118
4 Applications of Nitrile Imine Derivatives
with the biomolecule in question. The accumulation of the bis-carboxylate species
known as fumarate can often be indicative of the poor regulation of metabolism,
a common characteristic of some cancers. Investigations by Meier have elucidated
that 1,3-dipolar cycloaddition between an NI and the dipolarophilic fumarate can
also form highly fluorescent pyrazolines, representing a quantitative approach for
the detection of fumarate levels in a biological system via an off → on chemical
probe (Scheme 4.20) [64, 65]. While initial studies employing hydrazonyl chlorides
observed a near 100-fold increase in fluorescence in the presence of excess fumarate,
this was driven to a 400-fold increase when 2,5-tetrazoles were applied [65].
Lin has also exemplified the self-reporting properties of the NI-fumarate cycloaddition, using 2,5-tetrazoles augmented with a BODIPY chromophore [66]. In contrast
to the cycloaddition products employed in earlier reports, the BODIPY-pyrazoline
fumarate adducts do not exhibit a fluorophore, as the fluorescence of the BODIPY
moiety is quenched by the pyrazoline, and vice versa. The starting 2,5-tetrazole, however, is highly fluorescent in the absence of the pyrazoline, making this an example
of an on → off chemical probe (Scheme 4.20). The authors also demonstrated that
some of the fluorescent signal could be recovered following the addition of hydrogen
peroxide, which oxidises the pyrazoline quencher to the relatively inert pyrazole.
While most publications in this field focus on the photochemical properties of
the NI-cycloaddition products, these dipoles can also be employed in conjunction
with other visualising agents. In 2019, Wittman engineered the visualisation of complex carbohydrates using the cycloaddition of the NI and an unnatural mannosamine
monomer modified with an acrylamide unit [67]. In this case, however, the reaction
product was not analysed using the fluorescence signal from the resulting pyrazoline. Instead, biotin was incorporated within the 2,5-tetrazole species. Following
cycloaddition, the resulting adduct was treated with streptavidin modified with a
N
N
N
N
h
DMSO/PBS
rt, 2 min
O
O
O
O
fumarate
H
N
O
N 3
N
N
H
N
O
N 3
O
O
O
O
N N
N
N
S
S
O
OH
O
O
N
B
N
F F
N
N
S
S
O
OH
O
O
N
B
N
F F
O
O
O
O
h
OPTI-MEM serum
rt, 2 min
O
O
O
O
fumarate
quenching
OFF
ON
OFF
ON
Scheme 4.20 The application of NIs in the detection of the oncometabolite fumarate
4 Applications of Nitrile Imine Derivatives
with the biomolecule in question. The accumulation of the bis-carboxylate species
known as fumarate can often be indicative of the poor regulation of metabolism,
a common characteristic of some cancers. Investigations by Meier have elucidated
that 1,3-dipolar cycloaddition between an NI and the dipolarophilic fumarate can
also form highly fluorescent pyrazolines, representing a quantitative approach for
the detection of fumarate levels in a biological system via an off → on chemical
probe (Scheme 4.20) [64, 65]. While initial studies employing hydrazonyl chlorides
observed a near 100-fold increase in fluorescence in the presence of excess fumarate,
this was driven to a 400-fold increase when 2,5-tetrazoles were applied [65].
Lin has also exemplified the self-reporting properties of the NI-fumarate cycloaddition, using 2,5-tetrazoles augmented with a BODIPY chromophore [66]. In contrast
to the cycloaddition products employed in earlier reports, the BODIPY-pyrazoline
fumarate adducts do not exhibit a fluorophore, as the fluorescence of the BODIPY
moiety is quenched by the pyrazoline, and vice versa. The starting 2,5-tetrazole, however, is highly fluorescent in the absence of the pyrazoline, making this an example
of an on → off chemical probe (Scheme 4.20). The authors also demonstrated that
some of the fluorescent signal could be recovered following the addition of hydrogen
peroxide, which oxidises the pyrazoline quencher to the relatively inert pyrazole.
While most publications in this field focus on the photochemical properties of
the NI-cycloaddition products, these dipoles can also be employed in conjunction
with other visualising agents. In 2019, Wittman engineered the visualisation of complex carbohydrates using the cycloaddition of the NI and an unnatural mannosamine
monomer modified with an acrylamide unit [67]. In this case, however, the reaction
product was not analysed using the fluorescence signal from the resulting pyrazoline. Instead, biotin was incorporated within the 2,5-tetrazole species. Following
cycloaddition, the resulting adduct was treated with streptavidin modified with a
N
N
N
N
h
DMSO/PBS
rt, 2 min
O
O
O
O
fumarate
H
N
O
N 3
N
N
H
N
O
N 3
O
O
O
O
N N
N
N
S
S
O
OH
O
O
N
B
N
F F
N
N
S
S
O
OH
O
O
N
B
N
F F
O
O
O
O
h
OPTI-MEM serum
rt, 2 min
O
O
O
O
fumarate
quenching
OFF
ON
OFF
ON
Scheme 4.20 The application of NIs in the detection of the oncometabolite fumarate