Supporting Information

9-Azido Analogs of Three Sialic Acid Forms for Metabolic

Remodeling of Cell-Surface Sialoglycans

Bo Cheng,†,‡ Lu Dong,†,§ Yuntao Zhu,†,‡ Rongbing Huang,†,‡ Yuting Sun,†,‖

Qiancheng You,†,‡ Qitao Song,†,§ James C. Paton, ∇ Adrienne W. Paton,∇ and Xing
Chen*,†,‡,§,⊥,#

†
College of Chemistry and Molecular Engineering, ‡Beijing National Laboratory for
Molecular Sciences, §Peking−Tsinghua Center for Life Sciences,‖Academy for
⊥
Advanced Interdisciplinary Studies, Synthetic and Functional Biomolecules Center,
and #Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry
of Education, Peking University, Beijing 100871, China

∇Research Centre for Infectious Diseases, Department of Molecular and Biomedical

Science, University of Adelaide, Adelaide SA 5005, Australia

Page S1
Table of Contents:

Scheme S1.……………………………………………………….........……………. S3
Figure S1……………………………………………………..………..……………. S3
Figure S2……………………………………………………..………..…………… S4
Figure S3……………………………………………………..………..…………… S4
Figure S4……………………………………………………..………..…………… S5
Figure S5……………………………………………………..………..…………… S6
Figure S6……………………………………………………..………..…………….S7
Figure S7……………………………………………………..………..…………….S8
Figure S8……………………………………………………..………..…………….S9
Experimental Procedures……………………………….…........…………....S10-S27
Table S1………………………………………………..………..…………….S28-S48
Supporting Reference……………………………………………….......………...S49

Page S2
Scheme S1. Synthesis of 9AzNeu5Gc

Figure S1: a, b, c, d) Representative scatter plots (FSC vs. SSC) and histograms of flow
cytometry analysis on Vero cells treated with 9AzNeu5Gc (a, c) or 9AzNeu5Ac (b, d) at
varied concentrations. The scatter plots were gated based on the FSC (forward scatter) and
SSC (side scatter) signals for cell debris discrimination. The gated cells were used to
analyze cell-surface fluorescence. This figure is related to Figure 1b in the main text.

Page S3
Figure S2. Evaluation of metabolic incorporation of 9AzNeu5Gc and 9AzNeu5Ac
in CHO (a), HeLa (b), and Jurkat cells (c). The cells were treated with 9AzNeu5Gc
or 9AzNeu5Ac at varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and
stained with streptavidin−Alexa Fluor 488, followed by flow cytometry analysis.
MFI (mean fluorescence intensity) was normalized to that of control cells treated with
vehicle. Error bars represent mean ±SD from three independent experiments.

Figure S3. HeLa cells were treated with 9AzNeuAc or 9AzNeu5Gc at varied
concentrations for 24 h. The treated cells were reacted with DBCO-biotin and
stained with streptavidin−Alexa Fluor 647. The nuclei were stained with Hoechst
33342 (blue). The cells were imaged by confocal fluorescence microcopy. DIC,
differential interference contrast. Scale bar: 50 m.

Page S4
Figure S4. HPLC analysis of metabolic incorporation of 9-azido sialic acid analogs.
(a) CHO cells were treated with 9AzNeu5Ac, 9AzNeu5Gc, or 9AzKDN for 24 h.
(b) Vero cells were treated with 9AzNeu5Ac at varied concentrations for 24 h. (c)
MDCK II cells were cultured with 9AzNeu5Ac at varied concentrations for 24 h.
The treated cells were lysed, from which the proteins were precipitated and subjected
to acid hydrolysis to release ketosidically bound sialic acids. The released sialic
acids were then derivatized with DMB and analyzed by HPLC with fluorescence
detection. The symbol (*) indicates the peaks of DMB-azido sialic acid. The
dashed box indicates no peak of DMB-9AzNeu5Gc in the 9AzNeu5Ac-treated cells.
The quantified incorporation ratios were shown on the right.

Page S5
Figure S5. Evaluation of metabolic incorporation of 9AzKDN and 9AzNeu5Ac in
A375 (a), CHO (b), BJA-B K20 cultured with FBS in the medium (c), Daudi (d),
HeLa (e) and Vero cell (f). The cells were treated with 9AzKDN or 9AzNeu5Ac at
varied concentrations for 24 h, reacted with alkyne-PEG4-biotin and stained with
streptavidin−Alexa Fluor 488, followed by flow cytometry analysis. The zoomed
out bar graph inserted highlights 9AzKDN-labeling. MFI (mean fluorescence
intensity) was normalized to that of control cells treated with vehicle. Error bars
represent mean ±SD from three independent experiments.

Page S6
Figure S6: MS analysis of CMP-9AzKDN from CHO cells treated with 9AzKDN.
CHO cell was treated with 4 mM 9AzKDN for 24 h. The lysates were separated
using HPAEC-UV and the CMP-9AzKDN fraction corresponding to the peak eluting
at 29.4 min in figure 2a was collected and analyzed by MALDI-TOF mass
spectrometry using the cationic mode.

Page S7
Figure S7. HPAEC-UV/PAD analysis of CMP-9AzKDN from BJA-B K20 cells
treated with 4 mM 9AzKDN or vehicle for 24 h in medium containing FBS. Pure
CMP-9AzKDN was shown as the standard. Pure CMP-9AzKDN was co-injected
with the 9AzKDN-treated sample to further validate the CMP-9AzKDN peak.

Page S8
Figure S8. Competitive metabolic incorporation of azido sialic acid analogs with
Neu5Ac in CHO and PA-1 cells. (a) CHO cells were treated with 4 mM 9-azido
sialic acid analog (9AzKDN, 9AzNeu5Gc or 9AzNeu5Ac, respectively) and Neu5Ac
at varied concentration for 24 h. (b) PA-1 cells were treated with 4 mM 9-azido
sialic acid analog (9AzKDN or 9AzNeu5Ac, respectively) and Neu5Ac at varied
concentration for 24 h. The treated cells were reacted with alkyne-PEG4-biotin via
BTTAA-assisted CuAAC and stained with streptavidin−Alexa Fluor 488, followed by
flow cytometry analysis. MFI (mean fluorescence intensity) was normalized to that
of control cells treated with vehicle. Error bars represent mean ±SD from three
independent experiments.

Page S9
 Experimental Procedures

General materials. Reagents for chemical synthesis were obtained from

commercial sources and used as directly without further purification unless otherwise
noted. KDN1, Neu5Gc2, 9AzKDN3, 9AzNeu5Ac4, CMP-Neu5Ac5, CMP-
9AzNeu5Ac6and CMP-9AzKDN5 were synthesized as previously described. Biotin-
PEG4-alkyne, DBCO-biotin and Cy5 Alkyne were purchased from Click Chemistry
Tools (Scottsdale, AZ, USA), streptavidin-Alexa Fluor 488 and streptavidin-Alexa
Fluor 647 were purchased from Invitrogen (Carlsbad, CA, USA), BCA Protein Assay
Kit were obtained from Pierce. EDTA-free protease inhibitor cocktail and
Nutridoma SP were purchased from Roche. For metabolic incorporation
experiments, azido sialic acids were dissolved in PBS (500 mM) and pH was adjusted
to 7.4 using NaOH solution, the stock was kept at 4 oC. CMP-9AzKDN was dissolved
in PBS (10 mM) and kept at -80 oC. Oregon-Green-labelled SubAB (OG-SubAB)
was prepared according to a reported procedure.7

Cell culture. CHO cells (Chinese hamster ovary cell), HeLa cells, MDCK Ⅱ cells,
A375 cell and Vero cells were grown in DMEM (Dulbecco’s modified Eagle’s
medium) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin
and 100 µg/mL streptomycin. PA-1 cells were incubated in MEM supplemented
with 10% FBS (fetal bovine serum), 100 units /mL penicillin and 100 µg/mL
streptomycin. BJA-B K20 cells (a gift from Prof. Michael Pawlita), Daudi cells and
Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100
units /mL penicillin and 100 µg /mL streptomycin. All the cells were incubated at
37 oC under 5% CO2 in a water-saturated atmosphere. To obtain BJA-B K20 cell in
serum-free conditions, the cells were grown carefully for two passages in serum free
Page S10
medium (RPMI-1640 with 2 mM L-glutamine containing 1x Nutridoma SP, 50
units/mL penicillin, and 50 μg/mL streptomycin) to deplete endogenous sialic acid.

Flow cytometry analysis. After cells were incubated in 6-well plates with azido
sialic acids at the indicated concentrations for 24 h. The cells were harvested with
trypsin, transferred and distributed into 96-well V-bottomed plate, pelleted (800 g, 6
min, 4 oC), and washed with ice-chilled PBS containing 1% FBS for three times.
The pelleted cells were then suspended in PBS containing 0.5% FBS, 50 M alkyne-
PEG4-biotin, BTTAA-CuSO4 (50 M CuSO4, BTTAA: CuSO4 = 6:1), and 2.5 mM L-
sodium ascorbate at room temperature. After 5 min, the reaction was quenched by
adding 1 mM BCS (bathocuproine disulphonate). The cells were washed with cold
PBS containing 1% FBS for three times, and incubated with chilled PBS containing
1% FBS and 1 µg/mL Streptavidin-Alexa Fluor 488 for 30 min. After three washes
with chilled PBS containing 1% FBS, the cells were suspended in chilled PBS
containing 1% FBS and used for FACS (fluorescence-activated cell sorting) analysis
on a BD FACSCalibur Flow Cytometer system or BD AccuriTM C6 flow cytometer.

In-gel fluorescence scanning. The cells treated with sialic acid probes at the
indicated concentrations or vehicle for 24 hours were harvested by trypsin, washed
three times with ice-chilled PBS (800 g, 6 min). Then the pelleted cells were lysed
in modified RIPA lysis buffer (1% Nonidet P 40, 1% sodium deoxycholate, 0.1%
SDS, 50 mM triethanolamine, pH=7.4, 150 mM NaCl, EDTA-free Piercent HaltTM
protease inhibitor cocktail, 1 tablet/50 mL). Protein concentrations in the
homogenous lysis was normalized to 1 mg/ mL with lysis buffer. The normalized
samples were then reacted with 100 µM alkyne-Cy5 in a 60 µL reaction containing
premixed BTTAA-CuSO4 complex (50 µM CuSO4, BTTAA: CuSO4 in a 2:1 molar
ratio) and 2.5 mM L-sodium ascorbate (freshly prepared). The samples were
vortexed for 2 h at 4 °C and resolved on 10% SDS-PAGE gels. The gel was washed
in destaining solution (50% methanol, 40% H2O, 10% acetic acid) for 5 min, followed

Page S11
by washing in water for another 5 min. After that, the in-gel fluorescence was
scanned on a Typhoon FLA 9500 laser scanner (GE Healthcare, USA).

Sialylated glycoproteomic identification. For sialylated glycoproteomic

identification with three 9-azido sialic acid probes in PA-1 cells, we used a reported
procedure.8 Briefly, PA-1 cells treated with azido sialic acid were washed three
times in ice-chilled PBS, pelleted and lysed in RIPA buffer. Homogeneous cell lysis
was obtained by centrifugation (10,000 × g, 10 min) to remove cell debris. BCA
assay was used to determine protein concentration. 5 mg proteins in 5 mL RIPA
buffer were incubated with 100 μM alkyne-PEG4-biotin, premixed BTTAA-CuSO4
complex (100 μM CuSO4, BTTAA: CuSO4 at a molar ratio of 2:1), and 2.5 mM L-
sodium ascorbate (freshly prepared) for 1 h. After adding 40 mL ice-cold methanol,
the reaction was kept at - 30 °C overnight to precipitate proteins. The precipitated
proteins were centrifuged at 4,500 × g for 15 min, and washed twice with 20 mL ice-
chilled methanol. The protein pellet was resuspended in 4 mL resuspension buffer
(Solution A: 4% (wt/vol) SDS and 10 mM EDTA in H2O; Solution B: 1% (vol/vol)
Brij 97, 150 mM NaCl and 50 mM triethylamine in H2O, pH = 7.4; A: B =1:8
(vol/vol)). 100 μl of streptavidin beads (Thermo Scientific, USA) were washed three
times with 1 mL PBS, and added to the protein solution. The solution was incubated
at r.t. for 3 h, followed by washing the beads sequentially with 2% (wt/vol) SDS in
PBS, 8 M urea with 250 mM ammonium bicarbonate (ABC), 2.5 M sodium chloride
in PBS, 0.5 M ABC, 0.25 M ABC, and 0.05 M ABC. The beads were suspended in
20 μL 5 × loading buffer, heated for 10 min at 95 °C to release the enriched
sialoglycoproteins and resolved in 10% SDS-PAGE. Coomassie brilliant blue R-250
was used to display the distribution of the captured proteins in the gel. Each lane of
the SDS-PAGE gel was sliced into 8 fractions, and gel slices were washed with Milli-
Q water, destained with a 1:1 solution of 50 mM ABC/acetonitrile for 30 min at

Page S12
37 °C, and dehydrated in 100% acetonitrile. The gel slices were rehydrated with 10
mM DTT in 50 mM ABC, and incubated for 45 min at 56 °C, then the slices were
incubated with 55 mM iodoacetamide in 50 mM ABC for 45 min at r.t. in the dark.
After dehydrated in 100% acetonitrile, gel pieces were rehydrated in a trypsin solution

(2 ng/L) and incubated at 37 °C for 16 h. The peptides were eluted in 50%

acetonitrile in H2O with 5% (vol/vol) TFA (200 μL, twice), and SpeedVac dried.
Samples were then subjected to an Easy nLC 1000 coupled to an LTQ Velos Pro-
Orbitrap Elite mass spectrometer (Thermo Fisher). Peptides were pressure-loaded
onto a 100 μm diameter, 2 cm C18 precolumn and separated on a 75 μm diameter, 20
cm C18 capillary column with a gradient running from 95% buffer A (H2O with 0.1%
formic acid) and 5% buffer B (CH3CN with 0.1% formic acid) to 35% B over 60 min
at the flow rate of 300 nL/min, next ramping to 75% B over 2 min and holding at 75%
B for 10 min. One full MS scan (375-1600 M/z) was followed by ten data-
dependent scans of the nth most intense ions with dynamic exclusion enabled.
Peptides were identified using Mascot version 2.3.02 (MatrixScience) and were
searched against the SwissProt mouse sequence database to compile the data.

Electroporation of 9-azido sialic acid and CMP-9-azido sialic acid.

Electroporation was performed on a Gene Pulser Xcell Electroporation System (Bio-
Rad) according to manufacturer’s instructions. In brief, HEK 293T cells (~70%
confluence) were harvested by trypsin and washed with PBS for three times (500 × g,
6 min). The cells were suspended in electroporation buffer (PBS or PBS containing
2 mM 9-azido sialic acid or CMP-9-azido sialic acid) at a density of ~2 × 106 cells/
mL. 200 µL of cells were cooled on ice for 10 min and transferred into a 4-mm
electroporation cuvette. The cells were pulsed at 220 V for 25 msec and transferred
to a plate containing ~2.0 mL preheated media immediately after the pulse. The

Page S13
cells were cultured in the medium for another 48 hours before flow cytometry
analysis of cell-surface 9-Azido sialic acid as previously described.

HPLC analysis of DMB-sialic acid derivatives. To quantify sialic acids bonded on

cellular glycoproteins, cells treated with azido sialic acids (500 mM stock in PBS) or
PBS were harvested by trypsin digestion and washed with ice-chilled PBS for three
times (800 g, 6 min). To the centrifuge tube containing cell pellets was added
modified RIPA (1% Nonidet P 40, 1% sodium deoxycholate, 0.1% SDS, 50 mM
triethanolamine, pH=7.4, 150 mM NaCl, EDTA-free Piercent HaltTM protease
inhibitor cocktail) and the tube was kept shaking gently at 4 oC for 30 min to ensure
sufficient lysis. Then chilled alcohol (75%, V/V) was added into the homogeneous
cell lysis to precipitate the cellular proteins, and the mixture was kept at -80 oC
overnight. After that, the protein precipitates were collected by centrifugation and
washed four times with chilled alcohol/H2O (3/1, V/V) to wash off the free sialic
acids or CMP-sialic acids. The rinsed proteins (or sialic acid standard) were
dispersed in 2 M acetic acid and incubated for 3 hours at 80 oC to release the protein
bonded sialic acids. Then the hydrolysate was cooled to 4 oC and filtrated through a
3,000 MWCO ultrafilter and the filtrate was collected and directly used for DMB-
sialic acid derivatization. To quantify sialic acids on the cell-surface, BJA-B K20
cells was cultured with different sialic acid in serum-starved conditions for 48 hours,
harvested and washed three times with PBS. Then the collective cells was lysed by
hypotonic shock in 10 mM sodium phosphate (pH 7.5) containing ETDA-free
protease inhibitor cocktail at 4 oC for 15 min, after centrifugation at 100,000 g for 60
min, crude membrane fraction was pelleted. The pellet was washed three times with
double deionized water (dd H2O) and lyophilized. The lyophilized cell-membrane
fraction was treated with 2 M acetic acid to release ketosidically linked sialic acids as
described to extract glycoproteins bonded sialic acid. For sialic acid DMB

Page S14
derivatization, dd H2O, DMB, β-mercaptoethanol and Na2S2O4 was added to adjust
the final concentration of acetic acid, DMB, β-mercaptoethanol, Na2S2O4 was 1.4
mM, 7 mM, 0.75 M and 18 mM, respectively. Then the DMB-derivatization
mixture was kept at 50 oC for 2.5 hours in the dark.9 The fluorescent DMB-sialic
acid derivatives were analyzed by RP-HPLC (Aglient 1260, XDB-C18 column, 5 µm,

4.6 × 250 mm) with a fluorescence detector (ex=373 nm, em=448 nm). The flow
rate was 0.8 mL/min and the elution gradient used was as followed: T (0 min) 84%
H2O + 9% CH3CN + 7% CH3OH, T (14 min) 84% H2O + 9% CH3CN + 7% CH3OH,
T (22 min) 64% H2O + 18% CH3CN + 18% CH3OH, T (28 min) 64% H2O + 18%
CH3CN + 18% CH3OH, T (29 min) 84% H2O + 9% CH3CN + 7% CH3OH, T (30
min) 84% H2O + 9% CH3CN + 7% CH3OH. The incorporation efficiency of azido
sialic acid into glycoproteins was quantified by integration of peak areas.

HPAEC-PAD analysis of CMP-sialic acids. BJA-B K20 cells or CHO cells

cultured in 10 cm dishes were treated with vehicle or sialic acid probes at the
indicated concentrations for 24 hours, harvested by trypsin, centrifuged at 800 g for 6
min, then the cell pellets were washed three times with ice-chilled PBS and subjected
to ultrasonication using ultrasonic processor (Cole-Parmer Instruments, Vernon Hills,
Illinois, USA.) in 0.5 mL deionized water on ice for 1 minute (50% amplitude, 50 %
pulse). After centrifugation (1 5000 g, 10 min) at 4 oC, the debris was discarded and
the supernatant was collected and filtrated through an Amicon Ultra centrifugal filter
unit (Millipore, 10,000 MWCO) by centrifugation (1,5000 x g, 30 min) at 4 oC.
Then the filtrate was collected and 0.1 mL filtrate was injected for HPAEC analysis.
For HPAEC-UV/PAD (high pH anion exchange chromatography followed by UV and
pulsed amperometric detection) analysis of CMP-sialic acids, a Dionex CarboPacTM
PA1 column (4 x 250 mm, analytical) equipped on a DIONEX ICS-5000 HPAEC
system with the combined detection of UV and PAD was used. Two elution buffer

Page S15
A (140 mM NaOH in water) and B (140 mM NaOH + 600 mM NaOAc in water)
were applied in the separation process. To analyze CMP-sialic acids extracted from
CHO cells, the elution gradient used was as followed with a flow rate of 1 mL/min: T
(0 min) 20% B, T (10 min) 55% B, T (25min) 55% B, T (35 min) 80% B, T (40 min)
100% B, T (50 min) 100% B. To analyze CMP-sialic acids extracted from BJA-B
K20 cells, another elution gradient used was as followed with a flow rate of 1
mL/min: T (0 min) 4% B, T (45 min) 46% B, T (65min) 100% B, T (80 min) 100% B,
T (81 min) 4% B, T (90 min) 4% B.

MALDI-TOF mass spectrometry analysis of intercellular CMP-9AzKDN: CHO

cells cultured in 10 cm dishes (4 x) were treated with 4 mM 9AzKDN for 24 hours.
The cell was harvested and lysed, and the lysate was separated by HPAEC-UV as
described previously. The peak eluted at 29.4 min was collected, the collection was
neutralized with acetic acid and lyophilized. The lyophilized powder was treated

with methanol (150 L), the suspension was centrifuged (1,2000 x g, 2 min), the
supernatant was used for CMP-9AzKDN analysis with MALDI-TOF mass

spectrometry on Bruker Autoflex Ⅲ system (a matrix of -Cyano-4-

hydroxycinnamic acid and cationic mode was used).

Fluorescence microscopy. For confocal fluorescence experiments, the cells treated

with azido sialic acids were cultured in Lab-TekTM 8-well chamber slides for 24 h.
The cells were washed with ice-chilled PBS for three times, and then incubated with
PBS containing 50 M DBCO-biotin at room temperature for 30 min. The cells
were washed with chilled PBS containing 1% FBS for three times, and incubated with
chilled PBS containing 5 µg/mL Streptavidin-Alexa Fluor 647 on ice for 30 min.
Hoechst 33342 (5 g/mL) was added to the cells and incubated for another 15 min.
The cells were washed with chilled PBS containing 1% FBS for three times and
imaged on a Zeiss LSM 700 laser scanning confocal microscope equipped with a 63×
oil immersion objective lens (N.A. 1.4) with ZEN 2011 software. Two lines of
Page S16
solid-state lasers with 405-nm and 639-nm were used to excite Hoechst 33342
fluorescence and Alexa Fluor 647 fluorescence respectively, and the signal was
collected with a 405- to 587-nm band-pass filter (405-nm laser) or a 639-nm long-
pass filter (639 –nm laser).

Binding of SubAB to BJA-B K20 cell-surface sialic acids. BJA-B K20 cells
cultured in serum-free medium for two passages was treated with 2 mM sialic acid or
9-azido sialic acid for 48 hours, then the cells was harvested by centrifugation (400 x
g, 5 min) and counted. Equal amount of cells were pelleted and washed twice with
PBS. The pellet was suspended in serum-free culture medium (RPMI 1640 with 2
mM L-glutamine containing 1x Nutridoma SP, 50 U/mL penicillin, and 50 μg/mL
streptomycin) containing 1g/mL OG-SubAB (~3 x106 cell/mL), the suspension was
kept at 37oC for 30 mins, then pelleted and washed twice with PBS. After fixation in
PBS containing 4% paraformaldehyde (25 oC, 10 min), the OG-SubAB labeled cells
was washed and suspended in PBS, the Oregon Green fluorescence intensity was
quantified by FACS on a BD FACSCalibur Flow Cytometer system.

Page S17
Chemical Synthesis

5-acetoxyacetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic acid
(Neu5GcAc, 2)

4.12 g (14.7 mmol, 1.0 eq) N-Acetoxyacetate-D-

mannosamine (ManNGcAc, 1)10 and 8.08 g sodium
pyruvate (73.5 mmol, 5.0 eq) were dissolved in 80 mL
KH2PO4 buffer (50 mM), followed by the addition of
recombinant Escherichia coli K12 aldolase5. The reaction was kept shaking for 2
o
days at 37 C, the product was purified by ion-exchange chromatography eluted with
1 M formic acid in water to get 3.45 g Neu5GcAc (2) with a yield of 64%. Rf =0.1
1
(MeOH: DCM=1: 1, V/V). H NMR (500 MHz, D2O): 4.72 (s, 2H,
NHCOCH2OAc), 4.19 (m, 2H, H4+H6), 4.06 (t, J = 10.2 Hz, 1H, H5), 3.89 (d, J = 11.8
Hz, 1H, H9a), 3.80 (d, J = 7.7 Hz, 1H, H8), 3.72-3.64 (m, 1H, H9b), 3.59 (d, J = 9.1 Hz,
1H, H7), 2.42-2.34 (m, 1H, H3-eq), 2.25 (s, 3H, COCH3), 1.94 (t, J = 12.3 Hz, 1H, H3-
ax).
13
C NMR (125 MHz, D2O) δ 173.3, 170.8, 165.8, 95.4, 70.3, 70.2, 68.3, 66.5,
63.3, 63.0, 52.0, 39.0, 20.0. HRMS (ESI): Calcd for C13H21NO11Na [M+Na] +
390.09395, found 390.09344.

Page S18
1
H NMR of Neu5GcAc (2)

13
C NMR of Neu5GcAc (2)
Page S19
1-Methyl-5-acetoxyacetamido-3,5-dideoxy-D-glycero-D-galacto-2-nonulosonic
acid (1-Met-Neu5GcAc, 3)

To a stirred solution of 100 mL MeOH was added 3.45 g

(9.4 mmol, 1.0 eq) Neu5GcAc (2), then 1.5 mL TFA (20.2
mmol, 2.1 eq) was added dropwise over a period of 5
minutes. The solution was kept stirring at room
temperature overnight. The methyl ester product was purified by flash
chromatography to get 2.17 g white powder with a yield of 61%. Rf = 0.4 (DCM:
1
MeOH=4: 1, V/V). H NMR (500 MHz, D2O): 4.74 (s, 2H, NHCOCH2OAc), 4.25-
4.16 (m, 2H, H4+H6), 4.06 (t, J = 10.3 Hz, 1H, H5), 3.95-3.86 (m, 4H, COOCH3+H9a),
3.83-3.76 (m, 1H, H8), 3.69 (dd, J = 11.9, 6.4 Hz, 1H, H9b), 3.64-3.57 (m, 1H, H7),
13
2.43-2.35 (m, 1H, H3-eq), 2.26 (s, 3H,COCH3), 1.99 (t, J = 12.3, 1H, H3-ax). C
NMR (125 MHz, D2O): δ 173.4, 171.4, 170.8, 95.4, 70.3, 70.2, 68.3, 66.4, 63.3, 63.0,
53.6, 52.0, 38.8, 20.1. HRMS (ESI): Calcd for C14H24NO11 [M+H] + 382.13439,
found 382.13488.

Page S20
1
H NMR of 1-Met-Neu5GcAc (3)

13
C NMR of 1-Met-Neu5GcAc (3)
Page S21
1-Methyl-5-acetoxyacetamido-9-tosyl-3,5,9-trideoxy-D-glycero-D-galacto-2-
nonulosonic acid (1-Met-9-Ts-Neu5GcAc, 4)

To 40 mL anhydrous pyridine was added 1.15 g (3.0

mmol, 1.0 eq) 1-Met-Neu5GcAc (3), the mixture was
cooled to 0 oC in an ice-water bath under N2 atmosphere.
Then 0.86 g TsCl (4.5 mmol, 1.5 eq) was poured into the
chilled solution. The mixture was stirred overnight while allowing the water bath
warmed slowly to room temperature. The product was purified by flash
chromatography to get an off-white foam with a yield of 50%. Rf = 0.5 (DCM:
1
MeOH=9:1, V/V). H NMR (500 MHz, CD3OD) 7.82-7.78 (m, 2H,Ar-H), 7.46-7.42
(m, 2H, Ar-H), 4.62-4.53 (m, 2H, NHCOCH2OAc), 4.30 (dd, J=10.1, 2.3 Hz, 1H, H6),
4.09 (ddd, J=11.3, 10.2, 4.9 Hz, 1H, H4), 4.06-4.01 (m, 2H, H9a+H9b), 3.87-3.79 (m,
2H, H8+H5), 3.77 (s, 3H, COOCH3), 3.45 (dd, J = 9.1, 1.3 Hz, 1H, H7), 2.46 (s, 3H,
Ar-CH3), 2.21 (dd, J = 12.9, 4.9 Hz, 1H, H3-eq), 2.15 (s, 3H, COCH3), 1.87 (dd,
J=12.9, 11.5 Hz, 1H, H3-ax). 13
C NMR (125 MHz, MeOD): δ 171.3, 170.98, 170.90,
145.6, 133.5, 130.2, 128.3, 95.9, 73.0, 70.8, 69.1, 68.7, 66.8, 62.8, 53.3, 52.4, 39.9,
20.8, 19.7. HRMS (ESI): Calcd for C21H30NO13S [M+H] + 536.14388, found
536.14359.

Page S22
1
H NMR of 1-Met-9-Ts-Neu5GcAc (4)

13
C NMR of 1-Met-9-Ts-Neu5GcAc (4)
Page S23
5-acetoxyacetamido-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-2-nonulosonic
acid (9-N3-Neu5GcAc, 5)

0.98 g (15.1 mmol, 10.0 eq) sodium azide was added to

a stirred solution of 30 mL acetone and 10 mL H2O
containing 0.81 g (1.5 mmol, 1.0 eq) 1-Met-9-Ts-
Neu5GcAc (4) under N2. The mixture was heated at
80 oC to reflux for 2 days. The crude product was purified by flash to get 0.35 g
1
light yellow power with a yield of 59%. Rf = 0.1 (DCM: MeOH= 1:1, V/V). H
NMR (500 MHz, D2O) 4.73 (s, 2H, NHCOCH2OAc), 4.24-4.12 (m, 2H, H4+H6), 4.05
(t, J= 10.1 Hz, 1H, H5), 3.96 (ddd, J = 9.0, 6.0, 2.7 Hz, 1H, H8), 3.67 (dd, J = 13.5, 2.8
Hz, 1H, H9a), 3.59 (d, J = 9.1 Hz, 1H, H7), 3.53 (dd, J = 13.5, 5.8 Hz, 1H, H9b), 2.34
(dd, J = 12.9, 4.8 Hz, 1H, H3-eq), 2.26 (s, 3H,COCH3), 1.94 (t, J=12.2, 1H, H3-ax).
C NMR (125 MHz, D2O) δ 175.0, 173.4, 170.8, 96.0, 70.0, 69.2, 68.9, 66.7, 63.0,
13

53.9, 52.2, 39.2, 20.1. HRMS (ESI): Calcd for C13H20N4O10Na [M+Na] +
415.10716, found 415.10774.

Page S24
1
H NMR of 9-N3-Neu5GcAc (5)

13
C NMR of 9-N3-Neu5GcAc (5)
Page S25
9-Azido-9-deoxy-N-glycolylneuraminic acid (9AzNeu5Gc, 6)

To a solution of 0.39 g (1.0 mmol, 1.0 eq) 9-N3-

Neu5GcAc (5) in 20 mL MeOH was added 0.11 g
MeONa (2.0 mmol, 2.0 eq) and the mixture was stirred
for 15 minutes. Then H ion-exchange resin (Amberlite
IR 120) was added and the pH of the mixture was adjusted to 7.0, the mixture was
filtrated to get a yellow filtrate. The filtrate was concentrated in vacuum and the
residue was purified by flash to achieve the target product with a yield of 78%. Rf
1
=0.05 (DCM: MeOH =1: 1, V/V). H NMR (500 MHz, D2O) 4.27-4.17 (m, 4H,
H4+H6+NHCOCH2OH), 4.05 (t, J = 10.2 Hz, 1H, H5), 3.96 (ddd, J = 9.2, 6.0, 3.0 Hz,
1H, H8), 3.71-3.65 (m, 1H, H9a), 3.62 (d, J = 9.3 Hz, 1H, H7), 3.53 (dd, J = 13.2, 6.2
Hz, 1H, H9b), 2.38 (dd, J = 13.0, 4.9 Hz, 1H, H3-eq), 1.95 (t, J = 12.3 Hz, 1H, H3-ax).
C NMR (125 MHz, D2O): δ 175.7, 173.5, 95.5, 70.1, 69.1, 68.7, 66.5, 61.1, 53.9,
13

51.9, 39.0. HRMS (ESI): Calcd for C11H18N4O9Na [M+Na] + 373.09660, found
373.09702.

Page S26
1
H NMR of 9AzNeu5Gc (6)

13
C NMR of 9AzNeu5Gc (6)
Page S27
 Table S1: Identification of sialoglycoproteins with three 9-azido sialic acid
analogs in PA-1 cells

Protein Name Gene Name Spectral Counts High confidence

Blank 9AzNeu5Ac 9AzNeu5Gc 9AzKDN 9AzNeu5Ac 9AzNeu5Gc 9AzKDN

4F2 cell-surface antigen heavy chain SLC3A2 78 584 552 209 √ √

Cation-independent mannose-6- IGF2R 10 526 563 127 √ √ √

phosphate receptor

Podocalyxin-like protein 1 PODXL 39 526 601 313 √ √ √

Basigin BSG 15 473 506 104 √ √ √

Integrin beta-1 ITGB1 13 381 321 113 √ √ √

Golgi apparatus protein 1 GLG1 48 325 277 119 √ √

Neuroligin-4, X-linked NLGN4X 12 291 240 117 √ √ √

Alkaline phosphatase, tissue- ALPL 5 253 284 51 √ √ √

nonspecific isozyme

Solute carrier family 2, facilitated SLC2A3 58 252 330 92 √

glucose transporter member 3

Integrin alpha-6 ITGA6 13 236 340 38 √ √

Prolow-density lipoprotein receptor- LRP1 1 223 206 74 √ √ √

related protein 1

Carboxypeptidase D CPD 6 222 224 59 √ √ √

Transferrin receptor protein 1 TFRC 34 222 193 84 √ √

Dystroglycan DAG1 3 215 154 45 √ √ √

Neutral amino acid transporter B(0) SLC1A5 27 214 117 79 √

Chondroitin sulfate proteoglycan 4 CSPG4 0 210 207 43 √ √ √

Neuropilin-2 NRP2 7 203 201 79 √ √ √

Phospholipase D3 PLD3 14 194 146 92 √ √ √

Cadherin-6 CDH6 4 181 155 35 √ √ √

Tyrosine-protein kinase-like 7 PTK7 27 181 214 51 √ √

Page S28
 Integrin alpha-V ITGAV 3 176 157 34 √ √ √

Very low-density lipoprotein receptor VLDLR 1 174 197 49 √ √ √

Golgi integral membrane protein 4 GOLIM4 2 173 151 48 √ √ √

CD44 antigen CD44 2 166 216 81 √ √ √

Semaphorin-6A SEMA6A 14 156 191 62 √ √

Protogenin PRTG 13 148 150 35 √ √

Sodium/potassium-transporting ATP1B3 9 148 139 25 √ √

ATPase subunit beta-3

Versican core protein VCAN 0 137 155 72 √ √ √

Glypican-4 GPC4 0 134 69 32 √ √ √

Sulfhydryl oxidase 2 QSOX2 0 132 64 33 √ √ √

Cadherin-2 CDH2 0 126 113 19 √ √ √

Dipeptidyl peptidase 1 CTSC 9 122 149 17 √ √

Cation-dependent mannose-6- M6PR 0 121 117 13 √ √ √

phosphate receptor

Low-density lipoprotein receptor- LRP8 2 118 68 16 √ √ √

related protein 8

Receptor-type tyrosine-protein PTPRZ1 0 117 80 45 √ √ √

phosphatase zeta

Tyrosine-protein kinase ROR1 1 117 145 23 √ √ √

transmembrane receptor ROR1

Latrophilin-2 LPHN2 1 115 92 22 √ √ √

Amyloid beta A4 protein APP 4 114 50 34 √ √ √

Nicastrin NCSTN 4 112 67 25 √ √ √

Cell surface glycoprotein MUC18 MCAM 0 111 110 32 √ √ √

Receptor-type tyrosine-protein PTPRG 0 111 65 27 √ √ √

phosphatase gamma

C-type mannose receptor 2 MRC2 0 107 96 28 √ √ √

Page S29
 Ectonucleotide ENPP1 2 107 63 27 √ √ √

pyrophosphatase/phosphodiesterase

family member 1

Sortilin-related receptor SORL1 0 107 72 16 √ √ √

Desmoglein-2 DSG2 6 107 136 23 √ √

Coxsackievirus and adenovirus CXADR 0 105 112 12 √ √ √

receptor

Lysosome membrane protein 2 SCARB2 6 102 132 19 √ √

Basic fibroblast growth factor FGFR1 0 101 81 13 √ √ √

receptor 1

Lysosome-associated membrane LAMP1 2 101 105 32 √ √ √

glycoprotein 1

Choline transporter-like protein 2 SLC44A2 2 99 82 13 √ √ √

Lysosomal acid phosphatase ACP2 0 99 131 19 √ √ √

Tyrosine-protein kinase ROR2 0 99 120 14 √ √ √

transmembrane receptor ROR2

Endothelin-converting enzyme 1 ECE1 0 98 77 26 √ √ √

Polypeptide N- GALNT2 4 98 61 13 √ √

acetylgalactosaminyltransferase 2

Frizzled-7 FZD7 0 97 112 33 √ √ √

Alpha-mannosidase 2 MAN2A1 1 95 50 27 √ √ √

Trans-Golgi network integral TGOLN2 4 91 85 17 √ √

membrane protein 2

Junctional adhesion molecule A F11R 1 89 98 14 √ √ √

Prominin-1 PROM1 0 89 54 11 √ √ √

CD276 antigen CD276 1 87 128 17 √ √ √

Integrin alpha-5 ITGA5 0 87 84 15 √ √ √

Intercellular adhesion molecule 1 ICAM1 0 87 130 23 √ √ √

Protein sidekick-2 SDK2 0 87 100 11 √ √ √

Page S30
 Leucyl-cystinyl aminopeptidase LNPEP 0 84 99 16 √ √ √

Neuroplastin NPTN 0 84 119 13 √ √ √

Poliovirus receptor-related protein 2 PVRL2 0 83 76 5 √ √ √

Equilibrative nucleoside transporter 1 SLC29A1 6 82 75 21 √ √

Sodium-coupled neutral amino acid SLC38A2 6 81 133 36 √ √ √

transporter 2

Low-density lipoprotein receptor- LRP4 0 80 65 24 √ √ √

related protein 4

Platelet endothelial cell adhesion PECAM1 1 79 118 16 √ √ √

molecule

Endoplasmic reticulum mannosyl- MAN1B1 0 78 29 20 √ √ √

oligosaccharide 1,2-alpha-

mannosidase

Neural cell adhesion molecule L1 L1CAM 0 78 78 15 √ √ √

Tripeptidyl-peptidase 1 TPP1 2 78 82 8 √ √

Apolipoprotein E APOE 15 78 67 32 √

Golgi membrane protein 1 GOLM1 0 76 76 9 √ √ √

Sodium/potassium-transporting ATP1B1 2 76 66 13 √ √ √

ATPase subunit beta-1

Synaptophysin-like protein 1 SYPL1 4 76 97 10 √ √

Endoplasmic reticulum resident ERP44 5 74 50 23 √ √

protein 44

Leucine-rich repeat neuronal protein LRRN1 3 73 69 21 √ √ √

Transmembrane 9 superfamily TM9SF3 6 72 50 8 √ √

member 3

Insulin-like growth factor 1 receptor IGF1R 0 71 77 27 √ √ √

Low-density lipoprotein receptor LDLR 1 69 61 24 √ √ √

Protein ITFG3 ITFG3 0 68 31 6 √ √ √

Trophoblast glycoprotein TPBG 0 68 71 19 √ √ √

Bone marrow stromal antigen 2 BST2 3 65 122 25 √ √ √

Page S31
 Integrin alpha-3 ITGA3 0 64 76 12 √ √ √

Lysosome-associated membrane LAMP2 1 64 66 20 √ √ √

glycoprotein 2

Plasminogen activator inhibitor 1 SERPINE1 1 64 45 11 √ √ √

CD166 antigen ALCAM 0 62 47 14 √ √ √

Probable serine carboxypeptidase CPVL 3 62 83 20 √ √ √

CPVL

Prostaglandin F2 receptor negative PTGFRN 0 62 69 8 √ √ √

regulator

Discoidin, CUB and LCCL domain- DCBLD2 0 61 62 6 √ √ √

containing protein 2

Plexin-B2 PLXNB2 0 60 63 9 √ √ √

Receptor-type tyrosine-protein PTPRA 0 60 25 6 √ √ √

phosphatase alpha

Cell adhesion molecule 1 CADM1 0 59 51 18 √ √ √

High affinity cationic amino acid SLC7A1 7 59 64 16 √ √

transporter 1

Transmembrane protein 165 TMEM165 0 59 40 0 √ √

Kin of IRRE-like protein 1 KIRREL 1 57 54 10 √ √ √

Beta-galactosidase GLB1 9 54 53 17 √ √

Amyloid-like protein 2 APLP2 0 53 28 8 √ √ √

Voltage-dependent calcium channel CACNA2D1 0 53 51 7 √ √ √

subunit alpha-2/delta-1

Cell cycle control protein 50A TMEM30A 0 53 49 3 √ √

Plexin-D1 PLXND1 0 53 21 2 √ √

N-acetylgalactosaminyltransferase 7 GALNT7 0 52 41 12 √ √ √

Protein CASC4 CASC4 0 52 43 10 √ √ √

Tyrosine-protein kinase receptor AXL 0 52 46 6 √ √ √

UFO

Frizzled-2 FZD2 4 52 57 10 √ √

Page S32
Pituitary tumor-transforming gene 1 PTTG1IP 11 52 66 15 √

protein-interacting protein

Anoctamin-6 ANO6 0 51 30 5 √ √ √

Immunoglobulin superfamily member IGSF3 0 51 55 5 √ √ √

Niemann-Pick C1 protein NPC1 0 51 44 8 √ √ √

Polypeptide N- GALNT1 0 51 43 26 √ √ √

acetylgalactosaminyltransferase 1

V-type proton ATPase 116 kDa ATP6V0A1 1 51 37 4 √ √

subunit a isoform 1

5~-nucleotidase NT5E 0 50 48 5 √ √ √

Insulin receptor INSR 0 50 32 9 √ √ √

Matrix-remodeling-associated protein MXRA8 0 50 54 8 √ √ √

Zinc transporter ZIP14 SLC39A14 7 49 43 22 √ √

Poliovirus receptor-related protein 3 PVRL3 0 48 47 10 √ √ √

Sodium bicarbonate cotransporter 3 SLC4A7 0 48 26 17 √ √ √

CD63 antigen CD63 0 47 80 19 √ √ √

Cell adhesion molecule 4 CADM4 0 47 53 10 √ √ √

Neuronal cell adhesion molecule NRCAM 0 47 37 9 √ √ √

Collagen alpha-1(XVIII) chain COL18A1 4 47 37 12 √ √

Solute carrier family 12 member 2 SLC12A2 0 47 39 1 √ √

VWFA and cache domain-containing CACHD1 1 46 57 7 √ √ √

protein 1

Protein FAM3C FAM3C 6 46 30 8 √ √

Vesicular integral-membrane protein LMAN2 6 46 40 6 √ √

VIP36

Synaptic vesicle glycoprotein 2A SV2A 0 45 36 8 √ √ √

Protein disulfide-isomerase A6 PDIA6 44 45 69 231 √

Basal cell adhesion molecule BCAM 3 45 50 4 √ √

Page S33
 CD109 antigen CD109 0 44 23 2 √ √

Endothelial protein C receptor PROCR 0 44 32 4 √ √

Sodium-dependent multivitamin SLC5A6 0 43 23 9 √ √ √

transporter

Thy-1 membrane glycoprotein THY1 1 43 115 5 √ √ √

Lysosomal alpha-mannosidase MAN2B1 0 43 41 3 √ √

Ephrin-B1 EFNB1 1 42 48 9 √ √ √

Transmembrane protein 87A TMEM87A 0 42 27 9 √ √ √

Epidermal growth factor receptor EGFR 0 42 49 2 √ √

Myelin protein zero-like protein 1 MPZL1 0 41 27 2 √ √

Palmitoyl-protein thioesterase 1 PPT1 3 41 57 13 √ √

ADAMTS-like protein 4 ADAMTSL4 0 40 17 7 √ √ √

Alpha-1,6-mannosyl-glycoprotein 2- MGAT2 0 40 31 5 √ √ √

beta-N-

acetylglucosaminyltransferase

Integrin beta-5 ITGB5 0 40 33 6 √ √ √

Reversion-inducing cysteine-rich RECK 0 40 20 6 √ √ √

protein with Kazal motifs

Ephrin type-A receptor 7 EPHA7 0 40 38 3 √ √

Proactivator polypeptide PSAP 0 39 42 0 √ √

Coiled-coil domain-containing protein CCDC47 2 37 39 13 √ √ √

47

Vasorin VASN 0 37 36 9 √ √ √

Cystine/glutamate transporter SLC7A11 0 37 12 1 √ √

Tetraspanin-6 TSPAN6 0 37 41 4 √ √

Uronyl 2-sulfotransferase UST 0 37 33 4 √ √

Zinc transporter 1 SLC30A1 0 36 33 6 √ √ √

Sodium-coupled neutral amino acid SLC38A1 0 35 39 13 √ √ √

transporter 1

Ciliary neurotrophic factor receptor CNTFR 2 34 30 3 √ √

subunit alpha

Matrix metalloproteinase-14 MMP14 0 34 27 1 √ √

Page S34
Uncharacterized protein KIAA0319- KIAA0319L 0 34 18 4 √ √

like

Osteopetrosis-associated OSTM1 0 33 41 8 √ √ √

transmembrane protein 1

Sushi domain-containing protein 5 SUSD5 0 33 23 18 √ √ √

Galectin-3-binding protein LGALS3BP 0 33 21 2 √ √

Multidrug resistance-associated ABCC1 0 33 28 3 √ √

protein 1

Complement decay-accelerating CD55 0 32 36 7 √ √ √

factor

Sortilin SORT1 0 32 32 8 √ √ √

Carbohydrate sulfotransferase 14 CHST14 0 32 12 2 √ √

Glucosylceramidase GBA 3 32 41 4 √ √

Beta-type platelet-derived growth PDGFRB 0 32 35 0 √ √

factor receptor

N-acetylated-alpha-linked acidic NAALAD2 0 32 15 0 √ √

dipeptidase 2

Melanocyte protein Pmel 17 SILV 0 31 27 10 √ √ √

Solute carrier family 12 member 9 SLC12A9 0 31 12 4 √ √

Semaphorin-4D SEMA4D 0 30 20 6 √ √ √

Beta-hexosaminidase subunit beta HEXB 6 30 45 14 √ √

Keratinocyte-associated KCT2 0 29 34 6 √ √ √

transmembrane protein 2

Cationic amino acid transporter 3 SLC7A3 0 29 15 3 √ √

High affinity copper uptake protein 1 SLC31A1 0 29 32 1 √ √

Uncharacterized protein KIAA2013 KIAA2013 0 29 13 1 √ √

Lipid phosphate phosphohydrolase 1 PPAP2A 0 29 12 0 √ √

Protein jagged-1 JAG1 0 28 37 3 √ √

Xylosyltransferase 2 XYLT2 0 28 6 4 √ √

Junctional adhesion molecule C JAM3 0 28 16 0 √ √

Roundabout homolog 1 ROBO1 0 28 18 0 √ √

Crumbs homolog 2 CRB2 0 27 17 5 √ √ √

Integrin alpha-1 ITGA1 0 27 33 6 √ √ √

Page S35
 Membrane cofactor protein CD46 0 27 28 8 √ √ √

Beta-galactoside alpha-2,6- ST6GAL1 0 27 29 1 √ √

sialyltransferase 1

Immunoglobulin superfamily member IGSF8 0 26 39 8 √ √ √

Poliovirus receptor PVR 0 26 35 11 √ √ √

Alpha-N-acetylglucosaminidase NAGLU 0 26 24 4 √ √

CD81 antigen CD81 4 26 26 1 √ √

Choline transporter-like protein 1 SLC44A1 3 26 15 3 √ √

Plexin-A1 PLXNA1 0 26 21 1 √ √

Endoglin ENG 0 25 19 5 √ √ √

Alpha-galactosidase A GLA 1 25 24 3 √ √

Carbonic anhydrase 14 CA14 0 25 27 2 √ √

Neutral amino acid transporter A SLC1A4 0 25 19 4 √ √

Poliovirus receptor-related protein 1 PVRL1 0 25 15 1 √ √

Chloride transport protein 6 CLCN6 0 24 17 5 √ √ √

Feline leukemia virus subgroup C FLVCR1 0 24 24 0 √ √

receptor-related protein 1

N-acetylglucosamine-1- NAGPA 0 24 24 0 √ √

phosphodiester alpha-N-

acetylglucosaminidase

Protein tweety homolog 3 TTYH3 0 24 30 0 √ √

Receptor-type tyrosine-protein PTPRF 0 24 55 0 √ √

phosphatase F

Fibulin-1 FBLN1 0 23 43 5 √ √ √

Carbohydrate sulfotransferase 10 CHST10 0 23 20 1 √ √

Ectonucleotide ENPP4 0 23 16 3 √ √

pyrophosphatase/phosphodiesterase

family member 4

Leukocyte surface antigen CD47 CD47 0 23 23 2 √ √

Protein GPR107 GPR107 0 23 27 2 √ √

Protocadherin-1 PCDH1 0 23 14 1 √ √

Semaphorin-7A SEMA7A 2 23 25 5 √ √

Page S36
Sodium/myo-inositol cotransporter SLC5A3 1 23 12 2 √ √

CD82 antigen CD82 0 23 28 0 √ √

Transmembrane emp24 domain- TMED9 4 23 19 3 √

containing protein 9

N-acetylglucosamine-6-sulfatase GNS 2 22 45 10 √ √ √

Alpha-1,3-mannosyl-glycoprotein 2- MGAT1 0 22 10 1 √ √

beta-N-

acetylglucosaminyltransferase

Anthrax toxin receptor 1 ANTXR1 0 22 18 2 √ √

Low affinity cationic amino acid SLC7A2 0 22 19 0 √ √

transporter 2

Carcinoembryonic antigen-related CEACAM1 0 21 14 3 √ √

cell adhesion molecule 1

Low-density lipoprotein receptor- LRP6 0 21 17 1 √ √

related protein 6

Neogenin NEO1 0 21 13 3 √ √

NHL repeat-containing protein 3 NHLRC3 0 21 14 4 √ √

Zinc transporter ZIP10 SLC39A10 0 21 20 3 √ √

Carbohydrate sulfotransferase 6 CHST6 0 21 12 0 √ √

Fibroblast growth factor receptor 3 FGFR3 0 21 15 0 √ √

Beta-hexosaminidase subunit alpha HEXA 0 20 21 5 √ √ √

Alpha-(1,3)-fucosyltransferase FUT4 0 20 13 1 √ √

Beta-1,4-galactosyltransferase 5 B4GALT5 0 20 18 3 √ √

CD320 antigen CD320 0 20 18 1 √ √

Disintegrin and metalloproteinase ADAM10 0 20 27 3 √ √

domain-containing protein 10

Exostosin-like 2 EXTL2 0 20 12 1 √ √

N-acetyllactosaminide beta-1,3-N- B3GNT1 0 20 11 1 √ √

acetylglucosaminyltransferase

Transmembrane protein 9 TMEM9 0 20 23 1 √ √

Beta-1,4-galactosyltransferase 3 B4GALT3 0 20 8 0 √ √

Excitatory amino acid transporter 1 SLC1A3 0 19 4 5 √ √

CD151 antigen CD151 0 19 29 3 √ √

Integrin alpha-7 ITGA7 0 19 12 1 √ √

Page S37
 Receptor tyrosine-protein kinase ERBB2 0 19 27 1 √ √

erbB-2

Receptor-type tyrosine-protein PTPRJ 0 19 26 3 √ √

phosphatase eta

Beta-1,4-galactosyltransferase 1 B4GALT1 0 19 8 0 √ √

Matrix metalloproteinase-15 MMP15 0 19 8 0 √ √

Neuroligin-3 NLGN3 0 19 7 0 √ √

Receptor-type tyrosine-protein PTPRK 0 19 21 0 √ √

phosphatase kappa

Signal peptide peptidase-like 2A SPPL2A 0 19 7 0 √ √

Transmembrane protein 2 TMEM2 0 19 33 0 √ √

Neuroligin-2 NLGN2 0 19 0 0 √

N-acetylglucosamine-1- GNPTG 0 18 13 10 √ √ √

phosphotransferase subunit gamma

Seizure 6-like protein 2 SEZ6L2 0 18 11 8 √ √ √

Fibroblast growth factor receptor 2 FGFR2 0 18 25 3 √ √

HLA class I histocompatibility HLA-A 0 18 25 4 √ √

antigen, A-68 alpha chain

Immunoglobulin superfamily member IGSF1 0 18 10 1 √ √

LysM and putative peptidoglycan- LYSMD3 0 18 7 1 √ √

binding domain-containing protein

Nucleobindin-1 NUCB1 1 18 13 1 √ √

Multidrug resistance-associated ABCC4 0 18 9 0 √ √

protein 4

Probable lysosomal cobalamin LMBRD1 0 18 4 2 √

transporter

Attractin ATRN 0 17 25 5 √ √ √

Monocarboxylate transporter 1 SLC16A1 1 17 20 11 √ √ √

Scavenger receptor class B member 1 SCARB1 1 17 30 5 √ √ √

CD97 antigen CD97 0 17 12 0 √ √

Integral membrane protein 2B ITM2B 0 17 7 0 √ √

Page S38
 HLA class I histocompatibility HLA-A 0 17 0 0 √

antigen, A-69 alpha chain

Alpha-1,6-mannosylglycoprotein 6- MGAT5 0 16 13 4 √ √

beta-N-

acetylglucosaminyltransferase A

Disintegrin and metalloproteinase ADAM15 0 16 17 2 √ √

domain-containing protein 15

Nidogen-1 NID1 0 16 13 4 √ √

Occludin OCLN 2 16 17 4 √ √

Semaphorin-4C SEMA4C 0 16 13 1 √ √

Tetraspanin-13 TSPAN13 0 16 22 1 √ √

Acid ceramidase ASAH1 0 16 19 0 √ √

Transmembrane protein 41B TMEM41B 2 16 5 1 √

Clusterin CLU 0 15 18 1 √ √

Disintegrin and metalloproteinase ADAM9 0 15 15 1 √ √

domain-containing protein 9

Tetraspanin-3 TSPAN3 0 15 26 3 √ √

Ephrin type-B receptor 4 EPHB4 0 15 6 0 √ √

Folate receptor alpha FOLR1 0 15 9 0 √ √

Lactadherin MFGE8 0 15 11 0 √ √

Podocalyxin-like protein 2 PODXL2 0 15 5 0 √ √

Putative polypeptide N- WBSCR17 0 15 5 0 √ √

acetylgalactosaminyltransferase-

like protein 3

Solute carrier family 12 member 4 SLC12A4 0 15 6 0 √ √

T-cell immunomodulatory protein ITFG1 0 15 11 0 √ √

Tenascin TNC 2 15 16 0 √ √

Beta-mannosidase MANBA 1 14 20 6 √ √ √

Major prion protein PRNP 0 14 14 8 √ √ √

Endoplasmic reticulum-Golgi ERGIC3 0 14 10 1 √ √

intermediate compartment protein

N-acetylglucosamine-1- GNPTAB 0 14 11 3 √ √

phosphotransferase subunits

alpha/beta

Page S39
 Receptor expression-enhancing REEP5 1 14 13 2 √ √

protein 5

Transmembrane emp24 domain- TMED7 2 14 16 8 √ √

containing protein 7

HLA class I histocompatibility HLA-A 0 14 26 0 √ √

antigen, A-34 alpha chain

Neurogenic locus notch homolog NOTCH2 0 14 15 0 √ √

protein 2

Plexin-B1 PLXNB1 0 14 16 0 √ √

Sodium/potassium-transporting ATP1A2 0 14 24 0 √ √

ATPase subunit alpha-2

Tetraspanin-18 TSPAN18 0 14 9 0 √ √

Beta-1,3-galactosyltransferase 6 B3GALT6 0 14 4 1 √

Transmembrane 9 superfamily TM9SF1 0 14 4 0 √

member 1

Frizzled-1 FZD1 0 13 19 6 √ √ √

Inositol monophosphatase 3 IMPAD1 0 13 9 1 √ √

Leukosialin SPN 0 13 8 4 √ √

Lysosomal protein NCU-G1 C1orf85 0 13 11 2 √ √

Serine incorporator 3 SERINC3 0 13 11 1 √ √

Uncharacterized protein KIAA1467 KIAA1467 0 13 18 1 √ √

Ephrin type-A receptor 2 EPHA2 0 13 10 0 √ √

Integrin alpha-2 ITGA2 0 13 8 0 √ √

Solute carrier family 12 member 6 SLC12A6 0 13 6 0 √ √

Transmembrane emp24 domain- TMED4 0 13 9 0 √ √

containing protein 4

Solute carrier family 23 member 2 SLC23A2 0 13 1 0 √

CD59 glycoprotein CD59 0 12 11 5 √ √ √

Galectin-1 LGALS1 1 12 7 6 √ √ √

Acid sphingomyelinase-like SMPDL3B 0 12 12 1 √ √

phosphodiesterase 3b

Calumenin CALU 1 12 18 1 √ √

Glycerophosphoinositol GDPD2 0 12 9 1 √ √

inositolphosphodiesterase GDPD2

Page S40
Peptidyl-glycine alpha-amidating PAM 0 12 6 1 √ √

monooxygenase

Transmembrane protein 206 TMEM206 0 12 12 1 √ √

Alpha-N-acetylgalactosaminidase NAGA 0 12 14 0 √ √

Disintegrin and metalloproteinase ADAM23 0 12 7 0 √ √

domain-containing protein 23

Ephrin type-B receptor 2 EPHB2 0 12 10 0 √ √

Epithelial membrane protein 3 EMP3 0 12 12 0 √ √

Leucine-rich repeats and LRIG1 0 12 15 0 √ √

immunoglobulin-like domains

protein 1

N-acetyllactosaminide beta-1,6-N- GCNT2 0 12 11 0 √ √

acetylglucosaminyl-transferase

Neurosecretory protein VGF VGF 0 12 2 1 √

Fukutin-related protein FKRP 0 11 9 1 √ √

Latrophilin-3 LPHN3 0 11 7 2 √ √

Serine incorporator 1 SERINC1 0 11 19 1 √ √

Syndecan-2 SDC2 0 11 9 2 √ √

Amyotrophic lateral sclerosis 2 ALS2CR4 0 11 7 0 √ √

chromosomal region candidate gene

4 protein

Disintegrin and metalloproteinase ADAM17 0 11 11 0 √ √

domain-containing protein 17

Interferon gamma receptor 1 IFNGR1 0 11 10 0 √ √

Membrane protein FAM174B FAM174B 0 11 8 0 √ √

Soluble calcium-activated CANT1 0 11 8 0 √ √

nucleotidase 1

Tetraspanin-9 TSPAN9 0 11 9 0 √ √

Transmembrane protein 63B TMEM63B 0 11 5 0 √ √

Gremlin-1 GREM1 2 11 3 3 √

Putative sodium-coupled neutral SLC38A9 0 11 3 2 √

amino acid transporter 9

Acid phosphatase-like protein 2 ACPL2 0 11 4 0 √

Latrophilin-1 LPHN1 0 11 3 0 √

Protein-tyrosine sulfotransferase 1 TPST1 0 11 2 0 √

Page S41
 Protein HEG homolog 1 HEG1 0 10 22 10 √ √ √

Ephrin-B2 EFNB2 0 10 15 2 √ √

Phospholipid transfer protein PLTP 0 10 5 1 √ √

Syndecan-1 SDC1 0 10 10 3 √ √

Tetraspanin-31 TSPAN31 0 10 12 1 √ √

Tumor necrosis factor receptor TNFRSF21 0 10 9 1 √ √

superfamily member 21

Amphoterin-induced protein 2 AMIGO2 0 10 5 0 √ √

Extracellular matrix protein FRAS1 FRAS1 0 10 14 0 √ √

Killer cell lectin-like receptor KLRG2 0 10 6 0 √ √

subfamily G member 2

Natural resistance-associated SLC11A2 0 10 10 0 √ √

macrophage protein 2

Neurogenic locus notch homolog NOTCH3 0 10 18 0 √ √

protein 3

OX-2 membrane glycoprotein CD200 0 10 7 0 √ √

Sialin SLC17A5 0 10 7 0 √ √

Tumor necrosis factor receptor NGFR 0 10 9 0 √ √

superfamily member 16

Agrin AGRN 0 10 4 1 √

EGF, latrophilin and seven ELTD1 0 10 0 0 √

transmembrane domain-containing

protein 1

Melanotransferrin MFI2 0 10 3 0 √

CD99 antigen CD99 0 9 5 1 √ √

Neuronal membrane glycoprotein GPM6B 0 9 11 1 √ √

M6-b

Proton myo-inositol cotransporter SLC2A13 0 9 5 1 √ √

Sodium- and chloride-dependent SLC6A6 0 9 16 2 √ √

taurine transporter

Endothelin B receptor EDNRB 0 9 6 0 √ √

Granulins GRN 1 9 8 0 √ √

Lymphocyte function-associated CD58 0 9 9 0 √ √

antigen 3

Page S42
Major facilitator superfamily domain- MFSD8 0 9 8 0 √ √

containing protein 8

Protein sidekick-1 SDK1 0 9 12 0 √ √

SID1 transmembrane family member SIDT2 0 9 9 0 √ √

Solute carrier family 15 member 4 SLC15A4 0 9 7 0 √ √

Transmembrane protein 9B TMEM9B 0 9 8 0 √ √

Uncharacterized protein C1orf159 C1orf159 0 9 7 0 √ √

Small cell adhesion glycoprotein SMAGP 0 9 3 1 √

Calcium release-activated calcium ORAI1 0 9 3 0 √

channel protein 1

Solute carrier family 2, facilitated SLC2A10 0 9 0 0 √

glucose transporter member 10

Transmembrane protein 219 TMEM219 0 9 4 0 √

UDP-GlcNAc:betaGal beta-1,3-N- B3GNT2 0 9 4 0 √

acetylglucosaminyltransferase 2

Aspartyl/asparaginyl beta- ASPH 1 8 6 29 √ √ √

hydroxylase

Osteopontin SPP1 0 8 7 3 √ √

Porimin TMEM123 0 8 6 1 √ √

Transmembrane emp24 domain- TMED1 0 8 9 2 √ √

containing protein 1

Tumor necrosis factor receptor TNFRSF10C 0 8 5 2 √ √

superfamily member 10C

FRAS1-related extracellular matrix FREM2 0 8 11 0 √ √

protein 2

Gamma-glutamyltranspeptidase 1 GGT1 0 8 13 0 √ √

Leucine-rich repeat-containing G- LGR4 0 8 8 0 √ √

protein coupled receptor 4

Mucolipin-1 MCOLN1 0 8 7 0 √ √

Protein tweety homolog 1 TTYH1 0 8 9 0 √ √

UPF0458 protein C7orf42 C7orf42 0 8 10 0 √ √

V-type proton ATPase subunit S1 ATP6AP1 0 8 11 0 √ √

Adipocyte adhesion molecule ACAM 0 8 3 1 √

Cadherin-13 CDH13 0 8 4 1 √

Ephrin-B3 EFNB3 0 8 3 0 √

Page S43
 H(+)/Cl(-) exchange transporter 3 CLCN3 0 8 2 0 √

Probable cation-transporting ATPase ATP13A3 0 8 2 0 √

13A3

Solute carrier family 12 member 7 SLC12A7 0 8 4 0 √

Transmembrane protein 119 TMEM119 0 8 4 0 √

Cadherin-5 CDH5 0 7 18 1 √ √

Heparan sulfate 2-O-sulfotransferase HS2ST1 0 7 8 1 √ √

Kunitz-type protease inhibitor 2 SPINT2 0 7 7 1 √ √

Protein FAM20B FAM20B 0 7 7 2 √ √

Transmembrane protein 179B TMEM179B 0 7 11 3 √ √

G-protein coupled receptor family C GPRC5B 0 7 8 0 √ √

group 5 member B

Lysosomal acid lipase/cholesteryl LIPA 0 7 7 0 √ √

ester hydrolase

Transmembrane protein 132A TMEM132A 0 7 5 0 √ √

Zinc transporter ZIP1 SLC39A1 0 7 7 0 √ √

Anion exchange protein 2 SLC4A2 0 7 1 1 √

HLA class I histocompatibility HLA-A 0 7 3 3 √

antigen, A-25 alpha chain

Alpha-1,3-mannosyl-glycoprotein 4- MGAT4B 0 7 2 0 √

beta-N-

acetylglucosaminyltransferase B

Carbohydrate sulfotransferase 12 CHST12 0 7 3 0 √

Carboxypeptidase M CPM 0 7 2 0 √

Frizzled-6 FZD6 0 7 2 0 √

Uncharacterized protein C3orf21 C3orf21 0 7 2 0 √

V-type proton ATPase 116 kDa ATP6V0A2 0 7 3 0 √

subunit a isoform 2

Gamma-glutamyl hydrolase GGH 0 6 5 8 √ √ √

Group XV phospholipase A2 PLA2G15 1 6 11 4 √ √

Growth/differentiation factor 15 GDF15 0 6 7 3 √ √

V-type proton ATPase subunit C 1 ATP6V1C1 0 6 8 3 √ √

Battenin CLN3 0 6 9 0 √ √

Exostosin-like 3 EXTL3 0 6 10 0 √ √

Page S44
 Fibroblast growth factor-binding FGFBP3 0 6 7 0 √ √

protein 3

Intercellular adhesion molecule 3 ICAM3 0 6 6 0 √ √

Protein FAM3A FAM3A 0 6 5 0 √ √

Receptor-type tyrosine-protein PTPRM 0 6 5 0 √ √

phosphatase mu

Tissue factor F3 0 6 7 0 √ √

Transmembrane protein 106B TMEM106B 0 6 11 0 √ √

Proline-rich transmembrane protein 3 PRRT3 0 6 3 1 √

Claudin domain-containing protein 1 CLDND1 0 6 4 0 √

Glucoside xylosyltransferase 1 GXYLT1 0 6 4 0 √

HLA class I histocompatibility HLA-C 0 6 0 0 √

antigen, Cw-12 alpha chain

HLA class I histocompatibility HLA-C 0 6 0 0 √

antigen, Cw-6 alpha chain

Myelin protein P0 MPZ 0 6 4 0 √

Na(+)/H(+) exchange regulatory SLC9A3R2 0 6 1 0 √

cofactor NHE-RF2

NKG2D ligand 3 ULBP3 0 6 4 0 √

Phosphoinositide-3-kinase-interacting PIK3IP1 0 6 1 0 √

protein 1

Porcine endogenous retrovirus A GPR172A 0 6 2 0 √

receptor 1

Protein JTB JTB 0 6 4 0 √

RING finger protein 13 RNF13 0 6 3 0 √

Signal peptide peptidase-like 2B SPPL2B 0 6 2 0 √

Sodium- and chloride-dependent SLC6A9 0 6 3 0 √

glycine transporter 1

Sodium-dependent phosphate SLC20A2 0 6 4 0 √

transporter 2

Stimulated by retinoic acid gene 6 STRA6 0 6 4 0 √

protein homolog

Sulfhydryl oxidase 1 QSOX1 0 6 2 0 √

Tetraspanin-4 TSPAN4 0 6 1 0 √

Transmembrane protein 59 TMEM59 0 6 3 0 √

Page S45
 Tumor necrosis factor receptor TNFRSF10B 0 6 1 0 √

superfamily member 10B

Protein transport protein Sec61 SEC61B 0 5 7 7 √ √ √

subunit beta

Probable glutathione peroxidase 8 GPX8 0 5 8 2 √ √

Prostasin PRSS8 0 5 5 1 √ √

Sialidase-1 NEU1 0 5 6 1 √ √

Zinc transporter ZIP6 SLC39A6 0 5 12 1 √ √

Ceroid-lipofuscinosis neuronal CLN5 0 5 10 0 √ √

protein 5

Glycerophosphodiester GDPD5 0 5 5 0 √ √

phosphodiesterase domain-

containing protein 5

Heparan-sulfate 6-O-sulfotransferase HS6ST2 0 5 6 0 √ √

Neurogenic locus notch homolog NOTCH1 0 5 7 0 √ √

protein 1

Sodium-dependent glucose NAGLT1 0 5 6 0 √ √

transporter 1

Sphingosine 1-phosphate receptor 3 S1PR3 0 5 6 0 √ √

Tetraspanin-14 TSPAN14 0 5 7 0 √ √

Cathepsin B CTSB 2 5 11 5 √

Myeloid-associated differentiation MYADM 1 5 1 1 √

marker

Protein transport protein Sec61 SEC61A1 0 5 2 3 √

subunit alpha isoform 1

Surfeit locus protein 4 SURF4 1 5 0 2 √

Carbohydrate sulfotransferase 3 CHST3 0 5 1 0 √

Claudin-12 CLDN12 0 5 1 0 √

CMP-N-acetylneuraminate-beta- ST3GAL2 0 5 2 0 √

galactosamide-alpha-2,3-

sialyltransferase 2

Disrupted in renal carcinoma protein DIRC2 0 5 3 0 √

Metalloproteinase inhibitor 1 TIMP1 0 5 2 0 √

Protein Dos DOS 0 5 1 0 √

Page S46
 Proteinase-activated receptor 1 F2R 0 5 2 0 √

Reticulon-4 receptor-like 2 RTN4RL2 0 5 4 0 √

Sodium-driven chloride bicarbonate SLC4A10 0 5 3 0 √

exchanger

Type 2 lactosamine alpha-2,3- ST3GAL6 0 5 3 0 √

sialyltransferase

N-sulphoglucosamine SGSH 2 4 20 3 √

sulphohydrolase

Plasma alpha-L-fucosidase FUCA2 0 4 5 3 √

Autophagy-related protein 9A ATG9A 0 4 6 0 √

Ephrin type-A receptor 5 EPHA5 0 4 6 0 √

Frizzled-5 FZD5 0 4 6 0 √

Monoacylglycerol lipase ABHD12 ABHD12 0 3 1 5 √

Protein CYR61 CYR61 0 3 3 5 √

Secretory carrier-associated SCAMP4 1 3 6 1 √

membrane protein 4

Brother of CDO BOC 0 3 7 0 √

Cadherin EGF LAG seven-pass G- CELSR2 0 3 15 0 √

type receptor 2

Sodium-dependent phosphate SLC20A1 0 3 7 0 √

transporter 1

Sphingomyelin phosphodiesterase SMPD1 0 3 5 0 √

Anosmin-1 KAL1 0 2 10 1 √

Endoplasmic reticulum-Golgi ERGIC2 0 2 5 1 √

intermediate compartment protein

Endosialin CD248 0 2 6 1 √

Epididymis-specific alpha- MAN2B2 0 2 5 0 √

mannosidase

Protocadherin-18 PCDH18 0 2 7 0 √

Transmembrane 7 superfamily TM7SF3 0 2 6 0 √

member 3

Putative phospholipase B-like 2 PLBD2 0 1 15 5 √ √

Arylsulfatase E ARSE 0 1 0 42 √

Page S47
 Protein disulfide-isomerase TMX3 TMX3 1 1 1 7 √

Thioredoxin domain-containing TXNDC12 1 1 1 11 √

protein 12

Mammalian ependymin-related EPDR1 1 1 5 4 √

protein 1

Cell adhesion molecule-related/down- CDON 0 1 7 0 √

regulated by oncogenes

Contactin-associated protein 1 CNTNAP1 0 0 0 10 √

Laminin subunit beta-1 LAMB1 1 0 1 7 √

Pregnancy zone protein PZP 0 0 3 7 √

Protein disulfide-isomerase A5 PDIA5 0 0 0 5 √

Semenogelin-1 SEMG1 0 0 0 5 √

Sulfatase-modifying factor 1 SUMF1 0 0 1 6 √

Zymogen granule protein 16 homolog ZG16B 4 0 1 21 √

Ephrin type-A receptor 4 EPHA4 0 0 5 0 √

Epididymal secretory protein E1 NPC2 0 0 5 0 √

HLA class I histocompatibility HLA-B 0 0 7 0 √

antigen, B-41 alpha chain

Intercellular adhesion molecule 2 ICAM2 0 0 6 0 √

Proteinase-activated receptor 2 F2RL1 0 0 6 0 √

Receptor-type tyrosine-protein PTPRD 0 0 13 0 √

phosphatase delta

UPF0577 protein KIAA1324-like KIAA1324L 0 0 6 0 √

Page S48
Supporting reference

(1) Izumi, M., Shen, G. J., Wacowich-Sgarbi, S., Nakatani, T., Plettenburg, O., and Wong,
C. H. (2001) Microbial glycosyltransferases for carbohydrate synthesis: alpha-2,3-
sialyltransferase from Neisseria gonorrheae. J. Am. Chem. Soc. 123, 10909-10918.
(2) Pearce, O. M., and Varki, A. (2010) Chemo-enzymatic synthesis of the carbohydrate
antigen N-glycolylneuraminic acid from glucose. Carbohydr. Res. 345, 1225-1229.
(3) Kong, D. C. M., and von Itzstein, M. (1997) The chemoenzymatic synthesis of 9-
substituted 3,9-dideoxy-D-glycero-D-galacto-2-nonulosonic acids. Carbohydr. Res. 305, 323-
329.
(4) Han, S., Collins, B. E., Bengtson, P., and Paulson, J. C. (2005) Homomultimeric
complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1,
93-97.
(5) Yu, H., Yu, H., Karpel, R., and Chen, X. (2004) Chemoenzymatic synthesis of CMP-
sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of
three microbial CMP-sialic acid synthetases. Bioorg. Med. Chem. 12, 6427-6435.
(6) Mbua, N. E., Li, X., Flanagan-Steet, H. R., Meng, L., Aoki, K., Moremen, K. W.,
Wolfert, M. A., Steet, R., and Boons, G. J. (2013) Selective Exo-Enzymatic Labeling of N-
Glycans on the Surface of Living Cells by Recombinant ST6Gal I. Angew. Chem., Int. Ed.
(7) Chong, D. C., Paton, J. C., Thorpe, C. M., and Paton, A. W. (2008) Clathrin-dependent
trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum
chaperone BiP. Cell. Microbiol. 10, 795-806.
(8) Xie, R., Dong, L., Du, Y., Zhu, Y., Hua, R., Zhang, C., and Chen, X. (2016) In vivo
metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted
bioorthogonal reporter strategy. Proc. Natl. Acad. Sci. U. S. A. 113, 5173-5178.
(9) Hara, S., Takemori, Y., Yamaguchi, M., Nakamura, M., and Ohkura, Y. (1987)
Fluorometric high-performance liquid chromatography of N-acetyl- and N-
glycolylneuraminic acids and its application to their microdetermination in human and animal
sera, glycoproteins, and glycolipids. Anal. Biochem. 164, 138-145.
(10) Yu, H., Huang, S., Chokhawala, H., Sun, M., Zheng, H., and Chen, X. (2006) Highly
efficient chemoenzymatic synthesis of naturally occurring and non-natural alpha-2,6-linked
sialosides: a P. damsela alpha-2,6-sialyltransferase with extremely flexible donor-substrate
specificity. Angew. Chem., Int. Ed. 45, 3938-3944.

Page S49