James C. Anderson’s research while affiliated with University College London and other places

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Publications (132)


The structure of D‐Luciferin and transmission of various photon wavelengths through mammal skin.
Literature Luciferin analogues with different emission wavelengths.
a) General structure of ‘V’‐shaped luciferin. b) Target ‘V’‐shaped luciferin analogues.
The normalized absorption dash line and fluorescence emission spectra solid line in anhydrous DMSO at r.t..
The normalized spectra of luciferins with a) PpyLuc and b) FlucRed.

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Synthesis and Bioluminescence of ‘V’‐Shaped Firefly Luciferin Analogues Based on A Novel Benzobisthiazole Core
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October 2023

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43 Reads

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1 Citation

Chia‐Hao Chang

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Danielle M. Fontaine

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Sandra Gómez

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[...]

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James C. Anderson

The design of π‐extended conjugation ‘V‘‐shaped red shifted bioluminescent D‐luciferin analogues based on a novel benzobisthiazole core is described. The divergent synthetic route allowed access to a range of amine donor substituents through an SNAr reaction. In spectroscopic studies, the ‘V’‐shaped luciferins exhibited narrower optical band gaps, more red‐shifted absorption and emission spectra than D‐luciferin. Their bioluminescence characteristics were recorded against four different luciferases (PpyLuc, FlucRed, CBR2 and PLR3). With native luciferase PpyLuc, the ‘V’‐shaped luciferins demonstrated more red‐shifted emissions than D‐luciferin (λbl=561 nm) by 60 to 80 nm. In addition, the benzobisthiazole luciferins showed a wide range of bioluminescence spectra from the visible light region (λbl=500 nm) to the nIR window (>650 nm). The computational results validate the design concept which can be used as a guide for further novel D‐luciferin analogues based upon other ‘V’‐shaped heterocyclic cores.

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Figure 1 Representative synthetic D-luciferin analogues with their bioluminescence wavelengths
Bioluminescence, Photophysical, Computational and Molecular Docking Studies of Fully Conformationally Restricted Enamine Infraluciferin

March 2023

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84 Reads

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1 Citation

Organic & Biomolecular Chemistry

A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH2 and iLH2. With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH2 at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range.


FIGURE 4 Effect of Fluc mutations E354R and D357Y on activity and color with DL-iLH 2 . (A) Quantitative bioluminescence spectra showing that E354R leads to significant improvement in activity with DL-iLH 2 . (B) Normalized bioluminescence spectra of WT Ppy Fluc, E354R, and D357Y with D-LH 2 and DL-iLH 2 .
FIGURE 5 Activity of purified Flucs and mutants with both substrates. Specific activity and emission peak wavelengths of selected R354 and/or D357 mutants of WT, x5, and x11 Flucs. 150μM D-LH 2 or 15 μM DL-iLH 2 and 2 mM ATP were used to saturate 0.167 and 0.0167 μM Flucs, respectively, and light emission was captured using the PIO. The assay was then repeated with the blood phantom.
A higher spectral range of beetle bioluminescence with infraluciferin

August 2022

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115 Reads

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2 Citations

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin ( D -LH 2 ). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH 2 ). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH 2, including in human cells. iLH 2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH 2 : Lampyrid wild-type (WT) Photinus pyralis ( Ppy ) firefly luciferase, Ppy -based derivatives previously engineered to be thermostable with D -LH 2 , and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH 2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase ( D -LH 2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL -iLH 2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH 2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH 2 and engineer an improved infraluciferase (iluc) based on this mutant.


Applications of bioluminescence in biotechnology and beyond

March 2021

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3,394 Reads

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203 Citations

Chemical Society Reviews

Bioluminescence is the fascinating natural phenomenon by which living creatures produce light. Bioluminescence occurs when the oxidation of a small-molecule luciferin is catalysed by an enzyme luciferase to form an excited-state species that emits light. There are over 30 known bioluminescent systems but the luciferin–luciferase pairs of only 11 systems have been characterised to-date, whilst other novel systems are currently under investigation. The different luciferin–luciferase pairs have different light emission wavelengths and hence are suitable for various applications. The last decade or so has seen great advances in protein engineering, synthetic chemistry, and physics which have allowed luciferins and luciferases to reach previously uncharted applications. The bioluminescence reaction is now routinely used for gene assays, the detection of protein–protein interactions, high-throughput screening (HTS) in drug discovery, hygiene control, analysis of pollution in ecosystems and in vivo imaging in small mammals. Moving away from sensing and imaging, the more recent highlights of the applications of bioluminescence in biomedicine include the bioluminescence-induced photo-uncaging of small-molecules, bioluminescence based photodynamic therapy (PDT) and the use of bioluminescence to control neurons. There has also been an increase in blue-sky research such as the engineering of various light emitting plants. This has led to lots of exciting multidisciplinary science across various disciplines. This review focuses on the past, present, and future applications of bioluminescence. We aim to make this review accessible to all chemists to understand how these applications were developed and what they rely upon, in simple understandable terms for a graduate chemist.


Asymmetric synthesis of piperidines using the nitro-Mannich reaction

December 2020

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29 Reads

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2 Citations

Tetrahedron

A method for the synthesis of functionalized piperidines containing 3 contiguous stereocentres in the 2-,3- and 4- positions uses a diastereoselective nitro-Mannich to control stereochemistry. The nitro-Mannich reaction between a β-aryl/heteroaryl substituted nitroalkanes and glyoxylate imine provides β-nitro-amines with good selectivity (70:30 to >95:5) for the syn, anti-diastereoisomers. Reductive cyclisation with BF3.OEt2 and Et3SiH gave, after purification, stereochemically pure piperidines in 19–57% yield for ten examples with different 4-aryl/heteroaryl substituents.


Fig. 2 Chemical equilibria of firefly OL.
Fig. 3 Model analogues of firefly OL studied in this work.
Fig. 4 359 nm (3.45 eV) photoelectron spectra of M-phenolate-keto (red), M-phenolate-enol (purple) and M-phenol-enolate (light blue) generated by ESI of 1 mM MeOH solutions, plotted as a function of eBE (top). Photoelectron spectra of OL À generated by ESI from 1 mM dry MeOH (blue) and MeCN (green) solutions, plotted as a function of eBE (bottom). Combs mark EOM-IP-CCSD/aug-cc-pVDZ calculated VDEs of the trans conformers.
Fig. 5 Photoelectron spectra recorded at 294 nm (4.22 eV), 298 nm (4.16 eV), 310 nm (4.00 eV), 320 nm (3.87 eV), 346 nm (3.58 eV) and 359 nm (3.45 eV) for M-phenolate-keto, M-phenolate-enol, M-phenol-enolate and OL À sprayed from MeOH and MeCN. The spectra of the model analogues are normalised to their rising edges and the OL À spectra are normalised to the peaks around 2.6 eV (MeOH) and 3.3 eV (MeCN).
Shining light on the electronic structure and relaxation dynamics of the isolated oxyluciferin anion

August 2020

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129 Reads

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7 Citations

Physical Chemistry Chemical Physics

Firefly bioluminescence is exploited widely in imaging in the biochemical and biomedical sciences; however, our fundamental understanding of the electronic structure and relaxation processes of the oxyluciferin that emits the light is still rudimentary. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate the electronic structure and relaxation of a series of model oxyluciferin anions. We find that changing the deprotonation site has a dramatic influence on the relaxation pathway following UV photoexcitation of higher lying electronically excited states. The keto form of the oxyluciferin anion is found to undergo internal conversion to the fluorescent S1 state, whereas we find evidence to suggest that the enol and enolate forms undergo internal conversion to a dipole bound state, possibly via the fluorescent S1 state. Partially resolved vibrational structure points towards the involvement of out-of-plane torsional motions in internal conversion to the dipole bound state, emphasising the combined electronic and structural role that the microenvironment plays in controlling the electronic relaxation pathway in the enzyme.


A Divergent Synthetic Route to the Vallesamidine and Schizozygine Alkaloids: Total Synthesis of (+)‐Vallesamidine and (+)‐14,15‐Dehydrostrempeliopine

October 2019

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236 Reads

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30 Citations

The total synthesis of representative members of the schizozygine alkaloids, (+)‐vallesamidine and (+)‐14,15‐dehydrostrempeliopine, were completed from a late‐stage divergent intermediate. The synthesis took advantage of efficient nitro‐group reactions with the A/B/C ring skeleton constructed concisely on a gram scale through an asymmetric Michael addition, nitro‐Mannich/lactamisation, Tsuji–Trost allylation, and intramolecular C−N coupling reaction. Other key features of the synthesis are a novel [1,4] hydride transfer/Mannich‐type cyclisation to build ring E and a diastereoselective ring‐closing metathesis reaction to construct ring D. This approach gave access to a late‐stage C14,C15 alkene divergent intermediate that could be simply transformed into (+)‐vallesamidine, (+)‐14,15‐dehydrostrempeliopine, and potentially other schizozygine alkaloids and unnatural derivatives.


Investigation of the [1,5]-hydride shift as a route to nitro-Mannich cyclisations

October 2019

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58 Reads

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4 Citations

Tetrahedron

Conditions were found for the [1,5]-hydride shift nitro-Mannich reaction that led to the synthesis of 2,3-disubstituted tetrahydroquinolines. Two simple cyclic amine substrates gave diastereomerically pure rearranged products in 65 and 90% yields by refluxing in HFIP. A more general procedure used Gd(OTf)3 as a catalyst and successfully rearranged other cyclic and acyclic amines in 42–84% yield with diastereomeric ratios of 75:25 to >95:5 in favour of the anti-diastereoisomer (9 examples). Two examples of sulphur containing heterocycles gave lower yields of 9 and 25%. Electron withdrawing substituents were shown to have a deleterious effect on the success of the reaction. The results indicated the limitation of the [1,5]-hydride shift nitro-Mannich reaction with respect to the stability of the intermediate iminium ion.


Figure 1: Crystal structure of Firefly luciferase in complex with a iLH 2 analogue
Table 507 508
Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin

October 2019

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442 Reads

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52 Citations

eLife

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achievedin vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5'-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.


Fig. 4 (a) P(i-MAC) showed pH sensitive behaviour at a pH of 6.0. At this pH the hydrophilic protonated version (blue) becomes hydrophobic (red). (b) The nanoparticles from PEG-P(DMMDO-stat-i-MAC) disassembled into single polymer chains upon acidification from a pH of 4 and below. (c) The same nanoparticles were degraded upon the addition of esterase (porcine liver) as shown by the decay of the count rate in DLS.
Novel Monomers in Radical Ring-Opening Polymerisation for Biodegradable and pH Responsive Nanoparticles

September 2019

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47 Reads

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28 Citations

Polymer Chemistry

Responsive and biodegradable nanoparticles are essential for functional drug delivery systems. We herein report the first pH sensitive polyester from radical ring-opening polymerisation of novel amine-bearing cyclic ketene acetals (CKAs)....


Citations (65)


... Breaking the planarity in this way does not affect the dihedral angle S-C(thiazole)=C-C(benzene), and the differences ∆ o between the initial structure and that after molecular docking are 0.067 • , 0.039 • and 0.006 • for A-1, A-2 and A-3, respectively. Similar rotations of molecules when fitting into the aromatic cavity when forming active marker-protein complexes have been observed for other classes of compounds, such as luciferin derivatives [63]. A C=C double bond is not rigid and inflexible. ...

Reference:

Can a Small Change in the Heterocyclic Substituent Significantly Impact the Physicochemical and Biological Properties of (Z)-2-(5-Benzylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic Acid Derivatives?
Bioluminescence, Photophysical, Computational and Molecular Docking Studies of Fully Conformationally Restricted Enamine Infraluciferin

Organic & Biomolecular Chemistry

... Notably, this analog can provide different colors of light when paired with distinct luciferase enzymes. 48 Such spectral differences are advantageous for visualizing multiple cell populations in the same mouse. Toward this end, the authors showcased dual color imaging in mice via infa-luciferin application. ...

A higher spectral range of beetle bioluminescence with infraluciferin

... Although bioluminescence varies in different organisms, it is generally seen that luciferase is the enzyme living things use to produce light (Kim et al., 2012). The luciferase enzyme catalyzes a series of chemical reactions in the light production process and interacts with a substance called luciferin to create a reaction (Syed & Anderson, 2021). The luciferin enzyme is oxidized by the action of luciferase, releasing energy, and light emerges as a result of the reaction (Farusi & Watt, 2017). ...

Applications of bioluminescence in biotechnology and beyond

Chemical Society Reviews

... The resulting intermediate can be used for further modification and for obtaining various analogs of the final product. [107]. The diastereoselective Mannich reaction (first step) between functionalized acetals and imines is used to control the stereochemistry of piperidines, which is retained during reductive cyclization (second step). ...

Asymmetric synthesis of piperidines using the nitro-Mannich reaction
  • Citing Article
  • December 2020

Tetrahedron

... 35,36 Notably, the dual BLI studies performed to date have employed at least one ATP-dependent firefly or click beetle luciferase. 22,23,[37][38][39][40][41][42] In addition to engineering these luminescent-fluorescent biological light sources, significant progress has also been made in engineering orthogonal substrates for several luciferases, which has created an opportunity to envision a variety of multiplexed imaging strategies. 2,18,20,21,43 However, while orthologous substrates enable the specific detection and temporary resolution of signals emanating from distinct cell types both in vitro and in vivo, it necessitates a multi-dosing regimen wherein administration of the first substrate illuminates one cell type/population and this must be followed by a ''washout'' or active quenching period (few hours to days) before which a subsequent substrate can be administered and a different cell type/population can be imaged. ...

Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin

eLife

... [65] Additionally, amine containing CKAs able to polymerize into pH sensitive polyesters were presented by Gaitzsch. [66,67] In summary, CKAs have been extensively studied as cyclic monomers able to undergo rROP to produce degradable polymers for a variety of applications. However, work has to be done to overcome remaining limitations such as unavailability of these monomers on large scale, propensity to hydrolyze and the observed side reactions in homo and co-polymerization. ...

Novel Monomers in Radical Ring-Opening Polymerisation for Biodegradable and pH Responsive Nanoparticles

Polymer Chemistry

... Hydroboration-oxidation of olefin 14 with using cyclohexene and borane [14] followed by the oxidation with Dess-Martin reagent gave aldehyde intermediate 15. Due to its instability, the Corey-Chaykovsky reaction [15] was immediately performed to produce the epoxide 16 which provide the product in 19 % yield for 3 steps. ...

A Divergent Synthetic Route to the Vallesamidine and Schizozygine Alkaloids: Total Synthesis of (+)‐Vallesamidine and (+)‐14,15‐Dehydrostrempeliopine

... In other words, wavelength shifts that were normally only observed with D-LH 2 were observed at longer wavelengths with iLH 2 , demonstrating color tuning with a red-shifted analog. BLI in mice using D-iLH 2 methyl (Me) ester (or even DL-iLH 2 Me ester) with wild-type (WT) Photinus pyralis (Ppy) firefly luciferase (Fluc) and its thermostable/color derivatives previously allowed nIR BLI of detailed disease morphologies in small animal models of cancer (Jathoul et al., 2014) and allowed robust dual color imaging of T-cell effectors and cancer targets in a Chimeric Antigen Receptor (CAR) T-cell cancer therapy model in vivo (Stowe et al., 2019), showing its advantages for BLI compared to D-LH 2. However, pure WT Ppy Fluc enzyme produced much lower specific activity with DL-iLH 2 than with D-LH 2 , by over 3-orders of magnitude at pH 7.8 (Anderson et al., 2019). x11 Fluc (Jathoul et al., 2012a) was found to be our most active mutant, approximately 7fold brighter, and contained subsets of mutations previously engineered in Murray Lab ( Figure 1). ...

Synthesis and bioluminescence of electronically modified and rotationally restricted colour-shifting infraluciferin analogues
  • Citing Article
  • December 2018

Tetrahedron

... Chao (2007), [163] Kamimura (2007), [164] Petrini (2007), [165] Gribble (2007), [166] Li and Chen (2008), [167] Dixon (2008), [168] Johnston (2008), [169] Erica (2009), [170] Dixon (2009), [171] Kamimura (2009), [172] Danishefsky (2009), [173] Blay and Pedro (2010), [174] Danishefsky (2010), [175] Kim (2010), [176] Dixon (2011), [177] Shibata (2011), [178] Kim (2011), [179] Dixon (2011), [180] Luzzio (2011), [181] White (2011), [182] Dixon (2012), [183] Johnston (2012), [184] Dixon (2012), [185] Dixon (2012), [186] and Vaidyanathaswamy (2012). [187] [188] Thomson (2013), [189] Zhao (2013), [190] Hayashi (2014), [191] Pascal Jr. (2014), [192] Dixon and Ye (2015), [193] Lu (2015), [194] Gong and Meggers (2015), [195] Enders (2015), [196] Wolf (2016), [197] Dixon (2016), [198] Lu and Xiao (2016), [199] Gu and Tian (2016), [200] You (2017), [201] Hayashi (2017), [202] Ryan and Hyland (2017), [203] Anderson (2018), [204] Liang (2018), [205] Hayashi (2018), [206] Li (2018), [207] Xu and Yuan (2018), [208] Xu and Yuan (2018), [209] Hayashi (2019), [210] Ishikawa (2019) [211] and Ye (2019). [212] *Denitration was accompanied by a reduction of a xanthate ester moiety. ...

Radical cyclisation studies of β-nitroamines from the nitro-Mannich reaction
  • Citing Article
  • June 2018

Tetrahedron

... 6 Homopolymerisations of CKAs are known to generate polyesters (bio)degrading under basic or enzymatic conditions. 7,8 Until now, 2-methylene1,3-dioxepane (MDO), 2-methylene-1,3,6-trioxocane (MTC) and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) are the CKAs with be highest publication record. [9][10][11][12][13] These CKAs are generally characterised by an almost complete degree of ring-opening and a known structure in terms of the main repeating unit and branching positions from inter-or intramolecular H-transfer. ...

Revisiting Monomer Synthesis and Radical Ring Opening Polymerization of Dimethylated MDO Towards Biodegradable Nanoparticles for Enzymes
  • Citing Article
  • February 2018

European Polymer Journal