To help understand the red shift in bioluminescence emission from FLuc that is observed in its reaction with iLH₂, the X-ray crystal structure of FLuc in complex with iDLSA was resolved and is shown in Figure 1b (PDB ID: 6HPS). Data collection and refinement statistics (molecular replacement); and data collection, phasing and refinement statistics for mad (semet) structures can be found in Figure 1—figure supplement 1. iDLSA captures FLuc in the adenylation step of the light emitting reaction (¹H and ¹³C data spectra synthetic chemical compounds can be found in). The conformation of the iLH₂ heterocyclic rings with respect to the alkene, as drawn in Figure 1a, is confirmed to be as predicted by computational studies and is the most likely conformation of the light emitting form (Berraud-Pache and Navizet, 2016). This newly crystallised FLuc structure was aligned with the reported structure of FLuc with 5’-O-[(N-dehydroluciferyl)-sulfamoyl] adenosine (DLSA) (PDB ID: 4G36) (Sundlov et al., 2012). The structures show good alignment to each other, however there is evidence of a more open active site supported by a reduction in root-mean-squared (RMSD) score when aligned based on just the N-terminal domain of FLuc rather than the entire structure (RMSD = 0.688 and 0.783 respectively) (Figure 1c).

All FLuc residues in close proximity (4 Å) to DLSA were also found to be within the same distance to iDLSA, with the exception of Arg437, >4 Å away from iDLSA (Figure 1d and e). We noted that despite differences in the conformation of iDLSA compared to DLSA in both 4G36 and L. cruciata 2D1S (Nakatsu et al., 2006) structures, the positions of the phenolic groups are quite similar (~0.5 Å). The altered position of the benzothiazole ring and the greater size of iDLSA may be the cause of a series of small active site changes that affect residues Glu311, Arg337, Asn338, Gly339, and Thr343 resulting in a total of six differences in H-bonding interactions. When specific residues implicated in the light emitting reaction (Sundlov et al., 2012) were measured between the two structures differences ranged from 0.7 to 1.6 Å; with the biggest divergence being Lys529 (found in the C-terminal cap) which had a 2.4 Å difference in the nitrogen residue found in the side chain of the amino acid (Figure 1f). The resulting increase in active site polarity due to the rotation of the C-terminal cap, if maintained during the light emitting conformation, could contribute to the red-shift in light emission (Nakatsu et al., 2006), in addition to the increased π-conjugation through the chemical structure of the emitter. This X-ray structure will help the future design of more efficient FLuc-iLH₂ pairs.

A range of colour-shifted, thermo- and pH stable FLuc mutants were spectrally characterised in vitro with a comparative selection of LH₂ analogues proven to red-shift bioluminescence emission (CycLuc1– Evans et al., 2014; Aka-Lumine-HCL – Kuchimaru et al., 2016; and iLH₂– Jathoul et al., 2014). Two new luciferins NH₂-NpLH₂ and OH- NpLH₂ have also been shown to have near infrared emissions (Hall et al., 2018) but these were reported too late to include in this study. FLuc mutants were engineered to combine mutations reported to provide superior stability (Jathoul, 2012) and colour-shifting capability (Branchini et al., 2005) (stabilising and colour shifting FLuc mutations are detailed in Materials and methods). The Raji B lymphoma cell line engineered to express a FLuc mutant were spectrally imaged after addition of each substrate. These cell lines were subsequently used for all in vitro and in vivo testing. Both CycLuc1 and Aka-Lumine-HCL showed a consistent red-shift in peak bioluminescence emission wavelength to ~600 nm and ~660 nm respectively for all FLuc mutants, making these substrates unsuitable for dual colour BLI (Figure 2—figure supplement 1). The data confirmed that with LH₂ both FLuc_natural and FLuc_green have a peak emission of ~560 nm, whilst FLuc_red has a peak emission of ~620 nm (Figure 2—figure supplement 1) (Jathoul, 2012), (Branchini et al., 2005). When tested with iLH₂ all FLuc mutants were shifted >100 nm into the near infrared but maintained their relative spectral shift [FLuc_green ~ 680 nm, FLuc_natural ~ 700 nm and FLuc_red ~ 720 nm (Figure 2—figure supplement 1)]. From this, we progressed further with two FLuc mutants, FLuc_green and FLuc_red to explore their utility for dual-BLI.

The ability to spectrally unmix FLuc_green and FLuc_red (Figure 2a) in vitro was investigated by mixing the two FLuc_mutants expressed in the Raji B lymphoma cell line at various ratios followed by spectral imaging and unmixing with both LH₂ and iLH₂ (Figure 2b). As would be expected from accurate spectral unmixing, the top wells were classified as containing mostly FLuc_green signal, which gradually decreased down the plate in line with the decreasing proportions of FLuc_green expressing cells, with the bottom wells being largely classified as FLuc_red signal for both LH₂ and iLH₂. The percentage unmixed signal of FLuc_green and FLuc_red was plotted for each ratio of FLuc expressing cells (Figure 2c). Correlation analysis was performed on this data comparing input cellular proportions with unmixed signal, giving R² values of 0.9983 and 0.9972 for LH₂ and iLH₂ respectively. Even though all 18 bandpass filters equipped on the IVIS Spectrum were utilised for spectral unmixing in this in vitro testing, we appreciate that not all potential users of this novel dual bioluminescence methodology will have access to machines with such a wide selection of bandpass filters. Therefore, further analysis of our data showed that spectral unmixing could be achieved with high accuracy just using a subset of filters. For LH₂, the use of 3 bandpass filters (500 nm, 660 nm, 820 nm) gave an R² value of 0.9958; For iLH₂, the use of 3 bandpass filters (600 nm, 700 nm, 800 nm) gave an R² value of 0.9937. The highest accuracy of spectral unmixing we could achieve using just two filters were R² values of 0.9776 and 0.9775 for LH₂ (500 nm and 720 nm) and iLH₂ (600 nm and 720 nm), respectively. Additionally, an experiment was carried out where FLuc_green and FLuc_red have been expressed at different levels in the same cell. Spectral bioluminescence imaging and unmixing has subsequently been performed to successfully reflect these differing expression levels with both LH₂ and iLH₂ (Figure 2—figure supplement 2). This spectral imaging data show that both LH₂ and iLH₂ can be used for dual bioluminescence reporting in vitro.

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To investigate the use of FLuc_green and FLuc_red with LH₂ and iLH₂ for in vivo dual BLI three NOD scid gamma (NSG) tumour models, representing increasing tissue depth (subcutaneous, systemic and intracranial), were established with the Raji B lymphoma cell line expressing either FLuc_green or FLuc_red (as described for in vitro experiments). All tumour models were then spectrally imaged with both LH₂ and iLH₂ (Figure 3, Figure 3—figure supplement 1 and Figure 3—figure supplement 2) to obtain the normalised spectra and average radiance of each FLuc mutant with both luciferins in all three in vivo models (Figure 4). The normalised bioluminescence spectra for every mouse in each model when imaged with LH₂ is shown, with the total radiance for each mouse plotted to the right of the spectral plot (Figure 4a–c). The data show that when imaged with LH₂ both FLuc_green and FLuc_red had an average peak emission between 610–630 nm; meaning the peak emission for FLuc_red is maintained as in vitro whereas the peak emission of FLuc_green is red shifted by ~60 nm in vivo. FLuc_green also exhibited a bimodal spectral distortion, with a minor peak at ~560 nm. In contrast to LH₂, FLuc_green and FLuc_red had a ~ 20 nm separation of average peak emissions in all three animal models with iLH₂ (FLuc_green ~ 700 nm and FLuc_red ~720 nm) (Figure 4d–f).

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Figure 4

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In addition to the separation of peak emission wavelengths when imaged with iLH₂ in vivo, the relative intensities of FLuc_green and FLuc_red were more comparable when imaged with iLH₂ than with LH₂; With LH₂ FLuc_red had an average radiance that was 42 (subcutaneous), 4.12 (systemic) and 7.28 (intracranial) times brighter than FLuc_green (Figure 4a–c). Whereas, when imaged with iLH₂ the average radiance between FLuc_green and FLuc_red was 1.38 (subcutaneous), 1.2 (systemic) and 1.51 (intracranial) times different (Figure 4d–f). No statistically significant difference in relative intensities between FLuc_green and FLuc_red was found in tumour models imaged with iLH₂ (p=0.3414, 0.4594 and 0.6153 for the subcutaneous, systemic and intracranial tumour models respectively, T test). This comparability of relative intensities between FLuc_green and FLuc_red with iLH₂ means that if used as genetic reporters for dual imaging, the dynamic range of radiance values for both enzymes will be more similar, therefore giving a more accurate comparison of the processes being monitored.

To validate the ability to spectrally unmix FLuc_green and FLuc_red in vivo with iLH₂ a systemic Raji tumour model was established. Raji cell lines expressing the FLuc mutants were mixed in the following ratios: 90:10, 75:25 and 50:50 for FLuc_green: FLuc_red and vice versa. After spectral BLI with both substrates, animals were sacrificed and the bone marrow was extracted for flow cytometry analysis to confirm the proportions of engrafted Raji FLuc populations (representative examples of flow cytometry plots can be found in Figure 5—figure supplement 1). Spectral unmixing was performed using Living Image (Perkin Elmer) by creating library spectra of FLuc_green and FLuc_red with both LH₂ and iLH₂, established from the pure expressing populations obtained during in vivo spectral characterisation (Figure 4). Output images with both LH₂ (Figure 5a) and iLH₂ (Figure 5b) were generated for FLuc_green and FLuc_red, as well as a composite image for each substrate.

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The percentage signal unmixed as FLuc_green and FLuc_red with both LH₂ (Figure 5c) and iLH₂ (Figure 5d) was determined and correlated to the percentage population of each FLuc_mutant within the Raji cell population taken from extracted bone marrow samples and analysed using flow cytometry (Figure 5e). A correlation of 0.99 was found with iLH₂ (R² value, SD = 0.01). LH₂ had a correlation of 0.89 (R² value, SD = 0.06), which was significantly different from the R² values obtained by flow cytometry (p<0.0001, ONE-Way ANOVA with post hoc Tukey test). No significant difference was found between R² values determined by flow cytometry and unmixed bioluminescence signal using iLH₂. Additionally, significant differences were found between the percentage unmixed signal using LH₂ and cellular proportions determined by flow cytometry, with p values of < 0.0001, 0.003, 0.0042 and 0.0056 for the 90%, 75%, 50% and 25% FLuc_green conditions respectively. No significant difference was found between percentage unmixed signal using iLH₂ and cellular proportions determined by flow cytometry, except for the 90% FLuc_green condition (p=0.0257). Therefore, iLH₂ is superior to LH₂ for in vivo dual reporting applications.

The characterised and validated in vivo dual BLI system using FLuc mutants in combination with iLH₂ was then applied to track tumour burden and CAR T cell therapy within the same animal model. A Raji B lymphoma tumour cell line expressing FLuc_green was used, and healthy human donor T cells were engineered to express CD19 CAR and FLuc_red linked via a 2A peptide. Tumour cells were first systemically engrafted, followed by administration of CAR T cells 8 days later. A control animal received tumour only. Spectral BLI using iLH₂ was then performed at 3, 4 and 6 days post CAR T cell administration on the IVIS Spectrum.

Output images for the unmixed FLuc_green and FLuc_red signal, as well as the composite of the two unmixed images, for day six post CAR T cell administration were generated (Figure 6a). The radiance values from the unmixed FLuc_green and FLuc_red images for both tumour control and treatment (tumour + CAR T cells) at 3, 4 and 6 days post CAR T cell treatment were determined (Figure 6b). As would be expected, the tumour only control showed consistently high levels of FLuc_green signal which increased overtime compared to FLuc_red, which is likely due to the close proximity of Fluc_red photons emitted by the neighbouring treatment mouse. However, the CAR T cell treatment mice initially had a higher proportion of FLuc_green signal, which was then surpassed by FLuc_red by the final imaging. This represents the homing of CAR T cells expressing FLuc_red to the FLuc_green expressing Raji B lymphoma tumour, followed by the expansion of the CAR T cells and the reduction in the tumour growth in the treatment mice.

Figure 6

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All manipulations were routinely carried out under an inert (Ar or N₂) atmosphere. All reagents were used as received unless stated. For the purposes of thin layer chromatography (tlc), Merck silica-aluminium plates were used, with uv light (254 nm) and potassium permanganate or anisaldehyde for visualisation. For column chromatography Merck Geduran Si 60 silica gel was used. Butyl lithium solutions were standardised with diphenyl acetic acid.

Melting points are uncorrected and were recorded on a Griffin melting point machine. Infrared spectra were recorded using a Bruker Alpha ATR spectrometer. All NMR data were collected using a Bruker AMX 300 MHz or Bruker AVANCE III 600 MHz as specified. Reference values for residual solvents were taken as δ = 7.27 (CDCl₃), 2.51 (DMSO –d6), 3.30 (MeOD- d4) ppm for ¹H NMR and δ = 77.2 (CDCl₃), 39.5 (DMSO –d6), 49.0 (MeOD- d4) ppm for ¹³C NMR. ¹⁹F NMR spectra were measured using a Bruker DX300 spectrometer, referenced to trichlorofluoromethane. Coupling constants (J) are given in Hz and are uncorrected. Where appropriate COSY and DEPT experiments were carried out to aid assignments. Mass spectroscopy data were collected on a Micromass LCT Premier XE (ESI) instrument. Elemental analysis was performed on an Exeter Analytical Inc CE-440 CHN analyser.

6-(β-Methoxyethoxymethylether)benzothiazole (1), (Muramoto et al., 1999)

6-(β-Methoxyethoxymethyl ether)−2-formylbenzothiazole (2), (Anderson et al., 2017)

1-(4-methoxycarbonylthiazole)methyltriphenylphosphonium chloride (Hermitage et al., 2001; Old, 2008)

2’,3’-O-Isopropylidene-5’-O-sulfamoyladenosine (Heacock et al., 1996) were synthesised using procedures reported in the literature.

6-(β-Methoxyethoxymethoxy)−2-(2-(4-methoxycarbonylthiazol-2-yl)ethenyl) benzothiazole (3). A suspension of aldehyde 2 (200 mg, 0.748 mmol) and 1-(4-methoxycarbonylthiazole)methyltriphenylphosphonium chloride (680 mg, 1.50 mmol) in DMF (3.5 mL) was cooled to 0°C and treated with K₂CO₃ (348 mg, 2.52 mmol). The resultant solution was allowed to warm to rt and stirred for 16 hr. After this time H₂O (40 mL) was added and the solution extracted using EtOAc (2 × 40 mL). The organics were combined and washed with H₂O (40 mL), separated, dried (MgSO₄), filtered and concentrated in vacuo. Purification was achieved using flash column chromatography (60% Et₂O/Hexane) to give three as a mixture of cis and trans isomers (163 mg, 54%).

Cis: R_f = 0.52 (60 % EtOAc/Hexane); ¹H NMR (600 MHz, CDCl₃) δ 3.39 (3H, s, OCH₃), 3.58–3.59 (2H, m, OCH₂CH₂O), 3.86–3.89 (2H, m, OCH₂CH₂O), 4.00 (3H, s, OCH₃), 5.36 (2H, s, OCH₂O), 6.96 (1H, d, J = 12.8, CHC(N)S), 7.25–7.29 (2H, m, ArH, CHC(N)S), 7.62 (1H, d, J = 2.3, ArH), 8.06 (1H, d, J = 8.9, ArH), 8.28 (1H, s, CHS). In CDCl₃ solution the trans isomer was seen to isomerise to the cis isomer.

Trans: A pure sample of trans was by separated by column chromatography to give three as a yellow solid. m.p. 112–115°C; R_f = 0.48 (60 % EtOAc/Hexane); IR ν_max 3101, 2926 (ν_CH), 2889 (ν_CH), 2818 (ν_CH), 1716 (ν_CO), 1629, 1598, 1556, 1495, 1456, 1333, 1318, 1281, 1239, 1222, 1208, 1162, 1089, 1046, 978 cm^{-1; 1}H NMR (600 MHz, CDCl₃) δ 3.39 (3H, s, OCH₃), 3.58–3.59 (2H, m, OCH₂CH₂O), 3.86–3.88 (2H, m, OCH₂CH₂O), 4.00 (3H, s, OCH₃), 5.35 (2H, s, OCH₂O), 7.22 (1H, dd, J = 8.9, 2.4, ArH), 7.59 (1H, d, J = 2.4, ArH), 7.68 (2H, s, CHC(N)S), 7.92 (1H, d, J = 8.9, ArH), 8.21 (1H, s, CHS); ¹³C NMR (150 MHz, CDCl₃) δ 52.8 (CH₃), 59.2 (CH₃), 68.0 (CH₂), 71.7 (CH₂), 94.0 (CH₂), 107.5 (CH), 117.9 (CH), 124.1 (CH), 128.2 (CH), 128.7 (CH), 136.2 (C), 148.1 (C), 148.9 (C), 156.3 (C), 161.7 (C), 162.9 (C), 165.6 (C); m/z (ESI) 407 (100%, M⁺+H); HRMS C₁₈H₁₉N₂O₅S₂ calcd. 407.0735, found 407.0738.

(E)−6-(β-Methoxyethoxymethoxy)−2-(2-(4-carboxythiazol-2-yl)ethenyl) benzothiazole (4). A suspension of 3 (20 mg, 0.049 mmol) in THF (0.75 mL) and H₂O (0.37 mL) was treated with LiOH.H₂O (5.0 mg, 0.12 mmol) and stirred for 15 min. After this time H₂O (10 mL) and EtOAc (10 mL) were added and the layers separated. The aqueous layer was acidified with 2 M HCl and extracted using EtOAc (2 × 10 mL), organics dried over MgSO₄, filtered and concentrated in vacuo to give 4 (19 mg, quant.) as a yellow solid. m.p. 175–178°C; R_f = 0.20 (50 % EtOAc/MeOH); IR ν_max3101 (ν_OH), 2917 (ν_CH), 1680 (ν_CO), 1598, 1554, 1455, 1398, 1320, 1241, 1204, 1160, 1101, 1045, 938 cm^{-1; 1}H NMR (600 MHz, MeOD-d4) δ 3.33 (3H, s, OCH₃), 3.57–3.58 (2H, m, OCH₂CH₂O), 3.84–3.85 (2H, m, OCH₂CH₂O), 5.36 (2H, s, OCH₂O), 7.25 (1H, dd, J = 8.9, 2.4, ArH), 7.69 (1H, d, J = 2.4, ArH), 7.71 (1H, dd, J = 16.1, 0.6, CHC(N)S), 7.73 (1H, d, J = 16.1, CHC(N)S), 7.80 (1H, d, J = 8.9, ArH), 8.43 (1H, s, J = 7.5, CHS); ¹³C NMR (150 MHz, MeOD-d4) δ 59.1 (CH₃), 69.0 (CH₂), 72.8 (CH₂), 95.0 (CH₂), 108.7 (CH), 119.0 (CH), 124.7 (CH), 128.5 (CH), 129.1 (CH), 130.1 (CH), 137.5 (C), 149.8 (C), 150.0 (C), 157.7 (C), 163.8 (C), 164.6 (C), 166.7 (C); m/z (ESI) 393 (100%, M⁺+H), 300 (9%); HRMS C₁₇H₁₇N₂O₅S₂ calcd. 393.0579, found 393.0581; Anal. Calcd. for C₁₇H₁₆N₂O₅S₂: C, 52.03; H, 4.11; N, 7.14. Found C, 51.85; H, 4.29; N, 6.71%.

6-(β-Methoxyethoxymethoxy)−2-(2-(4-pentafluorophenoxycarbonylthiazol-2-yl)ethenyl) benzothiazole (5). A solution of 4 (30 mg, 0.077 mmol) in pyridine (3.80 mL) was treated with EDC (18 mg, 0.096 mmol) and pentafluorophenol (18 mg, 0.096 mmol) and stirred at rt for 16 hr. The reaction mixture was concentrated in vacuo. Purification was achieved using flash column chromatography (30% Et₂O/hexane) to give five as a yellow solid (37 mg, 86%). m.p. 87–91°C; R_f = 0.25 (30 % EtOAc/Pet. Ether); IR ν_max 2922, 2887, 2835, 1764 (ν_CO), 1599, 1554, 1486, 1470, 1454, 1321, 1290, 1260, 1242, 1211, 1199, 1180, 1137, 1123, 1100, 1060, 1010, 986 cm^{-1; 1}H NMR (600 MHz, CDCl₃) δ 3.40 (3H, s, OCH₃), 3.59–3.60 (2H, m, OCH₂CH₂O), 3.87–3.88 (2H, m, OCH₂CH₂O), 5.36 (2H, s, OCH₂O), 7.23 (1H, dd, J = 8.9, 2.4, ArH), 7.60 (1H, d, J = 2.4, ArH), 7.72 (1H, dd, J = 16.2, 0.6, CHC(N)S), 7.74 (1H, d, J = 16.2, CHC(N)S), 7.94 (1H, d, J = 9.0, ArH), 8.40 (1H, d, J = 0.4, CHS); ¹³C NMR (150 MHz, CDCl₃) δ 59.2 (CH₃), 68.0 (CH₂), 71.7 (CH₂), 94.0 (CH₂), 107.5 (CH), 117.9 (CH), 124.3 (CH), 124.9 (C), 127.3 (CH), 129.8 (CH), 131.4 (CH), 136.4 (C), 137.3 (C), 139.0 (C), 140.6 (C), 142.2 (C), 144.7 (C), 149.2 (C), 156.4 (C), 156.8 (C), 162.5 (C), 166.6 (C); ¹⁹F NMR (CDCl₃, 282) δ −150.0 (d, J = 16.9, ArF), −157.14 (app t, J = 19.7, ArF), −161.9 (dd, J = 19.7, 16.9, ArF); m/z (ESI) 559 (100%, M⁺+H); HRMS C₂₃H₁₅F₅N₂O₅S₂ calcd. 559.0415, found 559.0418.

2’,3’-O-Isopropylidene-5’-O-[N-(6-(β-methoxyethoxymethoxy)-dehydroinfraluciferyl)-sulfamoyl]adenosine (6). A solution of 2’,3’-O-Isopropylidene-5’-O-sulfamoyladenosine (20 mg, 0.052 mmol) in DMF (1.8 mL) was treated with DBU (11 mg, 0.076 mmol) and stirred at rt for 10 min. A solution of 5 (29 mg, 0.052 mmol) in DMF (0.2 mL) was then added dropwise. The reaction was stirred at rt for 16 hr. After this time pyridine (0.15 mL) was added and the solution stirred for 4 hr. The resultant solution was concentrated in vacuo and purified using flash column chromatography (5% MeOH/DCM) to give 6 (30 mg, 76%) as a yellow solid. m.p. 164°C, dec.; R_f = 0.32 (5 % MeOH/DCM); IR ν_max3290 (ν_OH), 2932 (ν_CH), 1644 (ν_CO), 1598, 1552, 1505, 1457, 1418, 1373, 1291, 1250, 1209, 1150, 1103, 1080, 1048, 985 cm^{-1; 1}H NMR (600 MHz, DMSO-d6) δ 1.34 (3H, s, C(CH₃)₂), 1.54 (3H, s, C(CH₃)₂), 3.21 (3H, s, OCH₃), 3.46–3.49 (2H, m, OCH₂CH₂O), 3.74–3.78 (2H, m, OCH₂CH₂O), 4.09 (1H, dd, J = 11.0, 4.9, CH₂OS(O)₂), 4.12 (1H, dd, J = 11.0, 4.9, CH₂OS(O)₂), 4.42–4.46 (1H, m, OCHCH₂), 5.09 (1H, dd, J = 6.1, 2.5, CHCHO), 5.36 (2H, s, OCH₂O), 5.41 (1H, dd, J = 6.1, 2.9, CHCHNO), 6.17 (1H, d, J = 2.9, CHCHNO), 7.22 (1H, dd, J = 8.9, 2.5, ArH), 7.35 (2H, br, NH₂), 7.71 (1H, d, J = 16.1, CHC(N)S), 7.79 (1H, d, J = 16.0, CHC(N)S), 7.79 (1H, d, J = 2.5, ArH), 7.95 (1H, d, J = 8.9, ArH), 8.11 (1H, s, CHS), 8.12 (1H, s, NCH(N)), 8.44 (1H, s, NCH(N)); ¹³C NMR (150 MHz, DMSO-d6) δ 25.2 (CH₃), 27.1 (CH3), 58.1 (CH₃), 67.3 (CH₂), 67.6 (CH₂), 71.0 (CH₂), 81.6 (CH), 83.5 (CH), 83.9 (CH), 89.3 (CH), 93.3 (CH2), 108.0 (CH), 113.2 (C), 117.4 (CH), 118.8 (C), 123.6 (CH), 125.2 (CH), 126.1 (CH), 128.5 (CH), 136.0 (C), 139.6 (CH), 148.6 (C), 149.0 (C), 152.8 (CH), 155.3 (C), 156.1 (C), 156.8 (C), 162.8 (C), 162.9 (C), 165.0 (C); m/z (ES⁺) 761 (100%, M⁺+H); HRMS C₃₀H₃₀N₈O₁₀S₃ calcd. 761.1482, found 761.1486.

6-Hydroxy-2-(4-1E,3E-(4-ethoxycarbonyl-4,5-dihydrothiazol-2-yl)buta-2,4-dienyl)benzothiazole (iDLSA). A solution of 6 (20 mg, 0.026 mmol) in TFA (0.32 mL) was stirred at rt for 2 hr and then H₂O (0.1 mL) added and the solution concentrated in vacuo. EtOH (2 mL) added and concentrated in vacuo to give the TFA salt of iDLSA (18 mg, 94%) as an orange solid. m.p. 68–70°C; IR ν_max3102 (ν_OH), 1667 (ν_CO), 1426, 1132 cm^{-1; 1}H NMR (600 MHz, DMSO-d6) δ 4.20–4.25 (2H, m, CHOH, CHOH), 4.53–4.59 (3H, m, CHOC, CH₂OS(O)₂), 5.96 (1H, d, J = 5.0, CHCHNO), 7.00 (1H, d, J = 2.4, ArH), 7.42 (1H, d, J = 2.5, ArH), 7.72 (1H, d, J = 15.9, CHC(N)S), 7.84 (1H, d, J = 8.8, ArH), 7.95 (1H, d, J = 15.8, CHC(N)S), 8.33 (1H, s, CHS), 8.53 (1H, s, NCH(N)), 8.59 (1H, s, NCH(N)). ¹³C NMR too weak due to poor solubility of compound. MS did not give M+ or meaningful fragment for accurate mass measurement.

Copies of all 1H and 13C NMR have been deposited at https://doi.org/10.5061/dryad.3j9kd51cs.

Approximately 0.6 mg of iDLSA was suspended in 500 µL of crystallisation buffer (25 mM Tris-Cl containing 200 mM AmSO4, 1 mM DTT, 1 mM EDTA) pH 7.85 at 21°C. The solution was vortexed vigorously and sonicated. Most of the solid was dissolved and the concentration was determined to be ~1 mM by UV absorbance (using an extinction coefficient of 8200 at 372 nm for this buffer and pH). A 20 mg/mL solution of P. pyralis luciferase (PpyWT that contains the N-terminal peptide GPLGS-) in the same buffer (500 µL) was mixed gently with the iDLSA at room temperature and then incubated at 15°C for 20 min. The concentration of iDLSA in the protein-inhibitor mixture was determined by UV absorbance to be 620 µM, giving an inhibitor:enzyme ratio of ~3:1, at this point the bioluminescence activity of the mixture was assayed and the enzyme was 85% inhibited. A small amount of a separate iDLSA solution (available from solubility trials) was added to bring the inhibitor:enzyme ratio to ~4:1, and based on activity the enzyme was 91% inhibited. Finally, the protein-inhibitor solution was added to ~0.5 mg of iDLSA and mixed gently and incubated at 15°C for 15 min. Based on activity, 99% of the enzyme was inhibited and based on UV absorbance the inhibitor:enzyme ratio was 5.6:1 (950 µM:170 µM) The protein-inhibitor solution was centrifuged and a very slight amount of inhibitor was evident. The supernatant was frozen in liquid nitrogen in ~18–50 µL aliquots and stored at −80°C. A single aliquot was thawed and the solution remained clear. The pH of this solution at 6°C should be 8.3, pH 8.17 at 10°C, and pH 7.9 at 21°C.

Approximately 0.6 mg of iDLSA was resuspended in 500 µL of buffer (25 mM Tris-Cl containing 200 mM (NH₄)₂SO₄, 1 mM DTT, 1 mM EDTA) pH 7.85 at 21°C to a concentration of 1 mM as determined by UV absorbance (extinction coefficient of 8200 at 372 nm). This solution was mixed with a 20 mg/mL solution of P. pyralis (inhibitor:enzyme ratio of ~3:1) at room temperature and then incubated at 15°C for 20 min. The inhibitor:enzyme solution was centrifuged and the supernatant was frozen in liquid nitrogen in ~18–50 µL aliquots and stored at −80°C for future crystallisations.

Crystallisations used the hanging drop vapour diffusion method. Drops containing 1–2 µl of inhibitor:enzyme solution were mixed with the same volume of well solution and equilibrated against 500 µl of well solution, incubated at 4°C, with crystals typically appearing within 48 hr. Glycerol was used as a cryoprotectant, in an optimised well solution of 150 mM (NH₄)₂SO₄, 50 mM HEPES pH 7.0, 2% PEG 1000.

Data were collected at the Diamond Light Source on beam line IO4-1, at wavelength 0.91587 Å, and 100 K. Processing and data reduction were carried out on site using CrysalisPro (Agilent Technologies), and synchrotron data sets were processed and scaled by using XDS, SCALA and XIA2 programs. Molecular replacement methods were used successfully to determine the relative orientation and position of the two monomers in the asymmetric unit using the PHASER program (McCoy et al., 2007). The starting dehydroinfraluciferin DLSA complex model was derived from the Firefly luciferase apo structure (PDB-ID 3IEP) with all solvent atoms and the luciferin removed. A simple rigid body refinement was sufficient to initiate refinement, with subsequent refinement and model building cycles performed using Refmac5 and Coot (Murshudov et al., 2011; Emsley et al., 2010).

The X-ray data collection and refinement statistics have been deposited at https://doi.org/10.5061/dryad.3j9kd51cs. 

FLuc mutants contained 11 pH and temperature stabilising mutations (F14R/L35Q/A105V/V182K/T214C/I232K/D234G/E354R/D357Y/S420T/F465R) (Jathoul, 2012). FLuc_green contained an additional three mutations (V241I/G246A/F250S), and FLuc_red has the red-shifting mutation S284T, as well as the mutation R354I which is required to maintain the red-shift in this stabilised FLuc backbone (Branchini et al., 2007). All FLuc mutants were codon optimised for mammalian expression and cloned into the MLV-based splicing gamma retroviral vector SFG. The Raji B lymphoma cell line used in all experiments was transduced to express a FLuc mutant, and subsequently flow-sorted for pure FLuc expressing populations using a co-expressed marker gene. For tumour cell lines FLuc.IRES was upstream of the marker gene CD34 or dNGFR as indicated. For T cells FLuc.2A_peptide was upstream of the CAR CD19-4G7_HL-CD8STK-41BBZ.

HEK-293T packaging cells were plated at a density of 200’000 cells/ml in 100 mm tissue culture dish ~24 hr prior to transfection. Transfections were performed when cells were 50–70% confluent. A bulk transfection mixture was prepared where 30 μl GeneJuice Transfection Reagent (Merck millipore) was added to 470 μl of plain RPMI for each supernatant to be produced. Following a 5 min incubation at room temperature, a total volume of 12.5 μg of DNA was added for each plate to be transfected (for retroviral transfection: 3.125 μg RDF RD114 env plasmid, 4.6875 μg PeqPam-env gagpol plasmid, 4.6875 μg SFG retroviral construct). Following addition of plasmid DNA, the mixture was incubated for a further 15 min at room temperature prior to dropwise addition to the HEK- 293 T cell culture. Plates were gently agitated following transfection. Supernatant harvested at 48 hr was stored at 4°C, and was then combined with the 72 hr harvest prior to aliquoting and storage at −80°C.

The day prior to transduction Raji B lymphoma cells (atcc ccl-86) (>90% viable) were diluted ~1 in 10 to ensure exponential growth for transduction; also a well of non-tissue culture treated 24 well plate was coated with 8 μg/ml retronectin (Lonza) for every plasmid to be transduced and left at 4°C overnight. The next day retronectin was aspirated and 250 μl of each retroviral supernatant for transduction was added to a well and incubated for 30 min at room temperature. Whilst incubating, Raji cells were harvested, counted and resuspended at a concentration of 600,000 cells/ml. supernatants were aspirated from wells of retronectin coated plate and 500 μl of cell suspension was added to each well followed by 1.5 ml of the same retroviral supernatant that was previously incubated in each well. Cells were spin transduced at 1000 RCF for 40 min then returned to incubator for 48 hr before harvest and expression testing.

Transduction efficiencies were assessed by flow cytometry, based on marker gene expression as indicated by antibody staining using the BD LSR FortessaX-20. If necessary Fluorescence Activated Cell Sorting (FACS) was performed to obtain pure expressing populations, also based on marker gene expression as indicated by antibody staining, using the BD FACS Aria Fusion. FACS was also use to sort populations of cells expressing differing levels of FLuc_green and FLuc_red within the same cell. Concentration of antibody used was guided by manufacturer’s instructions. Anti-human CD34-PE (clone 581), anti-human CD271-APC (clone ME20.4) and anti-mouse/human CD11B-PerCP/Cy5.5 (clone M1/70) (Biolegend). Anti-humanCD19-FITC (clone HIB19), anti-human CD20-eFluor450 (clone 2H7) and Viability APC-eFluor780 (eBioscience). Data were analysed using Flow Jo software (Tree Star Inc, Oregon, USA).

When bone marrow cells were required for flow cytometry analysis. Following animal sacrifice by CO₂ narcosis and cervical dislocation, the femurs were removed and transferred to PBS pending cellular harvest. The ends of the femur were snipped off using scissors and the bone was placed in an extraction tube (microfuge tube with holder made from 200 μl pipette tip inserted). Tubes were centrifuged at 1000 RCF for 60 s. Bone marrow pellet was resuspended in 50 μl Ammonium-Chloride-Potassium (ACK) lysing buffer (Lonza) and left for 60 s at room temperature before washing with PBS and passing through a 70 μm filter before pelleting. Samples were blocked 2.4G2 supernatant (rat anti-mouse CD32) supplemented with mouse Ig FcR blocking reagent (Miltenyl Biotec) for 30 min at room temperature. Cells were washed with PBS and pelleted, followed by each sample being transferred to a well of a U-bottomed 96 well plate before proceeding with antibody staining. An antibody master mix containing all antibodies to be used for staining was prepared in PBS to a total volume of 100 μl per sample. Samples were left to stain at room temperature in the dark for 30 min. Samples were washed once with PBS, pelleted and transferred to FACS tubes. Beckman Coulter Flow-Checkfluorospheres were used as a stopping gate for flow cytometry analysis. Beads are supplied at 1 × 10e⁶ beads/ml in an aqueous solution containing preservative surfactant. To prevent toxicity to cellular samples, beads were washed once with PBS prior to addition to samples. Following centrifugation (400 RCF for 5 min), beads were resuspended in an equal volume of PBS with 10 μl of beads added to each sample.

As six fluorophores were used for bone marrow analysis, compensation was performed prior to sample acquisition using OneComp eBeads (eBioscience). Events were kept between 2,000–5,000 events/second, with 1000 events being recorded per sample, using flow check beads as a stopping gates (10% each sample). Flow cytometry gating first identified the lymphocyte population (FSC-A vs SSC-A), exclusion of doublet cells (SSC-A vs SSC-W); antibodies detailed in Flow cytometry and Fluorescence Activated Cell Sorting were then used to gate on viable cells, exclude mouse monocyte cells (mCD11b), identify the Raji tumour cell population (CD19 and CD20) and finally co-expressed marker gene (dNGFR and dCD34).

On day one peripheral blood mononuclear cells were isolated from a healthy donor blood using Ficoll-paque density gradient media (GE Healthcare). Cells were resuspended at 2 × 10⁶/ ml and stimulated with 1 mg/ml PHA (Sigma). On day 2 cells were fed with IL-2 at a concentration of 100 U/ml (Genscript). On day 3 cells were transduced as described in transduction of cell lines, with IL-2 at a final concentration of 100 U/ml. On day 6 cells were harvested and resuspended at 1 × 10⁶/ml with 100 U/ml IL-2 and left to recover for at least 48 hr before in vivo injection. Transduction efficiency was measured using flow cytometry.

For spectrographic testing of FLuc, mutants were stably transduced in the mammalian Raji B-cell lymphoma cell line. For in vitro bioluminescence assays cells were harvested, counted and 1 × 10⁶ cells/well were resuspended in TEM buffer (1M Tris-acetate, 20 mM EDTA and 100 mM MgSO4 at pH 7.8) and added in triplicate to wells of a black 96-well plate (100 μl/well). If mixtures of cells were used, total cell number remained the same. For spectral testing the stage temperature of the IVIS Spectrum was set to 37°C (automatic acquisition mode, FOV 13.2, f/1). iLH₂ was synthesised by UCL Chemistry. Other substrates tested include D-luciferin (Regis Technologies), CycLuc1 (Merck Millipore) and Aka-Lumine-HCL (Wako Pure Chemical Industries). Substrates were dispensed into the wells using a multi-channel pipette (at a final concentration of 300 μM). A 2 min delay was allowed for stabilisation of light output. Images were acquired through all 18 bandpass filters on the IVIS Spectrum (20 nm bandpass, 490 nm to 840 nm). Living image software (Perkin Elmer) was used for ROI analysis of spectral images and spectral unmixing analysis. Image analysis involved placing a ROI over the signal in each well. If a series of spectral images was acquired, the same ROI was placed over the well in every image for each plate. For spectral unmixing analysis, guided spectral unmixing was first used on pure expressing FLuc_green and FLuc_red populations to create a library spectra for each mutant with each substrate. The relevant library spectra was then used to perform spectral unmixing on mixed FLuc_green and FLuc_red populations (or cellular populations expressing both enzymes). Data exported to Excel (Microsoft) and Prism (Graphpad) for further analysis. Spectra was normalised to peak emission for each FLuc mutant with each substrate. Due to the characterisation nature of these in vitro experiments, and the substantial amounts of precious chemicals needed to synthesise iLH₂, it was decided to repeat each in vitro experiment twice with three replicates.

All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Guidance for the Operation of Animals (Scientific Procedures) Act 1986 (Home Office, London, United Kingdom). All of the in vivo models used the severely immunocompromised NSG (NOD.Cg Prkdc^scidIl2rg^tm1Wji/SzJ) mouse model (JAX mouse strain, Charles River). Mice were male and aged between 6–8 weeks old. Due to the characterisation, or proof of concept, nature of these experiments, and the substantial amounts of precious chemicals needed to synthesise iLH₂, it was decided to engraft 4–5 mice for every condition in each model to ensure engraftment and survival in a least three animals for each condition. Also, no specific toxicity experiments were performed, but no adverse side effects were observed with iLH₂.

For engraftment of subcutaneous tumours, FLuc expressing Raji cell lines were counted and 2 × 10⁶ cells were pelleted for each animal to be injected. Cells were washed twice in PBS before being resuspended in plain RPMI 1% HEPES (Sigma-Aldrich) to a concentration of 2 × 10⁷ cells/ml and were kept on ice ready for injection. Cells were injected subcutaneously (100 μl bolus) into a shaved area of the flank. Mice were left at least 5 days for tumour development before imaging.

For engraftment of systemic tumours, FLuc expressing Raji cell lines were counted and 5 × 10⁵ cells were pelleted for each animal. If mixtures of two different FLuc expressing cell lines were used, total cell number per mouse remained at 5 × 10⁵ cells. Cells were washed twice in PBS before being resuspended in plain RPMI 1% HEPES to a concentration of 2.5 × 10⁶ cells/ml and were kept on ice ready for injection. Animals were transferred to a warming chamber set at 39–42°C to facilitate peripheral vasodilation prior to intravenous (IV) injections. Mice were placed in a restraint and cells were injected IV (200 μl bolus) into the tail vein. Mice were left at least 7 days for tumour development before imaging. For the CAR T cell model 5 × 10⁶ CAR positive T cells were injected IV (200 μl bolus) 8 days after Raji cell engraftment.

For engraftment of intracranial tumours, FLuc expressing Raji cell lines were counted and 2 × 10⁴ cells were pelleted for each animal. Cells were washed twice in PBS before being resuspended in PBS to a concentration of 1 × 10⁴ cells/μl and were kept on ice ready for injection. Intracranial injections were performed using a stereotaxic frame fitted with a hamilton syringe. Cells were injected (2 μl bolus) into the right striatum (from bregma 2 mm right, 1 mm anterior, 4 mm down). Mice were left at least 7 days for tumour development before imaging.

For imaging of in vivo models, LH2 and iLH2 were solubilised in sterile PBS and animals were administered with substrate (2 mg (or 100 mg/kg) of either LH₂ or iLH₂ in 200 μl or 400 μl bolus respectively) via intraperitoneal (IP) injection. Animals were anaesthetised using 2% Isofluorine (flow rate 1 L/min O₂). Spectral imaging was commenced 10 min post IP injection to allow stabilisation of light output. If the same animal was being imaged with both LH2 and iLH2, at least 24 hr was left between imaging to allow for full clearance of substrate. In vivo bioluminescent images were acquired using IVIS Spectrum (FOV 24, f/1, Medium (8)bin, automatic acquisition mode for imaging with LH₂, FOV 24, f/1, Medium (8)bin, 120 s acquisition, total imaging time 24 mins for imaging with iLH₂). These parameters are calculated to keep the binning, exposure time and f/stop within an optimal range for quantification. Up to five animals could be imaged at once and the stage was heated to 37°C. Open filter images were acquired prior to and post spectral imaging to confirm the stability of photon emission during spectral acquisition. Spectral imaging acquired images through 14 and 12 of the 20 nm bandpass filters on the IVIS Spectrum depending on substrate used (530–830 nm for LH2 and 590–830 nm for iLH2), starting from the lowest to the highest filter. It was not necessary to acquire images through all filters as the bioluminescent emissions of FLuc mutants did not cover the full spectral range from 490 to 850 nm. Living image software was used for ROI analysis of spectral images and spectral unmixing analysis. Radiance values for bioluminescence are shown using pseudo-colour scales detailed in each image. Image analysis involved placing an ROI over the tumour signal for every animal in each model. If a series if spectral images were acquired, the same ROI was placed over tumour signal in every image for each mouse. For spectral unmixing analysis, guided spectral unmixing was first used on pure expressing FLuc_green and FLuc_red populations from spectral characterisation experiments to create a library spectra for each mutant with each substrate. The relevant library spectra was then used to perform spectral unmixing on mixed FLuc_green and FLuc_red populations. Data exported to Excel (Microsoft) and Prism (Graphpad) for further analysis.

Where relevant means ± standard deviation of data given. Statistical tests used include T test and ONE-way ANOVA with post hoc Tukey’s test for multiple column comparison (Prism, Graphpad). Correlation analysis performed using Microsoft excel.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank Steven Pacman for the synthesis of infraluciferin, and the following masters students for their contributions to the synthesis of iDLSA, Laszlo Berencei (EPSRC vacation bursary) Boyuan Deng and Julia Holm (Erasmus), the MRC based at Imperial College London for the use of their IVIS Spectrum, especially to Dr Alex Sardini, and the core flow cytometry facility at UCL Cancer Institute for the sorting of cell lines. This work was partly funded by the National Science Foundation grant MCB-1410390, Force Office of Scientific Research grant FA9550-18-1-0017, Erba Diagnostics Mannheim and the EPSRC for an industrial CASE award EP/L504889/1 (SJP) and UCL.

Animal experimentation: All animal procedures were conducted in accordance with the Home Office Scientific Procedures Act (1986), within the guidelines of the relevant personal and project licences.

© 2019, Stowe et al.

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