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Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging

Multi-component bioluminescence imaging requires an expanded collection of luciferase–luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging.


Introduction
Bioluminescent enzymes (luciferases) are among the most popular reporters for imaging biological processes in vitro, in live cells, and in animal models. 1,2 Luciferases generate light via the oxidation of small molecule luciferins (Fig. 1a). Since no external light source is needed, bioluminescent probes offer high signal-to-noise ratios in heterogeneous environments. [3][4][5] The remarkable sensitivity, combined with the broad dynamic range, has made bioluminescence a go-to imaging technique for tracking cell movements, proliferation, and numerous other features in living organisms. [6][7][8] While widely used, bioluminescence has been slow to transition to imaging multiple targets simultaneously, owing to a lack of distinguishable probes. 9,10 Multiplexed imaging is possible using spectrally resolved luciferase-luciferin pairs. [11][12][13][14] Indeed, several luciferin analogs have been developed that emit different colors of light. [15][16][17] The emission proles can be further tuned with engineered luciferases 18,19 or luciferase-uorescent probe fusions. [20][21][22][23] In many cases, though, the broad spectral proles and overlapping emission maxima preclude routine multiplexing in vivo. Sensitive imaging in whole organisms further requires >650 nm light, as these wavelengths are signicantly less absorbed by tissue. 24 However, few bioluminescent probes emit substantial numbers of photons in the requisite far-red to near-infrared (NIR) range. The perceived color of emission in traditional bioluminescence detection also changes with the depth of the source, complicating the assignment of different colored probes. 25 Multiplexed bioluminescence imaging can also be achieved via substrate resolution-using luciferases that recognize different luciferin structures (i.e., orthogonal pairs). 10 Light is produced when complementary enzymes and substrates react, but minimized in all other cases. [26][27][28] A handful of such orthogonal probes have been co-opted for dual imaging in vivo, but applications in deep tissue remain challenging. 11,[29][30][31] This is due to a shortage of luciferins with both sufficient red emission and distinct molecular architectures. Candidate luciferins must also be sufficiently bright, bioavailable, and easy to synthesizecriteria that few existing probes meet. 32 Consequently, a set of three or more red-shied probes for routine multicomponent imaging remains elusive.
We surmised that unique classes of orthogonal, NIRemitting luciferins could be developed using uorescent scaffolds as guides (Fig. 1b). Our approach was inspired by previous reports of luciferin analogs comprising entirely new heterocycles, 33,34 including benzothiophene, 35 quinoline, 36 and coumarin derivatives. 37 We also took cues from recent efforts to develop red-shied luciferins by extending the pi-conjugation 38-40 of the scaffold and restricting conformational exibility (e.g., AkaLumine and infraluciferin). [41][42][43] Luciferins produced from these seminal studies emitted far-red light, but many were poor substrates for rey luciferase (Fluc). In some cases, light emission was recouped, although extensive enzyme engineering was required (e.g., generating Akaluc and related mutants). 38,43,44 We focused on a new class of luciferins (CouLuc-1s) comprising both an elongated pi-system and a 4-tri-uoromethylcoumarin unit (Fig. 1c). The coumarin uorophore is a well-established imaging agent, and structurally distinct from heterocycles found in existing red-shied luciferins. 45,46 Additionally, coumarins have been incorporated into other motifs to achieve bright NIR emission. [47][48][49][50][51] The small size of the coumarin core would also likely require only minimal enzyme engineering to identify complementary luciferases. Given the unique structure of the CouLuc-1s, we further anticipated that the analogs could be used for multi-component imaging with other red-emitting probes, including AkaLumine/Akaluc and furimazine/Antares.
Here we detail the synthesis and evaluation of the CouLuc-1 probes. We developed a two-step route to bridge the uorescent coumarin heterocycle with the key thiazoline unit necessary for luciferin bioluminescence. The resulting conjugates displayed red-shied emission. Complementary luciferases were identied via a parallel engineering approach. The resulting luciferaseluciferin pairs provided robust light outputs that were suitable for multiplexed imaging. Overall, these efforts provide a new class of easily accessible, long-wavelength bioluminescent pairs with signicant promise for orthogonal imaging in vivo.

Results and discussion
Design and synthesis of coumarin-linked luciferins We set out to prepare CouLuc-1 analogs bearing different electron donors (-NMe 2 , -NH 2 , and OH) at C7 of the coumarin heterocycle. These modications are commonly found in coumarin uorophores and are known to modulate the excited state properties. 45 A C4 triuoromethyl group was also included, as this motif is known to both red-shi the emission and increase the uorescence quantum yield of the parent coumarin. 52 Retrosynthetic analysis of the CouLuc-1 analogs revealed a key disconnection in the alkene linkage between the coumarin and thiazoline (Fig. 1c). We envisioned that if the olen bridge could be installed with a nitrile handle, subsequent cysteine condensation would afford the desired luciferins in a highly concise two-step sequence. Syntheses of related pi-extended or red-shied luciferins typically require 5-10 steps. 28,41,43,53,54 While methods to directly modify the carbonyl group on coumarins are rare, recent reports suggested that thiolactones are viable intermediates for synthesizing pi-extended coumarins. 47,48,55 Additionally, direct thiazoline formation via cysteine condensation has traditionally been limited to benzothiazole or otherwise activated nitriles; non-activated scaffolds require more steps. 15,40 With these synthetic challenges in mind, we set out to develop olenation conditions for installing a cyanomethylene moiety onto commercial coumarin starting materials. Aer examining several strategies, we found that cyanomethyl anions generated in situ from acetonitrile and n-BuLi react readily with coumarins 1a-c. 56 Subsequent treatment of the addition products with 0.5 M HCl afforded the desired cyanomethylene coumarins 2a-c as mixtures of E/Z isomers in good yield (Scheme 1). Exposing 2a-c (as a mixture of isomers) to Dcysteine and NaHCO 3 in ethanol generated CouLuc-1 analogs 3a-c in 3-5 days. Following cycloaddition, a single isomer was formed, and the geometry was veried by 2D NOSEY (Fig. S1 †). Overall, the route provided access to 3a-c in 26-32% yield in just two steps. This approach is among the shortest luciferin syntheses to date from readily available starting materials. To highlight the scalability of the route, we also developed a chromatography-free procedure to access 3a ( Fig. S2 and S3 †). Quick access to large quantities of luciferin is necessary for efficient identication of complementary luciferases.

In vitro characterization with native luciferase
With the CouLuc-1 analogs in hand, we rst examined their bioluminescent properties. All three analogs produced light when combined with Fluc and the necessary cofactors (Fig. 2a). Scaffolds with amino (CouLuc-1-NH 2 ) or dimethylamino (Cou-Luc-1-NMe 2 ) substituents exhibited stronger photon outputs than the hydroxy variant (CouLuc-1-OH, Fig. 2b). Similar boosts in brightness have been observed with AkaLumine and related luciferins. 15,42 Compared to the native Fluc substrate (D-luc), though, the CouLuc-1 derivatives produced lower levels of light ($1000-fold dimmer, Fig. S4 †). The reduced brightness was partly attributed to low binding affinities between Fluc and the coumarin analogs (Fig. S5 †). Similar trends have been observed with other sterically 26,57 and electronically modied luciferins 33,34 exhibiting comparable emission values (Fig. S6 †). While low, the photon outputs achieved with Fluc and the CouLuc-1 derivatives provided a clear starting point for evolving brighter luciferases.
In terms of color, CouLuc-1-NMe 2 and -OH exhibited peak emission wavelengths (l max ) at 620 and 625 nm, respectively. CouLuc-1-NH 2 was slightly blue-shied, with l max ¼ 597 nm. Compared to D-luc, all analogs were red-shied by 30-60 nm at 25 C (Fig. 2c). Nearly 40% of the emitted photons from CouLuc-1-NMe 2 and CouLuc-1-OH were >650 nm. For comparison, Fluc/ D-luc emits only 5% of photons >650 nm in vitro (8% in cellulo 58 ). For Akaluc/AkaLumine, a popular red-shied luciferase-luciferin pair, the value is 44% (in cellulo at 25 C). We further compared the CouLuc-1 bioluminescence spectra to the corresponding uorescence spectra. In aqueous media, the uorescence readouts exhibited similar trends, with the observed maxima red-shied by 70-100 nm compared to D-luc (Table S1 †). This strong agreement between CouLuc-1 uorescence and bioluminescence is consistent with other pi-extended and amino luciferins. 28,38,59 Engineering complementary luciferases for CouLuc-1s Aer establishing the CouLuc-1s as viable luminophores, we set out to improve photon outputs by engineering the luciferase enzyme (Fig. 3a). The ideal mutants would exhibit improved turnover, maintain red-shied emission, and be selective for the coumarin scaffolds. We initially focused on CouLuc-1-NMe 2 due to its robust activity with Fluc and desirable spectral properties. We used an established two-pronged approach to identifying mutants from library screens via (1) in silico design and (2) a semi-rational strategy. 38 Both methods have been used to generate complementary enzymes for synthetic luciferins. 26,38,44 In the rst approach, we sculpted the luciferase active site using RosettaDesign. This strategy is useful for identifying mutations unique to an analog without signicant prior knowledge or evolutionary starting point. 60,61 To computationally identify mutations suited for the CouLuc-1 scaffold, we used the Rosetta-Match algorithm 62,63 to dock CouLuc-1-NMe 2 with existing Fluc crystal structures. 64 The model oriented the coumarin heterocycle toward the pocket normally adjacent to C4 0 on D-luc. In this conguration, the dimethylamino substituent was predicted to clash with the backbone of some active site residues (Fig. S7 †), likely resulting in diminished turnover. We next employed RosettaDesign 62,63 to resolve this clash and optimize the packing interaction between the coumarin luciferin and surrounding residues. From this analysis, a total of 41 sites were mutated to create a complementary active site for CouLuc-1-NMe 2 (Fig. S8 †). These residues were then ranked for targeting based on their proximity and known biochemical data. 27,65-67 Twenty sites were ultimately selected for randomization via combinatorial codon mutagenesis (Fig. S8 †).
The resulting Rosetta-inspired library was introduced into bacteria, and the transformed colonies were sprayed with CouLuc-1-NMe 2 . Out of $7000 colonies screened on plate, $150 were light-emitting. These colonies were collected, and the mutants were veried in two secondary screens (Fig. S9 †). Variants with >10-fold improved photon output over Fluc were carried forward. Unique sequences were then validated in a second bacterial cell assay. From this workow, two hits were identied (Fig. 3b). Intriguingly, both variants contained a S347G mutation. We also screened the Rosetta-based library with CouLuc-1-OH. In this case, three hits were identied, with the point mutant S347G providing the highest photon outputs (Fig. S10 †). While only a subset of the Rosetta library was screened, the frequency of the S347G mutation among the 'hits' suggested that this residue is benecial for CouLuc-1 processing. S347G is also known to stabilize the open conformation of the luciferase active site 68,69 and process luciferins with steric bulk at C4 0 . 26,29 In parallel with the Rosetta approach, we screened CouLuc-1-NMe 2 against a focused library of 222 characterized Fluc mutants. 29 The luciferases comprise mutations conned to the luciferin binding pocket and exhibit unique preferences for sterically and electronically modied luciferins. 26,38 Screens of this library could provide additional information on residues underlying substrate specicity for the CouLuc-1 scaffolds. All three analogs were subjected to the focused library. Eleven hits were identied for CouLuc-1-NMe 2 (Fig. 3c). A similar number of mutants were found to exhibit enhanced light emission for the -NH 2 and -OH analogs (Fig. S11 †). The brightest mutants from these screens also comprised the S347G mutation, reinforcing the notion that residue 347 plays a pivotal role in CouLuc-1 processing. Another mutation common to multiple hits was F243M, a residue previously shown to aid in processing bulky luciferin analogs. 29,57 In vitro and in cellulo characterization of lead mutant From the screening hits, we selected the F243M/S347G mutant (dubbed Pecan) for additional characterization with CouLuc-1-NMe 2 . Pecan was particularly attractive for orthogonal probe development as it has been previously used in vivo. 29,31 When CouLuc-1-NMe 2 was incubated with puried Pecan, more intense light emission (77-fold) was observed compared to Fluc (Fig. S12 †). The boost in light output was likely due to the enhanced binding affinity of CouLuc-1s with Pecan, as revealed by kinetic analyses (Fig. 4a and S13 †). Importantly, robust emission in the NIR region was maintained. Approximately 30% of photons produced by CouLuc-1-NMe 2 were >650 nm, and the emission spectra for the other analogs were similarly red-shied (Fig. S14 †).
Aer examining Pecan/CouLuc-1-NMe 2 in vitro, we evaluated the pair in mammalian cells. Pecan and Fluc were transiently expressed in HEK293 cells. The cells were incubated with either the CouLuc-1 analogs or D-luc. Peak photon outputs for each enzyme-substrate combination were measured and normalized to a common transfection marker (GFP). As shown in Fig. 4b and S15 †, Pecan-expressing cells treated with CouLuc-1-NMe 2 emitted 14-fold more photons than Fluc-expressing cells. Similar improvements were observed when Pecan-expressing cells were incubated with either CouLuc-1-NH 2 or CouLuc-1-OH (Fig. S16 †). The robust emission from CouLuc-1 analogs was also recapitulated in Pecan-expressing DB7 cells (Fig. S17 †). Pecan/CouLuc-1-NMe 2 provided larger photon outputs than native Fluc/D-luc under standard cellular imaging conditions ( Fig. 4b and S15 †). Achieving such bright emission with a minimally modied luciferase is notable. Only two mutations to the Fluc scaffold were necessary. In our previous work to identify a complementary luciferase for a pi-extended luciferin, four rounds of engineering were required. The "winning" enzymes comprised 5-7 mutations and provided photon outputs that were only $2% of Fluc/D-luc emission. 38 Pecan/ CouLuc-1-NMe 2 also compares favorably to Akaluc/AkaLumine, a popular red-shied probe, in terms of photon output and color ( Fig. 4c and S18 †). Akaluc comprises 28 mutations and was identied aer 21 rounds of evolution. 44 Pecan/CouLuc-1-NMe 2 also emits signicant numbers of NIR photons (32% of photons >650 nm) in cells, on par with other state-of-the-art bioluminescent tools (Fig. S19 †). Collectively, these data suggest that the Pecan/CouLuc-1-NMe 2 is immediately useful for routine imaging.

Multicellular imaging with pecan and CouLuc-1-NMe 2
The optical properties of the CouLuc-1 analogs coupled with their unique structures made them promising candidates for multiplexed imaging. As noted earlier, resolving luciferases by substrate requires unique luciferin architectures. The more structurally diverse the luciferins, the better they can be distinguished by engineered enzymes. 29 Substrate unmixing and image processing algorithms can rapidly detect unique enzyme-substrate pairings within complex mixtures. 31,70 Multiple classes of orthogonal probes are available for multicomponent imaging via these methods, but only a few exhibit the necessary optical properties (NIR bioluminescence) for sensitive imaging. Given the unique structures and robust emission of Pecan/ CouLuc-1-NMe 2 , we reasoned that this pair would be useful for multiplexing with other red-shied probes. We were drawn to Akaluc/AkaLumine 44 and Antares/furimazine 71 as these pairs have been recently used to monitor tumor-immune interactions in vivo. 30 Pecan and Akaluc derive from the insect luciferase family and are thus inherently orthogonal to Antares, which uses a different mechanism for light emission. The CouLuc-1 scaffold is also structurally distinct from AkaLumine, suggesting that Pecan and Akaluc could be readily differentiated based on their substrate preference. Indeed, minimal crosstalk was observed when Pecan was treated with AkaLumine or when the CouLuc-1 luciferin was added to Akaluc (Fig. S20 †). The high level of orthogonality highlights the unique chemical space occupied by the coumarin analogs.
To test the multiplexed strategy, we plated Pecan-, Akaluc-, and Antares-expressing DB7 cells in varying ratios. Cell cultures containing a single reporter were also plated as controls (Fig. 5a). Each substrate was added sequentially, and an image was acquired aer each administration. The resulting images were analyzed using a linear unmixing algorithm, 31,72 and the relative abundance of each cell type was false colored in a composite image (Fig. 5a). Each luciferase-expressing cell was readily discerned, and the unmixed signals correlated with the number of cells present (Fig. 5b). The entire triple component imaging study was completed within 30 min, a notable improvement over traditional methods that require substrate clearance (multiple days). Altogether, these results suggest that Pecan/CouLuc-1-NMe 2 can be readily integrated with other engineered probes for rapid, multicellular imaging. Pecan,  Akaluc, and Antares also constitute the rst readily distinguishable, bioluminescent triplet probe set with NIR emission.

Conclusions
Despite the growing number of luciferin analogs, only a few are both chemically distinct and red-shied. We have developed and characterized a new panel of structurally unique and near infrared-emitting luciferins based on a modied coumarin scaffold. The substrates were synthesized in just two steps without chromatographic purication. The CouLuc-1 analogs were found to emit light with Fluc. While the emission levels were weak compared to existing bioluminescent systems, mutant luciferases were identied that afforded enhanced outputs. The brightest luciferase-CouLuc-1 pair exhibited luminescent signals outperforming native bioluminescent probes. Such robust emission suggests that the CouLuc-1 luciferins can be immediately adopted for biological imaging.
The unique structural and optical features of the CouLuc-1 analogs are well suited for multiplexed imaging. We demonstrated that the probes and their complementary enzymes could be used for rapid imaging of multiple targets. We also identied an easily distinguishable triplet set of NIR bioluminescent tools. Combinations of such red-emitting probes are necessary for applications in tissue and other scattering environments. More broadly, our approach to accessing novel luminophores from simple uorophores could spur the development of an expanded set of bioluminescent tools. Future work will also investigate whether more uorophore-luminophore hybrids can be accessed using the olenation strategy.

Conflicts of interest
Z. Y., D. R. C., A. C. L., M. J. S., and J. A. P. are on a provisional patent application, led through the NCI and UCI based on the results described here.