Alkylammonium Formate Mobile Phase Modifiers for Protein Liquid Chromatography

描述（由申请人提供）：我们的研究项目的主要目的是研究一类新的室温离子液体，甲酸烷基铵（AAFs），作为有机溶剂的流动相替代品，用于蛋白质的液相色谱（LC）。甲醇和乙腈等有机溶剂作为流动相改性剂，对反相LC分离的蛋白质的分辨率有明显的影响。合成了甲酸乙酯、正丙酯和正丁基甲酸铵等甲酸烷基铵，并对其粘度、极性、紫外吸光度和溶剂强度进行了表征。这些离子液体可以作为甲醇和乙腈的替代溶剂，用于紫外或荧光检测药物的LC分离。对这些离子液体中的蛋白质，特别是甲酸乙铵（EAF）的荧光研究表明，与甲醇和乙腈相比，疏水芳香氨基酸（如色氨酸）的展开和暴露较少。我们推测短链氨基酸如甲酸甲胺（MAF）和甲酸甲胺（EAF）与蛋白质的相容性最好。将研究MAF和EAF的合成和纯化，以将紫外背景降低到220 nm，从而可以利用圆二色性研究这些离子液体中细胞色素c、牛血清白蛋白和溶菌酶等模型蛋白的三级和二级结构。在缓冲液和MAF或EAF的混合物中，酶（如羟基类固醇脱氢酶）的活性应该比相应的缓冲液-有机溶剂溶液更稳定，允许它们用作分析试剂。用MAF和EAF作为流动相修饰剂对蛋白质的反相LC和疏水相互作用色谱（HIC）进行表征，并期望提高HIC的分辨率。根据所收集的组分的圆二色光谱，也预计将保持天然的蛋白质构象。这一建议与公共卫生的相关性在于，一类新的离子液体，甲酸烷基铵，已被发现比标准有机溶剂更适合用于蛋白质的纯化。在纯化所需的色谱分离过程中，蛋白质的天然构象保持不变。这将有利于制药行业和生物技术公司，以及对蛋白质和酶作为药物或试剂感兴趣的学术或政府研究项目，以促进医学研究。

DESCRIPTION (provided by applicant): The primary purpose of our research program is to investigate a new class of room temperature ionic liquids, alkylammonium formates (AAFs), as mobile phase alternatives to organic solvents for liquid chromatography (LC) of proteins. Organic solvents such as methanol and acetonitrile cause significant protein unfolding as mobile phase modifiers to control the resolution of proteins separated by reversed phase LC. Alkylammonium formates such as ethyl-, n- propyl-, and n-butyl-ammonium formates have been synthesized and characterized with respect to viscosity, polarity, UV absorbance, and solvent strength for LC. These ionic liquids can act as replacement solvents for methanol and aceonitrile for the LC separation of pharmaceuticals with UV or fluorescence detection. Fluorescence studies of proteins in these ionic liquids, particularly ethylammonium formate (EAF), have shown less unfolding and exposure of hydrophobic aromatic amino acids such as tryptophan as compared to methanol and acetonitrile. We hypothesize that the short chain AAFs such as methylammonium formate (MAF) and EAF will show the best compatibility with proteins. The synthesis and purification of MAF and EAF will be studied to reduce the UV background down to 220 nm so circular dichroism can be used to study the tertiary and secondary structures of model proteins such as cytochrome c, bovine serum albumin, and lysozyme in these ionic liquids. The activity of enzymes such as hydroxysteroid dehydrogenase should be more stable in mixtures of buffer and MAF or EAF than corresponding buffer-organic solvent solutions permitting their use as analytical reagents. Both reversed phase LC and hydrophobic interaction chromatography (HIC) of proteins will be characterized using MAF and EAF as mobile phase modifiers and improved resolution particularly for HIC is expected. Based on circular dichroism spectroscopy of collected fractions, it is also expected that the native protein conformations will be maintained. The relevance of this proposal to public health is that a new class of ionic liquids, alkylammonium formates, have been found to be more compatible than standard organic solvents for the purification of proteins. The native conformation of the protein is maintained during the chromatographic separation process required for purification. This will benefit the pharmaceutical industry and biotech companies as well as academic or government research programs with an interest in proteins and enzymes as drugs or reagents to facilitate medical research.