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: 7933114
• 负责人: NEIL D DANIELSON
• 金额: $ 3万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933114
• 关键词: Abbreviations; Acetonitriles; Ammonium; Aromatic Amino Acids; Biochemistry; Bovine Serum Albumin; Buffers; Chemistry; Chromatography; Circular Dichroism; Circular Dichroism Spectroscopy; 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; Reagent; Research; Resolution; Sodium Chloride; Solutions; Solvents; Structure; Students; Techniques; Temperature; Tryptophan; Viscosity; 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型室温离子液体，即甲酸烷基铵（AAF），作为蛋白质液相色谱（LC）有机溶剂的流动相替代品。甲醇和乙腈等有机溶剂作为流动相调节剂引起显着的蛋白质解折叠，以控制反相 LC 分离的蛋白质的分辨率。已经合成了甲酸烷基铵，例如甲酸乙酯、甲酸正丙酯和甲酸正丁酯，并对其粘度、极性、紫外吸光度和液相色谱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，用于通过紫外或荧光检测进行药物的液相色谱分离。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，疏水性芳香族氨基酸（如色氨酸）的解折叠和暴露较少。我们假设短链 AAF（例如甲酸甲酯铵 (MAF) 和 EAF）将表现出与蛋白质的最佳相容性。将研究MAF和EAF的合成和纯化，以将UV背景降低至220 nm，因此圆二色性可用于研究这些离子液体中的模型蛋白（例如细胞色素c、牛血清白蛋白和溶菌酶）的三级和二级结构。诸如羟基类固醇脱氢酶之类的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。蛋白质的反相 LC 和疏水相互作用色谱 (HIC) 将使用 MAF 和 EAF 作为流动相改性剂进行表征，并且预计会提高尤其是 HIC 的分辨率。基于收集的级分的圆二色光谱，还预计天然蛋白质构象将被保持。该提案与公共卫生的相关性在于，人们发现一类新型离子液体——甲酸烷基铵比标准有机溶剂更适合纯化蛋白质。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。这将使制药业和生物技术公司以及对蛋白质和酶作为药物或试剂促进医学研究感兴趣的学术或政府研究项目受益。

项目成果

Methylammonium formate as a mobile phase modifier for totally aqueous reversed-phase liquid chromatography.

• DOI: 10.1016/j.chroma.2008.09.064
• 发表时间: 2009-04-17
• 期刊: Journal of chromatography. A
• 作者: Grossman S;Danielson ND
• 通讯作者: Danielson ND

Fluorescence and circular dichroism spectroscopy of cytochrome c in alkylammonium formate ionic liquids.

• DOI: 10.1021/bm1008052
• 发表时间: 2011-02-14
• 期刊: BIOMACROMOLECULES
• 影响因子: 6.2
• 作者: Wei, Wenjun;Danielson, Neil D.
• 通讯作者: Danielson, Neil D.

The ionic liquid isopropylammonium formate as a mobile phase modifier to improve protein stability during reversed phase liquid chromatography.

• DOI: 10.1016/j.jchromb.2013.08.005
• 发表时间: 2013-12-01
• 期刊: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
• 影响因子: 3
• 作者: Zhou, Ling;Danielson, Neil D.
• 通讯作者: Danielson, Neil D.