Chemistry Reference
In-Depth Information
intensity. However, the emission maximum of the click product is at 422 nm with a
quantum yield of 0.29 when excited at 357 nm.
Metabolic labeling with alkynyl containing monosaccharides has also been
employed for glycoprotein identification and glycan mapping [83]. In this approach,
glycoconjugates of cells are metabolically labeled with an alkynyl sugar probe, which
can be selectively reacted with a biotin azide reagent using a Cu
I
-catalyzed [3
+
2]
azide-alkyne cycloaddition. Introduction of a biotin moiety permits enrichment of
glycoproteins by affinity capture with immobilized streptavidin. Tandem protein ID
and glycan site mapping were carried out on-bead. Thus, first, non-glycosylated
peptides were harvested by tryptic digestion, allowing protein identification. The
remaining resin bound glycopeptides were analyzed by treatment with
N
-glycosidase
F (PNGase), which hydrolyzes the amide bond between the biotinylated glycan and
Asn residue of the bound peptide. The shift from Asn to Asp at formerly glycosylated
sites can be identified by a differential modification of
1DaonAsninSEQUEST
searches of MS data. The approach, which was applied to analyze the sialylated
N
-linked glycoproteins of prostate cancer cells, offers a method to identify proteins
and map glycosylation sites of glycoproteins that carry a specific monosaccharide
such as sialic acid. However, it does not enable the elucidation of the nature of the
glycan at a particular glycosylation site.
+
8.6 STRAIN PROMOTED AZIDE-ALKYNE CYCLOADDITIONS (SPAAC)
Alkynes can be activated by ring strain and for example, cyclooctynes undergo [3
2]
cycloadditions with azides at ambient temperature without the need for a metal cata-
lyst (Fig. 8.4) [84-86]. The increased reactivity of cyclooctynes has been attributed
to ground-state destabilizing caused by the triple bond, however, this model has
been challenged with the introduction of the concept of dipole distortion [87-89].
The strain-promoted cycloaddition has been used for labeling biomolecules with-
out observable cytotoxicity [85]; however, the slow rate of reaction has limited the
scope of the approach [90]. Fortunately, it has been found that appending electron-
withdrawing groups to the cyclooctyne ring can increase rates of strain-promoted
cycloadditions. For example, reactions of difluorinated cyclooctyne (DIFO) with
azides proceed 60 times faster compared to an unsubstituted cyclooctyne [91]. The
rate enhancement by the
gem
-difluoro substitution has been attributed to the LUMO-
lowering effect of the fluoro substituent [91, 92]. DIFO linked to Alexa Fluor has
been successfully employed to investigate the dynamics of glycan trafficking and it
was found that after incubation for 1 hour, labeled glycans colocalize with markers
for endosomes and Golgi. DIFO-fluorophore conjugates have been used for imaging
azido-labeled biomolecules within complex biological systems such as living cells
[91], and zebrafish embryos [93] with low background fluorescence. Background
labeling has, however, been observed when samples were analyzed by Western blot-
ting, possibly due to nonspecific hydrophobic interactions or as yet uncharacterized
reactions with protein functions [33]. Therefore, a more hydrophilic azacyclooctyne
[94], which could be prepared in nine chemical steps from a glucose derivative, was
+
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