緩衝溶液60 mM、pH 8、添加20 %乙腈作為有機修飾劑。本研究第二部份以Tyr-Gly-Gly為模板分子，將其溶解於二甲基乙醯胺和水的混合液體中，加入N’N-亞甲基雙丙烯醯胺為交聯劑、2,2’-偶氮二異腈為起始劑，將此溶液於50℃水浴下攪拌20分鐘至完全溶解，隨後加入甲基丙烯酸作為聚合單體，室溫下攪拌10分鐘後以氮氣導入預先使用3-三甲氧基矽丙胺基氯化物進行矽烷化之內徑75 μm毛細管內，在65.5 ℃下聚合13.5分鐘，待聚合完成後，依序使用二甲基甲醯胺與水的混合液(v/v 1:1)、甲醇與醋酸混合液(v/v 10:1)萃取模板分子，萃取完成後將此管柱應用於十一種胜肽的分離，其最佳化條件為偵測波長210 nm，管柱長度60 cm(35 cm)以pH 4、30 mM之磷酸緩衝溶液作為動相，分析電壓為20 kV~30 kV的梯度施加。分離機制由淨電作用力、管柱孔洞大小、電泳淌度等所共同作用，而epitope approach所提供的高度選擇性則扮演了重要的角色。

In this work, I used peptide as template to make a MIP monolithic capillary and use this column to separate peptide samples in CEC. While trying to fabricate this column, the synthesis proceed encountered two limitation. The first one is that peptides are insoluble in organic solvents while the most often used MIP synthesis proceed were taken under organic environment. The second limitation is the size of template molecule. Using large molecule will give rise to steric effect and thermal disfavor. In order to overcome the first limitation, I tried to change the porogen and functional monomer to synthesize the MIP under water environment rather than to do derivation of peptide template. The second limitation was overcome by introducing the 『epitope approach』to MIP monolithic column formation that means we can use short-chain peptides as template instead of long-chain peptide. In the synthetic procedure, Tyr-Gly-Gly, methacrylic acid (as function monomer), N,N’-methylene bisacrylamide (as cross-linker) and α,α’-azobis(isobutyronitrile) (as initiator) were dissolved in porogen ( mixture of water and DMF [1:1, v/v] ) under 50 ℃water bath for 20 minutes. The solution was stirred for 10 minutes under room temperature, then it was filled in a fused silanized capillary. The mole ratio of template、functional monomer、cross-linker is 1：16.2：32.2. The filled capillary was polymerized by thermal initiated under 65.5℃ for 13.5 minutes to obtained a monolithic MIP column called Column-epitope . Column-epitope was washed by water / DMF(1:1)、MeOH / HAc(10:1) for two hours. The molecular recognition of Column-epitope due to epitope approach could be improved by separation 3 similar peptide, [Met5]-enkephalin, [Leu5]- enkephalin andβ-casomorphin Bovine. Other separation mechanisms such as relative mobility, hydrophobic force, electrostatic force…etc were also contributed to CEC separation. Column-epitope of 75 μm x 60 (35) cm with hydrostatic injection (10 cm x 10 sec), mobile phase of 5 % acetonitrile in 30 mM phosphate ( pH 4 ), applied voltage of voltage gradient 20 kV to 30 kV and detection UV 210 nm, eleven peptides, FRMF amide, β-casomorphin Human, β-casomorphin bovine, oxytocin, tocinoic acid, Angiotension I, Angiotension II, [Sar1,Thr8] Angiotension II, [Met5]-enkephalin, [Leu5]-enkephalin and template could be baseline separated within 40 minutes.