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was developed [72], which comprises a phosphine-tethered fluorophore (fluorescein)
that is quenched intramolecularly by an ester-linked F orster resonance energy trans-
fer (FRET) quencher disperse red 1. Upon Staudinger ligation, the ester is cleaved,
resulting in the release of the quencher, leading to a concomitant fluorescence increase
of the labeled molecule.
While fluorogenic phosphines have partially solved the problem of low signal-
to-noise ratios, the issue of tissue autofluorescence has complicated efforts to image
glycans of animals. In this respect, bioluminescence imaging (BLI) is gaining appreci-
ation for real-time animal imaging, since the total absence of tissue luminescence con-
fers negligible background. Recently, a phosphine-luciferin conjugate was reported
for real-time BLI of azidosugars of live cells [73]. Upon Staudinger ligation, ester
bound luciferin is released which can then freely diffuse into the cell where it is
converted into the bioluminescent oxyluciferin by luciferase.
8.5 Cu
I
-CATALYZED AZIDE-ALKYNE CYCLOADDITIONS (CuAAC)
The independent discovery by Sharpless [74] and Meldal [75] that the Huisgen's
1,3-dipolar cycloaddition of azides and alkynes can be catalyzed by Cu
I
has revolu-
tionized the field of bioorthogonal ligation. The resulting 1,4-disubstituted triazole
adducts are formed in high yields with exclusive regioselectivity and have excep-
tional high functional group tolerance making it possible to modify biomolecules
such as proteins, carbohydrates, lipids, and oligonucleotides. The rate of cycloaddi-
tion can be further accelerated by employing Cu
I
-stabilizing ligands such as tris[(1-
benzyl-1
H
-1,2,3-triazol-4-yl)methyl]amine (TBTA) [76] and the more polar tris(3-
hydroxypropyltriazolylmethyl)amine (THPTA) [77].
Despite many attractive features of CuAAC, the cytotoxicity of Cu
I
ham-
pers applications to living systems. To address this issue, a series of water-
soluble chelating ligands have been developed that make the CuAAC more bio-
compatible (Fig. 8.3) [78-82]. In particular, 2-4-(bis1-tert-butyl-1
H
-1,2,3-triazol-4-
yl)methylamino(methyl-1
H
-1,2,3-triazol-1-yl)ethanesulfonic acid (BTTES) exhibits
an appropriate balance between reactivity and solubility and is effective in promoting
the CuAAC in aqueous media [78]. While its bulky,
t
-Bu groups enhance the rate of
cycloaddition (three times faster than TBTA), the sulfate moiety minimizes cell per-
meability of the coordinated copper. BTTES-Cu
I
-catalyzed click reactions have been
successfully applied for the rapid detection of alkyne-modified sialylated glycans of
live cell surface with robust labeling achieved within one minute [78]. The biocom-
patibility of the BTTES-Cu
I
system was further demonstrated by
in vivo
imaging of
fucosylated glycans dynamic changes during zebrafish early development. However,
in this study some toxicity was observed and some of the treated embryos exhibited
minor developmental defects [78]. To overcome this shortcoming, Wu and coworkers
developed BTTAA [80], which is a carboxylic acid analog of BTTES, and the use of
this reagent did not lead to developmental defects. These new chelating ligands are
paving the way for rapid, noninvasive imaging of biomolecules in living organisms.
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