Synthesis and bioluminescence of electronically modified and rotationally restricted colour-shifting infraluciferin analogues

Article abstract of DOI:10.1016/j.tet.2018.11.061

Synthetic nIR emitting luciferins can enable clearer bioluminescent imaging in blood and tissue. A limiting factor for all synthetic luciferins is their reduced light output with respect to D-luciferin. In this work we explore a design feature of whether

Full text of DOI:10.1016/j.tet.2018.11.061

Accepted Manuscript
Synthesis and bioluminescence of electronically modified and rotationally restricted
colour-shifting infraluciferin analogues
James C. Anderson, Amit P. Jathoul, Aisha J. Syed
PII:
S0040-4020(18)31432-7
https://doi.org/10.1016/j.tet.2018.11.061
TET 29972
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 30 July 2018
Revised Date: 14 November 2018
Accepted Date: 27 November 2018
Please cite this article as: Anderson JC, Jathoul AP, Syed AJ, Synthesis and bioluminescence of
electronically modified and rotationally restricted colour-shifting infraluciferin analogues, Tetrahedron
(2019), doi: https://doi.org/10.1016/j.tet.2018.11.061.
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Synthesis and bioluminescence of
electronically modified and rotationally
restricted colour-shifting infraluciferin
analogues
Leave this area blank for abstract info.
James C. Anderson,a* Amit P. Jathoulb and Aisha J. Syeda
a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. E-mail: j.c.anderson@ucl.ac.uk
b School of Biosciences, University of Cardiff, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK

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Synthesis and bioluminescence of
electronically modified and rotationally
restricted colour-shifting infraluciferin
analogues
Leave this area blank for abstract info.
James C. Anderson,a* Amit P. Jathoulb and Aisha J. Syeda
a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. E-mail: j.c.anderson@ucl.ac.uk
b School of Biosciences, University of Cardiff, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK

1
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Tetrahedron
journal homepage: www.elsevier.com
Synthesis and bioluminescence of electronically modified and rotationally restricted
colour-shifting infraluciferin analogues
James C. Anderson,a* Amit P. Jathoulb and Aisha J. Syeda
a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK. E-mail: j.c.anderson@ucl.ac.uk
b School of Biosciences, University of Cardiff, Sir Martin Evans Building, Museum Avenue, Cardiff, CF10 3AX, UK
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Synthetic nIR emitting luciferins can enable clearer bioluminescent imaging in blood and tissue.
A limiting factor for all synthetic luciferins is their reduced light output with respect to D-
luciferin. In this work we explore a design feature of whether rigidification of an exceptionally
red synthetic luciferin, infraluciferin, can increase light output through a reduction in the degrees
of freedom of the molecule. A rigid analogue pyridobenzimidazole infraluciferin was prepared
and its bioluminescence properties compared with its non-rigid counterpart benzimidazole
infraluciferin, luciferin, infraluciferin and benzimidazole luciferin. The results support the
concept that synthetic rigidification of π-extended luciferins can increase bioluminescence
activity while maintaining nIR bioluminescence.
Available online
Keywords:
infraluciferin
bioluminescence
near infrared
synthesis
2009 Elsevier Ltd. All rights reserved
.
colour shifting
1. Introduction
removed the benzothiazole core and replaced it by extended π-
conjugation (5), which led to substantial increases in emission
wavelength (λmax = 675 nm).8 This molecule as the HCl salt has
been shown to be particularly effective for sensitive
bioluminescence deep tissue imaging9 and noninvasive imaging
in moving animals.10 More recently extending conjugation of the
benzothiazole by substituting for a suitable napthothiazole
resulted in very red shifted analogues 6a,b (λmax = 730 nm and
743) with mutant CBR2opt luciferases that proved particularly
useful for highly resolved deep tissue imaging and tomography.11
Modifications to the thiazoline portion of LH2 have shown poorer
results, with substitutions by lighter elements (7a and 7b)
resulting in no or slight emission and heavier elements (7c)
resulting in red-shifted weak emission (Fig. 1).12,13 Recently,
compound 8 and the HCl salt of 5 have shown brighter emission
than LH2 at low substrate concentrations.9,14 Luciferase mutants
have also been engineered to give brighter light for some
The Coleopteran Luciferin-Luciferase system is widely used
in bioluminescence imaging (BLI) to track disease and other
biological functions in-vitro and in-vivo.1 The most commonly
used substrate in this assay is the naturally occurring D-luciferin
(1, LH2, λmax = 558 nm). In the presence of molecular oxygen,
Mg2+ ions and ATP, LH2 is oxidised by luciferase to form
oxyluciferin (2), an excited-state ketone species that releases a
photon of light (558 nm) as it relaxes back to its ground-state
(Scheme 1).2
Scheme 1
analogues11,15 including compound
4 under sub-saturating
conditions.16 In 2014, we reported a π-extended analogue of
luciferin infraluciferin (9, iLH2) that exhibited nIR
bioluminescence (λmax = 706 nm).17,18 Like other red-shifted
synthetic luciferins, iLH2 (9) was less bright than LH2, but
because of the higher proportion of light output above λ = 600
nm there is less attenuation and scatter of the signal by blood and
tissue. This leads to better penetration because of a reduced
signal to noise effect caused by light scattering.17 We are still
interested in improving the light output of iLH2 and are
investigating mutated luciferases and alternative iLH2 structures
However, the light emitted by D-luciferin is strongly absorbed
by melanin, blood and tissue and this makes imaging in small
mammals particularly difficult. To allow better image resolution
the light emitted should be of higher wavelength in the near
Infra-red (nIR) region (600 – 750 nm). Fortunately, the luciferase
enzyme has shown considerable tolerance to different substrates
and several synthetic nIR luciferins have been made and tested.3-5
A pioneer in this area, White prepared the amino-luciferin
analogue 3 which had a red-shifted emission spectrum (λmax
=
593 nm) in-vitro compared to LH2,6 but was only about 10% as
bright as D-luciferin. Cyclic amino modifications (4) by Miller
and co-workers resulted in emissions greater than 600 nm.7 Maki
for wider applicability in
a
variety of challenging
bioluminescence imaging applications.

2
Tetrahedron
ACCEPTED MANUSCRIPTN
N
S
CO2H
N
CO2H
N
S
HO
S
S
HO
D-Luciferin (1, LH2)
Infraluciferin (9, iLH2) max = 706 nm
N
CO2H
N
X
HO
S
11 Hypothetical rigid infraluciferin structure
N
CO2H
N
N
S
CO2H
N
S
N
H
HO
N
H
HO
10 Benzimidazole luciferin (BILH2) 13 Benzimidazole Infraluciferin (BIiLH2)
N
CO2H
N
N
HO
S
12 Pyridobenzimidazole Infraluciferin (PBIiLH2)
Figure 1. Bioluminescence emissions of some synthetic red
Figure 2. Design hypothesis for rigid infraluciferins
shifted luciferins
2. Results and discussion
The relatively lower light output of iLH2 (9) compared to LH2
(1) could be due to a number of factors, such as catalytic
turnover, product inhibition and radiationless decay caused by
rotatable bonds. While we know that the Km values for iLH2 (9)
and LH2 (1) with certain Fluc enzymes is comparable, the
specific activity is 100 times less and the L-enantiomer of iLH2
(9) causes some enzyme inhibition,17 the question of radiation-
less decay has not been investigated. Comparing the two
molecules iLH2 (9) has more degrees of freedom compared to
LH2 (1) due to rotation about an extra single bond (Fig. 2). This
could allow more changes of the excited state geometry in the
enzyme’s active-site, causing greater radiation-less decay and a
lower light output than LH2 (1). In this study, we have
investigated whether synthesis of a more rigid infraluciferin
framework with the same number of rotational single bonds as
LH2 (1) affects bioluminescence light ouput.
2.1. Docking of luciferin analogues in Ppy-Luc
As these analogues scaffolds are different in size and shape
than LH2 (1), we have used docking studies to predict the
interaction between the adenylated luciferin structures and the
luciferase enzyme. For this purpose, the crystal structure of the
Photinus pyralis luciferase enzyme, bound to the adenylate
analogue 5′-O-[N-dehydroluciferyl)-sulfamoyl]- adenosine (LH2-
DLSA) (PDB accession number 4G36) was obtained.20 GOLD21
software version 5.4.1 was used for the docking studies. To test
our system, we first performed the docking of the energy
minimised structure of LH2-DLSA at the ATP-binding site.
Energy minimisation was carried out using the MM2 calculation,
Chem3D Pro software version 13.0.2.3021. Calculations were
ran on a PC with a 2.3 GHz-i5 processor and 8 GB RAM. The
docking solutions were viewed using Discovery Studio
Visualizer22 with the protein surface coloured by hydrophobicity.
The docking result for the crystallised ligand was overlayed with
the co-crystal structure and the calculated exhaustive root mean
squared deviation (RMSD) was 0.7453, indicating a good
prediction ability of our docking protocol. We decided to carry
out a rigid molecular docking due to close similarity in the
binding site between different crystallographic structures.
To rigidify the infraluciferin structure, it was thought that a
carbon bridge could be constructed to connect either of the
heterocycle rings to the alkene linker (Fig. 2). It is known that
substitution of the heteroatoms in the thiazoline ring to C or N
lead to inert compounds that give little or no light,12 Whereas
modification of the benzothiazole heterocycle has been reported
by Prescher for benzimidazole luciferin (10, BILH2) to give
bioluminescence emission more red-shifted (λmax = 578 nm) than
LH2 (1), but again with a lower quantum yield.19 Our
hypothetical design structure 11 has incorporated this
modification to allow a rigidifying linker in pyridobenzimidazole
infraluciferin (12, PBIiLH2), where X = N so that aromaticity and
conjugation (with the corresponding ketone from oxidation) can
be maintained in the final compound. Whilst not completely
rigid, this structure has the same degrees of freedom and rotatable
single bonds as LH2 (1). Benzimidazole infraluciferin (13,
BIiLH2) would serve as a comparison to PBIiLH2 (12) in the
bioluminescence studies to test our hypothesis.
Starting with the energy minimised structures of PBIiLH2-
DLSA and BIiLH2-DLSA, the GOLD wizard was used to carry
out the molecular modelling. The binding site was defined to
within 6 Å of the LH2-DLSA analogue and the docking was
carried out using the slow method to identify six different
solutions. By comparing the docking solutions to the binding
mode of LH2-DLSA, the pose of PBIiLH2-DLSA and BIiLH2-
DLSA which gave the best overlay was selected for the basis of
our docking model.
Both PBIiLH2 (12 Fig. 3A) and BIiLH2 (13 Fig. 3B) fitted into
the narrow and deep substrate binding site. Several proposed
favourable hydrophobic interactions and hydrogen bonds are
shown in Fig. 3C and 3D. This preliminary study showed that it
is structurally possible to locate the analogues in the luciferin
binding site and gave us added confidence that the real molecules
may bioluminesce.

3
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C
A
B
D
Fig. 1. - A and B - Pose prediction for PBIiLH2 and BIiLH2 in PpyLuc pocket; C and D - PBIiLH2 and BIiLH2 with predicted
interactions; Colour code: Pink – Hydrophobic interactions; Green – hydrogen bonds
2.2. Synthesis of luciferin analogues
Scheme 2. Appel’s salt=4,5-dichloro-1,2,3-dithiazol-1-ium
chloride.
To circumvent possible epimerisation of the carboxylic acid
forms of the infraluciferin analogues and accurate quantification
of enantiopurity, all substrates were prepared as racemic mixtures
using DL-cysteine. We have previously shown that DL-iLH2 (9)
is a competent substrate for bioluminescence and that the L-
enantiomer does not prohibit light production.18 The synthesis of
BILH2 has been previously reported.19, 23 The Prescher synthesis19
was repeated, but in our hands the demethylation of the phenol
ether was capricious and gave us poor yields of intermediate 17.
The route was modified by introducing a TBDMS protecting
group on the phenol 14 with TBDMS-Cl to give 15 in 95% yield.
The rest of the synthesis followed the Prescher route.19 The nitro
group in 15 was reduced by catalytic hydrogenation to give the
crude unstable diamine product, which was immediately reacted
with Appel’s salt in dry pyridine to give the desired
benzimidazole 16 in a 50% yield over 2 steps. The TBDMS
protecting group was readily cleaved with TBAF at room
temperature to obtain the key benzimidazole nitrile 17 in
excellent yield (90%). This was then condensed with DL-
cysteine hydrochloride to give racemic BILH2 (10) in 52% yield
(Scheme 2) to compare against the racemic infraluciferin
analogues.24
The conformationally restricted, PBIiLH2 12 was synthesised
in two steps. As 2-aminopyridine is known to condense with 1,4-
benzoquinone
(18)
in
acidic
conditions,25
the
pyridobenzimidazole core was synthesised in an analogous
fashion by a one-pot condensation/oxidation reaction between
1,4-benzoquinone (18) and 2-amino-4-cyanopyridine (19), to
give the nitrile 20 in 72% yield. DL-cysteine was then condensed
with nitrile 20 under refluxing conditions,26 after no product was
obtained at room temperature, to give PBIiLH2 12 in 38% yield
(Scheme 3).
Scheme 3.
For the synthesis of BIiLH2 (13) we followed a very similar
route to our published infraluciferin (9) synthesis.18 This required
the key aldehydes 24a,b, which we envisaged could be made
from the oxidation of the corresponding methyl precursor 22. The
synthesis of benzimidazole 22 was achieved by following a
literature route to an analogous compound27 with the addition
here of a protected hydroxyl group that could be unmasked in the
final stages of the synthesis. Phenol 14 was protected with the
triisopropylsilyl protecting group under standard conditions
(Scheme 4).28 Several other protecting groups were also trialled
(Boc, MEM, Tosyl) but these were incompatible with the various

4
Tetrahedron
stages of the final synthetic route. The triisopropylsilyl-
Scheme 4. SEMCl = 2-(trimethylsilyl)ethoxymethyl chloride;
ACCEPTED MANUSCRIPT
protected benzimidazole 22 was synthesised by a one-pot
reduction and cyclisation27 of 21 with H2, Pd/C catalyst in the
presence of triehtylorthoformate to give a single benzimidazole
product by NMR in 96% yield. The exact tautomeric form of the
benzimidazole was not determined and we have drawn 22 as a
representative structure. Oxidation of the aromatic methyl group
to an aldehyde was attempted on 22 using selenium dioxide and
TBHP,29 but this did not work and gave unidentified products.
Therefore 22 was protected using the SEM group as there is
literature precedent of it being cleaved from sensitive biological
substrates.30 We observed that the SEM group protected each
tautomer of the benzimidazole (23a:23b, 1:1) and these were
separated in 41% and 40% isolated yields respectively. Both
isomers were separated by column chromatography and each was
carried forward in the synthetic route. Oxidation of 23a,b was
successful and key aldehydes 24a (70%) and 24b (96%) were
obtained in good yields. Each aldehyde was reacted with key
TBHP = tert-butyl hydroperoxide; diethylaminosulfur
trifluoride; PLE = porcine liver esterase
2.3. Bioluminescence activity and spectra of analogues with
wild-type and thermostable luciferases.
Wild-type (WT) Photinus pyralis firefly luciferase (Fluc),
thermostable variants and also click beetle red were purified by
nickel NTA chromatography and their basic properties were
examined with compounds D-LH2 (1), DL-BILH2 (10), DL-iLH2
(9), DL-BIiLH2 (13) and DL-PBIiLH2 (12). Specific activities
were first collected by integrating light over the first 2 min of
emission using the PhotonIMAGER Optima (PIO) small animal
imager. Natural
D-LH2 (1) gave the highest activity with all
enzymes, followed by DL-BILH2 (10), which gave about 100-
times lower activity with the majority of enzymes, except CBR,
with which activity was 1000-times lower than with
D-LH2 (1)
(Fig. 4). With x2 and x11 Flucs DL-iLH2 (9) gave a similar level
of activity as with the non-extended DL-BILH2 (10),
demonstrating the efficacy of engineered Flucderivatives when
used with iLH2 (9), since they are more active and have less
decay of signal (SI Fig). Enzymes gave the lowest activity with
DL-BIiLH2 (13), which was ~10 000-times less bright than WT
phosphonate 25 in
a Horner-Wadsowrth-Emmons (HWE)
reaction from our previously published route for infraluciferin
(9).17 Our reported Masamune and Rouche conditions (DBU and
LiCl) 18,31 gave no product, in this case. A variety of bases were
screened (NaH and K2CO3) and the use of KOtBu in the HWE
reaction yielded the desired products 26a (70%) and 26b (35%)
in modest yields. The triisopropylsilyl group was easily cleaved
by TBAF at this stage, in good yields (27a 65%, 27b 93%), as
attempts at removing it at after the thiazoline had been
constructed were unsuccessful. The thiazoline, especially in the
π-extended analogues we have prepared is more prone to
Fluc and the majority of the other enzymes with
D-LH2 (1), but
~250 000 times less active with CBR. However, the rigidified
version DL-PBIiLH2 (12) showed an enhancement in
luminescence activity with WT Fluc compared to DL-iLH2 (9)
and also compared to DL-BIiLH2 (13) with each enzyme: 3-fold
with WT and x11 Flucs, 5-fold with x2 Fluc, 9-fold with x5 Fluc
and 20-fold with CBR. The activity of CBR with DL-PBIiLH2
oxidation than luciferin. As
a precaution all synthetic
manipulations, workups and purifications when the thiazoline
was formed were performed in degassed solvents. Cyclisation to
the thiazoline (28a 65%, 28b 67%) was achieved by treating
thioamides 27a,b with DAST at -78 °C. Finally, the SEM group
was cleaved with Tin (IV) chloride30 to give the methyl ester 29
in 95% yield. The ester was found to be unstable in base (LiOH)
and so was hydrolysed with porcine liver esterase17 to give
BIiLH2 (13). The ester and final compound BIiLH2 (13) were
again isolated as single benzimidazole tautomers by NMR, but
the exact structures were not determined.
(12) was of a similar level as with both DL-iLH2 (9) and DL
-
BILH2 (10). Therfore we can rationally improve the activity of
different luciferases with an electronically modified extended
infraluciferin backbone by making it more rigid and reducing its
degrees of freedom.
The emission colours of enzymes with each of the analogues
were assessed by acquiring spectra through successive 30nm
pass-band filters in the PhotonIMAGER Optima (PIO). With D-
LH2 (1) enzymes displayed their expected different colours with
WT, x5, x11, x2 and CBR giving λmax values ranging from
555nm (WT, x5, x11), 561nm (x2) and 614nm (CBR) (Table 1).
As previously described the emission colours (Fig. 5) of enzymes
with DL-iLH2 (9) also showed a colour-shifting effect, producing
λmax values red-shifted compared to with
D-LH2 (1) by 104nm
for CBR and 124-142nm for the Flucs. Interestingly, a colour-
shifting effect was also seen with DL-BILH2, (10) with both x2
Fluc displaying a 23nm red-shift compared to WT, x5 and x11
and CBR displaying a 53nm shift, which is similar to the colours
emitted with
D-LH2 (1), albeit CBR displayed a smaller blue-
shifted peak at ~560nm.

5
altered compound DL-BIiLH (13) since the emission peak
ACCEPTED MANUSCRIPT
2
wavelengths of enzymes were markedly blue-shifted compared to
those with DL-iLH2 (9) and were more akin to those emitted with
D-LH2 (1) or DL-BILH2 (10). This may be explained by twisting
of the light emitting oxy form of BIiLH2 in the active site of the
luciferase. However, a red-shift observed with CBR indicated
that a specific colour-shifting mechanism was also active for this
compound. Emission colours with DL-PBIiLH2 (12) were more
red-shifted and remarkably, we detected a >100nm red-shifted
secondary peak of emission x11 Fluc with DL-PBIiLH2 (12).
Although this peak was relatively unstable to time and
temperature, it indicated the possibility that due to the increased
rigidity of the light emitting oxy form it has access to relatively
redshifted colour forms compared to the light giving oxidized
Fig. 4. - Specific activities of wild-type (WT) Photinus pyralis form (ketone cf Scheme 1) of iLH2 (9). If PBIiLO (30) is
firefly luciferase and thermostable x2, x5 and x11 derivatives
and click beetle red luciferase (CBR). Enzyme activities were
measured immediately upon adding substrate for 2 min in the
PhotonIMAGER Optima (PIO).
conformationally stabilized to be relatively planar around the
only rotatable σ-bond in the active site of x11 Fluc, this red
shifted peak might be explained by the stabilization of resonance
forms accessible because of greater conjugation (Fig. 6). Bimodal
bioluminescence emission is not unusual and has been explained
by stablisation of each of the resonance forms of the anionic
ketone of the oxidized species.4,32 We speculate that x11 Fluc is
able to stabilize the enolate form leading to red emission.
Bioluminescene studies of x11 Fluc with DL-PBIiLH2 (12) as a
function of pH (Fig. 7) showed an increased bimodal emission
Table 1. Primary and secondary bioluminescence peak wavelengths
of enzymes with different compounds (λ/nm). Values acquired from
smoothing (spline fitting) of spectra acquired in bandpasses in the
PIO.
DL-iLH2 (9)
D
-LH2
DL-BILH2
DL-iLH2
DL-BIiLH2
DL-PBIiLH2
(1)
(13)
(9)
(10)
(12)
WT 555
567
590
561
697
691
679
561
572
555
614
596
596
x2
x5
561
555
x1
1
555
614
561
685
720
561
620
608, 714
620
CB
R
620, 555
D-LH2 (1)
DL-BIiLH2 (13)
1.0
The situation was different for the extended electronically
WT
-BIiLH
DL
2
2
x2 -BIiLH
DL
2
2
x5 -BIiLH
DL
0.5
0.0
I
x11 -BIiLH
DL
CBR
-BIiLH
DL
2
500
600
700
800
Wavelength (+/-15nm)
DL-BILH2 (10)
DL-PBIiLH2 (12)
1.0
1.0
WT
-BILH
DL
2
WT -PBIiLH
DL
2
x2
x5
-BILH
DL
2
x2 -PBIiLH
DL
2
2
-BILH
DL
0.5
0.0
I
2
x5 -PBIiLH
DL
0.5
0.0
I
x11 -BILH
DL
2
x11 -PBIiLH
DL
2
CBR
-BILH
DL
2
CBR -PBIiLH
DL
2
500
600
700
800
500
600
700
800
Figure 5. Bioluminescence spectra of WT, x2, x5 and x11 Flucs and CBR. Spectra acquired by integrating light for 1 min in different filters
Wavelength (+/-15nm)
and fitted with a spline smoothing function in PRISM.
Wavelength (+/-15nm)

6
Tetrahedron
ACCEPTED MANUSCRIPT
with increasing pH. Recent reports suggest an acidic H-bonding
°C). Commercially available solvents and reagents were used
network may stabilise the yellow-green emitting phenolate form
of luciferins.32 In the x11 Fluc/DL-PBIiLH2 (12) system
increasing the pH may remove this in favour of stabilization of
the red emitting enolate form.
without further distillation. The petroleum ether used in
purification procedures is the fraction that boils in the range of 40
– 60 °C. A solvent purification system (SPS) was used to obtain
anhydrous solvents where needed. Thin layer chromatography
(TLC) was carried out on Merck aluminium backed DC 60 F254
0.2 mm pre-coated plates. Visualisation of the plates was done
under ultraviolet light and then stained with potassium
permanganate solution where required. Flash column
chromatography was performed on Gedran® silica gel 60, 40-63
ꢀm.
N
N
O
O
N
S
N
S
N
N
O
O
30
PBIiLO anion (
)
Figure 6. Potential resonance forms of light emitting PBIiLO (30) in
All H and 13C NMR spectra reported were recorded on a
1
Bruker Avance III 600 MHz spectrometer, unless stated
otherwise. All 31P NMR spectra were recorded on a Bruker
Avance III 400 MHz spectrometer. The chemical shift (δ) for the
resonances in spectra have been reported in parts per million
(ppm) relative to the NMR solvent peaks, whilst coupling
constants (J) are quoted in Hertz (Hz). 13C NMR spectra were
recorded as proton decoupled spectra at 151 MHz, and DEPT,
COSY, NOESY and HSQC spectra were obtained to aid in
complete assignment. A Perkin Elmer Spectrum 100 FT/IR with
Spectrum 100 ꢀATR machine was used to record IR spectra.
Mass spectra were obtained on a ThermoMAT900 and an Accela
LC- Finnigan LTQ instruments. Melting points are uncorrected
and were recorded on Electrothermal IA9300 machine. iLH2 and
phosphonate 25 were synthesised as previously reported.18
Fig. 7. Bioluminescence emission of x11 Fluc with DL-PBIiLH2
(12) as a function of pH. Spectra acquired by integrating light for 1
min in different filters.
its deprotonated form.
4.2. Synthesis of luciferin analogues BILH2 (10), PBIiLH2 (12)
and BIiLH2 (13)
4.2.1. 4-(tert-butyldimethylsilyloxy)-2-nitroaniline
(15)
To a solution of 4-amino-3-nitrophenol (14) (3.00 g, 19.6
mmol) and imidazole (2.66 g, 39.2 mmol) in DMF (20 mL) was
added TBDMS-Cl (4.00 g, 26.5 mmol) at room temperature and
stirred for 24 h. The reaction was extracted with Et2O (3 x 20
mL), the organic layers combined, dried (MgSO4), and filtered.
After the volatile materials were removed in vacuo, the crude
product was used without further purification. The product 15
was obtained as a red solid (5.00 g, 95%); mp 100 ºC; (lit.34 98 –
3. Conclusion
The synthesis of a rigid analogue PBIiLH2 (12) of iLH2 (9)
has been achieved in two steps in an attempt to improve the
brightness of π-extended analogues of LH2 (1). Its
bioluminescence activity was compared with its non-rigid
counterpart BIiLH2 (13) which in turn was compared to its non π-
extended analogue BILH2 (10). The results show that both π-
extended analogues PBIiLH2 (12) and BIiLH2 (13) gave red
shifted bioluminescence emissions with respect to LH2 (1) with
wild type and thermostable luciferases ranging from 6-159 nm.
Most strikingly the rigid analogue PBIiLH2 (12) gave a
secondary emission of 714 nm with Fluc x11, comparable to the
leading red shifted π-extended analogues 6a,b and iLH2 (9)
reported.11,16,17 Although the rigid π-extended analogue PBIiLH2
(12) was not brighter than iLH2 (9) or its parent benzimidazole
luciferin BILH2 (10), it did show enhanced brightness compared
to its non-rigid analogue BIiLH2 (13) with each enzyme tested.
The greatest increase in activity was 20-fold with CBR which
also gave similar brightness for all three luciferins iLH2 (9),
BILH2 (10) and PBIiLH2 (12). The concept of reducing the
degrees in freedom in π-extended analogues of LH2 (1) by
synthetic rigidification has been demonstrated and we are
investigating the refinement of this strategy on other analogues to
investigate the brightness of red shifted luciferins.
1
100 ºC); Rf = 0.80 (80% EtOAc/hexane); H NMR (400 MHz,
CDCl3) δ 7.56 (1H, d, J = 8.0, ArH), 6.97 (1H, d, J = 4.0, ArH),
6.71 (1H, dd, J = 8.0, 4.0, ArH), 0.98 (9H, s, SiC(CH3)3), 0.19
(6H, s, OSi(CH3)2). The data was in agreement with the
literature.33
6-(tert-Butyldimethylsilyloxy)-1H-benzo[d]imidazole-2-
carbonitrile (16)
To a solution of 4-(tert-butyldimethylsilyloxy)-2-nitroaniline
(15) (1.75 g, 6.52 mmol) in MeOH (5 mL) was added 10%
palladium on carbon (0.550 g) as a slurry in MeOH (5 mL) at
room temperature. The solution was degassed with Ar, purged
with H2 3 times, and stirred for 7 h under a H2 atmosphere
(balloon). The mixture was filtered through celite and the filtrate
concentrated in vacuo to obtain the diamine product in crude
form as a purple oil. Without further purification, this was
dissolved in anhydrous pyridine (20 mL) and Appel’s salt (1.22
g, 5.85 mmol) added at 0 ˚C. The mixture was stirred at room
temperature for 24 h. The mixture was concentrated under
reduced pressure and the crude product was purified by flash
column chromatography (20% EtOAc/hexane) to give 16 (1.66 g,
60%) as a pale yellow solid; mp 178 ºC; Rf = 0.60 (20%
EtOAc/hexane); IR υmax (solution in CDCl3): 2953, 2930, 2892,
4. Experimental section
4.1. General
Experiments were carried out in flame dried glassware, in
anhydrous solvents and under a positive flow of nitrogen or
argon unless aqueous chemistry was involved. Temperatures of 0
°C were achieved using an ice-water bath. Cryogenic
temperatures were obtained using a solid CO2 – acetone bath (-78
1
2239, 1626 cm-1; H NMR (400 MHz, CDCl3) δ 7.59 (1H, d, J =
8.0, ArH), 7.05 (1H, s, ArH), 7.02 (1H, d, J = 8.0, ArH), 1.03
(9H, s, SiC(CH3)3), 0.25 (6H, s, OSi(CH3)2). 13C NMR (150

7
MHz, CDCl3) δ 155.8 (C), 125.2 (CH), 121.3 (CH),112.7 (CH),
25.1 (CH3), 19.0 (C), 4.4 (CH3); HRMS (ESI+) Calcd. for
C14H20N3OSi [M + H] + 274.1376, found 274.1375.
then dissolved in ice cold H2O (4.2 mL) and acidified with 2M
ACCEPTED MANUSCRIPT
HCl to pH 2. A precipitate was formed that was filtered and dried
to give 12 as a tan solid (100 mg, 0.32 mmol, 38%); mp 279 °C;
IR υmax (solid state) 3253 (br), 3063, 2927, 1719; H NMR (600
1
2-Cyano-6-hydroxybenzimidazole (17)
MHz, DMSO-d6) δ 13.11 (1 H, s, CO2H), 9.80 (1 H, br s, OH),
8.97 (1 H, d, J =7.1, ArCH), 7.84 (1 H, s, ArCH), 7.71 (1 H, d, J
=8.7, ArCH), 7.59 (1 H, s, ArCH), 7.32 (1 H, d, J =7.1, ArCH),
7.10 (1 H, dd, J =8.8, 2.0, ArCH), 5.39 (1 H, t, J =8.9, CHCO2H),
3.82 – 3.77 (1 H, m, CH2S), 3.72 – 3.68 (1 H, m, CH2S); 13C
NMR (151 MHz, DMSO-d6) 171.6 (C), 167.1 (C), 153.7 (C),
145.0 (C), 138.2 (C), 131.8 (C), 129.4 (C), 126.8 (CH), 120.0
(CH), 118.0 (CH), 117.1 (CH), 107.8 (CH), 96.5 (CH), 78.5
(CH), 35.4 (CH2S); m/z (ES+) 314 (100%, [M+H]+); HRMS for
C15H12N3O3S [M+H]+ calcd. 314.0599, found 314.0587.
To a solution of silyl ether 16 (0.200 g, 0.732 mmol) in THF
(10 mL) was added 1M tetrabutylammonium fluoride (0.16 mL,
˚
1.4 mmol) at 0 C. The reaction was stirred for 2 h at room
temperature. The reaction mixture was then concentrated under
reduced pressure and the crude product purified by flash column
chromatography (80% EtOAc/hexane) to give 17 (0.130 g, 90%)
as a yellow solid; mp 178 oC; Rf = 0.25 (80% EtOAc/hexane); 1H
NMR (400 MHz, Methanol-d4) δ 7.51 (1H, d, J = 8.0, ArH), 6.96
(1H, dd, J = 8.0, 4.0, ArH), 6.92 (1H, d, J = 4.0 Hz, ArH). The
data is in agreement with the literature.15
4.2.3. 2-nitro-4-((triisopropylsilyl)oxy)aniline (21)
2-(6-hydroxy-1H-benzo[d]imidazol-2-yl)-4,5-dihydrothiazole-
4-carboxylic acid (10)
To a solution of 4-amino-3-nitrophenol (1.00 g, 6.48 mmol) in
CH2Cl2 (30 mL) was added imidazole (0.70 g, 10.3 mmol). The
mixture was cooled to 0 °C, triisopropylsilyl chloride (1.78 mL,
8.32 mmol) added, the mixture allowed to warm to room
temperature and stirred for 16 h. The resultant mixture was
diluted with CH2Cl2 (30 mL), washed sequentially with a
saturated solution of NaHCO3 (20 mL), water (20 mL) and brine
(20 mL), and then the organic layer dried (MgSO4), filtered and
concentrated under reduced pressure to give the crude compound
as a bright red gelatinous solid which was purified by passing
through a silica plug (50% EtOAc/hexane) to give 21 as a bright
red crystalline solid (2.00 g, 6.48 mmol, 99%); mp 92-94 °C; Rf
0.80 (30% EtOAc/hexane); IR υmax (solid state) 3500, 3346,
2939, 2861, 1561 cm-1; 1H NMR (600 MHz, CDCl3) δ 7.60 (1 H,
d, J = 2.9, ArCH), 7.03 (1 H, dd, J = 8.9, 2.9, ArCH), 6.71 (1 H,
d, J = 8.9, ArCH), 5.81 (2 H, br s, NH2), 1.27 – 1.23 (3 H, m,
CH(CH3)), 1.10 (18 H, d, CH(CH3) J = 7.4); 13C NMR (151
MHz, CDCl3) δ 146.8 (C), 139.7 (C), 131.9 (C), 130.1 (CH),
119.7 (CH), 114.5 (CH), 18.0 (CH3), 12.6 (CH); m/z (ES+) 311
(100%, [M+H]+); HRMS C15H27N2O3Si [M+H]+ calcd. 311.1791,
found 311.1792.
To a solution of nitrile 17 (60.0 mg, 0.377 mmol) in MeOH (2
mL) and H2O (1 mL) was added DL-cysteine hydrochloride
monohydrate (70.1 mg, 0.399 mmol), K2CO3 (54.0 mg, 0.391
mmol) at room temperature and the suspension was stirred for 30
min. The mixture was acidified with 3 M HCl to pH = 3. The
MeOH was removed in vacuo, and the remaining solution was
extracted with EtOAc (3 x 100 ml). The organic layers were
combined, dried (MgSO4), filtered and concentrated under
reduced pressure to obtain the crude product that was purified by
flash column chromatography (100% EtOAc
-
1%
AcOH/EtOAc). The product 10 was obtained as a yellow solid
1
(98 mg, 93%) (lit. [15] 82%). m.p. 203 ºC; H NMR (400 MHz,
Methanol-d4) δ 7.48 (1H, d, J = 8.9, ArH), 6.94 (1H, d, J = 2.3,
ArH), 6.85 (1H, dd, J = 8.9, 2.3, ArH), 5.27 (1H, app t, J = 9.0,
C=NCH(COOH)CH2), 3.71 (2H, d, J = 9.0, CH2S). The data is in
agreement with the literature.15
4.2.2. 8-hydroxybenzo[4,5]imidazo[1,2-a]pyridine-
3-carbonitrile (20)
To a solution of 1,4-benzoquinone 18 (540 mg, 5.00 mmol) in
2-methyl-6-((triisopropylsilyl)oxy)-1H-benzo[d]imidazole (22)
AcOH (1.25 mL) was added
a solution of 2-amino-4-
To a stirred solution of 21 (2.00 g, 6.45 mmol) in MeOH (25
mL) were added AcOH (7 drops), triethyl orthoacetate (2.36 mL,
12.9 mmol), and 10% palladium on carbon (0.200 g) as a slurry
in MeOH (8 mL). The flask was evacuated and then purged with
H2 gas three times before leaving to stir under an atmosphere of
H2 overnight at room temperature. On completion the pale brown
solution was filtered through Celite® and the Celite® washed with
EtOAc (70 mL). The filtrate was concentrated under reduced
pressure to yield a crude pale-brown oil which was purified by
flash column chromatography (90% EtOAc/hexane) to give 22 as
an orange oil (1.88 g, 6.16 mmol, 96%); Rf 0.26 (90%
EtOAc/hexane); IR νmax (neat) 3112 , 2940, 2863, 1630, 1588 cm-
1; 1H NMR (600 MHz, CDCl3) δ 9.90 (1 H, br s, NH), 7.36 (1 H,
d, J = 8.6, ArCH), 7.03 (1 H, d, J =2.3, ArCH), 6.81 (1 H, dd, J
=8.6, 2.3, ArCH), 2.58 (3 H, s, N=CMe), 1.29 – 1.21 (3 H, m,
CHMe2), 1.09 (18 H, d, J =7.4, CHMe2); 13C NMR (151 MHz,
CDCl3) δ 152.1 (C), 151.2 (C), 138.9 (C), 134.0 (ArC), 115.9
(CH), 114.9 (CH), 104.4 (CH), 18.1 (CH3), 15.0 (CH3), 12.8
(CH); m/z (ES+) 305 (100%, [M+H]+); HRMS C17H29N2OSi
[M+H]+ calcd. 305.2049, found 305.2043.
cyanopyridine (290 mg, 2.50 mmol) in AcOH (0.75 mL). The
mixture was diluted with H2O (0.75 mL) and boiled for 5 mins
until the mixture turned red. The mixture was cooled quickly to
room temperature in an ice bath and then acidified with 6M HCl
(1.25 mL) and diluted with H2O (15 mL). The aqueous layer was
extracted with Et2O (3 x 15 mL), cooled to 0 °C and basified to
pH 8 with solid Na2CO3. The crude, brown precipitate formed
was filtered, dried under vacuum, and purified by flash column
chromatography (10% MeOH/EtOAc) to give 20 as a yellow
solid (379 mg, 1.81 mmol, 72%); mp 171-173 ºC; Rf 0.62 (10%
MeOH/EtOAc); IR υmax (solid) 3500 (br), 3086, 2229, 1641 cm-1;
1H NMR (600 MHz, Methanol-d4) δ 8.77 (1 H, dd, J =7.1,
ArCH), 8.05 (1 H, s, ArCH), 7.68 (1 H, d, J =8.9, ArCH), 7.43 (1
H, d, J =2.3, ArCH), 7.14 (1 H, dd, J =8.9, 2.3, ArCH), 7.02 (1
H, dd, J =7.1, ArCH); 13C NMR (151 MHz, Methanol-d4) δ 156.5
(C), 146.5 (C), 139.8 (C), 131.1 (C), 128.7 (CH), 124.9 (CH),
121.5 (C), 119.6 (CH), 118.6 (CH), 112.9 (C), 111.3 (CH), 97.2
(CH); m/z (ES-) 208 (60%, [M-H]-); HRMS for C12H6N3O [M-
H]- calcd. 208.0511, found 208.0501.
2-(8-hydroxybenzo[4,5]imidazo[1,2-a]pyridin-3-yl)-4,5-
2-methyl-6-((triisopropylsilyl)oxy)-1-((2-
dihydrothiazole-4-carboxylic acid (12)
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazole (23a) and
2-methyl-5-((triisopropylsilyl)oxy)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H- benzo[d]imidazole (23b)
To a suspension of nitrile 20 (177 mg, 0.85 mmol) in EtOH (5
mL) were added D, L-cysteine (205 mg, 1.69 mmol) and
NaHCO3 (286 mg, 3.40 mmol) and the mixture heated at reflux
for 48 h. The solvent was removed in vacuo and the remaining
residue washed with ice-cold Et2O (3 x 10 mL). The residue was
Solid KH (30% dispersion in mineral oil, 0.738 g, 5.52 mmol)
was cooled to 0 °C. Compound 22 (1.35 g, 4.45 mmol) was

8
Tetrahedron
dissolved in THF (20 mL) and added drop-wise to the cooled
(CH), 73.3 (CH2), 66.3 (CH2), 17.9 (CH3), 17.7 (CH2), 12.6 (CH),
ACCEPTED MANUSCRIP
T
KH over a period of 25 mins. Care was taken to ensure that the
internal temperature of the reaction mixture did not exceed 5 °C.
The mixture was stirred for 1 h at 0-5 °C before 2-
(trimethylsilyl)ethoxymethyl chloride (0.982 mL, 5.52 mmol)
was added drop-wise at 0 °C to the thick, sandstone coloured
suspension. After stirring at room temperature for 19 h, the
reaction mixture was cooled to 0 °C and water (15 mL)
cautiously added drop-wise to quench the reaction. The aqueous
layer was extracted with EtOAc (25 mL x 3). The organic layers
were combined, dried (MgSO4), filtered and concentrated under
reduced pressure to yield the crude product as a brown oil.
Purification by flash column chromatography (30%
EtOAc/petroleum ether) gave in order of elution compound 23a
as an off-white solid (0.766 g, 1.76 mmol, 40%), followed by
compound 23b as an off-white solid (0.795 g, 1.83 mmol, 41%).
-1.6 (CH3); m/z (ES+) 449 (65%, [M+H]+) HRMS C23H41N2O3Si2
[M+H]+ calcd. 449.2656, found 449.2643.
5-((triisopropylsilyl)oxy)-1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-benzo[d]imidazole-2-carbaldehyde (24b)
Procedure as that for aldehyde 24a on a 0.97 mmol scale to
give 24b as a pale yellow oil (0.420 g, 0.936 mmol, 96%); Rf
0.38 (10% EtOAc/petroleum ether); IR νmax (solution in CHCl3)
2947, 2867, 1693, 1618 cm-1; 1H NMR (600 MHz, CDCl3) δ
10.07 (1 H, s, CHO), 7.48 (1 H, dd, J =8.9, 0.4, ArCH), 7.36 (1
H, dd, J =2.3, 0.4, ArCH), 7.13 (1 H, dd, J =8.9, 2.3, ArCH), 6.00
(2 H, s, NCH2O), 3.55 (2 H, t, J =8.2, OCH2CH2Si), 1.33 – 1.29
(3 H, m, CHMe2), 1.12 (18 H, d, J =7.5, CHMe2), 0.88 (3 H, t, J
=8.2, CH2Si), -0.08 (9 H, s, SiMe3); 13C NMR (151 MHz, CDCl3)
δ 184.8 (C), 153.9 (C), 146.4 (C), 143.8 (C), 131.6 (C), 122.8
(CH), 112.3 (CH), 110.3 (CH), 73.4 (CH2), 66.6 (CH2), 18.0
(CH3), 17.9 (CH2Si), 12.7 (CH), -1.4 (CH3); m/z (ES+) 481
(100%, [M+CH3OH+H]+), 449 (4%, [M+H]+); HRMS
Data for Compound 23a: mp 58 – 59 °C; Rf 0.28 (40% EtOAc
/petroleum ether); IR νmax (neat solid) 2947 – 2867 (C-H), 1622
(C=N); 1H NMR (600 MHz, CDCl3) δ 7.20 (1 H, d, J =8.6,
ArCH), 7.18 (1 H, d, J =2.2, ArCH), 6.85 (1 H, dd, J =8.6, 2.2,
ArCH), 5.41 (2 H, s, NCH2O), 3.50 (2 H, t, J =8.4, OCH2CH2Si),
2.62 (3 H, s, N=CMe), 1.30 – 1.26 (3 H, m, CHMe2), 1.10 (18 H,
d, J =7.5, CHMe2), 0.88 (2 H, t, J =8.4, CH2Si), -0.07 (9 H, s,
SiMe3); 13C NMR (151 MHz, CDCl3) δ 152.7 (C), 152.4 (C),
143.3 (C), 130.6 (C), 116.6 (CH), 109.5 (CH), 109.3 (CH), 73.0
(CH2), 66.7 (CH2), 18.3 (CH3), 14.2 (CH2), 13.0 (CH3), -1.1
(CH3); m/z (ES+) 435 (100%, [M+H]+), 421 (28%, [M+ -CH3]);
HRMS C23H43N2O2Si2 [M+H]+ calcd. 435.2863, found 435.2858.
C23H41N2O3Si2 [M+H]+ calcd. 449.2656, found 449.2652.
Methyl (E)-(3-(6-((triisopropylsilyl)oxy)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)prop-2-
enethioyl)serinate (26a)
A solution of phosphonate 25 (0.313 g, 1.00 mmol) in THF (5
mL) was cooled to 0 °C and solid KOt-Bu (0.150 g, 1.33 mmol)
added. The mixture was allowed to warm to room temperature
and stirred for 1 h. The mixture was cooled to 0 °C and a solution
of aldehyde 24a (0.301 g, 0.67 mmol) in THF (3 mL) added
drop-wise and the mixture stirred at room temperature for 3 h.
The mixture was poured into brine (5 mL) and the organic layer
diluted with EtOAc (10 mL). The aqueous layer was further
extracted with EtOAc (2 x 10 mL). The organic extracts were
combined and further washed with brine (10 mL) and then dried
(MgSO4), filtered and concentrated under reduced pressure to
give the crude compound as a deep yellow oil which was purified
by flash column chromatography (gradient elution: 20% EtOAc/
hexane – 50% EtOAc/hexane) to give 26a as a deep yellow oil
(0.212 g, 0.35 mmol, 35%); Rf 0.17 (50% EtOAc/petroleum
ether); IR νmax (solution in CHCl3) 3245, 2944, 2863, 1739, 1615
Data for Compound 23b: mp 59 – 60 °C; Rf 0.15 (40% EtOAc
/petroleum ether); IR νmax (neat solid) 2945 – 2867 (C-H), 1621
(C=N); 1H NMR (600 MHz, CDCl3) δ 7.49 (1 H, d, J =8.6,
ArCH), 6.87 (1 H, d, J =2.2, ArCH), 6.81 (1 H, dd, J =8.6, 2.2,
ArCH), 5.38 (2 H, s, CH2), 3.50 (2 H, t, J =8.2, OCH2CH2Si),
2.61 (3 H, s, N=CMe), 1.29 – 1.25 (3 H, m, CHMe2), 1.10 (18 H,
d, J =7.5, CHMe2), 0.88 (2 H, t, J =8.2, CH2Si), -0.06 (9 H, s,
SiMe3); 13C NMR (151 MHz, CDCl3) δ 152.5 (C), 151.3 (C),
137.1 (C), 136.1 (C), 119.3 (CH), 115.8 (CH), 100.2 (CH), 72.8
(CH2), 66.5 (CH2), 18.1 (CH3), 18.0 (CH2), 14.0 (CH), 12.8
(CH3), -1.4 (CH3); m/z (ES+) 435 (25%, [M+H]+); HRMS
C23H43N2O2Si2 [M+H]+ calcd. 435.2863, found 435.2837.
cm-1; H NMR (600 MHz, CDCl3) δ 9.09 (1 H, d, J =7.5, NH),
1
7.95 (1 H, d, J =14.7, HC=C), 7.61 (1 H, d, J =14.7, C=CH), 7.55
(1 H, d, J =8.6, ArCH), 6.93 – 6.89 (2 H, m, ArCH), 5.54 (2 H, s,
NCH2O), 5.48 – 5.44 (1 H, m, HN-CH-CO2Me), 4.34 (1 H, dd, J
=11.5, 2.7, CHaHbOH), 4.22 (1 H, dd, J =11.5, 2.9, CHaHbOH),
3.82 (1 H, br s, OH), 3.68 (3 H, s, OMe), 3.53 (2 H, t, J =8.1,
OCH2CH2Si), 1.30 – 1.26 (3 H, m, CHMe2), 1.12 (18 H, d, J
=7.4, CHMe2), 0.90 (2 H, t, J =8.1, CH2Si), -0.07 (9 H, s, SiMe3);
13C NMR (151 MHz, CDCl3) δ 193.9 (C), 170.3 (C), 154.2 (C),
148.4 (C), 137.2 (C), 136.4 (CH), 133.3 (CH), 127.2 (CH), 120.0
(C), 118.4 (CH), 100.5 (CH), 72.5 (CH2), 66.8 (CH2), 61.8 (CH2),
60.5 (CH), 52.8 (CH3), 18.1 (CH3), 17.9 (CH2), 12.8 (CH), -1.3
(CH3); m/z (ES+) 608 (100%, [M+H]+); HRMS C29H50N3O5SSi2
[M+H]+ calcd. 608.3010, found 608.3010.
6-((triisopropylsilyl)oxy)-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-benzo[d]imidazole-2-carbaldehyde (24a)
To a suspension of selenium dioxide (0.394 g, 3.55 mmol) in
1,4-dioxane (10 mL) was added 5.0 M tert-butyl hydroperoxide
solution in decane (0.850 mL, 8.16 mmol) at room temperature
and the suspension stirred at 90 °C for 30 mins. A solution of
compound 23a (0.796 g, 1.83 mmol) in 1,4-dioxane (4 mL) was
added and the mixture was stirred at 110 °C for 5 h. The mixture
was allowed to cool to room temperature and concentrated in
vacuo to get a brown oil, to which sat. NaHCO3 solution (15 mL)
was added. The aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The organic layers were combined, dried (MgSO4), filtered
and concentrated in vacuo to afford the crude compound as a
brown oil which was purified by flash column chromatography
(10% Et2O/ petroleum ether) to give 24a as a pale yellow oil
(0.451 g, 1.23 mmol, 67%); Rf 0.64 (10% EtOAc/petroleum
ether); IR νmax (solution in CHCl3) 2946, 2866, 1693, 1617 cm-1;
1H NMR (600 MHz, CDCl3) δ 10.01 (1 H, s, CHO), 7.76 (1 H, d,
J = 8.9, ArCH), 7.04 (1 H, d, J =2.3, ArCH), 7.01 (1 H, dd, J
=8.9, 2.3, ArCH), 5.97 (2 H, s, NCH2O), 3.54 (2 H, t, J = 8.2,
OCH2CH2Si), 1.34 – 1.28 (3 H, m, CHMe2), 1.12 (18 H, d, J
=7.5, CHMe2), 0.88 (2 H, t, J = 8.2, CH2Si), -0.07 (9 H, s,
SiMe3); 13C NMR (151 MHz, CDCl3) δ 184.1 (C), 156.3 (C),
146.0 (C), 138.0 (C), 137.7 (C), 122.8 (CH), 119.6 (CH), 100.9
Methyl(E)-(3-(5-((triisopropylsilyl)oxy)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)prop-2-
enethioyl)serinate (26b)
Procedure as that for thioamide 26a on a 0.96 mmol of
phosphonate 25 gave 26b as a deep yellow oil (0.206 g, 0.34
mmol, 53%); Rf 0.25 (40% EtOAc/petroleum ether); IR νmax
1
(solution in CHCl3) 3183 (br), 2941, 2626, 1706, 1600 cm-1; H
NMR (600 MHz, CDCl3) δ 8.82 (1 H, d, J =7.5, NH), 7.96 (1 H,
d, J =14.7, HC=C), 7.66 (1 H, d, J =14.7, C=CH), 7.32 (1 H, d, J
=8.8, ArCH), 7.18 (1 H, d, J =2.3, ArCH), 6.97 (1 H, dd, J =8.8,
2.3, ArCH), 5.60 (2 H, s, NCH2O), 5.45 (1 H, apt. dt, J =7.5, 3.1,

9
HN-CH-CO2Me), 4.27 (1 H, dd, J =11.5, 3.0, CHaHbOH), 4.22 (1
methyl (E)-2-(2-(6-hydroxy-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)vinyl)-
4,5-dihydrothiazole-4-carboxylate (28a)
A
CCEPTED MANUSCRIPT
H, dd, J =11.5, 3.2, CHaHbOH), 3.75 (3 H, s, OMe), 3.54 (2 H, t,
J =8.2, OCH2CH2Si), 1.33 – 1.28 (3 H, m, CHMe2), 1.13 (18 H,
d, J =7.5, CHMe2), 0.91 (3 H, t, J =8.2, CH2Si), -0.07 (9 H, s,
SiMe3); 13C NMR (151 MHz, CDCl3) δ 193.2 (C), 169.9 (C),
153.2 (C), 148.7 (C), 142.8 (C), 133.7 (CH), 130.1 (C), 126.5
(CH), 118.7 (CH), 110.2 (CH), 108.2 (CH), 72.1 (CH2), 66.5
(CH2), 61.5 (CH2), 60.3 (CH), 52.5 (CH3), 17.7 (CH3), 17.4
(CH2), 12.4 (CH), -1.8 (CH3); m/z (ES+) 608 (100%, [M+H]+);
HRMS C29H50N3O5SSi2 [M+H]+ calcd. 608.3010, found
608.3014.
A solution of compound 27a (0.021 g, 0.047 mmol) in CH2Cl2
(1.0 mL) was cooled to -78 °C and diethylaminosulfur trifluoride
(33 ꢀL, 0.24 mmol) added. The mixture was stirred at -78 °C for
90 mins. The mixture was quenched with a sat. solution of
NaHCO3 (2 mL) at -78 °C, allowed to warm-up slowly to room
temperature and extracted with CH2Cl2 (3 x 5 mL). The organic
extracts were combined, dried (MgSO4), filtered and
concentrated under reduced pressure to give the crude compound
as red oil which was purified by flash column chromatography
(70% EtOAc/ hexane) to give 28a as a deep yellow oil (0.013 g,
0.030 mmol, 65%); Rf 0.19 (70% EtOAc/ hexane); IR νmax
(solution in CHCl3) 3163 (br), 2949, 2923, 1731, 1619 cm-1; 1H
NMR (600 MHz, CDCl3) δ 7.64 (1 H, d, J =15.8, HC=C), 7.55 (1
H, d, J =8.7, ArCH), 7.24 (1 H, d, J =15.8, HC=C), 6.91 (1 H, d,
J =2.0, ArCH), 6.87 (1 H, dd, J =8.7, 2.0, ArCH), 5.46 (2 H, s,
NCH2O), 5.26 (1 H, t, J =9.1, CHCO2Me), 3.83 (3 H, s, OMe),
3.70 – 3.66 (1 H, m, CH2S), 3.63 – 3.59 (1 H, m, CH2S), 3.53 (2
H, t, J =8.1, OCH2), 0.90 (2 H, t, J =8.1, CH2Si), -0.06 (9 H, s,
SiMe3); 13C NMR (151 MHz, CDCl3) 171.1 (C), 169.3 (C), 153.7
(C), 148.9 (C), 130.3 (C), 129.7 (C), 129.2 (CH), 126.2 (CH),
114.9 (CH), 110.4 (CH), 104.7 (CH), 78.4 (CH), 72.6 (CH2), 66.9
(CH2), 53.1 (CH3), 35.1 (CH2), 17.8 (CH2), -1.3 (CH3); m/z (ES+)
434 (55%, [M+H]+); HRMS C20H28N3O4SSi [M+H]+ calcd.
434.1570, found 434.1573.
Methyl (E)-(3-(6-hydroxy-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-benzo[d]imidazol-2-yl)prop-2-enethioyl)serinate (27a)
A solution of 1.0 M solution of TBAF in THF was added to a
solution of compound 26a (0.055 g, 0.090 mmol) in THF (1.5
mL) at 0 °C. The mixture was left to stir at 0 °C for 10 mins and
then quenched with a saturated solution of NH4Cl (6 mL) at 0 °C,
allowed to warm to room temperature, extracted with EtOAc (3 x
15 mL). The organic extracts were combined and washed with a
saturated solution of NH4Cl (3 x 20 mL), dried (MgSO4), filtered
and concentrated under reduced pressure to give the crude
compound as red oil which was purified by flash-column
chromatography (gradient elution: 90% EtOAc/petroleum ether –
neat EtOAc) to give 27a as a yellow oil (0.029 g, 0.064 mmol,
65%) as a mixture of E and Z isomers that inter-convert in
solvent; Rf 0.31 (90% EtOAc/hexane); IR νmax (solution in
CHCl3) 3242, 2953, 2895, 1739, 1621 cm-1; 1H NMR (600 MHz,
CDCl3) δ δ 13.69 (0.6 H, br s, -NH for Z-isomer), 8.85 (1 H, br s,
-NH for E-isomer), 7.82 (1 H, d, J =14.7, HC=C for E-isomer),
7.52 – 7.35 (2.4 H, m), 6.90 – 6.73 (4 H, m), 6.51 (0.6 H, d, J
=13.5, HC=C for Z-isomer), 5.42 (2 H, s, NCH2O; 1.6 H, HN-
CH-CO2Me), 5.34 (1.2 H, s, NCH2O), 4.38 (1 H, dd, J =11.7, 3.3,
CH2OH), 4.22 (2.2 H, m, CH2OH), 3.83 (1.8 H, s, OMe for Z-
isomer), 3.75 (3 H, s, OMe for E-isomer), 3.50 (3.2 H, m,
OCH2CH2Si), 2.55 (1 H, br s, -OH), 0.88 (5.4 H, m, CH2Si,
ArOH, CH2OH, OH), -0.07 (14.4 H, overlapping singlets,
SiMe3); 13C NMR (151 MHz, CDCl3) δ 193.8 (C), 192.0 (C),
170.6 (C), 170.3 (C), 154.0 (C), 153.7 (C), 148.7 (C), 147.9 (C),
142.7 (C), 141.7 (C), 137.7 (CH), 133.9 (CH), 129.7 (CH), 129.1
(CH), 115.7 (CH), 113.7 (CH), 110.9 (CH), 110.4 (CH), 104.4
(CH), 104.0 (CH), 72.6 (CH2), 72.3 (CH2), 67.0 (CH2), 66.9
(CH2), 62.2 (CH2), 62.0 (CH2), 53.0 (CH3), 53.0 (CH3), 18.1
(CH2), 17.8 (CH2), -1.3 (CH3); m/z (ES+) 452 (55%, [M+H]+);
HRMS C20H30N3O5SSi [M+H]+ calcd. 452.1675, found 452.1686.
Methyl (E)-2-(2-(5-hydroxy-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-2-yl)vinyl)-
4,5-dihydrothiazole-4-carboxylate (28b)
Procedure as that for thiazoline 28a on a 0.030 mmol scale to
give 28b as a yellow oil (0.010 g, 0.22 mmol, 67%); Rf 0.23
(90% EtOAc/petroleum ether); IR νmax (solution in CHCl3) 3279
(br), 2953, 2854, 1741, 1621 cm-1; 1H NMR (600 MHz, CDCl3)
δ 7.68 (1 H, d, J = 15.8, HC=C), 7.30 – 7.26 (2 H, m, ArCH,
C=CH), 7.23 (1 H, d, J =2.2, ArCH), 6.94 (1 H, dd, J = 8.7, 2.2,
ArCH), 5.54 (2 H, s, NCH2O), 5.26 (1 H, t, J = 9.1, CHCO2Me),
3.83 (3 H, s, OMe), 3.68 (1 H, dd, J =11.1, 9.0, CH2S), 3.60 (1 H,
dd, J = 11.0, 9.1, CH2S), 3.54 (2 H, t, J = 8.1, OCH2), 0.92 – 0.88
(3 H, m, CH2Si, OH), -0.06 (9 H, s, SiMe3); 13C NMR (151 MHz,
CDCl3) δ 171.1 (C), 169.3 (C), 153.5 (C), 149.0 (C), 143.8 (C),
130.5 (C), 129.2 (C), 126.3 (CH), 114.8 (CH), 110.4 (CH), 104.9
(CH), 78.5 (CH), 72.5 (CH2), 66.9 (CH2), 53.0 (CH3), 35.1 (CH2),
17.8 (CH2), -1.3 (CH3); m/z (ES+) 434 (100%, [M+H]+); HRMS
C20H28N3O4SSi [M+H]+ calcd. 434.1570, found 434.1571.
Methyl (E)-(3-(5-hydroxy-1-((2-(trimethylsilyl)ethoxy)methyl)-
1H-benzo[d]imidazol-2-yl)prop-2-enethioyl)serinate (27b)
Methyl (E)-2-(2-(6-hydroxy-1H-benzo[d]imidazol-2-yl)vinyl)-
Procedure as that for 27a on a 0.069 mmol gave to afford the
title compound as a yellow oil (0.029 g, 0.064 mmol, 93%); Rf
0.17 (90% EtOAc/hexane); IR νmax (solution in CHCl3) 3252,
3037, 2949, 2850, 1737, 1620 cm-1; 1H NMR (600 MHz, CDCl3)
δ 9.32 (1 H, s, NH), 7.81 (1 H, d, J =14.6, HC=C), 7.50 (1 H, d, J
= 14.6, C=CH), 7.22 (1 H, d, J = 8.7, ArCH), 7.17 (1 H, s,
ArCH), 6.89 (1 H, d, J = 8.7, ArCH), 5.45 (2 H, s, NCH2O), 5.39
– 5.36 (1 H, m, HN-CH-CO2Me), 4.20 – 4.11 (2 H, m, CH2OH),
3.71 (3 H, s, OMe), 3.53 (2 H, t, J = 8.2, OCH2CH2Si), 2.76 (1H,
4,5-dihydrothiazole-4-carboxylate (29)
A solution of compound 28a or 28b (13 mg, 0.030 mmol) in
CH2Cl2 (2 mL) was cooled to 0 °C and 1.0 M tin(IV) chloride
solution in CH2Cl2 (0.15 mL, 0.15 mmol) was added. The
mixture was stirred at 0 °C for 3 h, after which an additional
volume of 1.0 M tin(IV) chloride solution in CH2Cl2 (0.15 mL,
0.15 mmol) added and left to stir at 0 °C for 1 h. The mixture was
quenched at 0 °C with a 0.1 M solution of HCl (5 mL). The
CH2Cl2 layer was separated and the aqueous layer was
neutralised to pH=7 with a saturated solution of NaHCO3 and
extracted with EtOAc (3 x 10 mL). The organic extracts were
combined, dried (MgSO4), filtered and concentrated under
reduced pressure to give the crude compound as a waxy solid
which was purified by trituration with CHCl3 (3 x 0.5 mL) and
the residue dried to give the ester 29 as a brown solid (8.6 mg,
0.028 mmol, 95%); mp 175-176 °C; Rf 0.07 (2% MeOH/
EtOAc); IR νmax (solid state) 3297 (br), 2923, 2853, 1719, 1639
br s, OH), 0.92 (2 H, t, J = 8.2, CH2Si), -0.06 (9 H, s, SiMe3); 13
C
NMR (151 MHz, CDCl3) δ 193.9 (C), 170.3 (C), 154.1 (C),
148.8 (C), 142.8 (C), 141.7 (C), 129.8 (CH), 126.6 (CH), 115.3
(CH), 111.0 (CH), 104.2 (CH), 72.4 (CH2), 67.0 (CH2), 60.7
(CH2), 53.0 (CH3), 17.8 (CH2), -1.3 (CH3); m/z (ES+) 452 (65%,
[M+H]+); HRMS C20H30N3O5SSi [M+H]+ calcd. 452.1675, found
452.1678.

10
Tetrahedron
ACCEPTED MANUSCRIPT
1
cm-1; H NMR (600 MHz, MeOD-d4) δ 7.63 (1 H, d, J =
3. Branchini, B. R.; Ablamsky, D. M.; Davis, A. L.; Southworth, T.
L.; Butler, B.; Fan, F.; Jathoul, A. P.; Pule, M. A. Anal. Biochem.
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9.0, ArCH), 7.59 (1 H, d, J = 16.5, HC=C), 7.26 (1 H, d, J
= 16.5, HC=C), 7.16 (1 H, dd, J = 9.0, 2.2, ArCH), 7.08 (1
H, d, J = 2.2, ArCH), 5.42 (1 H, t, J = 9.1, CHCO2Me),
3.83 (3 H, s, OMe), 3.78 (2 H, dd, J =12.6, 9.1, CH2S); 13C
NMR (151 MHz, MeOD-d4) δ 170.5 (C), 168.7 (C), 157.8 (C),
143.9 (C), 133.9 (CH), 133.1 (C), 125.2 (C), 122.7 (CH), 119.1
(CH), 115.9 (CH), 98.8 (CH), 79.4 (CH), 53.3 (CH3), 36.1 (CH2);
m/z (ES+) 304 (2%, [M+H]+); HRMS C14H14N3O3S [M+H]+
calcd. 304.0756, found 304.0777.
4. Branchini, B. R.; Southworth, T. L.; Murtiashaw, M. H.; Magyar,
R. A.; Gonzalez, S. A.; Ruggiero, M. C.; Stroh, J. G. Biochemistry
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Nakamura, M.; Amano, Y.; Kojima, S.; Hirano, T.; Maki, S.;
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9. Kuchimaru, T.; Iwano, S.; Kiyama, M.; Mitsumata, S.;
Kadonosono, T.; Niwa, H.; Maki, S.; Kizaka-Kondoh, S. Nat.
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10. Iwano, S.; Sugiyama, M.; Hama, H.; Watakabe, A.; Hasegawa, N.;
Kuchimaru, T.; Tanaka, K. Z.; Takahashi, M.; Ishida, Y.; Hata, J.;
Shimozono, S.; Namiki, K.; Fukano, T.; Kiyama, M.; Okano, H.;
(E)-2-(2-(6-hydroxy-1H-benzo[d]imidazol-2-yl)vinyl)-4,5-
dihydrothiazole-4-carboxylic acid (13)
A solution of ester 29 (10.0 mg, 0.0330 mmol) was dissolved
in phosphate buffer (10 mL, pH=7.8), and treated with PLE
lyophilised powder (9.2 mg) and incubated at 37 °C for 24 h.
Solvent was removed in vacuo and the residue obtained
suspended in a mixture of 1:1 MeOH/CHCl3. The resultant
precipitate was isolated by filtration and washed with a mixture
of 1:1 MeOH/CHCl3 (10 mL). The washings were combined and
concentrated in vacuo to give 13 as a yellow solid (6.0 mg, 0.021
mmol, 63%); mp 190-191 °C; IR νmax (solid state) 3400 (br),
Kizaka-Kondoh, S.; McHugh, T. J.; Yamamori, T.; Hioki, H.;
Maki, S.; Miyawaki, A. Science 2018, 359, 935.
11. Hall, M. P.; Woodroofe, C. C.; Wood, M. G.; Que, I.; Root, M
van’t.; Ridwan, Y.; Shi, Ce.; Kirkland, T. A.; Encell, L. P.; Wood,
K. V.; Löwik, C.; Mezzanotte, L. Nature Comm. 2018, 9, 132.
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A.; Imoto, M.; Nishiyama, S. Chem. Eur. J., 2016, 22, 9930.
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Angew. Chem. Int. Edit. 2012, 51, 3350.
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3123, 2953, 1737, 1619 cm-1; H NMR (600 MHz, THF-d8) δ
1
7.83 (1 H, d, J =8.8, ArCH), 7.36 (1 H, d, J = 16.1, HC=C), 7.33-
7.28 (2 H, m, HC=C and ArCH), 6.99 (1 H, dd, J =8.8, 2.4,
ArCH, 5.22 (1 H, t, J =8.9, CHCO2H), 3.69 (1 H, d, CH2S), 3.63
(1 H, d, CH2S); 13C NMR (151 MHz, THF-d8) δ 171.6 (C), 166.7
(C), 160.1 (C), 148.0 (C), 136.9 (C), 133.7 (CH), 128.2 (CH),
124.2 (CH), 116.3 (CH), 106.1 (CH), 78.8 (CH), 34.6 (CH2); m/z
(ES+) 242 (100%), 597 (10%, [2M+Na]+); HRMS
[C26H18N6O6S2+Na]+ calcd. 597.0627, found 597.0650.
17. Jathoul, A. P.; Grounds, H.; Anderson, J. C.; Pule, M. A. Angew.
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A. J. Am. Chem. Soc. 2012, 134, 7604.
4.3 Enzyme assays
To prepare the compounds for assays, D-LH2 K+ salt (Regis
Tech. IL, USA), DL-BILH2, DL-PBIiLH2 and DL-iLH2 were
dissolved and made up to 10 mg/mL in TEM buffer (100 mM
Tris-acetate, 2 mM ethylenediaminetetraacetic acid (EDTA) and
10 mM magnesium sulphate (MgSO4)).DL-BIiLH2 (3.3 mg) was
dissolved in 50 µL dimethylsulfoxide (DMSO) to aid solubility,
before making up to 10 mg/mL in TEM buffer. These stocks
were stored dark protected at -20oC. Subsequently, each
compound was diluted to 600 µM as a working stock prior to its
use. 200 µM compounds were assayed with 0.167 µM WT, x2,
x5, x11 Flucs or click beetle red (CBR) in TEM buffer at pH 7.8.
Reactions were initiated by the addition of 2 mM saturating ATP
to obtain specific activities, kinetics and bioluminescence spectra
using the PhotonIMAGER Optima at 29oC. Each substrate was
assayed separately in separate plates and the assays were repeated
starting from the 600 µM working stocks (stored at -20oC)
20. Sundlov, J. A., Fontaine, D. M., Southworth, T. L., Branchini, B.
R., Gulick, A. M., Biochemistry, 2012, 51, (33), 6493.
21. Cole, J. C.; Nissink, J. W. M.; Taylor, R. Protein-Ligand Docking
and Virtual Screening with GOLD. Virtual Screening in Drug
Discovery (Eds. B. Shoichet, J. Alvarez), Taylor & Francis CRC
Press, Boca Raton, Florida, USA (2005).
22. Dassault Systèmes BIOVIA, Discovery Studio Modeling
Environment, Release 4.5, San Diego: Dassault Systèmes, 2015.
23. Woodroofe, C. C., Meisenheimer, P. L., Klaubert, D. H., Kovic,
Y., Rosenberg, J. C., Behney, C. E., Southworth, T. L., Branchini,
B. R., Biochemistry, 2012, 51, 9807.
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27. Liu, Z.; Li, H.; Zhao, Q.; Shen, J; Heterocycles; 2008, 75, 1907.
28. Greene, T. W.; Wuts, P. G. M.; Protective Groups in Organic
Synthesis; John Wiley & Sons, Inc., 1999; 3rd Edition; pp 247.
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Acknowledgments
We thank University College London (AJS) and the Welsh
Government for a Ser Cymru II Award (80762-CU-063) (APJ)
for funding, and Dr K. Karu for mass spectra.
30. Chandra, T.; Broderick, W. E.; Broderick, J. B. Nucleosides,
Nucleotides Nucleic Acids; 2010, 29, 132.
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Supplementary data
32. Branchini, B. R.; Southworth, T. L.; Fontaine, D. M.; Murtiashaw,
M. H.; McGurk, A.; Talukder, M. H.; Qureshi, R.; Yetil, D.;
Sundlov, J. A.; Gulick, A. M. Photochem. Photobiol. 2017, 93,
479.
Supplementary data (NMR spectra, bioluminescence kinetics
graphs) for this article can be found at
33. One of the reviewers suggested that the increase in brightness may
be due to the alkylation of the NH in BIiLH2. We had no SEM
alkylated 28a,b left, so attempted the preparation of N-Me-
BIiLH2. We repeated the synthesis as in Scheme 4, alkylating with
Me-I instead of SEM-Cl. Unfortunately the DAST cyclisation step
was unsuccessful, yielding degraded products. This possibility
References and Footnotes
1. Contag, C. H.; Bachmann, M. H; Annual Review of Biomedical
Engineering ; 2002; 4, 235.
2. Gomi, K.; Kajiyama, N. J. Biol. Chem 2001, 276, 36508.

11
will be explored at a later date and we thank the reviewer for their
insightful comment.
ACCEPTED MANUSCRIPT
34. Yoshikawa K.; Yoshino T.; Yokomizo Y.; Uoto K.; Naito H.;
Kawakami K.; Mochizuki A.; Nagata T.; Suzuk M.; Kanno H.;
Takemura M. Ohta T.; Bioorganic & Medicinal Chemistry Letters;
2011, 21, 2133.

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C
A
B
D
Fig. 3. - A and B - Pose prediction for PBIiLH2 and BIiLH2 in PpyLuc pocket; C and D - PBIiLH2 and BIiLH2 with
predicted interactions; Colour code: Pink – Hydrophobic interactions; Green – hydrogen bonds