A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging

Article abstract of DOI:10.1002/anie.201405955

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

Full text of DOI:10.1002/anie.201405955

Angewandte
Chemie
DOI: 10.1002/anie.201405955
Near-Infrared Bioluminescence
A Dual-Color Far-Red to Near-Infrared Firefly Luciferin Analogue
Designed for Multiparametric Bioluminescence Imaging**
Amit P. Jathoul, Helen Grounds, James C. Anderson,* and Martin A. Pule*
Abstract: Red-shifted bioluminescent emitters allow improved
in vivo tissue penetration and signal quantification, and have
led to the development of beetle luciferin analogues that elicit
red-shifted bioluminescence with firefly luciferase (Fluc).
However, unlike natural luciferin, none have been shown to
emit different colors with different luciferases. We have
synthesized and tested the first dual-color, far-red to near-
infrared (nIR) emitting analogue of beetle luciferin, which,
akin to natural luciferin, exhibits pH dependent fluorescence
spectra and emits bioluminescence of different colors with
different engineered Fluc enzymes. Our analogue produces
different far-red to nIR emission maxima up to lmax = 706 nm
with different Fluc mutants. This emission is the most red-
shifted bioluminescence reported without using a resonance
energy transfer acceptor. This improvement should allow
tissues to be more effectively probed using multiparametric
deep-tissue bioluminescence imaging.
resulting in less attenuation of red light. This wavelength
range is within what is termed the “bio-optical window” and
there has been much focus on engineering red-shifted Fluc
enzymes that have maximum emission wavelengths in this
range,[10–15] but these have peaked at wavelengths less than l =
645 nm.
The most red-shifted luciferin analogues to date[16] are
based upon amino derivatives (Figure 1b), for example cyclic
aminoluciferin (3a: lmax = 599 nm; 3b: lmax = 607 nm),[17]
seleno-d-aminoluciferin (4: lmax = 600 nm),[18] and a rationally
designed 4-(dimethylamino)phenyl derivative conjugated to
a thiazoline group (5: lmax = 675 nm).[19] In particular cyclic
aminoluciferin derivative 3a has been shown to give
improved bioluminescence imaging compared to luciferin
(LH2; 1) at dilute concentrations where the intracellular
concentration of the luciferin or analogue is limiting.[20] Near-
infrared emission has been detected with an aminoluciferin
B
ioluminescence imaging (BLI) has revolu-
tionized molecular genetic imaging in biomed-
ical research as a cheap and easy means to
longitudinally image the genetic behavior of
life and disease processes in whole mam-
mals.[1–4] As they produce the brightest form
of bioluminescence,[5] genes from coleopterans
are commonly used to localize, track, and
quantify cells and molecular or functional
events in vivo.[6–8] In a well-studied reaction,[9]
beetle luciferin (1, Figure 1a) is adenylated by
firefly luciferase (Fluc) and this reacts with
molecular oxygen to produce an excited state
species, oxyluciferin* (2), which decays to
release a photon with a high quantum yield
(lmax = 558 nm).[5] However, absorption of visi-
ble light by hemoglobin (Hb) and melanin
Figure 1. a) Bioluminescence of luciferin (1) catalyzed by luciferase. b) Structures of red-
shifted bioluminescent amino-luciferin analogues. c) New luciferin analogue iLH2 6 that
exhibits near-infrared bioluminescence with mutant Fluc. ATP=adenosine triphosphate.
restricts image resolution and signal penetration at this
wavelength. Between l = 600–800 nm, the absorption of
light by Hb decreases by a factor of approximately 50,
Cy5 conjugate, but this is due to bioluminescence resonance
energy transfer (BRET),[21] meaning that the conjugate
cannot be used for multiparametric imaging.
[*] Dr. A. P. Jathoul, Dr. M. A. Pule
metastasis model, Jorge M. Cardoso, Rupinder Ghatrora for imag-
ing assistance, and Mark F. Lythgoe, Arnold Pizzey (UCL, London
(UK)) and Laurence Tisi (Lumora Ltd., Ely, Cambs (UK)) for helpful
discussions.
Department of Haematology, UCL Cancer Institute and
NIHR University College London Hospitals Biomedical Research
Centre, London, WC1E 6BT (UK)
E-mail: martin.pule@ucl.ac.uk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201405955.
Dr. H. Grounds, Prof. J. C. Anderson
Department of Chemistry, University College London
London, WC1E 6BT (UK)
ꢀ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited.
E-mail: j.c.anderson@ucl.ac.uk
[**] We thank the BBSRC (BB/I014357/1) and the NIHR UCLH
Biomedical Research Centre for financial support. We thank Peter
Johnson for preparation and imaging of the orthotopic liver
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To date, despite a red-shift in emission, no analogues have
been reported with the other desirable properties of LH2 1,
such as a high quantum yield and the ability to produce more
than one color with different Fluc mutants. Considering this,
and the likely mechanisms of color tuning in Fluc biolu-
minescence, we describe the design, synthesis, and in vitro and
in vivo testing of the first far-red to nIR multicolor-emitting
analogue, which can produce the most red-shifted form of
true bioluminescence reported to date. Additionally, our far-
red-shifted analogue infra-luciferin (6, iLH2; Figure 1c)
produces distinct bioluminescent colors with different
enzymes akin to native luciferin, and could be of great
benefit to multiparametric deep-tissue and tomographic
bioluminescence in vivo imaging.
Despite a number of theories, the exact mechanism
regulating color tuning in Fluc bioluminescence has not
been solved.[11–13] Current measurements and calculations
suggest that color modulation is due to perturbing interac-
tions in the microenvironment surrounding the anionic
phenolate of excited-state oxyluciferin (2) in the Fluc active
site.[22–28] Additionally, p–p overlap between the benzothia-
zole and thiazolone heterocycles in 2 also appears to be
important.[29–31] Maki and co-workers demonstrated the
importance of extended p-conjugation in luciferin derivatives
which led to the development of 5.[19,32] In our design we
proposed that increasing the conjugation of LH2 1, and thus 2,
by addition of an alkene linker between the benzothiazole
and thiazoline fragments would lead to a red-shifted luciferin
analogue (6, Figure 1c) that would be amenable to color
modulation with different Fluc mutants. Extended conjuga-
tion should reduce the HOMO–LUMO energy gap in the
light-emitting phenolate of 7, which would lead to red-shifting
of the emitted light. Our design, in contrast to other
established red-shifted luciferin analogues (Figure 1),
retained the 6’-hydroxy group. This design was chosen in an
attempt to capitalize on the microenvironment effect of
different Fluc mutants to generate different bioluminescence
emission wavelengths that are essential for multiparametric
imaging. We also believed that the increase in the overall
shape of the molecule by only one alkene unit may be
tolerated by Fluc mutants to facilitate multiwavelength
emission.
Scheme 1. Synthesis of infra-luciferin 6. a) BnBr (1.2 equiv), K2CO3
(2.8 equiv), acetone, room temperature, 16 h, 85%; b) nBuLi (1.93m in
hexanes, 1.1 equiv), THF, À788C, 15 min then DMF (4.1 equiv), 1 h,
96%; c) (Carbethoxymethylene)triphenylphosphorane (3 equiv), PhMe,
reflux, 3 h, 92%; d) NaOH (1m), iPrOH, 16 h, quantitative yield;
e) Et3N (2.4 equiv), DMF, amino acid (aa; 1.2 equiv), 08C then BOP
(1.2 equiv) in CH2Cl2, 2 h (R=Me, 80%, R=Et, 82%); f) Ph3PO
(1.3 equiv), Tf2O (2.7 equiv), CH2Cl2, 08C, 30 min added to benzothia-
zole in CH2Cl2, 08C, 10 min, (R=Me, 65%, R=Et, 74%); g) penta-
methylbenzene (4.4 equiv), BCl3 (1m in CH2Cl2, 3 equiv), CH2Cl2,
À788C, (R=Me, 79%, R=Et, 72%); h) PLE, buffer, 378C, in situ.
Bn=benzyl; Tf2O=trifluoromethanesulfonic anhydride; Trt=triphenyl-
methyl; BOP=(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate; PLE=pig liver esterase.
588 nm, with a red shift of 58 nm compared to 1. Furthermore,
the fluorescence excitation and emission spectra are pH de-
pendent, as measured for 1. In contrast, the Maki analogue 5
had pH independent fluorescence spectra, producing only
one fluorescence excitation color (see Figures S1a–c in the
Supporting Information). This highlights the importance of
retaining the 6’-hydroxy group for color modulation.[22–28]
In vitro bioluminescence spectra of iLH2 6 ethyl ester
(saponified with PLE (pig liver esterase) in situ immediately
prior to use) with purified wild-type (WT) Fluc, the x5 Fluc
mutant (a thermostable Fluc with similar properties to WT
but with higher quantum yields),[35,36] and the x5 S284T Fluc
mutant (a bright red-shifted point mutant of x5)[37,38] showed
marked red-shifted peak maxima of 100 nm magnitude
compared to the lmax of each enzyme with 1 (Figure 2,
Table 1). This effect is remarkable considering that these
mutants were originally engineered for different emission
The molecule iLH2 6 was synthesized in 10 steps from
commercially available starting materials (Scheme 1). During
the synthesis we found that once the thiazoline ring had been
formed the molecule was incredibly sensitive to epimerization
next to the carboxy group. Both the methyl and ethyl ester of
iLH2 6 could be isolated in enantiopure form, but isolation of
the free acid after saponification was found to be extremely
difficult with epimerization and formation of the thiazole
detected. To maximize light output we decided to test the
enantiopure esters in vitro and in vivo as it has been shown
that esters of LH2 1 are active in live cells and living mice,[33,34]
as they are saponified by esterases. We also synthesized the
Maki analogue 5,[19] the most red-shifted bioluminescent
analogue reported to date, to compare its properties to
iLH2 6.
The fluorescence spectra of iLH2 6 compared to LH2 1
Figure 2. Bioluminescence spectra of native 1 and 6 with x5 and
showed that at pH 7, the emission maximum of 6 was lmax
=
x5 S284T Fluc mutants.
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Table 1: Bioluminescence spectral properties of 1, 5, and 6 with purified
enzymes.
purified enzymes, a difference of approximately 100-fold
was seen in the specific activity of x5 Fluc from LH2 1 and
iLH2 6, whereas 5 displayed a 3-fold lower specific activity
than 1. Apparent kinetic parameters indicate that the affinity
(Km) of x5 Fluc for infra-luciferin (6) are more similar to
LH2 1 than 5, though the turnover (kcat), and therefore the
overall catalytic efficiency (kcat KmÀ1), of 6 was approximately
285-fold lower than 1, compared to 25-fold lower for 5. A
similar magnitude of a decrease in the kcat value has been
reported for commercially available thermostable luciferase
mutants compared to WT Fluc with 1.[36] The full width at half
maximum (FWHM) of emission of both x5 Fluc and x5 S284T
are markedly wider with infra-luciferin (6) than with LH2 1,
indicating less emitter specificity of the x5 mutant framework
(Table 1). It may be possible to engineer Fluc mutants with
enhanced activities, kinetic parameters, and/or emitter spe-
cificity with infra-luciferin (6).[15,35,40,41] Despite 6 having
lower activity, the benefit of 6 is that it exhibits near-infrared
bioluminescence that is susceptible to color modulation by
mutated Fluc enzymes, unlike 5, and thus shows promise for
multiparametric imaging.
Cells transduced with Fluc readily showed biolumines-
cence activity upon treatment with iLH2 6. With this in mind,
we established a number of mouse models of cancer with the
aim of detecting and imaging these using iLH2 6 methyl ester
and comparing in vivo light yields to native luciferin 1
(Figure 3a–c).[42] A subcutaneous (sc) model was induced by
sc injection of 5 ꢀ 106 LS174T cells (colon carcinoma cell line)
expressing WT Fluc into Nude (MFI NuNu) mice (Fig-
ure 3a). After four days post-inoculation, mice were intra-
peritoneally (ip) injected with LH2 1 ethyl ester (2 mg) or
iLH2 6 methyl ester and imaged in the Photon Imager. Light
emission was readily apparent in mice administered with
iLH2 6 methyl ester. In vivo spectra showed that the max-
imum emission wavelength for WT Fluc with LH2 1 is within
the l = 590–640 nm bp filter, likely as a result of the
bathochromic shift of WT Fluc at physiological temperature
and also attenuation of the true spectrum by Hb in mouse
tissues (Figure S3).[39] However, the in vivo spectrum of
WT Fluc with iLH2 6 displays a lmax in the l = 700–750 nm
bp filter and its shape appears much less attenuated by mouse
tissues. We also found iLH2 6 methyl ester was active and
detectable in a systemic lymphoma mouse model of cancer
(Figure 3b). In this case, the same mice were imaged on
consecutive days with LH2 1 ethyl ester or iLH2 6 methyl
ester. One minute background luminescence images were
acquired prior to imaging to ensure there was no remaining
activity from previous sessions. These images (Figure 3b)
show that 6 gives a more even intensity across the whole
animal, likely because of the consequence of less
Fluc Mutant
WT Fluc
Luciferin Substrate
lmax [nm]
FWHM [nm][a]
1
5
6
1
5
6
1
5
6
558
652
670
554
652
646
605
658
706
76
76
74
62
76
92
56
78
81
X5 Fluc
X5 S284T
[a] FWHM=bioluminescence full width at half maximum. See Support-
ing Information for experimental conditions.
colors with LH2 1. Our analogue showed bioluminescence
with lmax = 706 nm with Fluc mutant x5 S284T, significantly
red-shifted compared to any reported natural or unnatural
bioluminescence system. This suggests that Fluc mutants emit
different color forms with 1 and 6 in line with the micro-
environment effect of the Fluc active sites.[12,22–28] In contrast,
bioluminescence of the Maki analogue 5 with different Fluc
mutants occurred with almost identical emission wavelength
maxima and only very small nonspecific shifts between Fluc
mutants (Figure S2a),[19] supporting the theory that the
phenolic hydroxy group is crucial for the color-tuning
mechanism of luciferin.[22–28] These results suggest that
iLH2 6 would be of use for in vivo multiparametric imaging,
whereas the Maki analogue 5 would not be. The other red-
shifted luciferin analogues (Figure 1) may also not be suitable
because of the substitution of the phenolic hydroxy group for
an amine.
The proportion of light output above l = 600 nm is a key
determinant for transmission efficiency through mammalian
tissue.[39] Bioluminescence data were acquired using different
band-pass (bp) filters in a Photon Imager and showed that
most light from the x5 Fluc mutant with 6 was collected
through a l = 670–720 nm bp filter and from the x5 S284T
mutant through a l = 700–750 nm bp filter. The proportion of
light emitted over l = 600 nm with LH2 1 was 32%
(WT Fluc), 18% (x5 Fluc), and 68% (x5 S284T Fluc), respec-
tively. For iLH2 6, this increased dramatically to 95%, 97%,
and 100%, respectively (Figure S2b). This should have
a significant impact on the amount of transmitted light
detected during in vivo imaging (see below).
To compare the in vitro specific activity of the x5 Fluc
mutant with the saponified esters (PLE) of LH2 1, the Maki
analogue 5, and iLH2 6, data was acquired using an IVIS 200
instrument—Caliper Life Sciences, USA (Table 2). With
Table 2: In vitro activity and apparent kinetic parameters of the x5 Fluc mutant with
the saponified esters of 1, 5, and 6.[a]
attenuation by mouse tissues due to the higher
proportion of light output above l = 600 nm (Fig-
ure S4). Finally, iLH2 6 was employed to image liver
metastases in an orthotopic model in nude mice
(Figure 3c, Figure S5), a particularly challenging
organ to get meaningful data from because of blood
and tissue attenuation. In this case, mice were
imaged 15 minutes after ip injection of iLH2 6
methyl ester (4 mg), or with LH2 1 potassium salt
Substrate Apparent
Apparent kcat
Apparent kcat/Km Specific Activity/cpm
Km [mm] [RLU sÀ1 ꢁ1015] [sÀ1 mmÀ1 ꢁ1015]
[cmÀ2 mgÀ1 ꢁ1015]
1 Et Ester
5 Me Ester
6 Me Ester
2.0
16.7
6.0
40.0
13.3
0.4
200
8.0
0.7
60.9
19.7
0.6
[a] Data recorded in triplicate. See Supporting Information for experimental
conditions. cpm=counts per minute. RLU=relative light units.
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bright does not appear to be the absolute
requirement for better penetration and
resolution because of a reduced signal to
noise effect caused by light scattering.
Scatter of light is proportional to the
reciprocal of wavelength to the fourth
power and this leads to enhanced signal
penetration of iLH2 6 through blood when
compared to luciferin (Figure 4).
Firefly luciferase (Fluc) produces true
near-infrared
bioluminescence
with
iLH2 6, and the emission color can be
tuned with different Fluc mutants in the
wavelength range of the bio-optical
window of mammalian tissues, currently
up to a maximum of l = 706 nm. As 95%
of the emitted light of iLH2 6 with Fluc has
an emission maximum greater than l =
600 nm (Figure S2b), there is less attenu-
ation in blood (Figure 4) and in vivo (Fig-
ure 3c) than with LH2 1. These results
suggest that this bright, red-shifted form of
bioluminescence has potential for deep-
tissue multiparametric BLI and could
provide a more detailed assessment of
in vivo cellular and molecular processes.
We are in the process of testing a number
of further analogue designs and are
exploring the possibility of creating Fluc
mutants with enhanced activity with
iLH2 6.
Figure 3. In vivo imaging with 6 in mouse cancer models expressing firefly luciferase (Fluc).
Left column of images: no substrate; middle column: mice imaged with LH2; right column:
mice imaged with iLH2 Me ester. Inset graphs show the relative in vivo light yields from mice
with different substrates and imaged for equivalent times. In vivo spectra are displayed in
Figure S4 in the Supporting Information.
(2 mg), also given ip. The relative in vivo light yield for 1 is
Received: June 5, 2014
Revised: September 3, 2014
Published online: September 29, 2014
identical to those measured for the methyl ester, showing
saponification is complete in the mouse.[33,34] After just
15 seconds, the image acquisitions (Figure 3c) show that 6
detailed the non-uniform nature of the metastatic tumor
burden whilst images with 1 neared saturation and showed
little definition because of its intensity.
Please note: Minor changes have been made to this manuscript since
its publication in Angewandte Chemie Early View. The Editor.
Keywords: imaging agents · luciferase · luminescence ·
.
Over the same time interval there was an average of
200 times less light from 6 compared to 1. Ironically, being
multiparametric imaging · structure–activity relationships
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