Alkylammonium Formate Mobile Phase Modifiers for Protein Liquid Chromatography

用于蛋白质液相色谱的甲酸烷基铵流动相改性剂

• 批准号: 7252276
• 负责人: NEIL D DANIELSON
• 金额: $ 21.3万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7252276
• 关键词: Abbreviations; Acetonitriles; Aromatic Amino Acids; Biochemistry; Bovine Serum Albumin; Buffers; Chemistry; Chromatography; Circular Dichroism; Circular Dichroism Spectroscopy; Class; Compatible; Detection; Development; Drug Industry; Enzymes; Fluorescence; Formates; Government; Hydrophobic Interactions; Hydroxysteroid Dehydrogenases; Liquid Chromatography; Liquid substance; Marketing; Medical Research; Methanol; Modeling; Molecular Conformation; Muramidase; Occupations; Organic solvent product; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Problem Solving; Process; Protein Conformation; Proteins; Public Health; Purpose; Reagent; Research; Resolution; Sodium Chloride; Solutions; Solvents; Standards of Weights and Measures; Structure; Students; Techniques; Temperature; Tryptophan; Viscosity; ammonium formate; aqueous; base; carboxylate; cytochrome c; design; enzyme activity; ethylammonium; fundamental research; improved; interest; medical schools; monomethylammonium ion; programs; protein purification; small molecule; tetrahydrofuran

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.

描述（由申请人提供）：我们研究计划的主要目的是研究一类新的室温离子液体，甲酸烷基铵（AAF），作为蛋白质液相色谱（LC）有机溶剂的移动的替代品。有机溶剂如甲醇和乙腈作为移动的相调节剂引起显著的蛋白质解折叠，以控制通过反相LC分离的蛋白质的分辨率。已经合成了甲酸烷基铵，例如甲酸乙基铵、甲酸正丙基铵和甲酸正丁基铵，并就粘度、极性、UV吸光度和用于LC的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，用于药物的LC分离与UV或荧光检测。在这些离子液体中的蛋白质的荧光研究，特别是乙基铵甲酸（EAF），已经显示出较少的展开和暴露的疏水性芳香族氨基酸，如色氨酸相比，甲醇和乙腈。我们推测，短链AAF如甲酸甲铵（MAF）和EAF将显示最好的兼容性与蛋白质。MAF和EAF的合成和纯化将被研究以将UV背景降低到220 nm，因此圆二色性可以用于研究模型蛋白质如细胞色素c、牛血清白蛋白和溶菌酶在这些离子液体中的三级和二级结构。酶如羟基类固醇脱氢酶的活性在缓冲液和MAF或EAF的混合物中应该比相应的缓冲液-有机溶剂溶液更稳定，从而允许它们用作分析试剂。使用MAF和EAF作为移动的相改性剂，蛋白质的反相LC和疏水相互作用色谱（HIC）都将被表征，并且预期特别是HIC的分辨率将得到改善。基于收集的级分的圆二色光谱，还预期将保持天然蛋白质构象。这一提议与公共卫生的相关性在于，已经发现一类新的离子液体，甲酸烷基铵，比用于纯化蛋白质的标准有机溶剂更相容。在纯化所需的色谱分离过程中，蛋白质的天然构象得以保持。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计划，以促进医学研究。