Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over d-luciferin

Article abstract of DOI:10.1021/ja505795s

Firefly luciferase adenylates and oxidizes D-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. D-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of D-luciferin in live luciferase-expressing cells. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.

Full text of DOI:10.1021/ja505795s

Article
pubs.acs.org/JACS
Open Access on 08/28/2015
Aminoluciferins Extend Firefly Luciferase Bioluminescence into the
Near-Infrared and Can Be Preferred Substrates over D‑Luciferin
David M. Mofford, Gadarla Randheer Reddy, and Stephen C. Miller*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street,
Worcester, Massachusetts 01605, United States
*
S Supporting Information
ABSTRACT: Firefly luciferase adenylates and oxidizes D-luciferin to
chemically generate visible light and is widely used for biological assays
and imaging. Here we show that both luciferase and luciferin can be
reengineered to extend the scope of this light-emitting reaction. D-Luciferin
can be replaced by synthetic luciferin analogues that increase near-infrared
photon flux >10-fold over that of D-luciferin in live luciferase-expressing cells.
Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins
with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity
over the natural luciferase substrate. These new luciferin analogues thus pave
the way to an extended family of bioluminescent reporters.
INTRODUCTION
In nature, D-luciferin is the only luminogenic substrate for
■
beetle luciferases. Over the past few years, many new luciferin
analogues have been described, including several that yield peak
Fireflies are beetles that have evolved the remarkable ability to
emit visible light based on a chemical reaction. Instead of a
photon of light, firefly luciferase uses adenosine triphosphate
and the chemical energy of oxygen to convert its substrate D-
4
−9
light emission well into the red.
Synthetic luciferins thus
have the potential to extend bioluminescence imaging to
wavelengths where tissue is more transparent to light. However,
there is already a significant red and near-infrared component
to luciferase emission with D-luciferin, and shifting the peak
wavelength does not necessarily mean that the overall level of
luciferin into an excited-state molecule of oxyluciferin (Figure
1
1
A). This bioluminescent reaction has been widely used as a
2
3
biological reporter both in vitro and in vivo. Although
bioluminescence has much lower background than fluorescence
and is more sensitive for in vivo imaging, it has been limited by
the relative lack of luciferases and luciferins compared to the
broad palette of fluorescent probes.
9
−11
red light has actually increased.
The synthetic luciferin
CycLuc1 (Figure 1) performs better than D-luciferin for
bioluminescence imaging in live mice, primarily due to its
improved ability to access the luciferase rather than a red-shift
12
in light emission. Therefore, substrates that combine this
ready access to intracellular luciferase in live cells with a red-
shift in total emission are expected to be candidates to further
13
improve in vivo performance.
Structural differences between luciferins could also poten-
tially allow the creation of orthogonal luciferases. Although
mutant firefly luciferases that exhibit luciferin selectivity have
14
been reported, D-luciferin remains a light-emitting substrate.
Surprisingly, recent work has identified the Drosophila fatty
acyl-CoA synthetase CG6178 as a latent luciferase that emits
1
5
light with CycLuc2 but not D-luciferin.
While this
demonstrates that it is possible to retain luciferase activity in
beetle luciferase homologues that are unresponsive to D-
luciferin, higher rates of photon emission are desirable for use
as reporters.
With all of these considerations in mind, we synthesized
compact, rigid aminoluciferins modeled after CycLuc1 and
4
,14
Figure 1. (A) Firefly luciferase catalyzes the adenylation and oxidation
of its native substrate D-luciferin to emit a photon of light. (B)
Previously synthesized aminoluciferin analogues. (C) New amino-
luciferins from this work.
CycLuc2 (Figure 1).
We found that these new substrates
Received: June 9, 2014
©
XXXX American Chemical Society
A
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could greatly increase the total photon flux of near-IR light
from live luciferase-expressing cells over D-luciferin. Moreover,
high photon flux was observed from a newly identified mutant
luciferase that gave virtually no light emission with the natural
substrate. Chemical modification of the luciferin substrate can
thus extend the scope of bioluminescence beyond what is
using the same general synthetic route as CycLuc1−CycLuc4
(
Figure 1C and Scheme 3).
Scheme 3. Synthesis of CycLuc7−CycLuc10
13
possible with D-luciferin.
RESULTS AND DISCUSSION
■
Synthesis of New Rigid Aminoluciferins. The synthetic
aminoluciferins CycLuc1 and CycLuc2 have 5′,6′-fused five-
membered indoline rings (Figure 1B). To evaluate the effect of
this ring fusion on bioluminescence, we synthesized new
luciferin analogues with fused six-membered rings of varying
composition. Luciferin analogues CycLuc3 and CycLuc4,
containing a six-membered oxazine ring, were readily accessed
following a slight modification of the CycLuc1 synthesis
4
paradigm (Scheme 1).
Scheme 1. Synthesis of CycLuc3 and CycLuc4
Surprisingly, attempts to access the 5′,6′-fused ring
differing from CycLuc1 by only a single methyleneprimarily
yielded the 6′,7′-fused product in a >8:1 ratio. Nonetheless,
elaboration of 32b to the 5′,6′-fused cyclic alkylaminoluciferins
CycLuc7 and CycLuc8 proceeded uneventfully. The 6′,7′-fused
product 32a was similarly converted into 6′,7′-fused cyclic
alkylaminoluciferins CycLuc9 and CycLuc10. Finally, 5′,6′-
fused cyclic aminoluciferins CycLuc11 and CycLuc12 were
synthesized as bulkier and more lipophilic analogues of
CycLuc7 and CycLuc8, where the gem-dimethyl substituents
also direct exclusive formation of the 5′,6′-fused isomers
CycLuc5 and CycLuc6 were designed to emit light at longer
wavelengths and to test the scope of substrates that could be
accommodated by the luciferase (Scheme 2). These bulky,
(
Figure 1C and Scheme 4).
Scheme 2. Synthesis of CycLuc5 and CycLuc6
Scheme 4. Synthesis of CycLuc11 and CycLuc12
lipophilic analogues incorporate 2,2,4-trimethyl-dihydroquino-
line, a scaffold that has been widely used to red-shift the
emission of many classes of fluorophores, including coumar-
Luciferase Activity in Vitro. At the outset of this work, it
was anticipated that some subset of the rigid luciferin analogues
would not be well accommodated by the luciferase. However,
all of the new aminoluciferin analogues are substrates, further
underscoring the generality of the light emission chemistry and
the tolerance of luciferase for modifications.
When purified firefly luciferase is treated with D-luciferin or
an aminoluciferin, a high initial rate of light emission (burst) is
observed in the first few seconds, which is then followed by a
substantial decrease in the rate of sustained light output. This
effect is more pronounced for high-affinity aminoluciferins than
for D-luciferin itself, and all of the new substrates exhibited this
same general behavior under saturating substrate conditions
(Figure 2 and Supplementary Figure 3). Relative to D-luciferin
with the wild-type (WT) luciferase, the initial rate of photon
1
6
16,17
18
ins, rhodamines,
and oxazines. Attempted synthesis of
intermediate 16 by trifluoroacetylation of the corresponding
nitroarene as in Scheme 1 failed, presumably due to a
1
9
combination of steric hindrance and electronic deactivation.2
0
We therefore synthesized the Boc-protected compound 14,
which suffers the same steric hindrance but was anticipated to
be less electronically deactivated. Gratifyingly, trifluoro-
19
acetylation of this intermediate was successful, and sub-
sequent TFA deprotection readily afforded 16. Elaboration as
shown in Scheme 2 afforded the desired luciferin analogues
CycLuc5 and CycLuc6.
The corresponding saturated tetrahydroquinoline amino-
luciferin analogues CycLuc7 and CycLuc8 were synthesized
B
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luciferin (Supplementary Figure 2). Bioluminescence is red-
shifted by 50−75 nm from the substrate fluorescence in
phosphate-buffered saline, which is expected since the oxy-
luciferin emitter produced in the luciferase has increased
conjugation with respect to the substrate.
The fluorescence emission of acyclic monoalkylated amino-
23
luciferins generally ranges from 520 to 540 nm, more red-
shifted than that of 6′-aminoluciferin (517 nm) but less so than
for dialkylated substrates such as 6′-Me NLH (554 nm).
2
2
CycLuc6 fluoresces at still longer wavelength (567 nm), while
CycLuc10 is the most red-shifted of all the luciferin analogues
(
576 nm). While this was initially surprising, Atkins and Bliss
have described similar behavior for aminocoumarin deriva-
tives.
24
Mutation of Luciferase Modulates Emission. We have
previously found that the luciferase active-site mutant R218K
increases the rate of light emission from CycLuc1, CycLuc2, 6′-
1
4
MeNHLH , and 6′-Me NLH . This effect is not particularly
2
2
2
selective, as it is observed with all of the aminoluciferins
Figures 4 and 5, Supplementary Figure 3). For instance,
(
Figure 2. Initial and sustained bioluminescence from luciferin
analogues (250 μM) with purified WT firefly luciferase (0.2 nM):
(
A) initial rates of emission relative to D-luciferin and (B) relative
emission 1 min after substrate addition.
flux ranged from a high of 43% for CycLuc4 to a low of 2% for
CycLuc11. This initial rate is high compared to luciferin
8
,21,22
analogues in which the benzothiazole has been replaced.
However, aminoluciferins have reduced emission 1 min after
substrate addition, consistent with product inhibition as the
primary factor limiting the light emission in vitro (Figure 2 and
Supplementary Figure 3).
Bioluminescence Emission Wavelengths. Peak bio-
Figure 4. Light emission from D-luciferin and CycLuc7 (250 μM)
treated with equal amounts of purified WT and mutant luciferases (0.2
nM). The assay was performed in triplicate and is represented as the
mean ± SEM.
4
,23
luminescence emission for the aminoluciferins ranged from
594 to 642 nm (Figure 3, Supplementary Table 1, and
Figure 3. Bioluminescence emission spectra for WT luciferase with
selected substrates.
Supplementary Figure 1). As expected, CycLuc6 yielded
strongly red-shifted bioluminescence (636 nm), exceeding
11
that of red-emitting mutant firefly luciferases, railroad worm
1
0
(
beetle) luciferase, and the red-shifted emission from 6′-
4
Me NLH . However, we were surprised to find that CycLuc10
2
2
yielded an even more red-shifted peak (642 nm). To determine
whether these differences are inherent to each luciferin, we
measured the fluorescence emission wavelengths of the
substrates. The fluorescence and bioluminescence emission
wavelengths of aminoluciferins were found to be strongly
correlated, suggesting that the bioluminescence wavelength is
primarily dictated by the photophysical properties of the
Figure 5. Initial and sustained bioluminescence from each luciferin
(250 μM) with equal amounts of three different purified luciferases
(0.2 nM, see text): (A) peak intensity within the first 2 s of substrate
addition and (B) sustained light emission 1 min post-injection. The
assay was performed in triplicate and is represented as the mean ±
SEM.
C
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Figure 6. Photon flux from luciferase-expressing CHO cells. (A) Live cells expressing the indicated luciferase and treated with high or low doses of
luciferin. (B) Comparison of total and near-IR photon flux from WT luciferase with the indicated luciferins. (C,D) Comparison of photon flux from
WT and triple-mutant luciferase with D-luciferin or CycLuc7 in live cells (C) or lysed cells (D). All assays were performed in triplicate and are
represented as the mean ± SEM.
CycLuc7 exhibits higher initial and sustained emission rates
with R218K compared to WT (Figure 4). The R218K mutant
also resulted in a slight red-shift in bioluminescence for most
substrates, pushing the maximal emission wavelength for
CycLuc10 to 648 nm (Supplementary Table 1 and
Supplementary Figure 1).
Toward Orthogonal Luciferases. In principle, chemical
and structural differences in luciferin substrates could be
exploited to create new orthogonal luciferases. However, active-
initial rate of D-luciferin with WT luciferase. This result
demonstrates that high luciferase activity can be maintained in a
luciferase mutant that is essentially unresponsive to the native
substrate D-luciferin. Furthermore, product inhibition has
largely been eliminated, as there is little diminution in flux
after the initial burst (Figures 4 and 5 and Supplementary
Figure 3).
Luciferin Emission in Live Luciferase-Expressing Cells.
We next compared the aminoluciferins to D-luciferin in live WT
luciferase-expressing Chinese Hamster Ovary (CHO) cells.
Under these conditions, the luciferin substrate must cross the
cell membrane to access the luciferase. In marked contrast to
what is observed in vitro, almost all of the alkylated
aminoluciferins yield higher flux than D-luciferin when assayed
at a concentration <30 μM (Figure 6 and Supplementary
Figure 4). At high substrate concentration (250 μM), the
relative emission from D-luciferin is still equaled or exceeded by
those of CycLuc1, CycLuc10, and CycLuc12 (Figure 6).
However, if the cell membrane is removed by cell lysis, D-
luciferin is the superior substrate at both high and low doses,
suggesting that substrate access is the primary factor limiting
luciferins in live cells (Supplementary Figure 5).
site mutations such as R218K and L286M raise the K of D-
m
14
luciferin but do not prevent its utilization as a substrate.
A
more selective point mutant, S347A, raises the K and lowers
m
the rate of emission from D-luciferin, possibly because it
removes a hydrogen-bonding interaction with the benzo-
thiazole nitrogen that may be more important for D-luciferin
1
4,25
binding and orientation than for aminoluciferins.
Yet D-
luciferin remains a substrate for this mutant luciferase as well.
Thus, previous work has not established whether high luciferase
activity can be retained in luciferase mutants that do not yield
light emission from D-luciferin.
Here we find that the combination of R218K, L286M, and
S347A renders D-luciferin essentially inactive. The purified
triple-mutant luciferase dramatically reduced both the initial
and sustained rates of photon emission from D-luciferin by >10
Near-IR Photon Flux in Live Cells. The peak emission
wavelengths of aminoluciferins are red-shifted in vitro. To
assess the near-IR emission from each luciferin in live cells, we
measured the relative photon flux passing through a Cy5.5 filter
(695−770 nm). All of the aminoluciferins exhibited greater
relative photon flux in the near-IR than D-luciferin (Figure 6B
and Supplementary Figure 6). For every substrate except 6′-
aminoluciferin and CycLuc3, this translated to a higher total
000-fold (Figure 4 and Supplementary Figure 3). Gratifyingly,
this is not due to a lack of luciferase activity, as the photon flux
from CycLuc2, CycLuc7, and CycLuc11 actually increased
compared to that of the WT enzyme (Figure 5 and
Supplementary Figure 3). CycLuc7 is the optimal substrate
for the triple-mutant luciferase in vitro, achieving 46% of the
D
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near-IR photon flux from live cells than D-luciferin under both
low-dose and high-dose conditions (Supplementary Figure 6).
CycLuc10 gave the greatest fraction of near-IR light emission:
D-luciferin altogether in favor of two or more synthetic
substrates.
1
3.9% of the total photon flux, >10-fold higher than that of D-
CONCLUSION
■
luciferin (Figure 6B). However, total near-IR flux of CycLuc10
was slightly exceeded by that of CycLuc12. Thus, the substrate
that yields the highest cellular near-IR light emission is a
function of substrate access (affinity and cell permeability) as
well as wavelength.
Mutant Luciferases in Live and Lysed Cells. Trans-
fection of CHO cells with R218K luciferase instead of WT
luciferase improved relative photon flux from synthetic
luciferins compared to D-luciferin (Figure 6 and Supplementary
Figure 4). CycLuc2 yielded the highest signal in live cells, and
most alkylated aminoluciferins were superior to D-luciferin at
both low and high substrate concentrations (Figure 6 and
Supplementary Figure 4). Further highlighting the importance
of factors other than peak emission wavelength on the total
emission of red-shifted photons in live cells, CycLuc2 yielded
the highest Cy5.5-filtered signal at 250 μM, while CycLuc6 was
best at low concentration (Figure 6 and Supplementary Figure
There is reason to expect that substrate performance in live
cells, rather than with purified protein or cell lysates, is more
predictive of in vivo behavior. In recent collaborative work we
have found that CycLuc1 allows dramatically improved
bioluminescence imaging in live mice compared to the standard
imaging conditions with D-luciferin. Tumor cells can be
imaged with 20−200-fold less substrate than D-luciferin, and
luciferase expression deep in the brain that cannot be detected
12
12
with D-luciferin is detectable with CycLuc1. Many of the
substrates described here provide higher total and red-shifted
photon flux in live cells, suggesting that they may also have
superior properties for in vivo imaging. Differences in substrate
affinity, lipophilicity, and functionality are also anticipated to
affect the pharmacokinetics of the luciferins in vivo, perhaps
allowing tuning of bioluminescent half-lives and/or tissue
distribution. Moreover, we have found that mutation of
luciferase can essentially eliminate light output from the native
D-luciferin substrate while retaining or improving light emission
from one or more aminoluciferin substrates to levels
comparable to or, in live cells, superior to that of D-luciferin
with the WT luciferase. Thus, these synthetic luciferins and
mutant luciferases not only expand the palette of luminogenic
molecules but transcend the emission properties of D-luciferin
and firefly luciferase. They are therefore expected to have
significant potential for bioluminescence imaging applications
both in vitro and in vivo.
6
). The longer emission wavelength of CycLuc6 (Figure 3) and
its higher cell permeability likely lead to its superior flux at low
concentrations, while the higher maximal rate of photon
emission for CycLuc2 ultimately prevails at high substrate
concentration (Figure 5). Thus, the best-performing substrates
are context-dependent.
No signal above background could be measured from live
cells expressing the triple-mutant luciferase after treatment with
D-luciferin (Figure 6C). In contrast, CycLuc2, CycLuc7, and
CycLuc11 achieve high photon flux (Figure 6). The triple
mutant possesses lower affinity for its substrates compared to
WT or R218K luciferase, yielding reduced photon flux at low
substrate concentration (Figure 6 and Supplementary Figure
ASSOCIATED CONTENT
■
*
S
Supporting Information
4
). However, in lysed cells, the lower affinity of the triple
Experimental procedures; compound characterization, includ-
ing all NMR spectra; Supplementary Table 1; and Supple-
mentary Figures 1−7. This material is available free of charge
via the Internet at http://pubs.acs.org.
mutant improved the signal from CycLuc7 and CycLuc11 due
to its lessened product inhibition (Figure 6D and Supple-
mentary Figure 5). CycLuc7 is the best substrate in cell lysates,
achieving ∼20% of the D-luciferin signal with the WT luciferase
(Figure 6D and Supplementary Figure 5). Mutant and WT
AUTHOR INFORMATION
■
luciferase protein expression is equivalent in transfected cells by
Western blot (Supplementary Figure 7), and the differences
between luciferases in cell lysates (Supplementary Figure 5)
generally mirrors what is observed with equal concentrations of
purified proteins in vitro (Figure 5). The triple-mutant
luciferase is thus a significant step toward orthogonal
bioluminescent reporters of gene expression in both live and
lysed cells, as it yields high light output with (alkylated)
aminoluciferins but little to no signal with D-luciferin. We
speculate that the triple mutant primarily discriminates between
substrates by lowering substrate affinity and removing an
interaction important for orienting the native substrate (S347).
Luciferin analogues possessing high affinity for luciferase and an
alternative “handle” for proper orientation remain effective
Corresponding Author
stephen.miller@umassmed.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(
This work was funded by the National Institutes of Health
R01EB013270 to S.C.M.).
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