Lanthanide tags for luminescence applications and paramagnetic NMR spectroscopy

The lanthanide elements have long fascinated scientists due to their intriguing combination of chemical and physical characteristics. Utilisation of these properties has seen lanthanide-based reagents applied in a plethora of fields, including medical diagnostics, optical imaging, catalysis and opto-electronics. This thesis sought to further exploit the optical and magnetic properties of these ions through the development of novel “clickable” lanthanide-based reagents for luminescence imaging and assay applications, and through the development of new lanthanide-binding tags for structural studies of biomolecules by paramagnetic NMR spectroscopy. In the first instance, a series of conjugatable luminescent terbium(III) and europium(III) complexes were designed and synthesised to provide a new route to the highly site-specific, “bio-orthogonal” tagging of proteins and other bio-macromolecules with a luminescent label. These tags feature azido groups that allow covalent attachment to alkyne- and cycloalkyne-bearing molecules via copper(I)-catalysed and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, respectively. A new amine-conjugatable tag was also synthesised, then converted to a “clickable” form via reaction with an appropriate cyclooctyne derivative. The azido tags were successfully conjugated to a simple “model” alkyne (phenyl propargyl ether) in the presence of a Cu(I) catalyst, or to a water-soluble cyclooctyne derivative in the case of those complexes that were found to be susceptible to trans-metallation. Detailed spectroscopic measurements (absorbance and emission maxima, extinction coefficient, quantum yield, luminescence lifetime) indicate that a number of the complexes exhibit favourable photophysical properties for imaging/assay applications; the Tb(III) complexes generally exhibit higher quantum yields (up to 45%), while a number of the Eu(III) complexes display a “switch-on” effect upon “clicking”. A number of the most promising “clickable” tags were conjugated to a small test protein, human ubiquitin, bearing an alkyne or azide moiety on its surface, demonstrating their utility for luminescent labelling of proteins. The alkyne or azide moieties were introduced either biosynthetically via installation of a 4-propargyloxy-L-phenylalanine or 4-azido-L-phenylalanine residue using engineered orthogonal aminoacyl-tRNA synthetase/tRNA pairs, or chemically via reaction of surface lysines with a cyclooctyne-PEG-succinimidyl ester. Several of the new tagging reagents, as well as an additional set of lanthanide-chelating agents based on diethylenetriaminepentaacetic acid (DTPA), were also successfully coupled to adenosine to generate a series of luminescent probes that may be employed as tools for studying the pharmacology of adenosine-binding GPCR receptor (AR) proteins. A number of these were found to be potent AR agonists (log IC50 ≤ -7.17 ± 0.26). The second major aspect of this work involved the development of a new series of lanthanide-binding tags for application in protein structural studies via paramagnetic NMR spectroscopy. The cysteine-conjugatable tags represent hybrids of the amino acid, L-cysteine, and the well-known metal chelators, iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA). The single stereocentre present in these structures was found to be sufficient to limit the corresponding protein-bound lanthanide complexes to a single stereoisomeric form, and immobilisation of the lanthanide ions was found to be adequately rigid to produce sizable pseudo-contact shifts (PCSs) in the NMR spectra of two model proteins – the Ala28-Cys mutant of human ubiquitin (UbiqA28C) and the N terminal domain of the E. coli arginine repressor (ArgN); these PCSs were successfully characterised and shown to be appropriate for structure refinement. Rigidification of a lanthanide ion with respect to the protein frame is highly desirable to obtain both long and close range effects and was aided by having either a metal-binding aspartic acid (Asp) residue or, in the case of the NTA tag, a second copy of the tag in the i + 4 position of an α-helix. Similarly, a cobalt(II) ion could also be bound using pairs of the IDA tag in an i, i + 4 α-helical arrangement. Perhaps the most significant discovery to be made was the capacity of an IDA tag and Asp residue to be used in combination to provide a rigid, yet kinetically-labile lanthanide-binding site. The exchange rate between metal-bound and -unbound states was found to be sufficiently fast to produce definitive exchange cross-peaks, enabling the rapid assignment of both small and extraordinarily large PCSs by 15N heteronuclear exchange spectroscopy, without recourse to a structural model. This is the first time such a result has been achieved with a synthetic tag and promises to greatly extend the scope of paramagnetic NMR spectroscopy within the field of structural biology

Lanthanide tags for luminescence applications and paramagnetic NMR spectroscopy

The lanthanide elements have long fascinated scientists due to their intriguing combination of chemical and physical characteristics. Utilisation of these properties has seen lanthanide-based reagents applied in a plethora of fields, including medical diagnostics, optical imaging, catalysis and opto-electronics. This thesis sought to further exploit the optical and magnetic properties of these ions through the development of novel “clickable” lanthanide-based reagents for luminescence imaging and assay applications, and through the development of new lanthanide-binding tags for structural studies of biomolecules by paramagnetic NMR spectroscopy. In the first instance, a series of conjugatable luminescent terbium(III) and europium(III) complexes were designed and synthesised to provide a new route to the highly site-specific, “bio-orthogonal” tagging of proteins and other bio-macromolecules with a luminescent label. These tags feature azido groups that allow covalent attachment to alkyne- and cycloalkyne-bearing molecules via copper(I)-catalysed and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions, respectively. A new amine-conjugatable tag was also synthesised, then converted to a “clickable” form via reaction with an appropriate cyclooctyne derivative. The azido tags were successfully conjugated to a simple “model” alkyne (phenyl propargyl ether) in the presence of a Cu(I) catalyst, or to a water-soluble cyclooctyne derivative in the case of those complexes that were found to be susceptible to trans-metallation. Detailed spectroscopic measurements (absorbance and emission maxima, extinction coefficient, quantum yield, luminescence lifetime) indicate that a number of the complexes exhibit favourable photophysical properties for imaging/assay applications; the Tb(III) complexes generally exhibit higher quantum yields (up to 45%), while a number of the Eu(III) complexes display a “switch-on” effect upon “clicking”. A number of the most promising “clickable” tags were conjugated to a small test protein, human ubiquitin, bearing an alkyne or azide moiety on its surface, demonstrating their utility for luminescent labelling of proteins. The alkyne or azide moieties were introduced either biosynthetically via installation of a 4-propargyloxy-L-phenylalanine or 4-azido-L-phenylalanine residue using engineered orthogonal aminoacyl-tRNA synthetase/tRNA pairs, or chemically via reaction of surface lysines with a cyclooctyne-PEG-succinimidyl ester. Several of the new tagging reagents, as well as an additional set of lanthanide-chelating agents based on diethylenetriaminepentaacetic acid (DTPA), were also successfully coupled to adenosine to generate a series of luminescent probes that may be employed as tools for studying the pharmacology of adenosine-binding GPCR receptor (AR) proteins. A number of these were found to be potent AR agonists (log IC50 ≤ -7.17 ± 0.26). The second major aspect of this work involved the development of a new series of lanthanide-binding tags for application in protein structural studies via paramagnetic NMR spectroscopy. The cysteine-conjugatable tags represent hybrids of the amino acid, L-cysteine, and the well-known metal chelators, iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA). The single stereocentre present in these structures was found to be sufficient to limit the corresponding protein-bound lanthanide complexes to a single stereoisomeric form, and immobilisation of the lanthanide ions was found to be adequately rigid to produce sizable pseudo-contact shifts (PCSs) in the NMR spectra of two model proteins – the Ala28-Cys mutant of human ubiquitin (UbiqA28C) and the N terminal domain of the E. coli arginine repressor (ArgN); these PCSs were successfully characterised and shown to be appropriate for structure refinement. Rigidification of a lanthanide ion with respect to the protein frame is highly desirable to obtain both long and close range effects and was aided by having either a metal-binding aspartic acid (Asp) residue or, in the case of the NTA tag, a second copy of the tag in the i + 4 position of an α-helix. Similarly, a cobalt(II) ion could also be bound using pairs of the IDA tag in an i, i + 4 α-helical arrangement. Perhaps the most significant discovery to be made was the capacity of an IDA tag and Asp residue to be used in combination to provide a rigid, yet kinetically-labile lanthanide-binding site. The exchange rate between metal-bound and -unbound states was found to be sufficiently fast to produce definitive exchange cross-peaks, enabling the rapid assignment of both small and extraordinarily large PCSs by 15N heteronuclear exchange spectroscopy, without recourse to a structural model. This is the first time such a result has been achieved with a synthetic tag and promises to greatly extend the scope of paramagnetic NMR spectroscopy within the field of structural biology

Engineering of a bis-chelator motif into a protein α-helix for rigid lanthanide binding and paramagnetic NMR spectroscopy

Attachment of two nitrilotriacetic acid-based ligands to a protein α-helix in an i, i + 4 configuration produces an octadentate chelating motif that is able to bind paramagnetic lanthanide ions rigidly and with high affinity, leading to large pseudocont

DOTA-Amide Lanthanide Tag for Reliable Generation of Pseudocontact Shifts in Protein NMR Spectra

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin

DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin

Extending the Excitation Wavelength of Potential Photosensitizers via Appendage of a Kinetically Stable Terbium(III) Macrocyclic Complex for Applications in Photodynamic Therapy

The development of viable photodynamic therapy protocols is often hindered by photosensitizers that require high-energy UV irradiation that has limited potential for clinical use due to its low tissue penetration. Herein, we report a strategy for extending the excitation wavelength of potential photosensitizers via the covalent attachment of a terbium­(III)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate complex (<b>DO3A-Tb</b>). The method was systematically demonstrated with a series of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, anthracene, pyrene, and fluoranthene) to prepare six new complexes (<b>Tb1</b>–<b>Tb6</b>) with bathochromic shifts that extended into the visible region. Determination of their quantum yields for singlet oxygen (¹O₂) production at 350 and 420 nm showed significant enhancements from the parent molecule in all cases. Cell viability studies on cervical cancer cells (HeLa) and noncancerous MRC-5 cells showed no measurable cytotoxicity for all complexes prior to light irradiation. However, after irradiation at 420 nm (20 min, 9.27 J cm^–2), <b>Tb3</b>–<b>Tb6</b> were phototoxic to HeLa cells with IC₅₀ values between 14.3–32.3 μM. Cell morphological studies and fluorescence microscopy with live/dead cell stains confirmed these findings. In addition, these complexes were highly stable in human blood plasma, with no significant degradation observed after 96 h at 37 °C. This excellent phototoxicity profile and high stability in blood plasma, coupled with the moderately lipophilic nature of the complexes, favorably indicate the potential of <b>DO3A-Tb</b> as a heavy atom-bearing moiety for modification of potential photosensitizers into ideal phototherapeutic drug candidates with longer excitation wavelengths for in vivo application