Anthranilic Acid as a Versatile Fluorescent Tag and Linker for Functional Glycomics

Article abstract of DOI:10.1021/acs.bioconjchem.8b00678

The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.

Full text of DOI:10.1021/acs.bioconjchem.8b00678

Article
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/bc
Anthranilic Acid as a Versatile Fluorescent Tag and Linker for
Functional Glycomics
Yuyang Zhu, Xueyun Liu, Ying Zhang, Zhongfu Wang,‡ Yi Lasanajak, and Xuezheng Song*
†
†
†,‡
†
,†
†Department of Biochemistry, Emory Comprehensive Glycomics Core, Emory University School of Medicine, Atlanta, Georgia
0322, United States
3
‡
Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest
University, Xi’an 710069, P. R. China
*
S Supporting Information
ABSTRACT: The advancement of glycoscience is critically
dependent on the access to a large number of glycans for their
functional study. Naturally occurring glycans are considered a
viable source for diverse and biologically relevant glycan
libraries. A mixture of free reducing glycans released from
natural sources can be fluorescently tagged and separated by
chromatography to produce a natural glycan library.
Anthranilic acid (AA) has been widely used to fluorescently
tag reducing glycans for HPLC or LC/MS analysis. However,
AA conjugated glycans are not efficiently immobilized on
microarray slides due to the lack of a primary alkylamine
functional group. In this study, we have developed simple and
efficient chemistry for bioconjugation and further functionalization of glycan−AA conjugates. This new approach enables quick
preparation of glycan microarrays and neoglycoproteins from glycan−AA conjugates, which can be separated by weak anion
exchange (WAX) and C18 reversed-phase HPLC.
INTRODUCTION
specialized laboratories, setting a high hurdle to detail their
biological roles.
■
13
Glycans have crucial biological functions in many aspects of
biology, including protein folding, cell−cell adhesion and
trafficking, cell signaling, host−pathogen recognition and
An alternative route to access complex glycan structures is to
isolate them from natural sources. Diverse and biologically
relevant natural glycans, including N- and O-glycans linked to
glycoproteins, glycans linked to glycosphingolipids, and others,
can be released using chemical and/or enzymatic ap-
1
immune response, etc. With growing interest in glycans and
their functions in modern biomedical research, functional
glycomics, a systematic study of the glycome, has witnessed
significant development in the past decades. High perform-
ance liquid chromatography (HPLC), mass spectrometry
MS), and online LC-MS have greatly advanced our ability
to determine glycan structures. For functional studies, glycan
microarray has become an indispensable technology for
screening protein-glycan interactions.
14−17
2
proaches.
Because most glycans lack chromophores and
are poorly ionized, the released glycans are often derivatized to
facilitate HPLC, MS, or online LC-MS analysis. Fluorescent
tagging at the reducing end through quantitative reductive
amination has been commonly used to increase their
hydrophobicity and amplify the detection limit in both
normal-phase and reversed-phase HPLC. Among various
(
3
−9
In a glycan micro-
array experiment, multiple glycan structures are immobilized
on solid surfaces such as microscope slides for interrogation
with glycan binding proteins (GBPs), providing a high
throughput method to probe specific protein-glycan inter-
actions. The value of a glycan microarray is dictated by the
number, diversity, and biological relevance of the immobilized
glycans, and the lack of these resources is currently a major
obstacle in glycoscience. Unlike DNA, RNA, or proteins,
biosynthesis of glycans is not template-driven but is an
orchestrated process involving multiple glycosyltransferases
with distinct substrate and linkage specificities. Despite recent
advancements in chemoenzymatic synthesis of highly complex
18
fluorescent tags used in reductive amination of glycans,
anthranilic acid or 2-aminobenzoic acid (AA) is widely used,
and many glycan−AA conjugates are commercially available as
HPLC and MS standards, albeit at high prices. Meanwhile,
anthranilic acid has received much attention in biochemical
19,20
analyses as a fluorophore in FRET systems.
That the AA
fluorescent tag holds one negative charge makes it highly
flexible for HPLC separations such as hydrophilic interaction
liquid chromatography (HILIC), mixed-mode HILIC/anion
Received: September 25, 2018
Revised: October 30, 2018
Published: November 1, 2018
1
0−12
glycan structures,
the access to biomedically relevant
glycans is inadequate and limited to a number of very
©
XXXX American Chemical Society
A
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Figure 1. Strategy of the preparation of glycan−AA conjugates and versatile derivatizations of glycan−AA conjugates for further functionalization
and microarray preparation.
Figure 2. 2-AA tagged-LNnT conversion with different amines. a) Scheme of LNnT reacting with 2-AA tag and then amidating with three different
amines; b) reversed-phase HPLC profile (150 × 4.6 mm C18 column) of LNnT−AA conjugated with different amines; c) MALDI-TOF-MS of
+
+
+
purified fluorophore tagged conjugates of LNnT. Only [M + Na] ions are labeled although [M + H] and [M + K] ions are also present.
exchange, and weak anion exchange (WAX) chromatography
separations and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) analysis
a primary alkylamine for efficient immobilization on activated
solid phases including microarray slides. AEAB and AA
conjugations provide similar UV range fluorescence with
similar intensity and detectability. The installation of an AEAB
tag, however, reduces the negative charges on glycans and
affects their separation based on anion exchange chromatog-
raphy.
For preparative scale isolations of glycans, anion exchange
chromatography is an important separation mechanism. While
HILIC provides the best separation of glycans at analytical
scale (μg), the high organic content of the mobile phase poses
a significant challenge due to the low solubility of glycans. This
problem has been largely overlooked since large quantities of
18
in both positive and negative modes. A comprehensive
review discussed the pros and cons among commonly used
18
florescent tags on glycans. One major drawback of glycan−
AA conjugates is the low immobilization efficiency on amine-
reactive surfaces due to the lack of a primary alkylamine
functional group, limiting their use in glycan microarray
preparation. To address this problem, we have developed
bifunctional fluorescent tag 2-amino-N-(2-aminoethyl)-
2
1,22
benzamide (AEAB),
which can specifically conjugate
with free reducing glycans via the aromatic amine, retaining
B
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Figure 3. AA tagged-3SL and -6SL conversions under different reaction conditions. (HPLC peaks are labeled with retention time, min, and base
+
peak ion [M + Na] (m/z).) a) Scheme of two SL glycans reacting with an AA tag and then amidating with EDA; b) reversed-phase HPLC profile
(
150 × 4.6 mm C18 column) of a 6SL−AA tagged glycan conversion, bottom (black): purified 6SL−AA HPLC profile, middle (red:) conversion
using EDA solution at pH = 6.5, upper (purple): conversion using EDA solution at pH = 8−9; c) structural analysis of peaks collected from (b) by
MALDI-TOF MS/MS; d) reversed-phase HPLC profile of a 3SL−AA tagged glycan conversion, bottom (black): purified 3SL−AA HPLC profile,
middle (red): conversion using EDA solution for 3 h, upper (purple): conversion using EDA solution for 5 h; e) MALDI-TOF-MS analysis of
peaks collected (d); f) chemical structure of intraesterification product from 3-SL−AA when treated with EDA for an extended time.
complex glycans are generally not available. With more effort
directed toward generating larger amounts of natural glycans,
the solubility of glycans becomes an issue. For example, gram-
quantity isolation of sialylglycopeptide from hen egg yolk and
milligram-level separation of asparagine-linked
fluorescent tags, we re-explored the widely used AA tag. We
envisioned that besides being a fluorescent tag for HPLC and
MS, AA can serve as a versatile linker if the reactivity of the
carboxylic acid functional group can be efficiently harnessed
(
Figure 1). Here we report simple chemistry for selective
2
3
amidation of the AA tag, enabling facile immobilization of
glycans to solid phases for preparation of glycan microarray
and for synthesis of neoglycoproteins from glycan−AA
conjugates.
Glc Man GlcNAc have been reported.
We recently
1
9
2
developed a novel method called oxidative release of natural
glycans (ORNG) for large scale release of glycans linked to
proteins and glycosphingolipids directly from kilograms of
24,25
natural materials using household bleach.
To separate
RESULTS AND DISCUSSION
grams of natural glycans, anion exchange and C18 reversed-
phase chromatography are more desired due to the high water
content of the mobile phases. In searching more compatible
■
Amidation of Carboxylic Acid Group on Glycan−AA
Conjugates. The condensation reaction between carboxylic
C
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Figure 4. HPLC separation of chicken egg N-glycan AEAB and AA conjugates. a) Analytical HILIC-HPLC profiles of chicken egg N-glycan−AA
conjugates (bottom, black) and −AEAB conjugates (upper, red) on an amino column, a1−3 and a′1−3 are three well separated groups of glycans;
b) analytical WAX-HPLC profiles of chicken egg N-glycan−AA conjugates (bottom, black) and −AEAB conjugates (upper, red) on an amino
column, b1−3: three well separated groups of glycans; c) the elution profile (UV 330 nm) of crude chicken egg N-glycan−AA conjugates on
preparative WAX-HPLC (50 × 250 mm amino column). Fractions were collected based on the UV profile (flow rate: 50 mL/min).
acid and amine to form an amide bond is widely used in
bioconjugation. Many reagents have been developed to
activate carboxylic acid including carbodiimides, and additives
such as N-hydroxysuccinimde (NHS) and hydroxybenzotria-
zole (HOBt) can reduce epimerization of activated amino
Δ28 Da shift is likely due to fragmentation by N expulsion,
2
which has been reported previously on MS analysis of azido
2
8−30
compounds.
This facile amidation of glycan−AA
conjugates can quickly introduce new functionalities to glycan
structures for further bioconjugation using either amine-
reactive or clickable biomolecules or solid surfaces, making
AA a versatile linker for glycan derivatizations (Figure S3).
Selective Amidation of Carboxylic Acid Group on
Aromatic Ring of Sialylated Glycan−AA Conjugates.
Natural glycans occurring in animal tissues often contain
terminal sialic acids such as N-acetylneuraminic acid
(Neu5Ac), which play important roles in biological and
2
6,27
acids.
Our study suggests that the combination of 1-ethyl-
3
-(3-(dimethylamino)propyl)carbodiimide (EDC) and HOBt
can efficiently convert glycan−AA conjugates to aromatic
amide tagged glycans. As shown in Figure 2a, lacto-N-
neotetraose (LNnT, Galβ1−4GlcNAcβ1−3Galβ1−4Glc) as
the model compound reacted with AA to generate LNnT−AA
via reductive amination. The purified LNnT−AA conjugate
then reacted with excess amines (5% w/v amines in 0.5 M 2-
31,32
immunological as well as pathological processes.
Chemical
(
N-morpholino)ethanesulfonic acid (MES) aqueous buffer,
derivatizations on sialic acids such as permethylation,
33−37
pH = 6.5) in the presence of EDC and HOBt in DMSO
solution for 3 h at room temperature. The reaction mixture was
precipitated in 10 volumes of acetonitrile, and the precipitates
were analyzed using C18 reversed-phase HPLC (Figure 2b,
color: magenta, green, and red). HPLC profiles show that
LNnT−AA is efficiently amidated with three selected amines,
propargylamine, 2-azidoethylamine, and ethylenediamine
amidation, and esterification
have been developed to
facilitate MS analysis, but these derivatizations would likely
abolish their biological activities. In our effort to install
functional groups to glycan−AA conjugates, such as the
conversion of glycan−AA to glycan−AEAB, it is vital to
selectively amidate the benzoic acid but maintain the structural
integrity of sialic acid (Figure 3). Many examples show
evidence that conjugated carboxylic groups with a π bond or an
(
EDA), forming LNnT−PYAB, LNnT−AZAB, and LNnT−
38
AEAB, respectively. All reactions yields are greater than 95%
based on HPLC analysis. Since amidation of LNnT−AA with
ethylenediamine produces LNnT−AEAB (overall yield greater
than 90%), direct reductive amination of LNnT with AEAB·
aromatic ring diminish the rate of the reaction. However,
using the same reaction condition described for LNnT−AA
amidation, Neu5Acα2,6Galβ1,4Glc (6-SL)-AA with ethyl-
enediamine afforded a diamidation product (Figure 3b, red,
retention time = 13.42 min) showing a MALDI-MS peak at m/
z = 861.31 (Figure 3c) along with a sialic acid monoamidation
product (Figure 3b, red, retention time = 21.27 min) showing
a MALDI-MS peak at m/z = 819.30 (Figure 3c). After
screening the reaction condition, using 5% v/v EDA in 1.0 M
MES aqueous solution at pH 8−9, the reaction generated the
favored benzoic acid monoamidation product, i.e. 6-SL−AEAB
(Figure 3b, purple, retention time = 9.65 min). The ratio of
benzoic acid monoamidation:sialic acid monoamidation:dia-
2
2
HCl was used to confirm conversion product identity (Figure
21
b, color: black, yield greater than 98%). The amidation
products were purified by HPLC and analyzed by MALDI-
TOF-MS in positive ionization mode (Figure 2c). As expected,
corresponding masses were observed for LNnT−AEAB (M +
+
+
Na , m/z = 893.31), LNnT−AZAB (M + Na , m/z = 919.39),
+
and LNnT−PYAB (M + Na , m/z = 888.29). Interestingly, the
azido product LNnT−AZAB showed more complex MALDI-
+
MS including a minor peak at m/z = 891.34 (M + Na ). This
D
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Figure 5. Plant lectin binding of a) Concanavalin A (Con A), b) Ricinus communis agglutinin I (RCA 1), c) Erythrina cristagalli (ECL), and d)
Sambucus nigra (SNA) on the glycan microarray of chicken egg N-glycan−AEAB conjugates converted from −AA conjugates. The X-axis represents
glycan ID, and the Y-axis represents the relative fluorescence unit of bound proteins. Error bars represent standard deviation of 6 replicates.
mindation is ∼7:1:0.3, with conversion of 6SL−AA being
greater than 90%. The two different monoamidation products
can be easily separated by C18 RP-HPLC with a retention time
of 9.65 and 21.51 min, respectively, and can be distinguished
using MS/MS analysis (Figure 3c). The desired benzoic acid
monoamidation product (6SL−AEAB) produced a character-
istic fragment ion at m/z 528 confirming the conversion of AA
to AEAB (Figure 3c, bottom). The fragment ions at m/z 486
indicate the underivatized AA, and the fragment ions at m/z
with a gram or higher scale glycan mixture. Anion exchange
and C18 chromatography, on the other hand, use an aqueous
mobile phase and can be scaled up relatively easily. Using N-
glycans released from chicken eggs by ORNG, we compared
the separation of glycan−AEAB and glycan−AA conjugates on
an amine column using both HILIC and WAX modes at
analytical scale. As shown in Figure 4a,b, while both chicken
egg N-glycan−AA and −AEAB conjugates showed good
separation using the HILIC mode, only glycan−AA conjugates
can be well resolved using the WAX mode. This is not
surprising since the installation of AEAB on glycans
significantly reduces the overall negative charge of sialylated
glycans. Therefore, we employed AA as a fluorescent tag for
chicken egg N-glycans and performed preparative scale WAX-
HPLC separation of the AA conjugates (Figure 4c). Neutral,
monosialylated and disialylated glycans are well separated into
three groups. The preparative WAX-HPLC profile of chicken
egg N-glycan AA conjugates (Figure 4c) showed a very similar
resolution compared to the analytical profile (Figure 4b,
bottom), proving that WAX-HPLC on an amino column can
be used for large scale glycan separation. Interestingly, the
excess AA tag was eluted later than disialylated glycans during
a preparative run, while it was eluted before the disialylated
glycans during the analytical run. It is interesting to note that
although three groups of glycans are well separated in the
WAX mode (Figure 4c), suggesting their different charge
statuses, neutral glycans were also observed in the b2 group.
This is apparently due to the installation of the AA tag, which
may dramatically change the charge density of glycans.
3
56 indicate monoamidated Neu5Ac (Figure 3c, middle). The
selective AA amidation of Neu5Acα2,3Galβ1,4Glc (3-SL) was
achieved similarly to produce 3-SL−AEAB (Figure 3d,e).
However, for α2,3-linked sialic acid, an intraesterification
byproduct of 3-SL−AEAB (Figure 3f) with an m/z peak at
8
01.34, can be observed (Figure 3d, top, retention time 14.70
min) upon extension of the reaction time. The typical reaction
time was then set to 3 h to avoid this further esterification
product. Similar intraesterification of 3-linked sialic acids was
36
also reported by other groups.
Preparative 2D-HPLC Purification of AA-Tagged N-
Glycans Released from Chicken Eggs by ORNG. Lack of a
large scale method to release N- and O-glycans from
glycoproteins is still a roadblock for functional glycomics as
traditional enzymatic and chemical methods are only practical
1
3
in analytical scale. We recently reported the oxidative release
of natural glycans (ORNG) using sodium hypochlorite
(
NaOCl) to release free reducing N-glycans from different
natural sources including animal/plant tissues or commercial
protein isolates at kilogram scale to acquire gram scale or larger
2
4,25
amounts of glycans.
The released glycan mixture, however,
Fractions were collected from preparative WAX-HPLC and
subjected to preparative C18 reversed-phase HPLC for second
dimensional purification (Figure S1 and Table S1). After
obtaining purified AA tagged N-glycans, a selection of
individual glycan−AA conjugates, including both neutral and
needs to be tagged and separated by HPLC to yield purified,
individual glycans. While HILIC normally provides the best
separation at analytical scale, its use of high organic content
mobile phases creates a serious solubility issue when dealing
E
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sialylated glycans, was converted to glycan−AEAB conjugates
under the optimized reaction condition described above for
LNnT−AA (neutral glycans) or 3-SL−AA (sialylated glycans).
A total of 30 glycan−AEAB conjugates were purified for
microarray preparation.
various amines, they can potentially be conjugated directly
with amino groups on proteins (Figure 6a) to form
Microarray Printing and Binding Assay. To compare
the immobilization efficiency between AA tagged glycans and
AEAB tagged glycans, LNnT−AA and its conversion product
LNnT−AEAB and 2′-fucosyllactose−AA (2′-FL−AA) and its
conversion product 2′-FL−AEAB were printed on an NHS-
activated slide and assayed with two lectins (Figure S2). The
relative binding of the lectins AAL and ECL to glycans 2′-FL
and LNnT, respectively, is in agreement with the known
binding specificities, and binding decreases with decreasing
concentration of printed glycan. The converted AEAB tagged
glycans clearly showed stronger binding signals than AA tagged
glycans, indicating a much better printing efficiency, which is
consistent with the higher reactivity of primary alkylamine on
21
AEAB and is consistent with our previous study. Using this
facile chemistry to convert glycan−AA to glycan−AEAB, we
printed a microarray of 30 chicken egg N-glycans. This
microarray was interrogated with four plant lectins: ConA,
RCA-1, ECL, and SNA (Figure 5 and Table S2). As expected,
ConA showed binding to many N-glycans. However, there are
a number of glycans that showed no binding, presumably tri-
and tetraantennary ones. Interestingly, 4 N-glycans with
composition of H3N7 were printed. While glycan ID #2, 5,
Figure 6. BSA conjugation with LNnT−AA: a) scheme and reaction
condition of BSA conjugation with LNnT−AA and b) MALDI-TOF
profiles of BSA (bottom) and BSA−LNnT conjugation product.
6
showed no binding to ConA, glycan ID #10 showed clear
binding, suggesting the occurrence of isomeric structures. Both
with H5N7 composition, glycan ID #8 showed no binding,
while glycan ID #26 showed clear binding to ConA. SNA
showed bindings to two sialylated glycans. Both ECL and
RCA-1 are known to bind terminal Galβ4GlcNAc. One major
difference is that terminal 6-sialylation on the Galβ4GlcNAc
motif abolishes binding to ECL but does not affect RCA-1
binding. As expected, all ECL bound glycans also showed
binding to RCA-1. Glycan ID 28 showed binding to RCA-1
but not ECL (chart ID 28) and also showed clear binding to
SNA, consistent with a disialylated structure. On the other
hand, glycan ID 22 showed bindings to RCA I, ECL, and SNA,
consistent with a monosialylated structure. Glycan ID #1, 7,
and 16, with the same H4N8 composition, all showed clear
binding to ECL and RCA-I, suggesting a terminal Galβ4Glc-
NAc. On the other hand, while glycan ID #8, 17, and 20 all
have the same composition H5N5, they show none, weak, and
strong binding to ECL and RCA-1, respectively, suggesting
distinct structures. The binding results provide important
structural information to the printed glycans. While more study
is required to further elucidate the glycan structures and is not
the focus of this study, the results clearly demonstrated that
glycan−AA conjugates can be easily converted to glycan−
AEAB conjugates for microarray preparation.
neoglycoproteins. To explore this possibility, LNnT−AA was
activated by EDC/HOBt followed by addition of bovine serum
albumin (BSA). When the product was analyzed by MALDI-
TOF-MS, the attachment of glycans onto proteins is evident
(
Figure 6b). The resulting neoglycoprotein BSA-LNnT
showed increased mass and wider molecular weight distribu-
tion. By estimation based on the mass shift from unconjugated
BSA, more than 5 molecules of LNnT per BSA molecule are
attached on average. This procedure provides a straightforward
way to prepare neoglycoproteins.
CONCLUSION
■
In this study, we have developed facile chemical methods to
greatly expand the use of AA as a fluorescent tag and
bifunctional linker. Glycan−AA conjugates prepared from
natural glycans can be separated using WAX-HPLC and C18-
HPLC at large scale. Besides being a sensitive fluorescent tag
for glycan analysis and preparation, the AA tag can be easily
and selectively amidated with various amines to convert
glycan−AA conjugates into various forms of functionalized
glycans. These functionalized glycans can be adopted for
further conjugation by click chemistry, microarray printing,
and neoglycoprotein preparation. Because of its simplicity,
versatility, and the current wide use of AA tags for glycan
derivatization, we believe that this method can contribute
greatly to functional glycomics.
Preparation of Neoglycoproteins from Glycan−AA
Conjugates. We predicted that glycan−AA conjugates can
also be linked to proteins to produce neoglycoproteins using
similar chemistry. Neoglycoproteins prepared by linking
glycans to proteins have many potential applications, including
microarray preparation and carbohydrate-based vaccine
EXPERIMENTAL SECTION
■
Materials. All chemicals and HPLC solvents were
purchased from Sigma-Aldrich, St. Louis, MO and Fisher
Scientific, Pittsburgh, PA. Milli-Q water was used to prepare all
aqueous buffers. All solutions were prepared freshly to use.
Mass Spectrometry (MS). A Bruker Daltonics UltraflexII
MALDI-TOF/TOF system and an anchorchip target plate
were used for MS analysis. A reflective positive mode was used
3
9−41
development.
Previously we reported using p-nitrophenyl
anthranilate (PNPA) as a heterobifunctional linker to prepare
3
9
neoglycoproteins. The glycan−PNPA conjugate, as an
activated benzoic ester, is only stable for short-term storage.
Since glycan−AA conjugates can be converted to amides using
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for glycans. 2,5-Dihydroxybenzoic acid (DHB) (10 mg/mL in
quench the reaction and adjust the pH to 4.8. The final
reaction mixture, ∼10.7 L, was then centrifuged to remove
undissolved material. The supernatant was then concentrated
to ∼500 mL in vacuo. The further process followed the
5
0% acetonitrile with 0.1% trifluoroacetic acid) was used as
matrix.
Synthesis of 2-Azidoethanamine. 2-Chloroethylamine
25
hydrochloride (2.32 g, 20 mmol) and sodium azide (NaN , 2.6
procedure described in our previous publication.
3
g, 40 mmol) were dissolved in 40 mL of Milli-Q water. After
stirring for 12 h at 80 °C, 10 mL of 1 M sodium hydroxide
solution was added to adjust the reaction to alkaline condition.
After stirring for another 10 min at room temperature, the
reaction was extracted with dichloromethane (3 × 10 mL).
The organic layer was dried with magnesium sulfate and then
concentrated in vacuo at 0 °C to give product (yield: 60%).
Synthesis of Glycan−AA. In a 1.5 mL snap-cap
microcentrifuge tube, 100 μg of free reducing glycan was
mixed with 50 μL of 60 mg/mL 2-AA in DMSO/AcOH (7:3)
High Performance Liquid Chromatography (HPLC)
Analyses. A Shimadzu HPLC CBM-20A system with UV
detector SPD-20A and fluorescence detector RF-10Axl was
used for analytical HPLC profiles. A Shimadzu HPLC LC-8a
preparative system with UV detector SPD-10Av was used for
preparative HPLC separation. UV absorption at 330 nm or
fluorescence at 330 nm excitation and 420 nm emission was
used for detection of anthranilic acid (AA) and 2-amino-N-(2-
aminoethyl)benzamide (AEAB). Phenomenex amino columns
were used for both HILIC and WAX HPLC. Phenomenex C18
columns were used for reversed-phase HPLC separation. For
WAX HPLC, the mobile phase gradient was from 0 to 200
mM ammonium acetate (pH 4.5) containing 5% acetonitrile
over 50 min (analytical run) or 120 min (preparative run). For
reversed-phase HPLC, the mobile phase gradient was from 2%
to 15% acetonitrile containing 0.1% trifluoroacetic acid over 30
or 45 min.
solution and 50 μL of 64 mg/mL NaCNBH in DMSO/AcOH
3
(
7:3) solution and vortexed for 1 min. After 2 h at 65 °C, the
reaction solution was quenched with 10X volume of
acetonitrile. The cloudy reaction mixture was centrifuged for
2
min at 10000 × g. The supernatant was decanted, and the
pellet was dried under vacuum and dissolved into 100 μL of
Milli-Q water for further analysis and purification.
The synthesis of glycan−AEAB by reductive amination is
essentially the same as that for glycan−AA conjugates except
that 84 mg/mL AEAB in DMSO/AcOH (7:3) solution was
used instead.
2D-HPLC Separation. Preparative WAX HPLC separa-
tion: 1.8 g of a white solid powder containing AA conjugated
glycans was dissolved into 10 mL of H O. Then the 10 mL
2
solution was injected into a preparative amino HPLC column
(column size: 250 × 50 mm). Fractions (0.4 min) were
collected, and selected fractions were analyzed using MALDI-
TOF-MS. C18 reversed-phase HPLC separation: fractions
collected from preparative WAX-HPLC separation were
individually lyophilized, redissolved in water, and then applied
to preparative C18 reversed-phase HPLC (column size: 250 ×
LNnT−AA Conversion via Amidation Reaction. In a
.5 mL snap-cap microcentrifuge tube, 20 μg of LNnT−AA
1
was mixed with 50 μL of 10 mg/mL EDC in DMSO solution
and 50 μL of 10 mg/mL HOBt in DMSO solution. Then 10
μL of 5% (v/v) amine (EDA, propargylamine, or 2-
azidoethanamine) and 0.5 M MES buffer (pH = 6.5) were
added to the mixture and vortexed for 1 min. After 3 h at room
temperature, the reaction solution was quenched by 10X
volume acetonitrile. The cloudy reaction mixture was
centrifuged for 10 min at 10000 × g. The supernatant was
decanted, and white precipitate was dried under vacuum and
then dissolved into 100 μL of Milli-Q water for further analysis
and purification.
21.2 mm). Peaks were collected and characterized using
MALDI-TOF-MS.
LNnT-BSA Conjugation. In a 1.5 mL snap-cap micro-
centrifuge tube, 50 μg of LNnT−AA was mixed with 5 μL of
100 mg/mL EDC in DMSO solution, 5 μL of 100 mg/mL
HOBt in DMSO solution, and 5 μL of 0.5 M MES buffer (pH
5.5). After 15 min at room temperature, 5 μL of BSA (1 mg/
=
mL in water) and 5 μL of phosphate buffer (1.0 M, pH = 8.5)
were added to the mixed solution and reacted for 1 h under
room temperature. The reaction solution was then subjected to
a Sephadex G-25 column to remove salts and other impurities
before MALDI-TOF-MS analysis.
α-2,6-Linked Sialylated glycan−AA Conversion to
AEAB Tagged Glycan. In a microcentrifuge tube, 10 μg of α-
2
,6-linked sialylated glycan−AA was mixed with 50 μL of 10
mg/mL EDC in DMSO solution and 50 μL of 10 mg/mL
HOBt in DMSO solution. Then 10 μL of 5% (v/v) EDA and
Microarray Printing, Binding Assay, and Scanning.
Contact printing on NHS-activated slides was carried out using
an Aushon’s 2470 microarrayer. N-Glycans were printed at 5
μM to 100 μM in 100 mM sodium phosphate (pH = 8.5) in
replicates of 6. Biotinylated AAL, ECL, SNA, ConA, and RCA
were assayed on the microarray slides at concentrations
described in the figures. Five μg/mL cyanine 5-streptavidin
1
.0 M MES aqueous solution (pH 8.2) was added to the
DMSO mixture solution and vortexed for 1 min. After 5 h at
room temperature, the reaction solution was dissolved with an
equal volume of Milli-Q water for further analysis and
purification.
α-2,3-Linked Sialylated glycan−AA Conversion to
AEAB Tagged Glycan. In a microcentrifuge tube, 10 μg of α-
(
Invitrogen) was used for the detection of lectin binding using
2
,3-linked sialylated glycan−AA was mixed with 50 μL of 10
a fluorescent scanner (Innopsys).
mg/mL EDC in DMSO solution and 50 μL of 10 mg/mL
HOBt in DMSO solution. Then 10 μL of 5% (v/v) EDA and
1
.0 M MES aqueous solution (pH 8.2) was added to the
ASSOCIATED CONTENT
■
DMSO mixture solution and vortexed for 1 min. After 3 h at
room temperature, the reaction solution was dissolved with an
equal volume of Milli-Q water for further analysis and
purification.
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.8b00678.
Release of N-Glycans from Chicken Eggs by ORNG.
Figures of HPLC profiles of chicken egg glycan−AA
purification, lectin binding to a microarray of LNnT−AA
and LNnT−AEAB at multiple concentrations, and click
Forty-eight chicken eggs (contain about 310 g proteins) were
mixed with 7.1 L of H O and 3.6 L of 6% NaOCl under open
air and stirred for 30 min. Concentrated HCl was added to
2
G
DOI: 10.1021/acs.bioconjchem.8b00678
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Bioconjugate Chemistry
Article
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13) Seeberger, P. H. (2015) The Logic of Automated Glycan
chemistry validation of LNnT−AZAB with propargyl-
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compositions included in microarray printing and lectin
binding data to the glycan microarray (PDF)
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(14) Plummer, T. H., Jr., Elder, J. H., Alexander, S., Phelan, A. W.,
and Tarentino, A. L. (1984) Demonstration of peptide:N-glycosidase
F activity in endo-β-N-acetylglucosaminidase F preparations. J. Biol.
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AUTHOR INFORMATION
Corresponding Author
E-mail: xsong2@emory.edu. Phone: +1-404-727-3664. Fax:
1-404-727-2738.
(15) Tarentino, A. L., Gomez, C. M., and Plummer, T. H., Jr. (1985)
Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase
F. Biochemistry 24, 4665−4671.
■
*
(16) Mulagapati, S., Koppolu, V., and Raju, T. S. (2017) Decoding
+
of O-Linked Glycosylation by Mass Spectrometry. Biochemistry 56,
ORCID
1
218−1226.
Zhongfu Wang: 0000-0003-1616-5056
Xuezheng Song: 0000-0003-1751-2785
(17) Li, Y.-T., Chou, C.-W., Li, S.-C., Kobayashi, U., Ishibashi, Y.-h.,
and Ito, M. (2009) Preparation of Homogenous Oligosaccharide
Chains from Glycosphingolipids. Glycoconjugate J. 26, 929−933.
Notes
(
18) Ruhaak, L. R., Zauner, G., Huhn, C., Bruggink, C., Deelder, A.
The authors declare the following competing financial
interest(s): X.S. is a cofounder of NatGlycan LLC, which is
commercializing the ORNG technology.
M., and Wuhrer, M. (2010) Glycan Labeling Strategies and Their Use
in Identification and Quantification. Anal. Bioanal. Chem. 397, 3457−
3481.
(
19) Narawane, S., Budnjo, A., Grauffel, C., Haug, B. E., and Reuter,
ACKNOWLEDGMENTS
N. (2014) In Silico Design, Synthesis, and Assays of Specific
Substrates for Proteinase 3: Influence of Fluorogenic and Charged
Groups. J. Med. Chem. 57, 1111−1115.
■
This work was supported by NIH Common Fund Glyco-
science (U01GM116254) and partially by a STTR grant
R41GM122139. We thank Dr. David F. Smith for discussions
and comments on the manuscript. We also acknowledge
Emory Comprehensive Glycomics Core (ECGC), which is
subsidized by the Emory University School of Medicine and is
one of the Emory Integrated Core Facilities.
(
20) Schulz-Fincke, A.-C., Tikhomirov, S. A., Braune, A., Girbl, T.,
Gilberg, E., Bajorath, J., Blaut, M., Nourshargh, S., and Gutschow, M.
2018) Design of an Activity-Based Probe for Human Neutrophil
Elastase: Implementation of the Lossen Rearrangement To Induce
Fo
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̈
rster Resonance Energy Transfers. Biochemistry 57, 742−752.
(21) Song, X., Xia, B., Stowell, S. R., Lasanajak, Y., Smith, D. F., and
Cummings, R. D. (2009) Novel Fluorescent Glycan Microarray
Strategy Reveals Ligands for Galectins. Chem. Biol. 16, 36−47.
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