Ingratta Mark, Duhamel Jean
Institute for Polymer Research, Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
J Phys Chem B. 2008 Jul 31;112(30):9209-18. doi: 10.1021/jp8021248. Epub 2008 Jul 9.
Two series of pyrene-labeled poly(glutamic acid) (Py-PGA) were synthesized utilizing two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA). Several Py-PGAs were synthesized for each series with pyrene contents ranging from 4 to 15 mol %. Py-PGA forms a rigid alpha-helix in DMF that effectively locks the backbone in place, thus enabling only side-chain or linker motions to be monitored by time-resolved fluorescence. Time-resolved fluorescence decays were acquired for the pyrene monomer of the Py-PGA constructs and the fluorescence blob model (FBM) was used to quantify the dynamics of the different linkers connecting pyrene to the backbone. Nitromethane was used to shorten the lifetime of the pyrene monomer, in effect controlling the probing time of the pyrene group, from 50 to 155 ns for PGA-PBA and from 50 to 215 ns for PGA-PMA. The FBM analysis of the fluorescence decays led to the conclusion that excimer formation around the rigid alpha-helix backbone takes place in a compact environment. The number of glutamic acid units within a blob, N blob, decreased only slightly with decreasing probing time for both Py-PGA constructs as a result of the compact distribution of the chromophores around the alpha-helix. The PGA alpha-helix was modeled using Hyperchem software and the ability of two pyrene groups to encounter was evaluated as they were separated by increasing numbers of amino acids along the alpha-helix. The number of amino acids required for two pyrenes to lose their ability to overlap and form excimer matched closely the N blob values retrieved using the FBM.
利用两种不同的芘连接基团，即1-芘甲胺（PMA）和1-芘丁胺（PBA），合成了两个系列的芘标记聚谷氨酸（Py-PGA）。每个系列合成了几种芘含量在4%至15%（摩尔）之间的Py-PGA。Py-PGA在二甲基甲酰胺中形成刚性α-螺旋，有效地将主链固定在原位，因此通过时间分辨荧光仅能监测侧链或连接基团的运动。获取了Py-PGA构建体中芘单体的时间分辨荧光衰减，并使用荧光斑点模型（FBM）来量化连接芘与主链的不同连接基团的动力学。使用硝基甲烷缩短芘单体的寿命，实际上控制芘基团的探测时间，对于PGA-PBA从50纳秒到155纳秒，对于PGA-PMA从50纳秒到215纳秒。荧光衰减的FBM分析得出结论，在刚性α-螺旋主链周围的准分子形成发生在紧凑环境中。由于发色团在α-螺旋周围的紧凑分布，对于两种Py-PGA构建体，随着探测时间的减少，斑点内谷氨酸单元的数量N斑点仅略有下降。使用Hyperchem软件对PGAα-螺旋进行建模，并评估两个芘基团沿着α-螺旋被越来越多的氨基酸隔开时相互相遇的能力。两个芘失去重叠并形成准分子能力所需的氨基酸数量与使用FBM获得的N斑点值密切匹配。
