Changes in metabolic chemical reporter structure yield a selective probe of O -GlcNAc modification

Article abstract of DOI:10.1021/ja504063c

Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N- acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.

Full text of DOI:10.1021/ja504063c

Article
pubs.acs.org/JACS
Open Access on 08/20/2015
Changes in Metabolic Chemical Reporter Structure Yield a Selective
Probe of O‑GlcNAc Modification
Kelly N. Chuh,†,# Balyn W. Zaro,†,# Friedrich Piller,‡ Veronique Piller,‡ and Matthew R. Pratt*,†,§
́
§
†Department of Chemistry and Department of Molecular and Computational Biology, University of Southern California, Los
Angeles, California 90089-0744, United States
‡Centre de Biophysique Molec
ulaire, CNRS UPR4301, Universite d’Orleans and INSERM, F45071 Orleans Cedex 2, France
́ ́ ́ ́
S
* Supporting Information
ABSTRACT: Metabolic chemical reporters (MCRs) of
glycosylation are analogues of monosaccharides that contain
bioorthogonal functionalities and enable the direct visual-
ization and identification of glycoproteins from living cells.
Each MCR was initially thought to report on specific types of
glycosylation. We and others have demonstrated that several
MCRs are metabolically transformed and enter multiple
glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that
6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and
comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz)
revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination
of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the
corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro
experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme
phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated
proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data
demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the
biosynthesis of natural and unnatural carbohydrates.
modifications are unknown; however, limited biochemical
analyses demonstrate that O-GlcNAc modification can change
protein localization, stability, molecular interactions, and
activity. Critically, O-GlcNAcylation is also misregulated in
Alzheimer’s disease and cancer. For example, in neuro-
degenerative disorders such as Alzheimer’s disease, O-
GlcNAcylation levels are diminished directly leading to protein
aggregation and cell death,10 and we have demonstrated that it
likely plays a similar role in Parkinson’s disease.11 Finally,
higher levels of O-GlcNAc modification are a common feature
of many cancers and are necessary for tumorigenesis and
proliferation.12−15
To identify and characterize O-GlcNAc modifications,
complementary chemical methods have been developed.16,17
In general, these technologies take advantage of bioorthogonal
chemistries, such as the copper(I)-catalyzed azide−alkyne
cycloaddition (CuAAC or “click chemistry”, Figure 1A).18−21
This reaction relies upon small, abiotic chemical reporters
(azides and alkynes) that can be selectively reacted with alkyne-
and azido-probes, respectively, for the installation of visual-
ization and affinity tags. One of these methods, initiated by the
Bertozzi laboratory, takes advantage of monosaccharide
INTRODUCTION
■
There are three common types of protein glycosylation that
modify large numbers of protein substrates in mammalian cells.
Proteins localized to the secretory pathway and the cell surface
or secreted into the extracellular space can be modified by
oligosaccharide structures, such as N-linked glycosylation
(linked through asparagine) or mucin O-linked glycosylation
(linked through serine and threonine). Additionally, cytoplas-
mic, nuclear, and mitochondrial proteins can be substrates for
the addition of the single monosaccharide N-acetyl-glucos-
amine, termed O-GlcNAc modification (O-GlcNAcylation,
linked through serine and threonine).1−4 Unlike other forms
of glycosylation, O-GlcNAcylation is dynamic. It is added to
protein substrates by one of three isoforms of O-GlcNAc
transferase (OGT) and removed by two isoforms of O-
GlcNAcase (OGA).5 The expression of these enzymes is also
required for embryonic development in mice and Drosophi-
la.6−8 O-GlcNAc modification displays significant crosstalk with
other posttranslational modifications (PTMs), most signifi-
cantly phosphorylation and ubiquitination, setting up O-
GlcNAcylation as a key regulator of cellular pathways.9
A
wide variety of proteins have been shown to be O-GlcNAc
modified, including regulators of transcription and translation,
cytoskeletal proteins, signaling proteins, and metabolic
enzymes. The specific consequences of most of these
Received: April 23, 2014
© XXXX American Chemical Society
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that MCRs are converted in the same manner.27 Upon careful
characterization, it was demonstrated that GlcNAz can be
readily transformed to N-azidoacetyl-galactosamine (GalNAz,
Figure 1B) and vice versa, resulting in the labeling of both O-
GlcNAcylated and mucin O-linked glycosylated proteins.26−29
Furthermore, we showed that GlcNAz treatment leads to
labeling of N-linked glycosylation.26 This can be overcome
using cellular fractionation;29 however, since we have complete
chemical control over the MCR, we predicted that structural
alterations can limit this “off-target” labeling and produce an O-
GlcNAcylation-specific reporter. Indeed, we previously dem-
onstrated that an alternative MCR, N-pentynyl-glucosamine
(GlcNAlk), which contains a larger functional group at the N-
acetyl position, is not converted to the galactosamine derivative
and therefore could not label mucin O-linked glycoproteins.26
Unfortunately, GlcNAlk was still incorporated into N-linked
glycans, preventing its use as a completely selective O-
GlcNAcylation reporter.
We report here the development and application of 6-azido-
6-deoxy-N-acetyl-glucosamine (6AzGlcNAc, Figure 1B and
Scheme S1 and Figure S7 in Supporting Information (SI)) as
a MCR in living cells. Cellular analysis of this MCR using
CuAAC and fluorescent probes demonstrated that, unlike
previous reporters, it is highly selective for O-GlcNAcylated
proteins, allowing for the robust visualization of O-GlcNAc
modifications using in-gel fluorescence scanning. Furthermore,
comparative proteomics using 6AzGlcNAc, GlcNAz, and
GalNAz confirmed the specificity of 6AzGlcNAc toward O-
GlcNAc modifications. 6AzGlcNAc-labeling resulted in the
enrichment of zero proteins, out of 367, which are annotated to
have exclusively extracellular or lumenal localization. In
contrast, GlcNAz and GalNAz identified 9 and 72 such
proteins, respectively. Finally, we also demonstrate that
6AzGlcNAc can bypass an assumed biosynthetic roadblock by
being phosphorylated by the enzyme phosphoacetylglucos-
amine mutase.
Figure 1. Metabolic chemical reporters (MCRs). (A) Copper(I)-
catalyzed azide−alkyne cycloaddition (CuAAC). (B) Peracetylated
MCRs used in this study.
analogues that directly incorporate azides or alkynes into their
structures.22 These analogues, termed metabolic chemical
reporters (MCRs),23 are taken up by cells through carbohy-
drate salvage pathways and subsequently feed into the
biosynthesis of nucleotide sugar-donors for use by glycosyl-
transferases. For example, the first O-GlcNAc-targeted MCR,
N-azidoacetyl-glucosamine (GlcNAz, Figure 1B), has been used
for the visualization and proteomic identification of labeled
proteins.24−26 Unlike other methods (e.g., Western blotting),
MCRs do not necessarily read-out on endogenous levels of O-
GlcNAcylation, as they must compete with GlcNAc in the cell.
However, because they must be metabolically transformed
before their incorporation onto proteins, they not only report
on O-GlcNAc modification but also on the integration of
upstream metabolic pathways. Additionally, they can be used
much like radioactivity to isolate new modification events and
subsequent rates of removal in pulse and pulse-chase labeling
experiments. Despite the clear utility of this technology, the
previous iterations have limitations. Until recently, GlcNAz and
other MCRs were presumed to label only one type of
glycosylation (i.e., GlcNAz treatment results in O-GlcNAcyla-
tion labeling), but several enzymatic pathways exist that can
interconvert different monosaccharides, raising the possibility
Figure 2. Ac36AzGlcNAc labels proteins in living cells. (A) NIH3T3 cells were treated with Ac4GlcNAz (200 μM), Ac36AzGlcNAc (200 μM), or
DMSO vehicle for 16 h, followed by CuAAC and analysis by in-gel fluorescence scanning. (B) NIH3T3 cells were treated with varying
concentrations of Ac4GlcNAz or Ac36AzGlcNAc for 16 h, followed by CuAAC and analysis by in-gel fluorescence scanning. (C) NIH3T3 cells were
treated with Ac36AzGlcNAc (200 μM), or DMSO vehicle for the times indicated and were tested for toxicity using an MTS assay. (D) Proteins
modified by 6AzGlcNAc were enriched from NIH3T3 cells treated with Ac36AzGlcNAc (200 μM) or DMSO vehicle using CuAAC with alkyne-azo-
biotin and analyzed by Western blotting. Error bars represent SEM from three biological replicates.
B
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different short-hairpin RNA vectors targeting GalK2 using
retroviral infection and then treated with Ac36AzGlcNAc (200
μM) for 16 h. Subsequent CuAAC with alk-rho and in-gel
fluorescent scanning showed no loss of fluorescent signal,
despite a clear reduction of GalK2 mRNA as measured by
semiquantitative RT-PCR (Figure S3 (SI)), suggesting that
GalK2 is not the enzyme responsible for 6AzGlcNAc
metabolism. To confirm this result, we subjected 6AzGlcNAc
(Scheme S3A and Figures S13−S15 (SI) for details of synthesis
and characterization) to in vitro phosphorylation by recombi-
nant GalK2.36 Specifically, GalK2 was incubated with 40 mM
concentrations of GalNAc, GlcNAc, or 6AzGlcNAc and
[32P]γATP (5 mM). At these elevated substrate-concentrations,
GalK2 readily phosphorylated both GalNAc and GlcNAc but
gave no detectable modification of 6AzGlcNAc (Figure 3A).
RESULTS AND DISCUSSION
■
6AzGlcNAc Is a Robust Metabolic Chemical Reporter
in Living Cells. Our previous data using MCRs demonstrated
that even small alterations in chemical structure can have
dramatic effects on the distribution of chemical reporters into
different types of glycosylation.26,30 Therefore, to find a specific
MCR of O-GlcNAcylation, we synthesized a small panel of O-
acetylated N-acetyl-glucosamine analogues bearing azides at
different positions; the acetate protecting-groups allow diffusion
across the cell membrane and are subsequently removed by
endogenous lipases/hydrolases. NIH3T3 cells were treated
with these compounds at 200 μM concentrations for 16 h,
followed by lysis, CuAAC with an alkyne-containing rhodamine
dye (alk-rho), and analysis by in-gel fluorescent scanning. One
of these compounds, Ac36AzGlcNAc (Figure 1B), gave a
protein-labeling pattern that was subjectively similar in both
intensity and pattern to Ac4GlcNAz (Figure 2A).
To further characterize this MCR, NIH3T3 cells were treated
with various concentrations of Ac36AzGlcNAc or Ac4GlcNAz
for 16 h before reaction with alk-rho. In-gel fluorescence
scanning revealed labeling of a wide-range of proteins in
concentrations as low as 50 μM and maximal labeling achieved
at approximately 200 μM (Figure 2B), consistent with our
other MCRs of glycosylation.26,30,31 To examine the toxicity of
Ac36AzGlcNAc, the viability of NIH3T3 cells was measured
after treatment with 200 μM Ac36AzGlcNAc for 16 or 72 h
using a MTS cell-proliferation assay. Only minimal loss of cell
growth/survival was seen even after 72 h of treatment (Figure
2C). To determine if 6AzGlcNAc could report on O-GlcNAc
modifications, we treated NIH3T3 cells with Ac36AzGlcNAc
(200 μM) for 16 h. The cells were then lysed and reacted with
an alkyne-containing cleavable affinity tag (alk-azo-biotin,
Scheme S2 and Figures S8−S12 (SI)) using CuAAC. Labeled
proteins were enriched using streptavidin beads before elution
with sodium dithionite. Enriched proteins were then subjected
to Western blotting using antibodies against the known O-
GlcNAc modified proteins NEDD4,26,32 pyruvate kinase,26 and
nucleoporin 62 (nup62).33 All three proteins were selectively
enriched using 6AzGlcNAc (Figure 2D and Figure S1 (SI)),
showing that the MCR does label known O-GlcNAcylated
proteins.
6AzGlcNAc Is Metabolically Incorporated by Bypass-
ing GlcNAc-6-Kinase. MCRs are enzymatically transformed
into their nucleotide sugar-donors by monosaccharide salvage
pathways. Previous O-GlcNAc MCRs are thought to largely
utilize the GlcNAc salvage pathway (Figure S2A (SI)).24 The
first step of this pathway is the phosphorylation of MCRs at the
6-position of the carbohydrate ring by N-acetylglucosamine
kinase (GNK). This is followed by enzymatic mutation of the
phosphate to the 1-position and conversion to the uridine-
diphosphate (UDP) sugar donor by N-acetylglucosamine-
phosphate mutase (AGM1) and uridine-diphosphate-N-acetyl-
glucosamine pyrophosphorylase (AGX1/2), respectively.
Although UDP-6AzGlcNAc is known to be accepted by
OGT,34 6AzGlcNAc cannot be phosphorylated at the 6-
position, as we have replaced the 6-hydroxyl functionality with
an azide. Therefore, we first took a candidate-based approach to
identify a kinase that could directly phosphorylated 6AzGlcNAc
at the 1-position and chose N-acetyl-galactosamine kinase
(GalK2), which performs this reaction on N-acetylgalactos-
amine (GalNAc) and poorly on GlcNAc.35 To test this
possibility, NIH3T3 cells were stably transformed with five
Figure 3. Investigation of 6AzGlcNAc metabolism. (A) The indicated
monosaccharides (40 mM concentration) were tested as substrates for
purified GalK2 in vitro. (B) Proposed mechanism by which AGM1
directly phosphorylates 6AzGlcNAc in the presence of GlcNAc-6-
phosphate. (C) Kinetic constants for the enzymatic production of
UDP sugar donors from GlcNAc-1-phosphate and 6AzGlcNAc-1-
phosphate by the enzyme UDP-N-acetylhexosamine pyrophosphor-
ylase (AGX1).
Next, we tested whether phosphoacetylglucosamine mutase
(AGM1) could directly generate 6AzGlcNAc-1-phosphate.
AGM1 typically converts GlcNAc-6-phosphate to GlcNAc-1-
phosphate during the biosynthesis of UDP-GlcNAc. As part of
its enzymatic cycle, AGM1 removes the 6-phosphate from
substrate sugars, resulting in a phosphoenzyme intermediate
(Figure 3B).37 Therefore, once loaded, phosphorylated AGM1
might be capable of phosphorylating 6AzGlcNAc. To test this
possibility, human AGM1 was heterologously expressed in
Escherichia coli and purified. The enzyme was then incubated
with 6AzGlcNAc (2.25 mM) with or without different
“cofactors” that could generate phosphorylated AGM1,
specifically glucose-6-phosphate or glucose-1,6-bisphosphate
or GlcNAc-6-phosphate (all at 1 mM). To isolate any
6AzGlcNAc-1-phosphate that had been produced, the
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enzymatic reactions were first subjected to copper-free click
chemistry with a fluorescein-conjugated cyclooctyne tag.
Fluorescein-labeled compounds (i.e., 6AzGlcNAc and 6AzGlc-
NAc-1-phosphate) were then separated from the phosphory-
lated-cofactors by paper chromatography. Finally, any fluo-
rescent-spots were eluted and analyzed by mass spectrometry
(LC−MS, Figure S4 (SI)). Incubation of AGM1 with
6AzGlcNAc alone, or with glucose-6-phosphate or glucose-
1,6-bisphosphate, resulted in no detectable formation of
6AzGlcNAc-1-phosphate. However, in the presence of
GlcNAc-6-phosphate as a cofactor, the formation of 6AzGlc-
NAc-1-phosphate was unambiguously detected. This demon-
strates that direct phosphorylation of 6AzGlcNAc by AGM1
represents one pathway that circumvents the GNK biosyn-
thetic-roadblock. However, because the conversion is very low
(<1% conversion to product based on ion-intensities in ESI-
MS), AGM1 may not be the only enzyme that can produce
6AzGlcNAc-1-phosphate in living cells. We next analyzed the
final enzyme in the biosynthetic pathway, UDP-N-acetylhexos-
amine pyrophosphorylase (AGX1), by first synthesizing
6AzGlcNAc-1-phosphate (Scheme S3B and Figures S16−S30
(SI) for synthetic details and characterization). Recombinant
AGX1 was then incubated with different concentrations of
GlcNAc-1-phosphate or 6AzGlcNAc-1-phosphate and
[3H]UTP. Subsequent Michaelis−Menten kinetic analysis
demonstrated that 6AzGlcNAc-1-phosphate is a substrate of
AGX1, although at a significantly lower efficiency than GlcNAc-
1-phosphate (Figure 3C). Taken together, these data suggest
that in living cells 6AzGlcNAc can be directly phosphorylated
by AGM1 and enter the remainder of the GlcNAc salvage
pathway to generate UDP-6AzGlcNAc (Figure S2B (SI)).
6AzGlcNAc Is a General and Dynamic Metabolic
Chemical Reporter in Living Cells. Next, to explore the
generality of 6AzGlcNAc as a MCR, we labeled a panel of
different cell lines. Specifically, Cos-7, H1299, HEK293, HeLa,
MCF7, mouse embryonic fibroblasts (MEFS), and NIH3T3
cells were treated with Ac36AzGlcNAc (200 μM) for 16 h. In-
gel fluorescence scanning after CuAAC with alk-rho showed
labeling in all the cell lines examined and a diversity of the
pattern and intensity of modified proteins (Figure 4). To
qualitatively compare 6AzGlcNAc to previous MCRs of O-
GlcNAcylation, the same panel of cell lines was treated with
200 μM Ac4GlcNAz or Ac4GalNAz for 16 h (Figure S5 (SI)).
In-gel fluorescence scanning showed incorporation of pre-
viously characterized MCRs in each cell line with varying
intensities and patterns, which were more pronouncedly
different for GalNAz, when compared to 6AzGlcNAc and
GlcNAz.
Figure 4. Indicated cell lines were treated with 200 μM
Ac36AzGlcNAc for 16 h before modified proteins were subjected to
CuAAC with alk-rho and analysis by in-gel fluorescence scanning.
were washed and fresh media containing Ac4GlcNAc (200 μM)
was added. Cells were collected after different lengths of time,
lysed, and subjected to CuAAC with alk-rho. In-gel
fluorescence scanning showed a steady loss of protein labeling
over the course of 48 h (Figure 5B). O-GlcNAcase (OGA) is
responsible for the dynamic removal of O-GlcNAc from
substrate proteins. To demonstrate that 6AzGlcNAc is
incorporated into O-GlcNAcylation and a substrate for OGA,
cells were first treated with Ac36AzGlcNAc (200 μM) or
DMSO for 5 h. Media was then exchanged for fresh media
containing 200 μM Ac4GlcNAc with or without Thiamet-G (10
μM), a potent and highly selective OGA inhibitor.38 After 12 h,
cells were harvested and subjected to CuAAC with alk-rho. In-
gel fluorescence scanning showed that cells that were treated
with Thiamet-G maintained higher levels of 6AzGlcNAc
labeling compared to those without (Figure 5C), demonstrat-
ing that 6AzGlcNAc is incorporated into O-GlcNAc
modifications that can be subsequently removed by OGA.
6AzGlcNAc Is a Specific Metabolic Chemical Reporter
of O-GlcNAc Modification. As noted above, previous MCRs
of O-GlcNAcylation are not selective for O-GlcNAc mod-
ifications because they are also incorporated into either N-
linked or mucin O-linked glycans or both.26,29 To determine if
6AzGlcNAc specifically modifies O-GlcNAcylated proteins, we
first took advantage of the chimeric, secreted protein GlyCAM-
IgG that contains both an N-linked and multiple mucin O-
linked glycosylation sites. NIH3T3 cells that stably express
GlyCAM-IgG via retroviral transformation were treated with
Ac36AzGlcNAc, Ac4GlcNAz, or Ac4GlcNAc at 200 μM
concentrations for 48 h. At this time, GlyCAM-IgG was
immunoprecipitated from the media using protein-A-con-
As stated above, MCRs only report on modifications that
occur during the labeling time, raising the possibility that they
can be used to isolate O-GlcNAcylation events in a short time
frame via a pulse-labeling experiment. To determine the
kinetics of protein labeling by 6AzGlcNAc, NIH3T3 cells
were treated with Ac36AzGlcNAc (200 μM) for different
lengths of time. The cells were then lysed, reacted with alk-rho
using CuAAC, and analyzed by in-gel fluorescence scanning
(Figure 5A). Modified proteins can be clearly visualized over
background in 2 to 4 h, slightly slower than the kinetics of
protein labeling by Ac4GlcNAz at 200 μM (Figure 5A). MCRs
also have the ability to read-out on the turnover of protein
modifications using a pulse-chase format. Accordingly, we
treated NIH3T3 cells with either Ac36AzGlcNAc or
Ac4GlcNAz at concentrations of 200 μM. After 16 h, the cells
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Figure 5. Characterization of Ac36AzGlcNAc. (A) NIH3T3 cells were treated with 200 μM Ac36AzGlcNAc or Ac4GlcNAz for the indicated times,
followed by CuAAC and analysis by in-gel fluorescence scanning. (B) NIH3T3 cells were treated with 200 μM Ac36AzGlcNAc or Ac4GlcNAz for 16
h, at which time media was exchanged for fresh media containing 200 μM Ac4GlcNAc. Cells were harvested after the indicated lengths of time,
subjected to CuAAC, and analyzed by in-gel fluorescence scanning. (C) HeLa cells were treated with 200 μM Ac36AzGlcNAc or Ac4GlcNAz for 5 h,
at which time media was exchanged for fresh media containing 200 μM Ac4GlcNAc and 10 μM of the OGA inhibitor Thiamet-G or DMSO. Cells
were harvested at the times indicated and subjected to CuAAC before being analyzed by in-gel fluorescence scanning.
jugated beads. In-gel fluorescence scanning, following CuAAC
with alk-rho, showed that while GlcNAz robustly labels
GlyCAM-IgG, as expected based on our previous results,26
6AzGlcNAc does not (Figure 6A and Figure S6A (SI) for the
full-gel image). This demonstrates that while GlcNAz does
label the major types of cell-surface glycosylation, 6AzGlcNAc
does not.
Next, to confirm that 6AzGlcNAc labels O-GlcNAcylated
proteins, we treated NIH3T3 cells that were stably transfected
with the FLAG-tagged transcription factor FoxO1 with
Ac36AzGlcNAc, Ac4GlcNAz, or Ac4GlcNAc at 200 μM
concentrations for 24 h. In contrast to GlyCAM-IgG, in-gel
fluorescence showed that both MCRs robustly labeled FoxO1
(Figure 6B and Figure S6B (SI) for the expanded-gel images of
replicate experiments), demonstrating that 6AzGlcNAc is a
highly selective MCR of O-GlcNAc modifications.
To rule out the possibility that 6AzGlcNAc was excluded
from GlyCAM-IgG but labeled other cell-surface glycoproteins,
NIH3T3 cells were treated with Ac36AzGlcNAc, Ac4GlcNAz,
or Ac4GalNAz at 200 μM for 16 h before being harvested and
submitted to copper-free click chemistry using commercially
available DBCO-biotin. After subsequent incubation with
FITC-conjugated avidin, cell-surface glycoprotein labeling by
each chemical reporter was analyzed using flow cytometry. No
labeling over background was observed with Ac36AzGlcNAc,
while labeling was observed with Ac4GlcNAz and Ac4GalNAz
(Figure 6C). Notably, this corroborates live-cell flow cytometry
data from the Bertozzi lab where they used the Staudinger
ligation to observe some cell-surface labeling with GlcNAz and
no labeling with 6AzGlcNAc.39
Direct Comparison of 6AzGlcNAc, GlcNAz, and
GalNAz as Metabolic Chemical Reporters of Glycosyla-
tion. To identify the proteins labeled by 6AzGlcNAc and
compare them to those enriched by the previous MCRs
GlcNAz and GalNAz, NIH3T3 cells were treated in triplicate
with either Ac36AzGlcNAc, Ac4GlcNAz, Ac4GalNAz, or
Ac4GlcNAc as a control (all at 200 μM) for 16 h. At this
time cells were lysed using denaturing conditions (4% SDS)
and subjected to CuAAC conditions with an alkyne-bearing
biotin tag. Equivalent amounts of the differentially labeled
proteomes were then reduced, alkylated, and subjected to
biotin-enrichment using streptavidin-conjugated beads. After
extensive washing to remove unlabeled proteins, on-bead
trypsinolysis afforded peptides that were analyzed using LC−
MS/MS, identified using Proteome Discoverer and Mascot, and
quantified by spectral counting. Labeled proteins were
identified as those that met the following threshold criteria:
First, proteins must have been identified by at least 1 unique
peptide in each of the three data sets and a total of 3 spectral
Figure 6. Glycoprotein specificity of 6AzGlcNAc. NIH3T3 cells stably
expressing either GlyCAM-IgG (A) or Flag-tagged FoxO1 (B) were
treated with the indicated MCRs or Ac4GlcNAc, followed by
immunoprecipitation, CuAAC, and analysis by in-gel fluorescence
scanning. Data is representative of two independent experiments. (C)
NIH3T3 cells were treated with Ac36AzGlcNAc, Ac4GlcNAz,
Ac4GalNAz, or Ac4GlcNAc (all at 200 μM) for 16 h, at which time
cells were harvested and subjected to copper-free click chemistry with
DBCO-biotin. After incubation with FITC-avidin, live-cell surface
labeling was analyzed by flow cytometry. Error bars represent SEM
from three biological replicates.
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Figure 7. Identification of O-GlcNAcylated proteins using 6AzGlcNAc. (A) NIH3T3 cells were treated with Ac36AzGlcNAc, Ac4GlcNAz,
Ac4GalNAz, or Ac4GlcNAc (all at 200 μM) for 16 h. At this time, the corresponding cell-lysates were subjected to CuAAC with alkyne-biotin,
enrichment with streptavidin-coated beads, and on-bead trypsinolysis. Proteins identified by LC−MS/MS are graphically presented as total number
of positive minus total number of control spectral counts. Three known O-GlcNAcylated proteins are annotated in black, and three known
extracellular/lumenal proteins are annotated in red. (B) Overlap between proteins identified using 6AzGlcNAc, GlcNAz and, GalNAz. (C) Graphical
representation of enriched proteins based on whether their localization is exclusively intracellular (i.e., cytoplasmic, nuclear, or mitochondrial),
exclusively extracellular or lumenal (i.e., ER, Golgi, lysosome), or have domains in both (e.g., transmembrane protein).
counts in the sum of three replicate data sets. Second, the sum
of spectral counts of the MCR-treated samples must be 3-times
greater than those in the GlcNAc labeled samples. Finally, the
number of spectral counts in the MCR treated sample
compared to the control must be statistically significant (p <
0.05, t test). Using these criteria, 366 proteins were identified as
being labeled by 6AzGlcNAc (Table S1 (SI)), including many
known O-GlcNAcylated proteins, such as the three annotated
in black in Figure 6A (MAP4, NEDD4, and HCF1). GlcNAz
and GalNAz labeling identified 359 proteins (Table S2 (SI))
and 348 proteins (Table S3 (SI)), respectively. In contrast to
6AzGlcNAc, these lists included both known O-GlcNAcylated
proteins and proteins that are exclusively localized to the
extracellular space or the lumen of the secretory pathway and
lysosome, such as the three annotated in red in Figure 7A
(fibronectin, calumenin, and α-glucosidase). Comparison of the
three proteomics lists showed that 6AzGlcNAc has greater
overlap with GlcNAz than GalNAz (Figure 7B), consistent with
with previous studies that show more efficient incorporation of
GalNAz versus GlcNAz into cell-surface glycoproteins.39,40
Importantly, many of the proteins that were identified by
6AzGlcNAc have been previously identified in other O-GlcNAc
proteomic studies (see Tables S1, S2, and S3 (SI) for
references).
proteins that can be localized to both the cytosol and
extracellular space or lumenal compartments (e.g., trans-
membrane proteins). In contrast, 9 and 72 exclusively
extracellular or lumenal proteins were found using GlcNAz
and GalNAz treatment, respectively (Figure 7C), reenforcing
the data demonstrating the nonspecific labeling of multiple
types of glycosylation by GlcNAz and GalNAz.
CONCLUSION
■
The use of MCRs for the visualization and identification of
protein glycosylation has expanded the ability to investigate
these key posttranslational modifications. However, recent
evidence from our lab and others has demonstrated that many
MCRs of protein glycosylation lack specificity, as they are
incorporated into multiple types of glycans.26,28,29 We
previously showed that small changes to the chemical structure
of MCRs can have a large impact on their distribution into
different glycans.26,30 Following this chemical-optimization
theme further, we identified a MCR (6AzGlcNAc) that
robustly labeled a variety of proteins in living mammalian
cells. Using a fluorescent alkyne tag, we compared 6AzGlcNAc
to the previous MCR, GlcNAz, and demonstrated that
6AzGlcNAc is efficiently incorporated onto proteins allowing
visualization in as little as 2 to 4 h after treatment (Figure 5A).
Furthermore, 6AzGlcNAc removal from proteins is dependent
on the activity of OGA, demonstrating that it is dynamically
incorporated into O-GlcNAcylated proteins (Figure 5C). Using
two reporter proteins, we next showed that while GlcNAz labels
both secreted glycoproteins and O-GlcNAcylated proteins,
We next annotated the proteins in our lists based on their
characterized localizations (Figure 7C). Proteins with unchar-
acterized localizations (Uniprot database) were omitted.
Consistent with specific labeling of O-GlcNAcylated proteins,
6AzGlcNAc treatment enriched 350 exclusively intracellular
proteins (i.e., nuclear, cytosolic, and mitochondrial) and 9
F
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6AzGlcNAc is specific for O-GlcNAc modifications (Figure 6A
and B). This is consistent with our flow cytometry data (Figure
6C) and previous reports that both showed essentially no cell-
surface labeling by 6AzGlcNAc and that chemically synthesized
UDP-6AzGlcNAc is a substrate for recombinant O-GlcNAc
transferase.34,39
Unlike GlcNAz, 6AzGlcNAc cannot be metabolized to the
corresponding UDP-sugar donor by the canonical GlcNAc
salvage-pathway (Figure S2 (SI)), as the first step involves
phosphorylation at the 6-hydroxyl of the monosaccharide.
Therefore, an alternative enzyme must directly phosphorylate
6AzGlcNAc at the 1-hydroxyl to bypass this roadblock. Taking
a candidate-based approach, we tested GalK2 and AGM1 in
vitro to determine if they could generate 6AzGlcNAc-1-
phosphate. We did not observe any product formation using
GalK2, and knockdown of GalK2 in living cells using shRNA
did not result in reduced protein-labeling by 6AzGlcNAc
(Figure S3 (SI)). Notably, however, we found that AGM1 is
capable of directly generating 6AzGlcNAc-1-phosphate when
its normal substrate, GlcNAc-6-phosphate, is added to the
reaction mixture (Figures 3B and S4 (SI)).
On the basis of the enzymatic mechanism, we conclude that
AGM1 removes the phosphate from GlcNAc-6-phosphate to
generate the known phosphoenzyme intermediate,37 followed
by binding of 6AzGlcNAc and phosphorylation of the 1-
hydroxyl. This is consistent with the reversible nature of
AGM1’s activity, where the monosaccharide substrates can bind
the active site with either the 1- or 6-hydroxyl groups oriented
toward the catalytic serine. However, given the low levels of
6AzGlcNAc turnover by AGM1, it is reasonable to assume that
additional enzymes may also phosphorylate 6AzGlcNAc in
living cells. Additionally, we also showed that once 6AzGlcNAc-
1-phosphate is formed it can be enzymatically transformed to
UDP-6AzGlcNAc by AGX1 (Figure 3C). We do not know if
UDP-6AzGlcNAc can be epimerized to UDP-6AzGalNAc in
cells; however, even if this metabolite is formed, previous
studies by Bertozzi and co-workers demonstrated that UDP-
6AzGalNAc is not a substrate for the polypeptide-N-acetyl-
galactosamine transferases.40 Together, these results suggest an
unappreciated metabolic flexibility in mammalian cells. AGM1
and potentially other, yet unidentified, small-molecule
phosphotransferases may contribute to the salvaging of natural
monosaccharides from the environment. Furthermore, they
have potentially important implications for the metabolism of
bacterial or abiotic carbohydrates that would otherwise be
assumed to not enter mammalian biosynthetic pathways.
Finally, our results challenge a dogma in MCR design, which
relies on well-established metabolic pathways and directly
resulted in the previous dismissal of 6AzGlcNAc as a viable
MCR in living cells.34,39
To further confirm the specificity of 6AzGlcNAc and
demonstrate any advantages over other MCRs previously
used to study O-GlcNAcylated proteins, we performed a
proteomics experiment using 6AzGlcNAc, GlcNAz, and
GalNAz in combination with alkyne-biotin and on-bead
trypsinolysis (Figure 7). We found that enrichment with
6AzGlcNAc resulted in the identification of essentially only
intracellular proteins that cannot contain glycans (e.g., N-linked
or mucin O-linked) that are added in the secretory pathway.
This confirms the high degree of specificity of 6AzGlcNAc for
O-GlcNAcylated proteins. Consistent with our fluorescence
data, GlcNAz was less selective, resulting in the enrichment of
28 transmembrane proteins and 9 exclusively extracellular or
lumenal proteins. Finally, GalNAz was the least selective, since
it enriched only 226 exclusively intracellular proteins and 72
proteins that are only extracellular or lumenal. We believe that
this lack of selectivity is one reason why a recent study using
GalNAz required subcellular fractionation and two-dimensional
electrophoresis to identify the potential O-GlcNAcylation of
the voltage-dependent anion-selective channel protein 2
(VDAC2), while the same protein was readily identified by
6AzGlcNAc labeling without any biochemical manipulations
(Table S1 (SI)). This specificity is an improvement over other
MCRs that require biochemical manipulations (e.g., cell
fractionation) to exclude cell-surface glycoproteins.29,41
Previous direct comparisons of the selectivity of different
glycoprotein MCRs are somewhat limited.26,28−30,39 The
Bertozzi lab reported that GalNAz has superior O-GlcNAc
labeling efficiency compared to GlcNAz due to more efficient
metabolic conversion of GalNAz to UDP-GalNAz and
subsequent epimerization to UDP-GlcNAz.29 Our in-gel
fluorescence data do not support these data, as GlcNAz and
GalNAz resulted in qualitatively similar levels of protein
labeling in a variety of cell lines (Figure S5 (SI)). Interestingly,
only a minority of cell lines show similar global-patterns of
labeling between GlcNAz and GalNAz (e.g., MCF7), while
most are significantly different. This also is true of 6AzGlcNAc,
which often shows different labeling patterns and intensities
from both GlcNAz and GalNAz (Figure 4 and Figure S5 (SI)).
This supports our results that each of the MCRs is incorporated
into different types of glycoproteins and utilizes independent
metabolic enzymes for the generation of the corresponding
UDP donor sugars. Notably, while 6AzGlcNAc is the most
selective reporter of O-GlcNAcylation, it requires longer
labeling-times to achieve the same signal-to-noise as GlcNAz
(Figure 5A), highlighting a potential trade-off between labeling
efficiency and specificity. However, based on our results, we
predict that any bottlenecks in the metabolism of 6AzGlcNAc
will not dramatically hamper the visualization and identification
of O-GlcNAcylated proteins. Furthermore, the extent of O-
GlcNAcylation and identity of modified proteins has been
shown to be dependent on the cellular concentration of UDP-
GlcNAc.42−44 Therefore, it could be advantageous to have
limited metabolic conversion of an MCR to minimize the
chances of altering the endogenous repertoire of O-
GlcNAcylated proteins, as long as the labeling is above the
detection limit. We are currently exploring if different
concentrations of MCR treatment change the overall levels of
O-GlcNAcylation. Despite the increased labeling efficiency of
GlcNAz and GalNAz compared to 6AzGlcNAc, approximately
the same total number of spectral counts were found in our
comparative proteomics experiment. We believe that this could
be due to an excess of input that exceeded the capacity of the
streptavidin beads, resulting in equal total levels of protein
enrichment prior to trypsinolysis and identification.
Coupled with the ever-growing toolkit of commercially
available and custom azide-reactive tags, including terminal
alkynes, cyclooctynes, and phosphines, we predict that
6AzGlcNAc will become the most powerful and readily used
MCR for the study of O-GlcNAcylation. In particular,
metabolic labeling strategies have the unique ability to isolate
time-resolved protein modifications that only occur during cell
labeling. Furthermore, MCRs can be used in pulse-chase
experiments to measure the dynamic removal of O-GlcNAc
modifications in living cells. Finally, the successful application
of synthetic chemistry to identify a selective MCR of O-
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mercaptoethanol, pH 6.8) was then added. The samples were boiled
for 5 min at 97 °C, and 40 μg of protein was then loaded per lane for
SDS-PAGE separation (Any Kd, Criterion Gel, Bio-Rad).
In-Gel Fluorescence Scanning. Following SDS-PAGE separa-
tion, gels were scanned on a Typhoon 9400 Variable Mode Imager
(GE Healthcare) using a 532 nm for excitation and 30 nm bandpass
filter centered at 610 nm for detection.
GlcNAcylation suggests that the same chemical strategy could
be used to create reporters that are specific for other types of
protein glycosylation. Coupled with new bioorthogonal
reactions (e.g., tetrazene clycoadditions), which enable more
diverse functional groups to be incorporated into MCRs,45−47
we predict that a library of MCRs can be created to enable the
specific visualization and identification of the several types of
glycosylation in mammals and other organisms.
Reverse Transcriptase-PCR. Information about primers used for
GALK2 and GAPDH available upon request. RNA from NIH3T3 cells
was isolated using the RNAeasy Kit (Qiagen). Concentrations of RNA
were obtained by UV−vis. PCR was conducted in an Eppendorf
Mastercycler thermocycler. To a 0.2 mL thermo-walled PCR tube was
added 2× Reaction Mix (SuperScript One-Step RT-PCR with
Platinum Taq, Invitrogen), template RNA from NIH3T3 cells (1000
ng), sense and antisense primers for GALK2 (10 μM), sense and
antisense primers for GAPDH (10 μM), water, and Taq enzyme
(SuperScript One-Step RT-PCR with Platinum Taq, Invitrogen). The
provided PCR cycle was used according to SuperScript One-Step RT-
PCR with Platinum Taq (Invitrogen) with an extension time of 32 s (1
min/kbp). Products were diluted with 6× sample loading dye (Bio-
Rad) and analyzed by electrophoresis on a 5% agarose gel (500 mg of
agarose in 1× TAE buffer, tris/acetic acid/EDTA, Bio-Rad). The gel
was subsequently visualized using a ChemiDoc XRS+ molecular
imager (Bio-Rad).
GalNAc Kinase 2 (GalK2) Assay. Recombinant human GalK2 was
prepared as previously described.48 Recombinant GalK2 (8 μg mL−1)
was incubated in triplicate with GalNAc, GlcNAc or 6AzGlcNAc (40
mM) in 25 μL reaction buffer (10 mM MgCl2, 50 mM Tris HCl pH
8.0) containing 1 mg/mL BSA and 5 mM [32P]γATP (1000 cpm/
nmol) for 60 min at 37 °C. After this time, reactions were terminated
by the addition of water (0.75 mL) and applied to a Dowex 1 × 8
(Cl‑) column (0.7 cm × 3.0 cm). Unreacted starting material was
eluted by washing with 2 mL of 25 mM NH4HCO3 before eluting the
sugar-1-phosphates with 100 mM NH4HCO3. The fractions (2 mL)
were counted in a liquid scintillation counter (μBeta, PerkinElmer).
Controls without acceptor substrates were treated in the same way.
Column profiles were compared to detect the presence of overlapping
radioactive peaks corresponding to degradation products. If present,
these peaks were subtracted from the assay chromatogram.
EXPERIMENTAL PROCEDURES
■
General Information. All reagents used for chemical synthesis
were purchased from Sigma-Aldrich, Alfa Aesar or EMD Millipore
unless otherwise specified and used without further purification.
DBCO-biotin was purchased from Click Chemistry Tools. FITC-
avidin was purchased from Sigma. All anhydrous reactions were
performed under argon or nitrogen atmosphere. Analytical thin-layer
chromatography (TLC) was conducted on EMD Silica Gel 60 F254
plates with detection by ceric ammonium molybdate (CAM),
anisaldehyde or UV. For flash chromatography, 60 Å silica gel
1
(EMD) was utilized. H spectra were obtained at 400, 500, or 600
MHz on a Varian spectrometers Mercury 400, VNMRS-500, or -600.
Chemical shifts are recorded in ppm (δ) relative to solvent. Coupling
constants (J) are reported in Hz. 13C spectra were obtained at 100,
125, or 150 MHz on the same instruments.
Cell Culture. COS-7, HEK293, HeLa and MCF7 cells were
cultured in DMEM media (Corning) enriched with 10% fetal bovine
serum (Atlanta Biologicals). AmphoPack-293 retroviral packaging cells
(Clontech) were cultured in DMEM media (Corning) enriched with
10% fetal bovine serum (HyClone, ThermoScientific). NIH3T3 and
MEF cells were cultured in high-glucose DMEM media (Corning)
enriched with 10% fetal calf serum (HyClone, ThermoScientific).
H1299 cells were cultured in RPMI media enriched with 10% fetal
bovine serum (HyClone, ThermoScientific). All cell lines were
maintained in a humidified incubator at 37 °C and 5.0% CO2.
Metabolic Labeling. To cells at 80−85% confluency, media
containing Ac4GlcNAc, Ac4GlcNAz, Ac4GalNAz, Ac36AzGlcNAc
(1000× stock in DMSO), or DMSO vehicle was added as indicated.
For chase experiments, existing media was replaced with media
supplemented with 200 μM Ac4GlcNAc (Sigma) or 200 μM
Ac4GlcNAc (Sigma) plus 10 μM Thiamet-G (1000 x stock in
DMSO) as indicated.
Expression of Phosphoacetylglucosamine Mutase (AGM1).
Homo sapiens phosphoacetylglucosamine mutase 3 (AGM1/PGM3)
cDNA was obtained from Biovalley (Marne-la-Vallee, France),
́
amplified by PCR, sequenced and cloned in pTrcHis A (Invitrogen).
The 6His-tagged PGM3 protein was expressed in Escherichia coli
DH5α (Invitrogen) cultured for 24 h at 18 °C in 2TY medium
supplemented with 1 mM IPTG and 2 mM MgCl2. Bacteria were lysed
in Y-Per (ThermoScientific) and the lysate diluted with 5 volumes of
50 mM phosphate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole,
0.1 mM PMSF and 0.1 mM TCEP. After application of the lysate on a
HisTrap FF 5 mL column (GE Healthcare), AGM1 was eluted with
250 mM imidazole in 25 mM phosphate buffer pH 8.0, 150 mM NaCl,
0.1 mM TCEP. AGM1 activity was checked in a coupled assay by 2 h
incubation at 37 °C with GlcNAc-6-phosphate (2 mM), in 75 mM
Tris HCl pH 8.8, 5 mM MgCl2, containing 0.1 mg BSA and 2 μL of
the PGM3 enzyme solution, and coupling with AGX1 (0.6−6.3 μg
mL−1, 2.2−22 mU mL−1) and yeast inorganic pyrophosphatase (1.6 μg
mL−1, 3 U mL−1) in the presence of [3H]UTP (PerkinElmer, 2 mM,
260 cpm nmol−1). After AGM1/AGX1/yeast inorganic phosphatase
heat denaturation, calf intestinal alkaline phosphatase (NEB, 80 U
mL−1) was added in order to hydrolyze all the UTP and UDP present,
and the products were monitored as described below for AGX1
enzymatic tests (separation on paper chromatography). Finally, the
production of UDP-GlcNAc was estimated by scintillation counting.
Under these conditions, the recombinant AGM1 activity was
estimated to 20 nmol h−1 μg−1 of the AGM1 enzyme solution.
Phosphoacetylglucosamine Mutase (AGM1) Assay. 6AzGlc-
NAc (2.25 mM final concentration) was incubated in 100 μL of 75
mM Tris HCl pH 8.8, 5 mM MgCl2, containing 0.1 mg BSA and 10 μg
of the PGM3 enzyme solution. Incubations were run at 37 °C for 1 h
30 min either without cofactor or in the presence of Glc-6P, Glc1,6-
Preparation of NP-40-Soluble Lysates. The cells were collected
by trypsinization and pelleted by centrifugation at for 4 min at 2000g,
followed by washing 2× with PBS (1 mL). Cell pellets were then
resuspended in 100 μL of 1% NP-40 lysis buffer [1% NP-40, 150 mM
NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete, Mini,
EDTA-free Protease Inhibitor Cocktail Tablets (Roche) for 20 min
and then centrifuged for 10 min at 10 000g at 4 °C. The supernatant
(soluble cell lysate) was collected and the protein concentration was
determined by BCA assay (Pierce, ThermoScientific).
Cu(I)-Catalyzed [3 + 2] Azide−Alkyne Cycloaddition
(CuAAC). Cell lysate (200 μg) was diluted with cold 1% NP-40
lysis buffer to obtain a desired concentration of 1 μg μL−1. Newly
made click chemistry cocktail (12 μL) was added to each sample
[alkynyl-rhodamine tag (100 μM, 10 mM stock solution in DMSO);
tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (1 mM, 50 mM
freshly prepared stock solution in water); tris[(1-benzyl-1-H-1,2,3-
triazol-4-yl)methyl]amine (TBTA) (100 μM, 10 mM stock solution in
DMSO); CuSO4·5H2O (1 mM, 50 mM freshly prepared stock
solution in water) for a total reaction volume of 200 μL. The reaction
was gently vortexed and allowed to sit at room temperature for 1 h.
Upon completion, 1 mL of ice cold methanol was added to the
reaction, and it was placed at −20 °C for 2 h to precipitate proteins.
The reactions were then centrifuged at 10 000g for 10 min at 4 °C.
The supernatant was removed, the pellet was allowed to air-dry for 15
min, and then 50 μL 4% SDS buffer (4% SDS, 150 mM NaCl, 50 mM
TEA pH 7.4) was added to each sample. The mixture was sonicated in
a bath sonicator to ensure complete dissolution, and 50 μL of 2× SDS-
free loading buffer (20% glycerol, 0.2% bromophenol blue, 1.4% β-
H
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diP or GlcNAc-6P (1.00 mM final concentration). Reactions were
stopped by freezing at −20 °C. After thawing, the reaction mixtures
were incubated for 1 h at 37 °C with bicyclo[6.1.0]nonyne-(POE)3-
NH-Dye495 conjugate (Synaffix, Oss, Netherlands). The reaction
mixtures which contain the azido sugars coupled through their azido
moiety to the BCN fluorescent Dye495, were then laid onto a 46 × 57
cm sheet of Whatman 3MM Paper (GE Healthcare) and run for
descending chromatography in ethyl acetate/formic acid/water
(70:20:10) for 5 h. After drying, fluorescent spots (Rf ∼ 0.8) were
cut out and the products eluted from the paper in 50% methanol. They
were further concentrated under a vacuum before mass spectrometry
analysis.
UDP-GalNAc Pyrophorylase (AGX1) Assay. Recombinant
human AGX1 was prepared as previously described.48 Recombinant
AGX1 (0.6−6.3 μg mL−1, 2.2−22 mU mL−1) was incubated with
GlcNAc-1P or 6AzGlcNAc-1P in 25 μL reaction buffer (1 mM MgCl2,
75 mM Tris HCl pH 8.8) containing 1 mg mL−1 BSA and 2 mM
[3H]UTP (260 cpm nmol−1) for 10 min at 37 °C. Yeast inorganic
pyrophosphatase (1.6 μg mL−1, 3 mU mL−1) was also added to inhibit
the reverse reaction. Reactions were terminated by heating for 6 min at
80 °C. To hydrolyze excess UTP and UMP, calf intestinal alkaline
phosphatase (New England Biolabs, 80 U/mL) was added to the
reaction mixture and incubated for 2 h at 37 °C. The reaction was
spotted onto Whatman 3MM chromatography paper and submitted to
descending chromatography in ethyl acetate/formic acid/water
(70:20:10) to remove [3H]Uridine (Rf = 0.25). Spots with
[3H]UDP-sugars (Rf = 0.01) were cut out of the chromatography
paper and counted in a liquid scintillation counter. Control samples
without sugar-1P received the same treatment in order to deduct
background radioactivity. All tests were run in triplicates and each
experiment was repeated three times. Km and Kcat were calculated
using the Enzyme Kinetic Module 1.3 of SigmaPlot 10, from plots
obtained with different concentrations of sugar-1P and different
amounts of AGX1.
MTS Assay. NIH3T3 cells were pretreated with 200 μM
Ac36AzGlcNAc or DMSO for 72 h prior to plating. NIH-3T3 cells
(1 × 104 cells) were plated per well in a 96-well, white bottom dish 24
h before treatment with 200 μM Ac4GlcNAc or Ac36AzGlcNAc for 16
h in triplicate. CellTiter 96 AQueous Non-Radioactive Cell
Proliferation Assay (Promega, Madison, WI) was used according to
the provided protocol. Absorbance at 490 nm was read using a BioTek
Synergy H4Multi-Mode Microplate reader.
Flow Cytometry of Cell-Surface Labeling with DBCO-Biotin.
NIH3T3 cells grown in 6-well plates at 80−85% confluency were
treated with 200 μM Ac4GlcNAc, Ac4GlcNAz, Ac4GalNAz or
Ac36AzGlcNAc in triplicate for 16 h at which time media was
removed and cells were gently washed with PBS before being detached
from the plate with 1 mM EDTA in PBS. Cells were collected by
centrifugation (5 min, 300g at 4 °C) and were washed three times with
PBS (5 min, 300g at 4 °C). Cells were then resuspended in 200 μL
PBS containing DBCO-biotin (Click Chemistry Tools, 60 μM) for 1
h, after which time they were washed three times with PBS (5 min,
300g at 4 °C) before being resuspended in ice-cold PBS containing
fluorescein isothiocynate (FITC) conjugated avidin (Sigma, 5 μg/mL,
30 min at 4 °C). Cells were then washed three times in PBS (5 min,
300g at 4 °C) before being resuspended in 400 μL PBS for flow-
cytometry analysis. A total of 10 000 cells [dead cells were excluded by
treatment with propidium iodide (2.5 μg mL−1 in water, 30 min)]
were analyzed on a BD SORP LSRII Flow Cytometer using the 488
nm argon laser.
lysate (1.5 mg) was diluted as necessary to a final volume of 1 mL with
1% NP-40 buffer with Complete Mini, EDTA-free Protease Inhibitor
Cocktail Tablets (Thermo Scientific). EZview Red ANTI-FLAG M2
affinity beads (30 μL, Sigma), prewashed with cold NP-40 buffer 2×
followed by cold PBS 2×, were added to each sample. The samples
were placed on a rotator for 2 h at 4 °C. Beads were collected by
centrifugation at 2000g for 2 min at 4 °C, and the supernatant was
carefully removed. Beads were then washed with cold PBS by rotating
for 5 min before centrifuging 2 min at 2000g. The final PBS wash was
carefully removed, and the beads were suspended in 40 μL of 4% SDS
buffer and boiled for 5 min at 97 °C. The appropriate amount of click
chemistry cocktail was added, and the reaction was allowed to proceed
for 1 h after which time 30 μL of 2× loading buffer was added.
Samples were boiled for 5 min at 97 °C. Protein samples (40 μg) were
then loaded per lane for SDS-PAGE separation (Any Kd Criterion Gel,
Bio-Rad) and imaged by in-gel fluorescence scanning.
GlyCAM-IgG labeling. NIH3T3 cells stably expressing GlyCAM-
IgG in 6-well dishes at 80−85% confluency were treated in DMEM
with 10% FCS and 200 μM Ac4GlcNAz, Ac36AzGlcNAc (1000× stock
in DMSO) or DMSO for 24 h. The media from each sample was
collected by centrifugation at 3000g for 10 min at 4 °C to remove cell
debris. The supernatant (1 mL) was incubated with 50 μL of
recombinant protein G sepharose beads (Invitrogen) in 100 mM TEA
pH 8 overnight. Beads were collected by centrifugation at 2000g for 2
min at 4 °C. Beads were washed 3× with 1 mL 100 mM TEA pH 8.
GlyCAM-Ig was eluted by addition of 50 μL of 4% SDS buffer (4%
SDS, 150 mM NaCl, 50 mM TEA pH 7.4) and boiling for 5 min at 97
°C. Protein concentration was determined by BCA assay (Thermo-
Scientific). Final SDS concentration was diluted to 0.5% by addition of
50 mM TEA pH 7.4. The appropriate amount of click chemistry
cocktail was added and the reaction was allowed to proceed for 1 h
after which time 4× loading buffer (200 mM Tris HCl, 4% SDS, 40%
glycerol, 0.4% bromophenol blue, 1.4% β-mercaptoethanol, pH 6.8)
was added. Samples were boiled for 5 min at 97 °C, and 50 μg were
loaded for SDS-PAGE separation (Any Kd Criterion Gel, Bio-Rad).
Western Blotting. Proteins were separated by SDS-PAGE before
being transferred to PVDF membrane (Bio-Rad) using standard
Western blotting procedures. All Western blots were blocked in TBST
(0.1% Tween-20, 150 mM NaCl, 10 mM Tris pH 8.0) containing 5%
nonfat milk for 1 h at rt. The blots were then incubated with the
appropriate primary antibody in blocking buffer for 1 h at rt. The anti-
FLAG antibody (Thermo) and anti-MAb414 antibody (Covance)
were used at a 1:5000 dilution and 1:1000 for detection of FoxO1 and
p62, respectively. The anti-NEDD4 antibody (Millipore) was used at a
1:10 000 dilution to detect NEDD4 and the anti-Pyruvate kinase
antibody (Abcam) was used at 1:1000. The blots were then washed
three times in TBST for 10 min and incubated with the horseradish
peroxidase (HRP)-conjugated secondary antibody for 1 h in blocking
buffer at rt. HRP-conjugated antimouse, antirabbit, antigoat and
antihuman antibodies (Jackson ImmunoResearch) were used at 1:10
000 dilutions. After being washed three more times with TBST for 10
min, the blots were developed using ECL reagents (Bio-Rad) and the
ChemiDoc XRS+ molecular imager (Bio-Rad).
Biotin Enrichment and On-Bead Trypsinolysis. NIH3T3 cell-
pellets labeled with Ac36AzGlcNAc, Ac3GlcNAz, Ac3GalNAz or
Ac4GlcNAc for 16 h were resuspended in 200 μL H2O, 60 μL
PMSF in H2O (250 mM), and 500 μL of 0.05% SDS buffer (0.05%
SDS, 10 mM TEA pH 7.4, 150 mM NaCl) with Complete Mini
protease inhibitor cocktail (Roche Biosciences). To this was added 8
μL Benzonase (Sigma), and the cells were incubated on ice for 30 min.
Then, 4% SDS buffer (2000 μL) was added, and the cells were briefly
sonicated in a bath sonicator followed by centrifugation (20 000g for
10 min at 15 °C). Soluble protein concentration was normalized by
BCA assay (Pierce, ThermoScientific) to 1 mg mL−1, and 10 mg of
total protein was subjected to the appropriate amount of click
chemistry cocktail containing alkyne-PEG3-biotin (5 mM, Click
Chemistry Tools) for 1 h, after which time 10 volumes of ice-cold
MeOH were added. Precipitation proceeded 2 h at −20 °C.
Precipitated proteins were centrifuged at 5200g for 30 min at 0 °C
and washed 3 times with 40 mL of ice-cold MeOH, with resuspension
FoxO1 Labeling. NIH3T3 cells stably expressing FLAG-tagged
FoxO1 were treated with 200 μM Ac4GlcNAz, Ac36AzGlcNAc (1000 x
stock in DMSO) or DMSO and allowed to incubate overnight. After
16 h, cells were washed with PBS, trypsinized and pelleted. Cell pellets
were resuspended in 100 μL of 1% NP-40 lysis buffer [1% NP-40, 150
mM NaCl, 50 mM triethanolamine (TEA) pH 7.4] with Complete
Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Thermo
Scientific) for 20 min and then centrifuged at 4 °C for 10 min at
10 000g. The supernatant was collected and the protein concentration
was determined by BCA assay (Pierce, ThermoScientific). Total cell
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of the pellet each time. The pellet was then air-dried for 1 h. To
capture the biotinylated proteins by streptavidin beads, the air-dried
protein pellet was resuspended in 2 mL of resuspension buffer (6 M
urea, 2 M thiourea, 10 mM HEPES pH 8.0) by bath sonication. To cap
cysteine residues, 100 μL of freshly made TCEP (200 mM stock
solution, Thermo) was then added and the mixture incubated for 30
min, followed by 40 μL of freshly prepared iodoacetamide (1 M stock
solution, Sigma) and incubation for a further 30 min in the dark.
Steptavadin beads (250 μL of a 50% slurry per sample, Thermo) were
washed 2× with 1 mL PBS and 1× with 1 mL of resuspension buffer
and resuspended in resuspension buffer (200 μL). Each sample was
combined with streptavadin beads and incubated on a rotator for 2 h.
These mixtures were then transferred to Mini Bio-Spin columns (Bio-
Rad) and placed on a vacuum manifold. Captured proteins were then
washed with agitation 5× with resuspension buffer (10 mL), 5× PBS
(10 mL), 5× with 1% SDS in PBS (10 mL), 30× with PBS (1 mL per
wash, vacuum applied between each wash), and 5× 2 M urea in PBS
(1 mL per wash, vacuum applied between each wash). Beads were
then resuspended in 2 M urea in PBS (1 mL), transferred to screw-top
tubes, and pelleted by centrifugation (2000g for 2 min). At this time,
800 μL of the supernatant was removed, leaving a volume of 200 μL.
To this bead-mixture was added 2 μL of CaCl2 (200 mM stock, 1 mM
final concentration) and 2 μL of 1 mg mL−1 sequence grade trypsin
(Promega) and incubated at 37 °C for 18 h. The resulting mixtures of
tryptic peptides and beads were transferred to Mini Bio-Spin columns
(Bio-Rad) and the eluent was collected by centrifugation (1000g for 2
min). Any remaining peptides were eluted by addition of 100 μL of 2
M urea in PBS followed by centrifugation as immediately above. The
tryptic peptides were then applied to C18 spin columns (Pierce)
according to manufacturer’s instructions, eluted with 70% acetonitrile
in H2O, and concentrated to dryness on a speedvac.
LC−MS/MS Proteomic Analysis. Peptides were desalted on a
trap column following separation on a 12 cm/75um reversed phase
C18 column (Nikkyo Technos Co., Ltd. Japan). A 3 h gradient
increasing from 10% B to% 45% B in 3 h (A: 0.1% Formic Acid, B:
Acetonitrile/0.1% Formic Acid) was delivered at 150 nL min−1. The
liquid chromatography setup (Dionex, Boston, MA, USA) was
connected to an Orbitrap XL (Thermo, San Jose, CA, USA) operated
in top-5-mode. Acquired tandem MS spectra (CID) were extracted
using ProteomeDiscoverer v. 1.3 (Thermo, Bremen, Germany) and
queried against the human Uniprot protein database using MASCOT
2.3.02 (Matrixscience, London, UK). Peptides fulfilling a Percolator
calculated 1% false discovery rate threshold were reported. All LC−
MS/MS analysis were carried out at the Proteomics Resource Center
at The Rockefeller University, New York, NY, USA. Excel files
containing identified proteins will be made available upon request.
Synthesis of Chemical Reporters and OGA Inhibitor. Known
chemical reporter Ac4GlcNAz39 was synthesized according to literature
procedures. The fluorescent detection tag alk-rho49 and the OGA
inhibitor Thiamet-G38 were also synthesized according to published
procedures.
(2 × 100 mL) and water (2 × 100 mL). Organic layer was dried over
sodium sulfate. The resulting crude mixture was purified by column
chromatography (65% ethyl acetate in hexanes) to afford 1.01 g of the
1
product in 60% yield over three steps as an alpha-beta mixture: H
NMR (400 MHz, CD3OD) of pure alpha-anomer δ (ppm) 6.18 (d, J =
3.7 Hz, 1H), 5.61 (d, J = 8.9 Hz, 1H), 5.25−5.10 (m, 2H), 4.46 (ddd, J
= 10.9, 9.0, 3.7 Hz, 1H), 3.94−3.92 (m, 1H), 3.38−3.32 (m, 1H), 3.29
(dd, J = 13.5, 5.5 Hz, 1H), 2.19 (s, 3H), 2.04 (d, J = 1.3 Hz, 6H), 1.92
(s, 3H).
Synthesis of Alkyne-azo-biotin. 4-(Prop-2-yn-1-yl)phenol (3).50
Hydroquinone (11.0 g, 0.910 mmol) and propargyl chloride (7.50 g,
0.101 mmol) were dissolved in ethanol (20 mL) under an argon
atmosphere in a three-neck flask equipped with an addition funnel.
The reaction was heated to reflux to dissolve all solids. KOH (0.10 M
in water) was added dropwise through the addition funnel. The
mixture was then stirred for 20 h upon which time the reaction was
cooled and solvent was removed by vacuum. The resulting crude
mixture was dissolved in CH2Cl2 and extracted with dilute, aqueous
KOH. The aqueous layer was then brought to a neutral pH by the
addition of 1 M HCl and subsequently extracted with CH2Cl2. The
organic layer was washed with water and dried over sodium sulfate,
filtered and concentrated. The crude mixture was purified by column
chromatography (10% ethyl acetate:hexanes) to afford the pure
1
product (4.07 g, 28%): H NMR (400 MHz, CDCl3) δ (ppm) 6.85
(dd, J = 9.1, 1.1 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.61 (dd, J = 2.4,
1.0 Hz, 2H), 2.50 (t, J = 2.4 Hz, 1H).
4-(Methoxycarbonyl)benzenediazonium chloride. To a suspen-
sion of methyl-4-amino-benzoate in 6 M HCl was added sodium
nitrite at 0 °C. The reaction was let stir for 30 min. The crude reaction
mixture was used in the subsequent reaction.
(E)-Methyl 4-((2-hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)-
benzoate (4). Compound 3 (1.30 g, 18.9 mmol) was dissolved in
water:THF (2:1) and cooled to 0 °C. Potassium carbonate (52.0 g,
376 mmol) was added and reaction let stir for 30 min upon which time
4-(methoxycarbonyl)benzenediazonium chloride was added dropwise.
The reaction was allowed to warm to rt and was stirred for 18 h. The
reaction was poured over water and extracted with ethyl acetate (3 ×
200 mL). The organic layer was dried over sodium sulfate, filtered, and
concentrated. The resulting crude mixture was purified by column
chromatography by first starting at 10% ethyl acetate:hexanes and
increasing to 20% ethyl acetate:hexanes to elute the product.
Concentration under decreased pressure affords the product as a
1
yellow oil (2.00 g, 90%): H NMR (400 MHz, CDCl3) δ (ppm) 8.20
(d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 3.1 Hz, 1H),
7.10 (dd, J = 9.1, 3.1 Hz, 1H), 7.00 (d, J = 9.1 Hz, 1H), 4.75 (d, J = 2.3
Hz, 2H), 3.97 (s, 3H), 2.56 (t, J = 2.4 Hz, 1H); 13C NMR (125 MHz,
CDCl3) δ (ppm) 166.25, 153.08, 150.83, 148.27, 137.01, 131.92,
130.80, 130.17, 128.36, 123.68, 122.02, 119.17, 111.60, 78.41, 75.86,
56.77, 52.38; MALDI-MS calculated for C17H15N2O3 [M + H]+
295.1083, found 293.9045.
(E)-4-((2-Hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)benzoic acid
(5). Compound 4 (0.124 g, 0.421 mmol) was dissolved in
tetrahydrofuran (2 mL). NaOH (0.758 mg, 1.90 mmol) dissolved in
water was added and reaction let stir 18 h. Upon completion, a color
change from purple to orange is seen. The reaction was neutralized by
the dropwise addition of acetic acid and subsequently concentrated
under reduced pressure to remove solvent. The resulting crude
mixture was dissolved in CH2Cl2 and washed with water (2 × 50 mL).
The organic layer was dried over sodium sulfate, filtered, and
concentrated. The crude mixture was column purified (8:1.5:0.5 ethyl
acetate:methanol:water) to afford the pure product as a bright orange
solid (0.825 g, 70%): 1H NMR (600 MHz, CD3OD) δ (ppm) 8.05 (d,
J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 3.0 Hz, 1H),
7.01 (dd, J = 9.2, 3.0 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 4.68 (d, J = 2.3
Hz, 2H), 2.87 (t, J = 2.4 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ
(ppm) 216.29, 178.12, 162.96, 160.46, 160.22, 148.17, 146.80, 140.14,
132.53, 132.07, 129.05, 115.05, 89.05, 88.03, 65.94, 40.41; MALDI-MS
calculated for C16H12N2O3Na [M + Na]+ 303.0740, found 302.9541.
(E)-2,5-Dioxopyrrolidin-1-yl-4-((2-hydroxy-5-(prop-2-yn-1-yl)-
phenyl)diazenyl)benzoate (6). To a solution of 5 (0.180 g, 0.642
Synthesis of Ac36AzGlcNAc. 6-O-p-Methylbenzenesulfonate-N-
acetyl-glucosamine (1). Commercially available N-acetyl-glucosamine
(1.00 g, 4.52 mmol) was dissolved in anhydrous pyridine under
nitrogen and cooled to −20 °C. p-Toluenesulfonyl chloride (1.04 g,
5.43 mmol) was dissolved in anhydrous pyridine (3 mL), and the
solution was added dropwise over 20 min to the above reaction
mixture. Upon completion of the addition, the reaction was warmed to
rt and stirred for 18 h. The mixture was concentrated by vacuum and
used without further purification.
1,3,4-Tri-O-acetyl-6-azido-6-deoxy-N-acetyl-glucosamine
(Ac36AzGlcNAc, 2).39 Compound 1 was resuspended in N,N-
dimethylformamide (20 mL) under a nitrogen atmosphere. Sodium
azide (1.47 g, 22.6 mmol) was added and the reaction was stirred for 3
d at 50 °C. The reaction mixture was then concentrated by vacuum
and resuspended in pyridine (30 mL). Acetic anhydride (10.0 mL, 66.0
mmol) was added and the mixture was stirred for 16 h at rt. Upon
completion, solvent was removed under reduced pressure and the
resulting mixture was redissolved in CH2Cl2 (200 mL) and washed
with 1 M HCl (2 × 100 mL), saturated aqueous sodium bicarbonate
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mmol) in THF under argon was added N-hydroxysuccinimide (0.177
g, 1.54 mmol) and N,N′-Dicyclohexylcarbodiimide (0.317 g, 1.54
mmol). The reaction was let stir for 18 h at rt at which time the
reaction was concentrated by vacuum. The mixture was dissolved in
ethyl acetate and filtered to remove solids. The flow-through was
concentrated and the crude product was purified by column
chromatography (1:10 ethyl acetate:CH2Cl2) to afford the product
as a dark red solid that was used in the subsequent reaction without
further purification.
(E)-4-((2-Hydroxy-5-(prop-2-yn-1-yl)phenyl)diazenyl)-N-(13-oxo-
17-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-
12-azaheptadecyl)benzamide (Alk-azo-biotin, 7). Compound 6
(0.040 g, 0.101 mmol) was dissolved in anhydrous N,N′-
dimethylformamide (1 mL) under argon. EZ-Link Amine PEG3-
Biotin (0.460 mg, 0.111 mmol) (Thermo Scientific) was added and
reaction let stir for 18 h upon which time solvent was removed by
vacuum. The resulting crude mixture was purified by RP-HPLC over a
C18 semipreparative column (The Nest Group) using a 5.5−44% B
linear gradient over 10 min before switching to a 44−100% B linear
gradient over 40 min, tR = 18 min (buffer A: 0.1% TFA in water, buffer
B: 0.1% TFA, 90% ACN in water) and lyophilized to afford the pure
product as an orange solid (0.022 g, 31%): MALDI-MS calculated for
C34H40N6O8S (oxidized at the biotin cysteine) [M + Na]+ 719.2839,
found 719.2656.
MHz, (CD3)2SO) δ 7.82 (d, J = 8.2 Hz, 1H), 7.38−7.28 (m, 5H), 4.71
(d, J = 3.5 Hz, 1H), 4.68 (d, J = 12.5 Hz, 1H), 4.31 (d, J = 12.5 Hz,
1H), 3.80 (q, J = 6.1 Hz, 1H), 3.70−3.64 (m, 2H), 3.55−3.47 (m,
2H), 3.18 (t, J = 9.02 Hz, 1H), 1.83 (s, 3H).
α-1-O-Benzyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucos-
amine (11).34 Compound 10 (2.36 g, 7.58 mmol) was coevaporated
from toluene and dissolved in anhydrous pyridine (20 mL) under an
argon atmosphere. The mixture was then cooled to −20 °C. p-
Toluenesulfonyl chloride (1.74 g, 9.10 mmol), freshly rescrystallized
from CH2Cl2, was dissolved in pyridine (7 mL) and added dropwise
over 20 min. The reaction was stirred at −20 °C for 1 h and the dry ice
bath replaced with an ice bath. The reaction was allowed to warm to
room temperature over 16 h. Upon completion, the mixture was
concentrated to remove pyridine and purified over silica gel (9:1:0.5
EtOAc:methanol:water) to afford product (1.85 g, 3.97 mmol, 52%
1
yield): H NMR (500 MHz, CD3OD) δ 7.85 (d, J = 10.4 Hz, 2H),
7.46 (d, J = 1.1 Hz, 2H), 7.36 (m, 5H), 4.76 (d, J = 4.5 Hz, 1H), 4.65
(d, J = 15 Hz, 1H), 4.45 (d, J = 14.8 Hz, 1H), 4.36 (dd, J = 2.6, 13.6
Hz, 1H), 4.27 (dd, J = 7.3, 13.6 Hz, 1H), 3.88 (dd, J = 4.6, 13.5 Hz,
1H), 3.81−3.78 (m, 1H), 3.71−3.66 (m, 1H), 3.35−3.31 (m, 1H),
2.46 (s, 3H), 1.97 (s, 3H).
3,4-Di-O-acetyl-α-1-O-benzyl-6-O-p-methylbenzenesulfonate-N-
acetyl-glucosamine (12).34 Compound 11 (1.85 g, 3.97 mmol) was
resuspended in pyridine (20 mL) and acetic anhydride (1.12 mL,
11.01 mmol). The reaction was stirred for 3 h at room temperature
after which time the reaction mixture was concentrated and purified
over silica gel (75% EtOAc in hexanes) to afford product in
quantitative yield (2.18 g, 3.97 mmol): 1H NMR (500 MHz,
CDCl3) δ 7.79 (d, J = 10.3 Hz, 2H), 7.37−7.29 (m, 7H), 5.63 (d, J
= 11.9 Hz, 1H), 5.20 (dd J = 11.7, 13.5 Hz, 1H), 4.96 (t, J = 12.1 Hz,
1H), 4.82 (d, J = 4.6 Hz, 1H), 4.67 (d, J = 14.7 Hz, 1H), 4.44 (d, J =
14.8 Hz, 1H), 4.26 (td, J = 4.6, 12.6 Hz, 1H), 4.07 (d, J = 5.0 Hz, 2H),
4.04−4.00 (m, 1H), 2.44 (s, 3H), 1.98 (d, J = 2.6 Hz, 6H), 1.86 (s,
3H).
3,4-Di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-glucos-
amine (13). Procedure adapted from published literature.34 Com-
pound 12 (969 mg, 1.76 mmol) was resuspended in methanol.
Pd(OH)2/C (10% Pd) was added and a balloon of H2 was attached.
The reaction was monitored by TLC (75% EtOAc in hexanes) and
stirred for 48 h to completion. The mixture was then filtered over a
pad of Celite and the flow-through evaporated to yield the product
(710 mg, 1.55 mmol) that was used in subsequent reactions with no
further characterization.
Diallyl(3,4-di-O-acetyl-6-O-p-methylbenzenesulfonate-N-acetyl-
glucosamine)-α-1-phosphate (14). Compound 13 (629 mg, 1.37
mmol) was coevaporated with toluene and resuspended in CH2Cl2 (10
mL) under an argon atmosphere. 5-(Ethylthio)-1H-tetrazole (1.07 g,
8.22 mmol) was added and the reaction stirred for 15 min. Diallyl-
N,N′-diisopropylphosphoramidite (1.00 g, 4.11 mmol) was added
dropwise, and the reaction stirred for 2 h until completed as
determined by TLC (5% methanol in CH2Cl2). At this time, the
reaction was cooled to −78 °C and freshly recrystallized m-
chloroperoxybenzoic acid was added (1.18 g, 6.85 mmol). The
reaction was allowed to proceed for 10 min after which time the dry
ice bath was replaced with an ice bath, and the reaction was slowly
warmed to room temperature over 1 h. Upon completion, the reaction
was diluted with CH2Cl2 (50 mL) and washed 2× each with saturated
sodium thiosulfate, saturated sodium bicarbonate, water and brine.
The organic layer was then concentrated and purified over silica gel
(35%−45% acetone in hexanes) to afford the product (717 mg,74%
yield over 2 steps): 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.4 Hz,
2H), 7.29 (d, J = 7.8 Hz, 2H), 6.06 (d, J = 9.4 Hz, 1H), 5.93−5.82 (m,
2H), 5.54 (dd, J = 3.3, 6.3 Hz, 1H), 5.32 (ddd, J = 17.1, 12.3, 1.4 Hz,
2H), 5.23 (ddd, J = 10.6, 9.6, 1.2 Hz, 1H), 5.12 (dd, J = 10.9, 9.4 Hz,
1H), 4.96 (dd, J = 10.3, 9.5 Hz, 1H), 4.54−4.49 (m, 3H), 4.25−4.21
(m, 1H), 4.19−4.15 (m, 1H), 4.04 (dd, J = 11.1, 2.6 Hz, 1H), 3.98
(dd, J = 11.1, 5.1 Hz, 1H), 2.39 (s, 3H), 1.94 (s, 3H), 1.93 (s, 3H),
1.86 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.08, 170.31, 169.06,
145.21, 132.23, 132.12, 132.07, 131.91, 131.86, 129.86, 128.05, 119.06,
118.93, 95.65, 95.60, 69.73, 69.37, 68.79, 67.67, 66.99, 51.69, 51.63,
Synthesis of 6AzGlcNAc and 6AzGlcNAc-1-phosphate. 6-O-
p-Methylbenzenesulfonate-N-acetyl-glucosamine (8). Commercially
available 2-deoxy-2-N-acetyl-glucopyranose (2.50 g, 11.3 mmol) was
coevaporated from toluene and dissolved in anhydrous pyridine (20
mL). The reaction mixture was cooled to −20 °C. p-Toluenesulfonyl
chloride (2.59 g, 13.6 mmol) was then dissolved is anhydrous pyridine
(5 mL) and added dropwise to the stirring mixture. Upon completion
of addition, the reaction was allowed to warm to room temperature
and stirred for 16 h under an argon atmosphere. To purify, the
reaction was concentrated under reduced pressure and the crude
mixture purified by column chromatography (7:1:0.5 ethyl acetate:-
1
methanol:water) to afford the product as a yellow oil (1.78 g): H
NMR (500 MHz, CD3OD) α-anomer δ 7.74 (d, J = 8.3 Hz, 2H), 7.26
(d, J = 8.2 Hz, 2H), 5.13 (d, J = 3.6 Hz, 1H), 3.99 (m, 1H), 3.90 (dd, J
= 2.2, 10.6 Hz, 1H), 3.73 (m, 1H), 3.55 (dd, J = 3.2, 13.3 Hz, 1H),
3.44 (dd, J = 5.4, 12.6 Hz, 1H), 3.38 (m, 1H), 2.39 (s, 3H), 2.01 (s,
3H). The product was used in the subsequent reaction with no further
characterization.
6-Azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc, 9).39 Com-
pound 8 (1.78 g, 4.73 mmol) was coevaporated from toluene and
dissolved in anhydrous N,N′-dimethylformamide (20 mL). Sodium
azide (1.54 g, 23.7 mmol) was then added and the reaction warmed to
50 °C. The reaction was stirred for 3 d after which time the reaction
was cooled and concentrated under reduced pressure. The crude
mixture was purified by silica gel chromatography (9:1:0.5 ethyl
acetate:methanol:water) to afford the product as a white solid (402
mg, 14% yield over 2 steps). The sugar was further purified by RP-
HPLC over a C18 semipreparative column (The Nest Group) using a
5−15% B linear gradient over 10 min, tR = 2.5 min (buffer A: 0.1%
1
TFA in water, buffer B: 0.1% TFA, 90% ACN in water): H NMR
(500 MHz, (CD3)2SO) α-anomer δ 7.69 (d, J = 8.3 Hz, 1H), 4.94
(app s, 1H), 3.78 (m, 1H), 3.62 (m, 1H), 3.50 (m, 2H), 3.37 (m, 1H),
3.10 (app t, J = 9.2 Hz, 1H), 1.83 (s, 3H); 13C NMR (125 MHz,
(CD3)2SO) β-anomer δ 169.39, 90.77, 71.85, 70.52, 70.17, 54.21,
51.60, 22.67.
α-1-O-Benzyl-N-acetyl-glucosamine (10).34 The procedure was
adapted from literature.51 Commercially available 2-deoxy-2-N-acetyl-
glucopyranose (5.00 g, 22.6 mmol) was suspended in benzyl alcohol
(50 mL) and concentrated HCl was added (1 mL). The solution was
warmed to 75 °C and stirred for 4 h after which time the reaction was
cooled and poured into diethyl ether (400 mL) with vigorous stirring.
A white precipitate was observed and the mixture left at 4 °C for 16 h.
The precipitate was then filtered and washed with diethyl ether (50
mL) to remove remaining benzyl alcohol. The filtrated was dried and
recrystallized in a minimal amount of isopropanol to afford the
product as white solid (2.48 g, 7.98 mmol, 35% yield): 1H NMR (500
K
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Article
22.87, 21.63, 20.58, 20.45; 31P NMR (500 MHz, CDCl3) δ −2.77;
APCI-HRMS calculated for C25H34NO13PSNa [M + Na]+ 642.1488,
found 642.1398
Author Contributions
#K. N. Chuh and B. W. Zaro contributed equally to this
manuscript.
Diallyl(6-O-p-methylbenzenesulfonate-N-acetyl-glucosamine)-α-
1-phosphate (15). Compound 14 (669 mg, 1.08 mmol) was
resuspended is methanol (10 mL). Freshly made NaOMe was added
dropwise until pH 9−10 was reached. The reaction was monitored by
TLC (10% methanol in CH2Cl2) and was determined complete after
1.5 h. Upon completion, the reaction was quenched with acetic acid
and concentrated to afford the crude. Silica gel chromatography (7%
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.N.C and B.W.Z are fellows of the National Science
Foundation Graduate Research Fellowship Program (DGE-
0937362). This research was supported by the Damon Runyon
Cancer Research Foundation (M.R.P), the Concern Founda-
tion (M.R.P), the Ligue Nationale contre le Cancer,
committees 44 and 45 (F.P. and V.P.), and in part by the US
National Cancer Institute of the US National Institutes of
Health (CCSG P30CA014089). LC−MS analysis was
performed at the mass spectrometry facility of the Centre de
1
methanol in CH2Cl2) yielded the product (282 mg, 57% yield): H
NMR (500 MHz, CD3OD) 7.79 (d, 2H, J = 8.3 Hz), 7.44 (d, J = 8.5
Hz, 2H), 6.02−5.92 (m, 2H), 5.59 (dd, J = 8.5, 8.5 Hz, 1H), 5.42−
5.37 (m, 2H), 5.29−5.26 (m, 2H), 4.60−4.55 (m, 4H), 4.32 (dd, J =
11.0, 1.9 Hz, 1H), 4.21 (dd, J = 11.0, 5.7 Hz, 1H), 3.93−3.89 (m, 1H),
3.86−3.82 (m, 1H), 3.64 (dd, J = 10.8, 8.8 Hz, 1H), 3.36 (t, J = 8.7 Hz,
1H), 2.46 (s, 3H), 1.98 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
173.75, 146.59, 134.05, 133.63, 133.58, 133.57, 133.53, 131.02, 129.09,
118.89, 118.87, 97.39, 97.34, 73.35, 71.15, 71.12, 70.22, 69.89, 69.84,
69.79, 69.75, 55.08, 55.01, 22.51, 21.62; 31P NMR (500 MHz, CDCl3)
δ −2.51; APCI-HRMS calculated for C21H30NO11PSNa [M + Na]+
558.1169, found 558.1154
́
Biophysique Moleculaire (CBM). LC−MS/MS proteomic
analysis was performed at the Rockefeller University
Proteomics Resource Center. Flow cytometry was performed
in the USC Flow Cytometry Core Facility. Measurement of the
MTS assay was performed at the USC Nano Biophysics Core
Facility. The authors thank J. M. Conn, J. Fernandez and H.
Molina for assistance with proteomics data analysis and C.
Colas and G. Gabant (CBM) for assistance with LC−MS
analysis.
Diallyl(6-azido-6-deoxy-N-acetyl-glucosamine)-α-1-phosphate
(16). Compound 15 (282 mg, 0.527 mmol) was coevaporated from
toluene and resuspended in N,N′-dimethylformamide (20 mL) under
an argon atmosphere. Sodium azide (172 mg, 2.63 mmol) was added,
and the reaction reaction warmed to 60 °C. The reaction proceeded
for 48 h after which time the reaction was concentrated and purified
over silica gel (7:2:1 EtOAc:methanol:water) to afford the product
1
(151 mg, 71% yield): H NMR (500 MHz, CD3OD) 6.07−5.73 (m,
2H), 5.40 (dd, J = 7.4, 3.3 Hz, 1H), 5.27−5.20 (m, 2H), 5.06−5.01
(m, 2H), 4.34−4.31 (m, 2H), 4.27−4.24 (m, 2H), 3.89−3.81 (m, 2H),
3.59 (dd, J = 10.6, 8.9 Hz, 1H), 3.49 (dd, J = 13.2, 2.5 Hz, 1H), 3.36−
3.31 (m, 2H), 3.22−3.21 (m, 1H), 1.91 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 173.81, 135.99, 135.92, 116.18, 116.15, 95.59, 95.54, 73.63,
72.48, 72.37, 67.33, 67.29, 67.17, 67.13, 55.33, 55.27, 52.63, 22.85; 31P
NMR (500 MHz, CDCl3) δ 0.66, −1.37.
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Compound 16 (50 mg, 0.123 mmol) was resuspended in 4 mL of
methanol:THF (1:1) under an argon atmosphere. p-Toluenesulfinic
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* Supporting Information
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Corresponding Author
matthew.pratt@usc.edu
67, 3057.
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