Visualizing proteins in living cells without perturbing biological function remains a key challenge in chemical biology. A chemical approach to this problem is the synthesis of small molecule fluorophores that react specifically with a protein of interest (POI). We have developed a site-specific labelling method based on a Fluorogenic Addition Reaction (FlARe). The FlARe probe’s fluorescence is quenched until it undergoes thiol addition with a small, genetically encoded dicysteine peptide tag fused to the POI. Recent blue coumarin probes were shown to be highly selective for target proteins over other cellular thiols; however, fluorogens that can label in the red and green channels of the fluorescence microscope are more desirable for cellular imaging, as red light is lower in energy and therefore less photo-toxic. In the work presented herein, we use DFT calculations to guide the design of red-shifted, PeT-quenched BODIPY based dimaleimide fluorogens. Driven by the preliminary results of a FlARe probe (YC29) that emitted in the red channel, we attempted to prepare the hit compound through a new synthetic approach to further evaluate kinetics and in cellulo labelling. Given the time available, this compound was unable to be synthesized through an SNAr or Pd-catalyzed approach. Alternatives probes lacking the red-shifting substituent were synthesized and evaluated in vitro and in cellulo. The fluorescent enhancement and reaction kinetics of these probes were evaluated in detail, in order to determine the suitability of their application to cellular labelling. A green-BODIPY fluorogen was synthesized that exhibits suitable kinetics for labelling and a dramatic fluorescent enhancement of ~800-fold upon tagging. This probe was successfully applied to the specific, fluorescent labelling of a nuclear histone protein in cellulo.

Peptide fusion tags are fundamental for the identification, detection, and capture of proteins in biological assays. Commonly used peptide fusion tags rely on temporary non-covalent interactions for binding, which can put constraints on assay sensitivity. Here, peptide fusion tags were developed that could specifically interact with protein binding partners via spontaneous and irreversible isopeptide bond formation. To develop covalently interacting peptide-protein pairs, outer-membrane proteins from Gram-positive bacteria that form autocatalyzed intramolecular isopeptide bonds were dissected to generate a short peptide fragment and a protein binding partner. Initially, the major pilin subunit Spy0128 from Streptococcus pyogenes was split to develop the 16 residue isopeptag peptide and the 31 kDa pilin-C protein partner. The isopeptag:pilin-C pair were able to react via spontaneous isopeptide bond formation between an Asn residue in isopeptag and a Lys residue in pilin-C without the requirement for any accessory factors, and with a yield of 60% after a 72 hr reaction. Reconstitution between the isopeptag:pilin-C pair was robust and occurred under all biologically relevant conditions tested, and also in the complex environment of a bacterial cytosol and on the surface of mammalian cells. A similar approach was also used to dissect the small CnaB2 domain that is part of the large FbaB fibronectin-binding protein from S. pyogenes. This led to the development of a more efficient peptide-protein pair, which was rationally modified to generate the highly optimized SpyTag:SpyCatcher pair. SpyTag is a 13 amino acid peptide with a reactive Asp that forms a spontaneous intermolecular isopeptide bond with a Lys present in the 12 kDa SpyCatcher binding partner. In a reaction with SpyTag, over 40% of SpyCatcher was depleted after 1 min and SpyCatcher could no longer be detected after 2 hr. The SpyTag and SpyCatcher reaction did not require any accessory factors and proceeded efficiently at a range of biologically relevant temperatures, pH values, concentrations, buffer compositions, and in the presence of commonly used detergents. The SpyTag:SpyCatcher technology was also used for specific cell surface labelling on mammalian cell membranes. SpyTag and SpyCatcher are both composed of the regular 20 amino acids and can therefore be genetically encoded as fusion constructs for a variety of in vitro and in vivo applications. Potential applications of the SpyTag:SpyCatcher technology include specific cell surface labelling, the development of novel protein architectures, and the covalent and irreversible capture of target proteins in biological assays.