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doi.org/10.1002/chem.202000780
Chemistry—A European Journal
venting false positive via simple Cu2+ reduction, competitive
ligand exchange or other non-bond cleavage-leading reac-
tions.[21] On the other hand, because of the irreversible nature
of the bond cleavage, the cleaved fluorophore can be accumu-
lated to result in a fluorescent signal despite potential compet-
itive binding to the copper by other molecules, which further
distinguishes the current design from other metal-based
probes that produce a fluorescent response via a simple
reversible binding with the target analyte.
guishes the present ascorbate sensing from most other current
designs of activity-based sensing that feature metal reactivity.
Ascorbate reactivity and selectivity
The ascorbate selectivity of 1-Cu, 2-Cu, 5-Cu and 6-Cu were
evaluated in more detail. Solutions of all the four complexes
are weakly emissive in aqueous buffer (pH 7.4) but showed a
rapid fluorescence turn-on upon treatment with ascorbate. For
example, 2-Cu showed a ca. 65-fold and 153-fold fluorescence
turn-on after 2 min and 30 min incubation with ascorbate, re-
spectively (Figure 2). In all cases, the ascorbate response is fast
with ca. 90% of the maximum fluorescence enhancement ob-
tained within 10–15 minutes under all the studied conditions.
For comparison, some activity-based probes for metal sensing
could have a reaction time as long as 1 to 2 hours.[18] Consider-
ing that the reaction rate will be dependent on the local con-
centrations of both the probe and ascorbate in different sam-
ples, a common 30-minute reaction time was adopted in sub-
sequent studies. In addition, all of the four compounds dis-
played good selectivity against a panel of biological and inor-
ganic reducing agents, including NADH, lipoic acid, a-
tocopherol (vitamin E), and uric acid. Incubation with other
common cellular or food components, such as sugars, amino
acids, other vitamins, and organic acids also resulted in no ap-
preciable fluorescent response (Figures 2 and S3). Importantly,
no fluorescence turn-on was observed when the probes were
treated with DHA, demonstrating a good selectivity for the re-
duced form (ascorbate).
Treatment of 1-Cu with ascorbate resulted in a 114-fold fluo-
rescence enhancement, showing the histidine brace mimic is
indeed responsive to ascorbate. Next, the responsive element
was modified to contain oxygen ligands to mimic the subset
of LMPOs that contain an axial tyrosine coordination in addi-
tion to the histidine brace.[15f] Consistent with previous re-
ports,[18,19] the reactivity of the copper complex was sensitive
to the coordination environment where 3-Cu (with a {bis(2-pyr-
idylmethyl)}(2-hydroxybenzyl)amine ligand) and 4-Cu (with a
(2-pyridylmethyl){bis(2-hydroxyethyl)}amine ligand) had no sig-
nificant fluorescent response (Figure 1 and S1).[22] On the other
hand, an increased level of fluorescence turn-on was observed
for 2-Cu, which contains a {bis(2-pyridylmethyl)}(2-methoxy-
benzyl)amine ligand. More detailed studies are necessary to
elucidate the roles and effect of different oxygen donors on
the ascorbate reactivity. To determine whether the counter ion
played an effect on the fluorescence enhancement, 1-Cu was
prepared from copper(II) chloride, perchlorate or tetrafluorobo-
rate, where no effect was observed (Figure S2). Finally, the as-
corbate reactivity of the copper(II) complex was evaluated
when the green emitting fluorescein dye (lem =510 nm) was
replaced by the cyan emitting 3-(benzothiazole-2-yl)-7-
hydroxycoumarin dye (lem =488 nm) (5-Cu and 6-Cu). The two
coumarin-based compounds were also found to be responsive
towards ascorbate, with a strong fluorescence turn-on, sug-
gesting that this activity-based sensing strategy is modular
(Figure 1). Together, these experiments demonstrate that the
bioinspired oxidation is an effective strategy for fluorescent as-
corbate sensing. It is worth noting that the selective triggering
of metal-based oxidative reactivity by neither the metal nor
the oxidant is indirect and non-trivial; thus, this further distin-
In addition to the tuning of the metal reactivity, varying the
coordination ligand in the ascorbate-responsive unit also gives
probes of different stability. For example, 5-Cu and 6-Cu differ
significantly in their stability against copper(II) dissociation, de-
spite their similar ascorbate reactivity and selectivity. Liquid
chromatography mass spectrometry (LCMS) showed complete
copper(II) dissociation from 5-Cu under the acidic chromato-
graphic conditions. On the other hand, 6-Cu elutes as an intact
complex with only partial copper(II) dissociation (Figure S14).
The higher stability of 6-Cu can be attributed to the weakly co-
ordinating methoxy group that chelates to the metal. Isother-
mal titration calorimetry experiments showed that binding of
the {bis(2-pyridylmethyl)}(2-methoxybenzyl)amine ligand to
Cu2+ is strong, with a measured Kd value of ca. 170 nm, sug-
gesting ca. 90% of the probe remained as the copper(II) com-
plex in a 10 mm probe solution (Figure S51). This result high-
lights the fact that careful design of the coordination ligand
can result in other favorable properties beyond sensitivity and
selectivity. Subsequent studies were therefore focused on 2-Cu
(AP-green) and 6-Cu (AP-cyan) because of their better stability.
To further evaluate the suitability of AP-cyan and AP-green for
fluorescent ascorbate detection in complex matrix, their fluo-
rescence response towards ascorbate was also measured in
the presence of other biological reducing agents. As shown in
Figure S4, a strong fluorescence enhancement was observed
when AP-cyan or AP-green was treated with 20 equiv. of ascor-
bate in the presence of other biological redox agents such as
NADH, a-tocopherol, CoQ10 and dopamine, albeit with a slight
Figure 1. Structures of copper(II) complex–fluorophore conjugates and their
fluorescent response towards twenty equivalents of ascorbate after a reac-
tion time of 30 min.
Chem. Eur. J. 2020, 26, 1 – 8
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