Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λmax=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate β-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for β-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.

A high yielding, scalable and convergent synthesis of infra-luciferins and investigation of their potential for near-infrared bioluminescence imaging.

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to nearinfrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to l max = 706 nm with different Fluc mutants. This emission is the most redshifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.Bioluminescence imaging (BLI) has revolutionized molecular genetic imaging in biomedical research as a cheap and easy means to longitudinally image the genetic behavior of life and disease processes in whole mammals. [1][2][3][4] As they produce the brightest form of bioluminescence, [5] genes from coleopterans are commonly used to localize, track, and quantify cells and molecular or functional events in vivo. [6][7][8] In a well-studied reaction, [9] beetle luciferin (1, Figure 1 a) is adenylated by firefly luciferase (Fluc) and this reacts with molecular oxygen to produce an excited state species, oxyluciferin* (2), which decays to release a photon with a high quantum yield (l max = 558 nm).[5] However, absorption of visible light by hemoglobin (Hb) and melanin restricts image resolution and signal penetration at this wavelength. Between l = 600-800 nm, the absorption of light by Hb decreases by a factor of approximately 50, resulting in less attenuation of red light. This wavelength range is within what is termed the "bio-optical window" and there has been much focus on engineering red-shifted Fluc enzymes that have maximum emission wavelengths in this range, [10][11][12][13][14][15] but these have peaked at wavelengths less than l = 645 nm.The most red-shifted luciferin analogues to date [16] are based upon amino derivatives (Figure 1 b), for example cyclic aminoluciferin (3 a: l max = 599 nm; 3 b: l max = 607 nm), [17] seleno-d-aminoluciferin (4: l max = 600 nm), [18] and a rationally designed 4-(dimethylamino)phenyl derivative conjugated to a thiazoline group (5: l max = 675 nm). [19] In particular cyclic aminoluciferin derivative 3 a has been shown to give improved bioluminescence imaging compared to luciferin (LH 2 ; 1) at dilute concentrations where the intracellular concentration of the luciferin or analogue is limiting.[20] Nearinfrared emission has been detected with an aminoluciferin Cy5 conjugate, but this is due to bioluminescence resonance energy transfer (BRET), [21] meaning that the conjugate cannot be used for multip...

The electronic structure and excited-state dynamics of the ubiquitous bioluminescent probe luciferin and its furthest red-shifted analogue infraluciferin have been investigated using photoelectron spectroscopy and quantum chemistry calculations. In our electrospray ionization source, the deprotonated anions are formed predominantly in their phenolate forms and are directly relevant to studies of luciferin and infraluciferin as models for their unstable oxyluciferin and oxyinfraluciferin emitters. Following photoexcitation in the range 357-230 nm, we find that internal conversion from high-lying excited states to the S(1ππ*) state competes efficiently with electron detachment. In infraluciferin, we find that decarboxylation also competes with direct electron detachment and internal conversion. This detailed spectroscopic and computational study defines the electronic structure and electronic relaxation processes of luciferin and infraluciferin and will inform the design of new bioluminescent systems and applications.