Chemistry Reference
In-Depth Information
processed by the GalNAc salvage pathway to form the intermediate uridine diphospho
UDP-GalNAz, which is utilized by a family of polypeptide GalNAc transferases in the
Golgi compartment, resulting in the formation of mucins that have a GalNAz moiety.
Modified fucose derivatives have been employed as a chemical reporter [58-60].
In this case, the salvage pathway converts the fucose analogs into the corresponding
GDP-fucose, which is then employed by fucosyltransferases for the biosynthesis of
glycoconjugates. It has been found that the salvage pathway and fucosyltransferases
tolerate modifications at C-6 of fucose. 6-Azido-6-deoxy-fucose was the first sugar
to be investigated as a chemical reporter, however, it was found that this derivative
exhibited some cytotoxicity. Fortunately, 6-alkynylfucose (FucAl) is accepted by
the fucose salvage pathway and exhibits lower toxicity. The alkyne group provides
a bioorthogonal reporter that can be chemoselectively labeled with azido-containing
probes using Cu
I
-catalyzed [3
2] cycloadditions, which will be described in detail
below [58-60]. It has also been found that 4-pentynoyl mannosamine (Ac
4
ManNAl)
is metabolically incorporated into cultured cells and mice with greater efficiency
than Ac
4
ManNAz [61]. This result underscores the sensitivity of sialic acid
biosynthetic enzymes to subtle differences in the
N
-acyl structures of ManNAc
analogs.
O
-GlcNAc-modified proteins, which occur in the cytosol and nucleus, have also
been labeled with bioorthogonal chemical reporters by using per-
O
-acetylated
N
-
azidoacetylglucosamine (Ac
4
GlcNAz) [62]. This azidosugar is modified by the Glc-
NAc salvage pathway enzymes to form UDP-GlcNAz, which is used as a substrate
by the cytosolic
O
-GlcNAc transferase. More recently, it was shown that GalNAz
can be converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz
and then epimerized to UDP-
N
-GlcNAz.
O
-GlcNAc transferase can then append the
azidosugar onto its native substrates, which can then be detected by covalent labeling
using azide-reactive chemical probes [63]. This approach was employed for affinity-
purification and identification of a large number of
O
-GlcNAc-modified proteins.
Another attractive approach to study
O
-GlcNAc modification of proteins exploits
an engineered galactosyltransferase enzyme to selectively label
O
-GlcNAc proteins
with an azide-biotin tag [64, 65]. The tag permits enrichment of low-abundance
O
-
GlcNAc species from complex mixtures and localization of the modification to short
amino acid sequences. Using this approach, changes in
O
-GlcNAc glycosylation on
several proteins involved in the regulation of transcription and mRNA translocation
were detected. Also, it provided evidence that
O
-GlcNAc glycosylation is dynami-
cally modulated by excitatory stimulation of the brain
in vivo.
Glycoconjugates containing azido or alkyne moieties can also be introduced in
living systems by enzymatic transformations, and, for example, Wu and coworkers
[66] took advantage of a mutant
Helicobacter pylori
+
(1,3)-fucosyltransferase, which
converts LacNAc to the Le
X
trisaccharide by transferring a fucose residue from GDP-
fucose to the C-3 hydroxyl of the GlcNAc in the LacNAc unit. This enzyme exhibits
promiscuity for GDP-fucose, and analogs containing a C-6 azido or alkyne moiety
at fucose are also well tolerated by this enzyme thereby providing opportunities for
installing these moieties in LacNAc-containing glycoconjugates. Lec2 cells, which
express complex and hybrid
N
-glycans mainly terminating in LacNAc residues,
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