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To establish a rational design strategy, we first conducted
a docking simulation of AL derivatives with firefly luciferase
using the MOE software package (Figure 1). The crystal
structure of firefly luciferase has been reported by several
groups, including Conti et al.[13] (PDB:1 LCI; first report but
without substrate) and Auld et al.[14] (PDB:3IES; complex
with luciferase inhibitor). Among them, we chose the one
reported by Nakatsu et al.[15] (PDB: 2DIS; complex of
luciferase from Luciola cruciate, Japanese name: Genji-
botaru, with a high-energy intermediate analogue, DLSA)
for our docking study because of the structural similarity of
the substrates. Therefore, we used this crystal structure as
a scaffold and changed the amino acid sequence to that of
Photinus pyralis, which was used in our work. We fixed the
peptide backbone of luciferase and the DLSA (aminoluci-
ferin) moiety, and searched for the most stable conformation,
freely rotating the side chains of amino acids and the
fluorophore-linker moiety of the substrate (Figure 1).
As shown in Figure 1, we found that in the most stable
conformation the linker moiety penetrates through the
luciferase molecule, and motion of the fluorophore and
linker moiety of the substrate in the luciferase complex would
appear to be highly restricted. If an AL derivative entered
from the right side and the adenosine moiety is placed like
a cap at the entrance of the pocket, as indicated by the orange
arrow in the figure, the bulky fluorophore moiety would have
to pass through the luciferase molecule, which seems very
unlikely. Therefore, based on this simulation, we hypothe-
sized that AL derivatives enter from the left side, as indicated
by the red arrow. If this is the case, a flexible linker might be
desirable, so that the fluorophore does not hinder the access
of the luciferin moiety to the active site of the luciferase. We
considered that it might be possible to employ relatively
bulky fluorophores, if we conjugated them by appropriate
linkers.
compound was compared at three minutes after the start of
the reaction (see Figure S6 for the choice of this time).
At first, fluorescein piperazine–AL was not luminescent
(Figure 2b). We also confirmed that fluorescein piperazine–
AL was not consumed by firefly luciferase (Figure S2). These
results support the idea that short and rigid linkers are
unsuitable, as we had expected from the docking simulation
with the crystal structure. Next, we focused on BODIPY FL–
AL and BODIPY FL X–AL, which differ only in the length of
the linkers. BODIPY FL X–AL, which has a longer linker,
was brighter than BODIPY FL–AL (Figure 2b), and this
tendency was also observed in other substrates (Figure S3).
Therefore, relatively long linkers appear to be favorable.
Focusing on 8-phenyl BODIPY X–AL, we found that
luminescence was retained if an appropriate linker was
selected (Figure 2b), even though the fluorophore is rela-
tively bulky (the BODIPY moiety and the benzene moiety
conjugated at the 8 position of BODIPY are orthogonal),
which is consistent with our hypothesis that the fluorophore
moiety does not pass through the luciferase molecule. We also
found that the nature of the fluorophore is not critical and
that excessively hydrophobic linkers and polyethylene glycol
(PEG) linkers conjugated near the luminophore are not
desirable (Figure S3). These results are consistent with the
docking simulation and the above discussion.
Therefore, our strategy of obtaining a range of AL–
fluorophore conjugates by using appropriate linkers (Fig-
ure S3) to conjugate various kinds of fluorophores, including
bulky ones, to AL with retention of luminescence appears to
be feasible.
Based on these findings, we designed and synthesized AL
derivatives bearing the NIR fluorophores Cy5, BODIPY 650/
665, SiR700,[16] and Cy7 as BRET acceptors to cover a broad
NIR window (Figure 3a). As expected, all the substrates
showed NIR bioluminescence (Figure 3b; see also the
Supporting Information, Figure S6). BRET efficiency was
very high (Figure S4), even if the overlap of the absorption
spectrum of the acceptor and the emission spectrum of the
original AL was small, this is probably because the BRET
donor and acceptor are sufficiently close to each other (the
BRET process is intramolecular). The BRETefficiency of our
system is comparable to those of reported BRET systems
using luciferase modified with NIR dye,[4] QDs,[6] or QRs,[7]
Taking these points into consideration, we next examined
the structure–luminescence intensity relationship of a number
of AL derivatives. (Figure 2; see also the Supporting Infor-
mation, Figures S2 and S3) To simplify the discussion, here we
focus on several AL–fluorophore conjugates in which BRET
does not occur because of the short absorption wavelength of
the fluorophores. The relative luminescence intensity of each
Figure 2. Example of the structure–luminescence intensity relationship of AL–fluorophore conjugates. a) Structure of each substrate. b) Compar-
ison of relative luminescence intensity of each substrate (% vs. AL). Luminescence intensity was compared at 3 min after the addition of
luciferase. Luminescence intensity of AL is about 30% of that of d-luciferin. N.D.=not determined.
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Angew. Chem. Int. Ed. 2013, 52, 1175 –1179