Lanthanide complexes for site-specific labelling of cysteine residues on proteins
Creators: name: Topping, Lydia
Publisher: name: Loughborough University
Year: 2024
Subjects: classificationCode: 349999
subject: Other chemical sciences not elsewhere classified

description: Lanthanide complexes are increasingly being studied and used in a number of applicatons within medical diagnostics, treatments, material sciences, industry, and agriculture sectors. Their unique luminescent propertes make them excellent luminescent dyes for use in bioassays and cellular imaging, and their paramagnetic propertes (partcularly of gadolinium complexes) make them ideal as MRI contrast agents for medical imaging. However, a rapidly growing applicaton of lanthanides is their use in protein structural studies. There are a number of lanthanide propertes that allow their use for protein structure determinaton to be so widely used. Their long emission lifetmes and line-like emission spectra make them excellent donors in FRET based experiments, and their paramagnetism allows for use in NMR and EPR spectroscopy.In order for successful use of lanthanides in the spectroscopic study of proteins, they need to be complexed to an organic ligand that fulfils certain criteria, which is as follows:To selectvely react with cysteine residues on the protein.To form a short and rigid tether between the lanthanide and protein.To be kinetcally and thermodynamically stable once attached to a protein.Once bound to a protein, the complex needs to form a single diastereoisomer in solution.The work presented in this thesis discusses the design, synthesis, and evaluation of novel lanthanide complexes as protein ‘tags’. Each complex has been designed to irreversibly react with specific sites on peptdes and proteins to facilitate their structural analysis. Throughout the design process, there were three macrocycles and three aromatic antennae designed and evaluated for their effects on the resulting complex’s emissive properties, and in certain cases their capabilities in paramagnetic NMR spectroscopy. Each lanthanide complex is designed to investigate any photophysical differences that occur upon reaction with cysteine, as well as its potential for use in at least one of the spectroscopic methods named above.The photophysical properties of a complex are heavily influenced by the choice of antenna chosen to sensitise the lanthanide. Chapters 2 and 3 discuss the design, synthesis and the photophysical evaluation of three different aromatic antennae designed for this project, each one attached to the same DO3A macrocycle to produce neutral lanthanide complexes. Each antenna was designed to investigate not only the UV and emission spectroscopic changes but also if the rate of reaction with cysteine can be increased, by increasing stabilisation of the intermediate during the SNAr reaction with cysteine.Paramagnetic NMR is one of the most powerful tools for protein structure determination and Chapter 4 is the evaluation of two new chiral lanthanide complexes, each bearing an overall 3+ charge and designed specifically for use as NMR protein tags. Both of these complexes bear three chiral pendant arms in order to ensure the formation of a single diastereoisomer once bound to the protein, which is paramount to successful interpretation of the NMR spectra.Chapter 5 discusses the design and synthesis of a very rigid two-armed complex, whereby 2-point attachment to peptides and proteins is evaluated. In principle, 2-point attachment holds the lanthanide complex closely and rigidly to the protein, making this a potentially very powerful paramagnetic tag. However, bearing two pyridine rings results in very low flexibility, which coupled with the required 2-point cysteine mutation to attach to the protein means that there is a risk of distorting the secondary and/or tertiary structure of a protein. This chapter describes the synthesis, photophysical characteristics and preliminary studies of the novel two-armed tag to successfully ‘staple' both linear and helical peptides.