Synthesis and Characterization of Versatile O-Glycan Precursors for Cellular O-Glycomics

Article abstract of DOI:10.1021/acssynbio.9b00168

Protein O-glycosylation is a universal post-translational modification and plays essential roles in many biological processes. Recently we reported a technology termed cellular O-glycome reporter/amplification (CORA) to amplify and profile mucin-type O-glycans of living cells growing in the presence of peracetylated Benzyl-α-GalNAc (Ac₃GalNAc-α-Bn). However, the application and development of the CORA method are limited by the properties of the precursor benzyl aglycone, which is relatively inert to further chemical modifications. Here we described a rapid parallel microwave-assisted synthesis of Ac₃GalNAc-α-Bn derivatives to identify versatile precursors for cellular O-glycomics. In total, 26 derivatives, including fluorescent and bioorthogonal reactive ones, were successfully synthesized. The precursors were evaluated for their activity as acceptors for T-synthase and for their ability to function as CORA precursors. Several of the precursors possessing useful functional groups were more efficient than Ac₃GalNAc-α-Bn as T-synthase acceptors and cellular O-glycome reporters. These precursors will advance the CORA technology for studies of functional O-glycomics.

Full text of DOI:10.1021/acssynbio.9b00168

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Article
Synthesis and Characterization of Versatile O-
glycan Precursors for Cellular O-glycomics
Qing Zhang, Zhonghua Li, Tatiana Chernova, Varma Saikam, Richard D.
Cummings, Xuezheng Song, Tongzhong Ju, David F Smith, and Peng G. Wang
ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00168 • Publication Date (Web): 22 Oct 2019
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ACS Synthetic Biology
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Synthesis and Characterization of Versatile O-glycan Precursors for
Cellular O-glycomics
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†§
‡§
‡
†
∆
Qing Zhang, Zhonghua Li, Tatiana Chernova, Varma Saikam, Richard Cummings, Xue-
‡
◊
‡
†
zheng Song, Tongzhong Ju,* David F. Smith* and Peng G. Wang*
†Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, USA
‡
Emory Comprehensive Glycomics Core, Department of Biochemistry, Emory University School of Medicine, At-
lanta, GA, USA
∆Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
◊Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration,
Silver Spring, MD, USA
ABSTRACT: Protein O-glycosylation is a universal post-
translational modification and plays essential roles in many
biological processes. Recently we reported a technology
termed Cellular O-Glycome Reporter/Amplification
(
CORA) to amplify and profile mucin-type O-glycans of liv-
ing cells growing in the presence of peracetylated Benzyl--
GalNAc (Ac GalNAc-α-Bn). However, the application and
3
development of the CORA method are limited by the prop-
erties of the precursor benzyl aglycone, which is relatively
inert to further chemical modifications. Here we described
a rapid parallel microwave-assisted synthesis of Ac GalNAc-
3
α-Bn derivatives to identify versatile precursors for cellular
O-glycomics. In total, 26 derivatives, including fluorescent
and bioorthogonal reactive ones, were successfully synthe-
sized. The precursors were evaluated for their activity as ac-
ceptors for T-synthase and for their ability to function as
3
CORA precursors. Several of the precursors possessing useful functional groups were more efficient than Ac GalNAc-α-Bn as T-
synthase acceptors and cellular O-Glycome reporters. These precursors will advance the CORA technology for studies of functional
O-glycomics.
Keyword: O-glycosylation, CORA, mucin-type O-glycans, O-glycan precursors, T-synthase
Protein glycosylation is the most common post-translational
modification and plays key roles in diverse fundamental
cellular processes . The highly regulated repertoire of cellular
that traverse the secretory apparatus, is one of the most
common types of protein glycosylation and is important in
many normal and pathologic settings . In contrast to N-
1
4-7
glycans are involed in many cellular mechanisms that
contribute to health and disease, largely through interactioins
with glycan binding proteins and sterically modulating
molecular interactions. Due to the inefficiency and the lack of
efficient and unbiased strategies for releasing O-glycans from
complex samples, current technologies for evaluating O-
glycans require relatively large amounts of biological samples
for detailed structural analysis, which is a major limitation for
functional glycomics. Hence, there is an urgent need to
develop a simple and sensitive method to obtain all of the
glycans, which can be released enzymatically, O-glycans
require chemical strategies for their release, primarily through
alkaline β-elimination, which is inefficient and may result in
O-glycan degradation. To address these problems, we recently
developed a technology termed Cellular O-glycome Re-
8
porter/Amplification (CORA) , which uses peracetylated Ben-
zyl-α-GalNAc (Ac GalNAc-α-Bn) as an O-glycan precursor to
3
amplify and profile the mucin-type O-glycome in living cells,
resulting in 100 to 1000-fold increase in sensitivity of detection
by mass spectrometry (MS) over traditional methods. Isotope-
Cellular O-glycome Reporter Amplification (ICORA) was sub-
sequently developed to quantitatively analyze the O-glycome
2
glycans synthesized by cells (the cellular glycome) for
investigations in functional O-glycomics.
9
from cells using stable isotopic labeling .
Mucin type O-glycosylation with GalNAc-α-O-linkage to
3
Ser/Thr/Tyr residues, which occurs on over 80% of proteins
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GalNAc-α-Bn at relatively high concentrations (2~5 mM) was
first used as an inhibitor of cellular mucin O-glycosylation in
provides the potential for covalent coupling the isolated O-
glycans to solid surfaces for production of glycan microarrays
that present the complete O-glycome of cultured cells for
functional O-glycomics.
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human colon cancer cells , and was also used as an acceptor
1
6-
substrate for in vitro T-synthase and Core3GnT activity assay
.
1
8
However, we successfully used less than 100 μM concentra-
Herein, we described a rapid parallel synthesis of cellular O-
glycan precursors via microwave assisted reaction. By this syn-
thesis method, a library of Ac GalNAc-α-Bn derivatives was
3
tions of its peracetylated form, Ac GalNAc-α-Bn, as the CORA
3
precursor which was converted by intracellular esterases to
Bn-α-GalNAc after being taken up by living cells, and then
presumably transported into Golgi lumen as a mucin type O-
glycosylation substrate to synthesize Bn-O-glycans with no
successfully synthesized, including fluorescent and other use-
ful functional groups. The utility of these precursors was de-
termined by assessing their activity as acceptors for T-syn-
thase and their ability to function as reporter precursors in liv-
ing cells in culture for profiling O-Glycomes. The new precur-
sors may significantly advance CORA applications, including
but not limited to more sensitive analysis, isolation of O-gly-
cans by HPLC with subsequent interrogation of O-glycan li-
braries by glycan-binding proteins on microarrays, and stud-
ies of O-glycosylation pathways by monitoring fluorescence in
vivo.
8
observed side effect on cellular properties or glycosylation .
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The precursor Ac GalNAc-α-Bn, however, is limited to MS
8
profiling of the O-glycome or to comparing and quantifying
9
O-glycans using ICORA . In order to broaden the application
of CORA, we synthesized a variety of differently modified ben-
zyl aglycones to increase the sensitivity of glycan detection by
using fluorescent derivatives that permitted us to monitor the
purification of the O-glycan derivatives from tissue cultured
cells by high performance liquid chromatography (HPLC). In
addition, the installation of functional groups to the aglycone
3
Figure 1. Structure of designed Ac GalNAc-α-Bn derivatives.
3
Scheme 1. Microwave assisted synthesis of Ac GalNAc-α-Bn
derivatives.

RESULTS AND DISCUSSION
Design and Synthesis of CORA precursor candidates. In
order to expand the potential applications of CORA, we de-
velop a variety O-glycan precursors that could not only func-
tion like Ac
tively detected and directly quantified. We designed 26
Ac GalNAc-α-Bn derivatives with active functional groups,
which are shown in Figure 1. The original Ac GalNAc-α-Bn
3
GalNAc-α-Bn, but also be more versatile, sensi-
3
3
3
Scheme 2. Synthesis of Ac GalNAc-α-Bn derivatives with az-
ido group.
was used as the standard for comparison with all of the deriv-
atives with respect to desired properties. These functional
groups on the derivatives are designed to be used by well-es-
tablished methods such as fluorescent labeling strategy (fluo-
19
rescent group; 20 and 21) , copper-catalyzed azide−alkyne cy-
cloaddition (azide and alkyne groups; 18, 23, 24, 25, 26 and
2
0
21
27) , Raman reporter strategy (nitrile group; 12) , IsoTaG
22
strategy (bromide; 2, 3, 4, 5, 7, 8, 10 and 22) , and Diels−Alder
reaction (alkene group; 18) .
23
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As shown in scheme 1, compounds 2-22 were synthesized by
microwave reaction described by Beau and co-workers to
Compound
RTAA*
1
Compound
RTAA
1
2
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1
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achieve α-glycosidation from galactosamine pentaacetate .
1.81±0.30
1.93±0.12
1.99±0.10
1.55±0.11
1.19±0.01
1.96±0.07
1.67±0.04
1.92±0.14
2.17±0.34
0.59±0.03
1.63±0.04
4.69±0.20
2.26±0.39
15
16
17
18
19
20
21
22
23
24
25
26
27
3.01±0.11
3.01±0.05
0
25
This one-step reaction avoids the use of 2-azido , 2,3-trans-
2
6
27
oxazolidinone , 4,6-O-benzylidenyl , 4,6-O-di-tert-butylsi-
28
lylene or other nonparticipating temporary groups. By con-
trolling the temperature and reaction time, we obtained the
desired products in acceptable yields (38~61%). We also tried
the reflux method developed by Du and co-workers , but this
method offered more complex mixture and lower-yield of de-
sired products. Microwave assisted reaction conditions per-
mitted shorter reaction time and better α-stereoselectivity
due to higher reaction temperature. Compounds with an az-
ido group cannot be achieved using scheme 1 directly, using
established conditions, because of azide degradation in the
1.64±0.17
11.00±0.05
13.38±0.45
1.91±0.11
3.85±0.09
1.34±0.01
1.59±0.02
1.29±0.01
1.28±0.01
2.15±0.03
29
10
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presence of Cu(OTf)
pared as outlined in scheme 2. Compound 10 was treated with
NaN in MeCN to give compound 23. The nitro group on com-
pounds 13-16 were modified to azido group to produce com-
2
. Therefore, compounds 23-27 were pre-
*
RTAA: relative T-synthase acceptor activity.
3
Testing of synthesized derivatives in CORA. Due to the
promising acceptor activity of most compounds, we tested
their CORA precursor activity in living cells. Since the O-gly-
come of human lung cancer cells has not been reported, we
chose the most commonly used non-small lung cancer cell
line A549 to test this advanced CORA. We grew A549 cells in
the presence of compounds 2-27 at a concentration of 50 µM
to see whether they can produce O-glycans corresponding to
3
0
pounds 24-27 .
Testing of synthesized derivatives as acceptors of T-syn-
thase. We reasoned that in order to produce Bn-O-glycans in
the CORA procedure, new precursors would also need to be
acceptors of T-synthase, the key enzyme in the pathway for
3
1-33
synthesis of most of complex O-glycan structures . Further-
more, cell lines without functional Cosmc, a molecular chap-
erone, do not possess a functional T-synthase and cannot se-
8
the known CORA precursor compound 1 (Figure 2).
8, 31, 34-35
cret Bn-O-glycans
. Therefore, we tested the T-synthase
acceptor activity of all compounds by using HPLC to separate
and quantify the product of T-synthase.
The acceptor activity was estimated based on the amount of
labeled disaccharide generated during the reaction. Each com-
pound shown in Figure 1 was deacetylated, assayed for accep-
tor activity, which was reported as activity relative to the con-
trol compound Bn-α-GalNAc that was set to a value of 1. The
relative T-synthase acceptor activity (RTAA) of compound 2-
27 are listed in Table 1. The data showed that most of com-
pounds were better acceptors for T-synthase than Bn-α-Gal-
NAc. Compounds bearing a nitro group (13, 14, 15 and 16)
seemed to be better substrates for T-synthase than com-
pounds bearing halogen (1-12), alkyne (18) or azide groups (23-
1
2, 18
27). The known T-synthase acceptors, pNP-α-GalNAc
(
Figure 2. Testing of designed compounds in CORA.
compound 19) and 4-MU-α-GalNAc19 (compound 20) were
the most active acceptors showing more than 10-fold higher
accepter activities compared to Bn-α-GalNAc. Compound 11,
with an RTAA value of 0.59 was considered a weak acceptor,
and compound 17, with an RTAA value of zero had no acceptor
activity for T-synthase. Since all of the synthesized com-
pounds possessed an -GalNAc, there was no obvious reason
for any of them not to be an acceptor for T-synthase. There-
fore, we investigated the possibility that compound 17 might
be an inhibitor of T-synthase. We used the fluorescence-based
The result showed that 20 of the 26 designed compounds
(compounds 2-9, 13-16, 18, 21-27) functioned as precursors for
CORA as demonstrated by their ability to produce O-glycan
MS profiles similar to that from compound 1, where the differ-
ences in m/z values were corresponding to the differences in
the mass of the different aglycones (Figure S3 and Figure 2).
As expected, compound 17, which was not an acceptor of T-
synthase in vitro did not function as a CORA precursor in vivo,
and compound 11, whose RTAA value was 0.59 also was not
converted to O-glycans (Figure S4). Interestingly, there were
T-synthase acceptor substrates that did not function as pre-
cursors for CORA; namely, compounds 10, 12, 19 and 20. Alt-
hough compound 19 and 20 were the most efficient substrates
for T-synthase in vitro assays, they generated only sialyl core 1
O-Glycan peaks with low abundance compared with the pro-
1
9
assay with 4-MU-α-GalNAc as an acceptor of T-synthase , and
determined that compound 17 was, indeed, an efficient inhib-
itor of T-synthase in vitro with IC50 value of 13.4 µM Figure
S2).
Table 1. T-synthase acceptor activity of compounds
3
file of O-glycans produced using Ac GalNAc-α-Bn (Figure S4).
These data indicate that these compounds either cannot be
transported into Golgi lumen, or cannot be processed further
in vivo. Another T-synthase substrate, compound 12, merely
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Page 4 of 7
produced a few and weak O-Glycan peaks (Figure S4). Similar
S3c and S5c), which suggested that the substituted benzyl
group was conversed to methyl group after permethylation re-
action due to nitro-derivation and azido-derivation of the
Benzyl group. Compounds 25 and 26 produced O-Glycans
mainly with a 15 Da mass increase instead of a 41 Da mass in-
crease (Figure S3d and S5d), which was presumably caused
by the conversion of the azido group (42 Da) to amine group
(16 Da).
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to compound 11, compound 10 was unable to produce any O-
Glycans (Figure S4). It is worth noting that the mass of nitro
group and azido group containing CORA precursor com-
pounds (compounds 13-16 and 23-27) were unstable during the
permethylation or MALDI-MS-TOF process (Figure S3c, S3d
and Figure S5c, S5d). For example, compound 13, 15 and 16
produced O-Glycans corresponding to Bn-O-Glycans with a
76 Da mass decrease instead of a 45 Da mass increase (Figure
0
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Figure 3. The MS profiles of O-glycans derived from Ac
3
GalNAc-α-Bn (panel a) and compound 22 (panel b) by A549 cells
respectively compared to the MS profile of O-Glycans derived from an equimolar mixture of Ac
(panel c) by A549 cells. The ratio of O-glycans MS area provided an estimate of the the relative CORA precursor activity (RCPA).
3
GalNAc-α-Bn and compound 22
In order to estimate the relative CORA precursor activity of
the compounds tested, we incubated A549 cells with an
equimolar concentration of designated compound and
Table s2) so that the RCPA reflects the difference in precursor
activities. Every analysis was repeated 3 times. The RCAP data
in Table 2 showed that compound 2-9, 13, 18, 21-27 possessed
better CORA precursor activity than standard compound 1
with RCPA values ranging from 1.23 to 6.04. The compounds
14-16 were less efficient than GalNAc-α-Bn, and the RCPA val-
ues of compounds 10-12, 17, 19, 20 were zero indicating that
they were not effective CORA precursors.
3
Ac GalNAc-α-Bn at 50 M for 3 days (Table 2). The O-glycans
in media were purified, permethylated, and analyzed by
MALDI-MS-TOF. A typical result is shown in Figure 3 where
the MS profile of the O-Glycan products generated from a
mixture of compound 22 & Ac
c. O-glycans derived from compound 22 and O-glycans de-
rived from the Ac GalNAc-α-Bn were easily distinguished by
3
GalNAc-α-Bn is shown in panel
3
Table 2. CORA precursor activity of compounds
their mass differences of 93 Da. To estimate CORA precursor
activity, the total MS area of O-glycan ions generated form
Ac
relative CORA precursor activity (RCPA) was defined as the
ratio of designated compound O-glycan area/ Ac GalNAc-α-
3
GalNAc-α-Bn and compound 22 were calculated and the
3
Bn O-glycan area. In the case, the RCPA of compound 22 was
6.04 and this value together with the calculations for all of the
designated compounds is shown in Table 2. To correct for dif-
ferential signal responses in MALDI-MS-TOF for different
aglycones, the MS area ratio of the designated compound to
Compound
RCPA*
1
3.08±0.57
3.09±0.50
3.98±0.86
Compound
RCPA
1
2
3
4
15
16
17
0.33±0.04
0.17±0.03
0
3
Ac GalNAc-α-Bn was used to normalize the ratios (Figure s5,
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2.55±0.46
3.44±0.39
4.27±0.53
2.80±0.35
2.92±0.32
0
18
19
20
21
22
23
24
25
26
27
1.88±0.07
this compound did not function as a precursor for CORA. As
1
2
3
4
5
6
7
8
9
0
0
mentioned above, compound 17 inhibited T-synthase in an in
vitro assay. However, when we incubated compound 17 at an
equimolar concentration with compound 1 with A549 cells in
culture, we observed that it had no effect on the MS profile of
the GalNAc-α-Bn derived glycans. These data suggest that alt-
hough compound 17 inhibits T-synthase in vitro, it was unable
to inhibit the extension of O-glycans on the GalNAc-α-Bn in
cells (Figure.s6), indicating an inability of compound 17 to
cross the Golgi or ER membrane.
Compounds 19 and 20 presented the best acceptor ac-
tivity for T-synthase in the in vitro assay, more than 10-fold
greater than the control Bn-α-GalNAc. Compound 19, PNP-α-
GalNAc, and compound 20, 4-MU-α-GalNAc, are substrates
1.65±0.79
6.04±0.59
1.59±0.18
1.51±0.71
1.89±0.74
1.68±0.63
2.72±1.97
10
11
0
0
1
1
2
3
1.23±0.52
0.25±0.10
14
*
RCPA: relative CORA precursor activity.
1
1
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Structure-Activity Relationships. The RTAA and the RCPA
values of all compounds are summarized in Figure 4. Com-
pounds 2-9, which have a halogen substitution on the aro-
matic ring, all performed stronger as acceptors for T-synthase
and as CORA precursors relative to the control compound 1,
which suggested that a halogen on the aromatic ring may con-
tribute to improved interactions with the glycosyltransferase
or more efficient uptake into cells or across membranes of the
ER and Golgi. Because bromine has a unique isotope pattern
in MS, the Br-labeled precursors may have useful applications
in identifying the O-glycome similar to IsoTaG glycoprote-
commonly used as substrate for T-synthase enzymatic in ac-
12, 18-19
tivity in vitro assays
. It was surprising that these com-
pounds possessed no activity as CORA precursors. Com-
pounds with T-synthase acceptor activity but without CORA
precursor activity are presumably not transported into the cell
or otherwise unable to access the O-glycan synthesis machin-
ery in the Golgi. Alternatively, they may be metabolized in cy-
tosol.
We were particularly interested in the properties of
compounds 18, 23, 24, 25, 26 and 27, all of which possess either
an alkyne (18) or azido (23-27) group, which are readily with
any functional group tag by click reaction. Compound 21 con-
tains a fluorescent aglycone, which could be an ideal fluores-
cent labeled precursor without any additional reaction. The
fluorescent O-glycans can be directly monitored during HPLC
separation permitting the isolation and purification of the in-
dividual components of the O-glycans of cultured cells. Fortu-
nately, all of these compounds demonstrated CORA precursor
activity that is slightly better than our control precursor
2
2
omics .
Derivatives 10 and 11 with bromomethyl and chlorome-
thyl groups, respectively, showed good substrate activity for
T-synthase, but don’t possess CORA activity; possibly due to
substitution reaction of benzyl bromide or chloride with high
concentrations of amine and thiol groups on proteins in cells.
Precursor 12 possessing a nitrile and compounds 13, 14, 15, 16
and 19 bearing a nitro group on phenyl ring showed better en-
zymatic acceptor activity than CORA precursor activity, which
might be due to these two groups being easy to reduce to
amine groups and being consequently metabolized inside the
3
Ac GalNAc-α-Bn. Compounds 22 and 27 showed better CORA
precursor activity and substrate activity than candidates 2-4
and 24-26, which suggested a longer linker between GalNAc
and the aromatic ring is better for glycosyltransferases, espe-
cially T-synthase, to access or bind to, and/or accommodate
the acceptor.
3
6-37
cell
.
Compound 17, which also possesses a nitro group and
an additional methoxy group, was the only tested compound
that was not recognized as an acceptor by T-synthase in spite
of having a terminal -linked GalNAc. It is not surprising that
Figure 4. Relative T-synthase acceptor activity (RTAA) and relative CORA precursor activity (RCPA) of all designed precurors.
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Page 6 of 7

CONCLUSION
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In summary, we designed a rapid parallel synthesis of a library
of cellular O-glycome precursors via microwave assisted reac-
tion. A total of 26 Ac GalNAc-α-Bn derivatives, containing flu-
3

ACKNOWLEDGMENT
This study was supported in part by the Emory Comprehen-
sive Glycomics Core (ECGC), which is subsidized by the
Emory University School of Medicine and is one of the Emory
Integrated Core Facilities.
orescent moieties and other functional groups, were success-
fully synthesized by this microwave assisted method. In addi-
tion, assays of their activity as acceptors for transfer of galac-
tose to GalNAc in vitro by T-synthase and their CORA activity
on living cell in culture were followed to evaluate the utility of
these derivatives. With these new functional precursors, ap-
plication of CORA can be expanded to IsoTaG type platforms,
direct fluorescence monitoring of O-glycans produced by cells
in tissue culture for investigations of O-glycosylation path-
ways, and post-synthetic derivatizations via click chemistry of
the O-glycans of cultured cells at the reducing end of glycans
for analysis and isolation of glycans on HPLC. Installation of
appropriate functional groups will also permit immobilization
of purified O-glycan derivatives as glycan microarrays for sub-
sequent interrogation by glycan-binding proteins. These
broad applications will provide useful tools to study functional
O-glycomics.

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Supporting Information. Detailed synthetic procedures and
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*
pwang11@gsu.edu (PGW), *dfsmith@emory.edu (DFS) and *
Tongzhong.Ju@fda.hhs.gov (TJ)
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