The sequencing of the human genome has allowed the identification of a vast number of putative genes. However, the function of only a small number of these genes can be inferred from their primary sequences. New techniques and agents are needed to cope with the task of assigning functional roles to these gene products. This implies determination of how, when and where they are involved in specific biochemical pathways. Ideally, these techniques and agents will allow the rapid screening of substantial subsets of the sum of a genome's products. Some methods have been designed for broad and rapid screening, but they are generally limited to in vitro application and do not necessarily provide information that is relevant to the function of proteins in living cells. Visualizing and monitoring specific proteins, with minimal disruption of their biological function and distribution, remains one of the foremost challenges in chemical biology. More powerful methods of detection of specific proteins and monitoring their localization and interactions inside living cells are urgently required.
Fluorescent labelling of a specific protein of interest (POI) is one of the most widely used methods for studying expression, localization and trafficking. Several labelling techniques have been developed that involve, for example, the use of fluorescent dyes bearing reactive functional groups such as succinimidyl esters or maleimides, known to react with amines or thiols (see, for example, Takaoka, Y. et al., Angew. Chem. Int. Ed. 2013, 52(15), 4088-4106). However these techniques are typically non-specific, as many such functional groups exposed on the surface of any protein may be labeled, and they do not provide a general means for gathering information on specific protein targets.
Several fluorescent probes for imaging in cell biology have been developed, including small organic dyes, quantum dots, intrinsically fluorescent proteins, small genetically encoded tags that can be complexed with fluorochromes, and combinations of these probes (Giepmans, B. N. et al., Science 2006, 312, 217-24). The most widely applied methods for specific protein labelling include the following: 1) fluorescent protein fusion; 2) small-molecule labelling using protein targeting sequences; 3) enzyme substrate fusion; 4) small-molecule labelling using unnatural amino acids; and 5) small-molecule labelling using peptide targeting sequences.
The first of these methods involves the genetic fusion of target proteins to fluorescent proteins such as jellyfish green fluorescent protein (GFP). This technique has seen broad application because of its intrinsic specificity (O'Hare, H. M. et al., Curr OPin Struc Biol 2007, 17, 488-494; Zhang, J. et al., Nat Rev Mol Cell Bio 2002, 3, 906-918). However, there are limitations to this method, including GFP's slow folding, tendency to aggregate (Tsien, R. Y. et al., Annu Rev Biochem 1998, 67, 509-544) and its steric bulk, all of which can perturb the native biology of a protein of interest.
The second method comprises the genetic fusion of an enzyme to a protein of interest. The pendant enzyme, for example, phosphopantetheine transferase (PPtase) (Yin, J. et al., J Am Chem Soc 2004, 126, 7754-7755 George, N. et al., J Am Chem Soc 2004, 126, 8896-8897), O6-alkylguanine-DNA alkyltransferase (ATG) (Keppler, A. et al., Proc Natl Acad Sci USA 2004, 101, 9955-9959) or mutant haloalkane dehalogenase (HALO) (Los, G. V. et al., Acs Chem Biol 2008, 3, 373-82) can then be irreversibly labelled with a fluorescent ligand. However, with this method background labelling of native enzymes can be problematic and the native biology of a labelled protein of interest can be seriously affected by the bulk of pendant enzyme. Moreover, attenuation of the fluorescent signal is still a problem for the ATG-based method, while the PPtase-based method is restricted to the labelling of cell surface proteins.
In the third method, a protein of interest in fused with a short peptide sequence that serves as a substrate for an enzyme that catalyzes the covalent labelling of the peptide tag with a substrate that bears a reactive functional group. This reactive group must then be subsequently attached to a fluorophore though bioorthogonal chemistry. A Q-tag/transglutaminase system (Lin, C. W. et al., J Am Chem Soc 2006, 128, 4542-4543) and a biotin acceptor peptide/biotin ligase system (Sueda, S. et al., Chembiochem 2011, 12, 1367-1375) are the most successful examples of this class of labelling methods. This method can only be used for cell surface proteins because of the incompatibility of the enzymes in intracellular labelling.
The fourth method is to introduce unnatural amino acids in a site-specific manner (Wang, L. and Schultz, P. G., Chem Commun (Camb), 2002, 1-11). These unnatural amino acids usually contain ketones, azides or alkynes, which can undergo reactions through hydrazone formation, Staudinger reaction or azide/alkyne cycloaddition to add a fluorophore. This method has some advantages with respect to bioorthogonality and versatility for small molecule labelling. However, unnatural amino acid mutagenesis is not yet widely applicable and is highly dependent on host cell type.
Finally, the fifth method mentioned above is the use of small organic fluorophores for labelling proteins that harbour a specific, genetically encoded motif, representing the development of powerful alternative labelling methods (Chen, I. et al., Curr Opin Biotech 2005, 16, 35-40). Among these, the “FlAsH” method developed by Tsien and co-workers employs certain organoarsenic compounds that have been shown to form specific complexes with a target sequence containing four proximal cysteine residues (Zhang, J. et al., Nat Rev Mol Cell Bio 2002, 3, 906-918). This method demonstrates specific labelling using a minimised, small molecule approach. However, several drawbacks have been noted, including the inherent toxicity of organoarsenic compounds, background staining that may persist despite extensive washing, the sensitivity of the tetracysteine motif to oxidizing extracellular environments, and its tendency to form inactive intermolecular disulfide-linked aggregates.
Maleimide groups have long been used in applications that exploit their propensity to react selectively with thiol groups, undergoing Michael addition reactions through their C2=C3 double bond (Kanaoka, Y. et al., Chem. Pharm. Bull. 1964, 12, 127). Maleimides are also known to quench fluorescence, probably due to their participation in a photoinduced electron transfer (PET), allowing non-radiative relaxation of the fluorophore's excited state. The thiol addition reaction breaks the conjugation of the maleimide group, altering the energy levels of its molecular orbitals and removing its capacity to quench fluorescence (Guy, J. et al., J. Am. Chem. Soc. 2007, 129, 11969). These properties were demonstrated in the characterization of a naphthopyranone derivative bearing a maleimide group whose fluorescence increased dramatically upon reaction with glutathione (Langmuir, M. E. et al., Tetrahedron Lett. 1995, 36, 3989).
Labelling techniques based on the use of fluorescent dyes bearing reactive functional groups like maleimides, known to react with thiols, have been described (Tsien, R. Y., Annu. Rev. Biochem. 1998, 67, 509-544). However, these methods are typically non-specific, labelling the surface-exposed functional groups of many different proteins. Based on this chemical reaction, we previously developed a strategy for protein labelling based on a reactive unit bearing two maleimide groups linked to a fluorophore, such that fluorescence is quenched by photoinduced electron transfer (PET) until both maleimide groups undergo specific thiol addition reactions (Keillor, J. W. et al., Org Biomol Chem 2011, 9, 185-197; Keillor, J. W. et al., J Am Chem Soc 2007, 129, 11969-11977; Girouard, S. et al., J Am Chem Soc 2005, 127, 559-566). Additionally, complementary alpha-helical peptide tags have been designed, bearing two cysteine residues whose thiol side chains are appropriately positioned to react with the novel fluorogens (Keillor, J. W. et al., Mol Biosyst 2010, 6, 976-987). Genetically fusing these helical peptides to test proteins of interest (POI), we were able to selectively label the target sequence.
These early dimaleimide dyes react with the di-cysteine peptide tag faster than with two equivalents of other thiols such as glutathione (GSH) because the second maleimide-thiol reaction with the tag is an intramolecular reaction, while the second reaction with other thiol compounds is an intermolecular reaction. However, due to the high (millimolar) concentration of GSH inside living cells, some non-specific reaction with GSH is still possible, leading to background fluorescence. This may limit the application of these first generation fluorogens to cell surface labelling (Keillor, J. W. et al., Mol Biosyst 2010, 6, 976-987), highlighting the need for more selective labelling agents that can be used for intracellular applications.
It was also reported previously that efficient quenching was observed with a dansyl-based fluorogen (Keillor, J. W. et al., Org Biomol Chem 2011, 9, 185-197). However, dansyl is an environment-sensitive fluorophore and its excitation and emission wavelengths do not correlate well with the filter sets of most fluorescent microscopes.