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(申请人提供)：我们研究计划的主要目的是研究一种新型的室温离子液体--烷基甲酸铵(AAFS)，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改进剂可使蛋白质大量展开，从而控制反相LC分离蛋白质的拆分。合成了烷基甲酸铵，如甲酸乙酯、正丙基甲酸铵和正丁基甲酸铵，并对它们的粘度、极性、紫外吸收和液晶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，用于药物的紫外或荧光检测的LC分离。对这些离子液体中蛋白质的荧光研究，特别是甲酸乙铵(EAF)，表明与甲醇和乙腈相比，色氨酸等疏水性芳香氨基酸的展开和暴露较少。我们推测，短链AAFs如甲酸甲铵(MAF)和电弧炉与蛋白质的相容性最好。将研究MAF和EAF的合成和纯化，使紫外光背景降低到220 nm，这样圆二色谱就可以用来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，羟基类固醇脱氢酶等酶的活性应该比相应的缓冲液-有机溶剂溶液更稳定，从而允许将其用作分析试剂。以MAF和EAF作为流动相改进剂，蛋白质的反相LC和疏水相互作用层析(HIC)都将得到表征，特别是HIC的分辨率有望得到提高。基于收集的组分的圆二色谱，还有望保持天然蛋白质的构象。这项提议与公众健康的相关性在于，一种新的离子液体--烷基甲酸铵--被发现比标准有机溶剂更适合于蛋白质的纯化。蛋白质的天然构象在纯化所需的层析分离过程中保持不变。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。

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.