C. Yan, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127085
showed the similar K
m
value compare to furimazine. Particularly,
compounds A4, A5, A6, A10 displayed smaller Km than furimazine,
which illustrated these compounds have higher affinity with NLuc. In
the aspect of Vmax, only A11 barely approached the Vmax of furimazine.
Furthermore, except compound A4, all the rest compounds had a much
longer half-decay life and could produce glow-type bioluminescence.
Especially, the half-decay life of compounds A2 over one hour. In
summary, compounds A2, A6 and A11 have the potential to become the
valuable substrates of NLuc system. In summary, we could conclude
that the introduction of a phenyl substituted or smaller group can have
a better affinity with NLuc, and the introduction of fluorine atom can
extend the half-life of bioluminescence.
Fig. 1. Chemical structures of furimazine and novel compounds in this article.
The chemiluminescence properties of furimazine and all new com-
pounds were also measured (Fig. S2 and Table S1).
Subsequently, all compounds were investigated with A549 cells that
expressing NLuc. Compounds A2, A6, A9 and A11 emerged decent
bioluminescent properties in cellular as shown in Figs. 3 and 4. In the
aspect of bioluminescence intensity, all the compounds were inferior to
furimazine, the best compound A11 displayed 60% bioluminescence
intensity compared to furimazine. In the area of the time-dependent
bioluminescence, A2, A6, A9 and A11 displayed better bioluminescence
persistent. Although furimazine performed superior to these com-
pounds in the ear period, after about 60 min these compounds sur-
passed furimazine. Meanwhile, the bioluminescence emitted by these
compounds could increase as the cell concentration increases. In ad-
dition, all compounds did not influence the cell viability when they
were used in the bioluminescence assay (Table S2).
Next, we evaluated the selectivity of all compounds for NLuc by
taking advantage of ES-2 cells that expressing Rluc. As shown in Fig. 5,
no compounds could bring out the detectable bright. This result in-
dicated that these compounds possessed the high selective for NLuc.
In view of the superior results in vitro, we further investigated the
bioluminescence properties of the best performing A6 and A11 in vivo.
We established the nude mice model transplanted with A549 cells that
expressing NLuc and chose lower (1 mM) and higher (5 mM) con-
centration for in vivo imaging. Representative images and quantification
of the bioluminescence imaging signal was provided on Figs. 6 and 7.
Compound A11 was greatly brighter than furimazine.
Scheme 1. Synthetic routes to analogues and structures. General procedures:
(
2
i) Zn, PdCl (PPh
3
)
2
, I
2
, DMF, THF, 85 °C, reflux, yield 90%; (ii) Rh
2
4
(OAC) ,
3
toluene, 100 °C, reflux, yield 70%; (iii) furfural, TMG, methanol, rt; (iv) Cs
Pd(PPh
Ac O, Et
°C, yield 39–57%.
2
CO
,
3
)
4
3
, 1,4-dioxane, 85 °C, yield 63%; (v) TFA, DCM, rt, yield 92–96%; (vi)
2
N, DMAP, DCM, 0 °C, yield 32–43%; (vii) NaBH , DCM, methanol,
4
0
group led to least bioluminescence, which revealed that the groups
were too large to combine and react with luciferase. Comparing A10
with A11, although A11 only attached benzyl hydroxyl group, A11
displayed a much higher intensity than A10, we thought this maybe
related with the hydrogen bonding and polarity. Moreover, all new
compounds had red-shifted emission compared to furimazine, as shown
in Fig. S1. The maximum peak of compound A8 was remarkably red-
shifted up to 55 nm.
The intensity of A11 was 1.4-fold brighter than furimazine at 1 mM,
and 2-fold stronger than furimazine at 5 mM. Although compound A6
was inferior to furimazine at 1 mM, it was 1.3-fold higher than fur-
imazine at 5 mM. Moreover, the bioluminescence of A6 and A11 could
last more than 1 h, which was longer than that of furimazine. Hence, we
can draw the conclusion that A11 is more suitable for bioluminescence
imaging in vivo. The reason why the compound A11 is brighter than
furimazine in vivo is probably that the red-shifted emission contributes a
lot to enhance bioluminescence performance. Moreover, the polarity of
To better express the kinetic characteristics of bioluminescence, we
conducted enzyme kinetic assays and acquired the Michaelis constant
K , the maximum rate Vmax and half-decay life by using the GraphPad
m
23,24,27
Prism software.
As shown in Table 1, Compounds A2 and A7
Fig. 2. (A) Bioluminescence imaging of all derivatives and furimazine with NLuc in 96-well plates; (B) The comparison of bioluminescence intensities of new
derivatives and furimazine at different concentrations with NLuc; (C) part amplification of (B).
2