Fluorinated Amino Acids for Nanobiosensing

用于纳米生物传感的氟化氨基酸

• 批准号: 1412978
• 负责人: Neal Zondlo
• 金额: $ 32.64万
• 依托单位: University of Delaware
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1412978&HistoricalAwards=false
• 关键词: Fluorinated; Amino; Acids; Nanobiosensing

Biological imaging has broad applications in basic research and in medicine, with new technologies capable of broad human and economic impact. The development of methods that can probe specific processes in complex solutions, in cells, or in organisms can substantially increase our understanding of complexity in biology and can be ultimately used to develop new approaches to identify changes that are associated with disease. In this work, new approaches to detect the functions of proteins and the structure of proteins are developed, via the incorporation of multiple fluorine atoms into proteins. Fluorine is exceptionally useful for imaging because it is not normally present in biological environments and because it can be specifically detected by magnetic resonance approaches such as MRI. Thus, addition of fluorine to molecules that are in complex solutions, such as inside cells, allows the specific identification of that fluorinated molecule in a sea of other non-fluorinated molecules that are not seen by fluorine magnetic resonance, as well as changes to those molecules that occur during biological processes. This proposal involves the development of highly fluorinated amino acids, to allow sensitive detection, and their incorporation in proteins and application to identify specific biological processes, including the actions of enzymes and other proteins, that are important in changes in cells that define development and disease. This approach should be broadly applicable to the identification of changes and functions of proteins by magnetic resonance. The technologies developed herein will be distributed to other researchers for maximum impact and may be commercialized, contributing to economic development. Undergraduate researchers will contribute significantly to all aspects of this project, training students who go on to careers in diverse areas and applications of science, including research, teaching, medicine, policy, business, and law. Undergraduate and graduate students trained in this project will have broad cross-disciplinary education, with preparation for diverse applications in their careers.With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Neal Zondlo of the University of Delaware to develop approaches to examine specific enzymatic activities and protein structure using F-19 NMR with fluorinated amino acids. Probing biological processes in real time in situ is a substantial challenge whose solution has broad potential applications in imaging and in understanding basic biology. The development of new and general approaches to imaging that allow the identification of specific intracellular or extracellular events will allow new explorations and interventions in basic and applied biological research. General methods to the application of fluorinated amino acids for the interrogation of biological processes in situ with high sensitivity will be developed. F-19 imaging has enormous potential because of its specificity (high signal to noise due to the absence of fluorine in vivo; application to detect specific molecular events), its high magnetic sensitivity comparable to proton, and its application using commercial proton NMR and MRI instruments. The potential of F-19 magnetic imaging is currently substantially limited by a need to achieve increased sensitivity for diverse applications and for the development of specific probes of defined biological processes. An ideal approach to enhance specificity and sensitivity of F-19 magnetic resonance spectroscopy would involve the incorporation of an intense fluorine signal in native ligands in a manner that is minimally disruptive of structure. Methods to incorporate perfluoro-tert-butyl groups into peptides as novel amino acids will be developed. Perfluoro-tert-butyl groups have 9 equivalent fluorines, and thus have a 9-fold increase in signal-to-noise over single fluorines. At least as importantly, perfluoro-tert-butyl groups are sharp singlets by NMR, further increasing signal-to-noise and operational simplicity, meaning that most existing proton-based instrumentation can readily be adjusted to detect peptides containing perfluoro-tert-butyl groups. This approach will be used to detect kinase activity in real time by F-19 NMR, with potential applications to imaging diverse biological processes. In addition, approaches to probe cis-trans isomerism by NMR in complex solutions will be developed, using 4,4-difluoroproline, an amino acid, which is a sensitive F-19 NMR probe of the isomerization state of proline amide bonds. The approaches developed may be broadly applicable to probing processes in solution, in cells, and potentially in vivo. While this work is initially focused on applications in kinases and cell signaling, it is also applicable to other enzymatic modifications of proteins, including proteases and cell surface ligands, as well as to protein-protein interactions. This work has the potential to open new and sensitive approaches to imaging.

生物成像在基础研究和医学中有着广泛的应用，其新技术能够对人类和经济产生广泛的影响。能够探测复杂溶液、细胞或生物体中特定过程的方法的发展，可以大大增加我们对生物学复杂性的理解，并最终可用于开发识别与疾病相关的变化的新方法。在这项工作中，通过将多个氟原子结合到蛋白质中，开发了检测蛋白质功能和蛋白质结构的新方法。氟对成像特别有用，因为它通常不存在于生物环境中，而且可以通过核磁共振等磁共振方法专门检测到。因此，将氟添加到复杂溶液中的分子中，例如细胞内的分子，可以在氟磁共振无法看到的其他非氟化分子海洋中具体识别该氟化分子，以及在生物过程中发生的这些分子的变化。这一建议涉及开发高度氟化的氨基酸，以便进行灵敏的检测，并将其纳入蛋白质和应用于确定特定的生物过程，包括酶和其他蛋白质的作用，这些过程在决定发育和疾病的细胞变化中很重要。该方法应广泛适用于磁共振鉴定蛋白质的变化和功能。在此开发的技术将分发给其他研究人员，以获得最大的影响，并可能商业化，促进经济发展。本科研究人员将为该项目的各个方面做出重大贡献，培养在不同领域和科学应用领域从事职业的学生，包括研究、教学、医学、政策、商业和法律。在这个项目中训练的本科生和研究生将获得广泛的跨学科教育，为他们的职业生涯中的各种应用做好准备。有了这个奖项，化学部门的生命过程化学项目资助特拉华大学的Neal Zondlo博士开发使用氟化氨基酸的F-19核磁共振检查特定酶活性和蛋白质结构的方法。实时原位探测生物过程是一项重大挑战，其解决方案在成像和理解基础生物学方面具有广泛的潜在应用。新的和通用的成像方法的发展，允许识别特定的细胞内或细胞外事件，将允许在基础和应用生物学研究中进行新的探索和干预。将开发利用氟化氨基酸进行高灵敏度生物过程原位检测的一般方法。F-19成像由于其特异性（体内不含氟，对噪声的信噪比高；可用于检测特定的分子事件）、可与质子相比较的高磁灵敏度以及可用于商用质子核磁共振和核磁共振仪器，因此具有巨大的潜力。F-19磁成像的潜力目前很大程度上受到限制，因为需要提高不同应用的灵敏度，以及开发特定生物过程的特定探针。提高F-19磁共振波谱的特异性和灵敏度的理想方法是将强氟信号以最小程度破坏结构的方式纳入天然配体中。将全氟叔丁基作为新型氨基酸纳入多肽的方法将得到发展。全氟叔丁基有9个等效的氟，因此比单个氟的信噪比增加了9倍。至少同样重要的是，全氟叔丁基是核磁共振的尖锐单线，进一步增加了信噪比和操作的简单性，这意味着大多数现有的基于质子的仪器可以很容易地调整以检测含有全氟叔丁基的肽。该方法将用于通过F-19核磁共振实时检测激酶活性，具有潜在的应用于多种生物过程成像。此外，将开发利用4,4-二氟脯氨酸（一种氨基酸，它是脯氨酸酰胺键异构化状态的敏感的F-19核磁共振探针）在复杂溶液中探测顺-反异构的方法。所开发的方法可能广泛适用于溶液中、细胞中以及潜在的体内探测过程。虽然这项工作最初集中在激酶和细胞信号传导方面的应用，但它也适用于蛋白质的其他酶修饰，包括蛋白酶和细胞表面配体，以及蛋白质-蛋白质相互作用。这项工作有可能为成像开辟新的、灵敏的方法。