Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose

Article abstract of DOI:10.1016/j.carres.2010.04.003

N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in human disease are hindered by limited sources of this ca

Full text of DOI:10.1016/j.carres.2010.04.003

Carbohydrate Research 345 (2010) 1225–1229
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Chemo-enzymatic synthesis of the carbohydrate antigen
N-glycolylneuraminic acid from glucose
*
Oliver M. T. Pearce, Ajit Varki
Department of Cellular and Molecular Medicine, University of California at San Diego, 9500 Gilman Dr. La Jolla, CA 92093-0687, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in
human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in
human disease are hindered by limited sources of this carbohydrate. Using a chemo-enzymatic approach,
Neu5Gc was accessed in six steps from glucose. The synthesis allows access to gram-scale quantities
quickly and economically and produces Neu5Gc in superior quality to commercial sources. Finally, we
demonstrate that the synthesized Neu5Gc can be incorporated into the cell glycocalyx of human cells,
which do not naturally synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or
in vivo use.
Received 19 February 2010
Received in revised form 1 April 2010
Accepted 6 April 2010
Available online 9 April 2010
Keywords:
Sialic acid
N-Glycolylneuraminic acid
Antigen
Ó 2010 Elsevier Ltd. All rights reserved.
Sialic acids (sias) are a family of acidic 9-carbon chain carbohy-
drates typically found in terminal positions on the cell glycocalyx
(the vast coating of glycans that covers all cell surfaces).1 Their bio-
logical functions include regulation of processes such as innate
immunity, inflammation,2 cell–cell interactions and neural plastic-
ity. They are also involved in tumour metastasis and pathogen
binding. In recent years the significance of this family of glycans
to human health and evolution is becoming ever clearer.3 However,
advancements have been hindered by a relative lack of available
sias and derivatives thereof with which to conduct these biological
studies. The sia N-glycolylneuraminic acid (Neu5Gc) is a non-hu-
man sialic acid, which is biosynthesized from N-acetylneuraminic
acid (Neu5Ac) via the enzyme CMP-Neu5Ac hydroxylase (CMAH)
encoded by the gene CMAH. Approximately 2–3 million years ago
the human CMAH gene was mutated and its product was no longer
able to hydroxylate CMP-Neu5Ac to CMP-Neu5Gc.4
Although humans are no longer able to make Neu5Gc, dietary
Neu5Gc can still be metabolically incorporated and displayed on
the glycocalyx of the human epithelia and associated carcinomas.5
In principle, Neu5Gc could be an important human-specific ‘xeno-
autoantigen’. Potential roles in tumourigenesis6 and vascular
pathologies7 have recently been identified.
To further investigate how dietary Neu5Gc is involved in hu-
man-specific disease requires synthetic access to Neu5Gc in large
quantities and high purity. Current commercial sources are limited
and expensive, contain 1–3% Neu5Ac which can interfere with
some biological assays, and their continued support and supply
have an uncertain future.8 We have developed a chemo-enzymatic
strategy to access Neu5Gc that is high yielding, suitable for use in
cell experiments requiring sterile conditions and allows quick
access to gram-scale quantities.
Synthetic methods to access the sias have been reported since
the 1980s.9 Because of the stereochemical considerations, most
methodologies take advantage of monosaccharide starting materi-
als. Auge et al.,9a synthesized Neu5Gc in milligram scale starting
from mannosamine hydrochloride, introducing the glycolyl moiety
using a benzyloxyacetic acid derivative followed by hydrogenation
to yield 2-deoxy-2-[(hydroxyacetyl)amino]-D-mannopyranose
(ManNGc), which was enzymatically converted to Neu5Gc.
This is an attractive route for small-scale synthesis, however, the
2-deoxy-2-amino-mannose, although commercially available, is
expensive and therefore impractical for use on large scale. Some
synthetic methods start from little or no chirality within the start-
ing materials.9b–d Kang et al. demonstrated a highly diastereoselec-
tive synthesis of Neu5Ac by stereoselective functionalization of
olefin starting materials.9b Some purely enzymatic synthetic routes
have also been described.10 These use enzymatic conversion of the
manno derivative, or the gluco derivative via a single or multiple
enzyme procedure. Wang et al. has recently demonstrated the syn-
thesis of Neu5Ac starting from N-acetyl-
immobilized enzymes, N-acetyl- -glucosamine 2-epimerase and
N-acetyl-
-neuraminic acid aldolase.10a In addition, bioreactors
D-glucosamine, via two
D
D
may also prove an important method to access neuraminic acids.
Feirfort et al. genetically engineered Escherichia coli to make exces-
sive quantities of sialylated glycans.11 Although further develop-
ment would be required here to isolate the pure sialic acid
monomer. Although a large body of work has already been under-
taken in the synthetic access of the neuraminic acids (examples are
summarized in Refs. 9–11), these studies have not been primarily
* Corresponding author. Tel.: +1 858 534 2214; fax: +1 858 534 5611.
E-mail addresses: varkiadmin@ucsd.edu, a1varki@ucsd.edu (A. Varki).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.04.003

1226
O. M. T. Pearce, A. Varki / Carbohydrate Research 345 (2010) 1225–1229
O
OH
OH
OH
OH
OH
NH2
O
HN
HO
HO
HO
HO
HO
HO
O
O
HO
CO2H
OH
NH
HO
HO
1
O
Neu5Gc
HO
HO
HO
PO
O
O
PO
OH
OP
PO
OH
Glucose
OH
Scheme 1. Retrosynthetic analysis of Neu5Gc synthesis reveals glucose as a suitable starting material. P = protecting group.
concerned with a quick, large-scale, high purity synthesis of the
Neu5Gc analogue.
Retrosynthetic analysis of Neu5Gc (Scheme 1) presents a syn-
thetic pathway from the readily available and cheap monosaccha-
Table 1
O-Linked hydrogenation in the presence of an amine, achieved using HCl(aq) within
the reaction mixture
Entry
Starting material
Catalyst
Solvent
Yielda (%)
ride
D-glucose. Orthogonal protection of the pyranose ring
1
2
3
4
4
3
4
4
Pd/C
MeOH
MeOH
MeOH
MeOH/HCl(aq)
ND
100
ND
100
followed by triflation prepares the molecule for selective stereo-
chemical inversion to the manno stereochemistry. Global deprotec-
tion and appending of the glycolyl moiety prepare the molecule for
final enzymatic conversion to the product Neu5Gc 1.
Scheme 2 outlines the synthetic route taken. Reaction of
cose with benzyl alcohol in the presence of acetyl chloride pro-
duced benzyl -glucopyranoside in 86% yield, which was
Pd(OH)2/C
Pd(OH)2/C
Pd(OH)2/C
a
The yield of the fully deprotected product (determined by TLC). ND = not
detected.
D-glu-
D
2
further modified to produce the 4,6-benzylidiene derivative 3 in
84% yield, by reaction with benzaldehyde dimethylacetyl and a
catalytic amount of (+)-camphor-10-sulfonic acid. Triflation of
the 2-OH group of 3 using triflic anhydride at ꢀ30 °C12 followed
by displacement with sodium azide selectively inverted the stereo-
chemistry to afford the 2-azido-2-deoxy-mannopyranoside 4 in
72% yield. A global deprotection strategy was envisaged using
Pd/C and H2(g), however, initial experiments showed only reduc-
tion of the azide moiety to form the amine product (Table 1, entry
1). No reduction of benzyl moieties occurred, thought to be due to
inhibition of the catalyst by the amine product (compare Table 1,
entries 1 and 2), an observation that has been made previously.13
It has been demonstrated that this problem can be overcome using
large amounts of Pd/C,14 however, this is only practical for small-
scale synthesis (5–10 mg). We initially changed catalyst to the
more rigorous Pearlmans catalyst, Pd(OH)2/C, which has been suc-
cessfully used to reduce more difficult O-benzyl moieties. How-
ever, this was unsuccessful (Table 1, entry 3). Access to 5 was
successfully achieved by addition of 1 M hydrochloric acid to the
reaction mixture (Table 1, entry 4), which afforded the product
after filtration through Celite in 89% yield. One previous example
of this has been demonstrated.15 Preparation of ManNGc 6 was
achieved in 94% yield by reaction with acetoxyacetyl chloride un-
der mild basic conditions and reduced temperature. Final conver-
sion of 6 to the target 1 was achieved using an aldolase isolated
from Pasteurella multocida, using a previously described method.16
Conversion of ManNGc to Neu5Gc using this methodology has pre-
viously been reported on a milligram scale.9a,16 Reaction of 6 with
the aldolase in the presence of an excess of pyruvate afforded
Neu5Gc 1 in 50% yield (Scheme 3). The starting material 6 could
be recovered during the purification step (see SI) and used again
as a substrate for the aldolase.
Comparison of the commercial Neu5Gc and synthesized ver-
sions by amperometric analysis (see SI Figs. S1–3) revealed the
methodology produced Neu5Gc which had no trace of Neu5Ac,
compared to approximately 3% seen in commercial sources.
Although humans can no longer make Neu5Gc, human tissue sam-
ples, including carcinomas, have been shown to have significant
levels of Neu5Gc on their surface.17 A likely explanation for this
is incorporation from dietary sources such as red meat products.
To test whether Neu5Gc could be incorporated into the cell glyco-
calyx of human cells, human monocytic cell line THP-1 was grown
in media containing either Neu5Ac, synthesized Neu5Gc, or
neither. After three days the cells were stained with a recently de-
scribed anti-Neu5Gc IgY18 and analyzed by flow cytometry (Fig. 1).
Neu5Gc fed cells showed incorporation of Neu5Gc into the cell sur-
face (1A). Moreover, the anti-Neu5Gc IgY staining seen in Neu5Gc
fed cells could be inhibited by addition of free Neu5Gc. Twenty
millimolar free Neu5Gc showed complete inhibition (Fig. 1B). In
addition, cells fed with the synthesized Neu5Gc showed compara-
ble cell proliferation to unfed cells (cell proliferation was measured
using a cytometer. Cells fed synthesized Neu5Gc, or commercial
HO
HO
HO
HO
HO
HO
a)
b)
Ph
O
O
O
O
O
OH
OBn
OBn
HO
OH
OH
2
3
OH
c), d)
O
N3
O
OH
OH
NH2.HCl
HN
f)
e)
Ph
O
HO
HO
HO
HO
HO
HO
O
O
O
OBn
OH
HO
6
5
4
Scheme 2. Synthesis of ManNGc. Reagents and conditions: (a) benzyl alcohol, acetyl chloride, 86%; (b) benzyl-dimethylacetyl, (+)-camphor-10-sulfonic acid, acetonitrile,
84%; (c) pyridine, triflic anhydride, dichloromethane; (d) sodium azide, dimethylformamide, 72% (over two steps); (e) Pd(OH)2/C(cat), H2(g), HCl(aq), methanol, 89%; (f)
acetoxyacetyl chloride, sodium bicarbonate, water, 94%.

O. M. T. Pearce, A. Varki / Carbohydrate Research 345 (2010) 1225–1229
1227
O
OH
OH
NaPyruvate
OH
OH
OH
HN
Pyruvate Lyase
HO
HO
O
HO
HO
HO
CO2H
NH
O
Tris.HCl pH 7.5.
37 ºC, 20 h
50 %
HO
O
Scheme 3. Enzymatic synthesis of Neu5Gc from ManNGc.
500
400
300
200
100
0
500
A
B
400
300
# Cells
200
# Cells
100
0
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
Cy5 / 670 nm
Cy5 / 670 nm
Figure 1. Synthetic Neu5Gc is metabolized by human THP-1 cells and displayed in terminal positions on the cell glycocalyx. (A) Synthesized Neu5Gc can be metabolized and
displayed on terminal positions of the cell glycocalyx of human cells. THP-1 cell, a human monocytic cell line that cannot synthesize Neu5Gc, was fed with synthesized
Neu5Gc, Neu5Ac, or no additional glycan. After three days the cells were fixed and stained for Neu5Gc using a primary IgY. Open black: IgY isotype control, closed black:
Neu5Gc fed, open dark grey: Neu5Ac fed, open light grey: not fed. (B) The binding of anti-Neu5Gc IgY was specific for Neu5Gc. Binding of the IgY to cells fed with Neu5Gc
could be blocked with ‘free’ Neu5Gc. Twenty millimolar ‘free’ Neu5Gc was sufficient to completely block IgY binding. Solid black: no ‘free’ Neu5Gc, solid dark grey: 0.2 mM
‘free’ Neu5Gc, solid light Grey: 2 mM free Neu5Gc, open light grey: 20 mM free Neu5Gc, open black: IgY isotype control.
Neu5Ac proliferated in a comparable manner to unfed cells) indi-
cating that the synthesized material contained no trace amounts
of cytotoxic materials.
formed using sorbsil C60 40/60 silica gel. Thin-layer chromatogra-
phy (TLC) was performed using Merck Kieselgel 60F254 pre-coated
aluminium-backed plates. Plates were visualized using UV Lamp
(kmax = 354 or 365 nm) or 5% sulfuric acid in MeOH. CH2Cl2 was
dried over molecular beads (8–12 mesh). Other anhydrous solvents
were purchased from Fluka and Sigma.
In summary, we have produced a chemo-enzymatic synthesis of
the human carbohydrate antigen Neu5Gc. The synthetic route is
quick and allows access to gram-scale quantities in high yield
and purity. Given the uncertain future of commercial sources and
the known Neu5Ac contamination issue, this methodology pro-
vides an alternative means to access this important sugar. We have
also demonstrated that the synthesized Neu5Gc is appropriate for
use in sterile in vitro conditions and can be loaded into the glyco-
calyx of human cells in vitro. We have also used it within in vivo
models with no reported atypical effects (unpublished data). We
envisage that the synthesis is flexible enough to allow a wide range
of ManNGc or Neu5Gc derivatives to be made which may prove
useful to other areas of glycobiology research.
1.2. Benzyl D
-glucopyranoside (2)19
Glucose (68 g, 0.38 mmol) was slurried in benzyl alcohol
(400 mL, 3.7 mol). Acetyl chloride (35 mL, 0.45 mmol) was added
slowly over 10 min. Reaction mixture was heated to 50 °C and stir-
red for 48 h, after which TLC (EtOAc/MeOH 9:1) showed significant
consumption of starting material (Rf 0.0) and formation of the
product (Rf 0.4). The reaction mixture was dried by vacuum distil-
lation (110 °C). The resulting crude product 2 was recrystallized
from acetonitrile to yield a mixture of anomers as a white amor-
phous material (88 g, 86%).
1. Experimental
1H NMR (CD3OD, 500 MHz) (assigned for the major anomer; al-
pha) d = 3.23–3.36 (m, 2H, H-6, H-60), 3.39 (dd, 1H, J1,2 3.7 Hz, J2,3
9.7 Hz, H-2), 3.64–3.69 (m, 2H, H-3, H-5), 3.76 (dd, J 2.3 Hz, J
11.8 Hz, 1H, H-4), 4.52 (d, 1H, Ja,b 11.7 Hz, PhHaHbO), 4.74 (d, 1H,
Ja,b 11.7 Hz, PhHaHbO), 4.86 (d, 1H, J1,2 3.7 Hz, H-1), 7.23–7.41 (m,
5H, Ph). m/z (ESI+) 288 (M+NH4þ), 100%), 293 (M+Na+, 35%). HRMS
m/z (ESꢀ) Calcd for C13H18O6 (MꢀH) 269.1020. Found 269.1019.
1.1. General methods
‘Petrol’ refers to the fraction of petroleum ether in the boiling
range of 35–60 °C. ‘Brine’ refers to a saturated aqueous solution
of sodium chloride. Proton nuclear magnetic resonance (dH) was
recorded on a Jeol ECA 500 (500 MHz). Five hundred megahertz
spectra were assigned using COSY. All chemical shifts are quoted
on the d-scale in ppm, using residual solvent as the internal stan-
dard. Low resolution mass spectra were recorded on a Micromass
Platform 1 spectrometer using electron spray (ES) ionization with
MeOH as carrier solvent. Flash column chromatography was per-
1.3. Benzyl 4-6-O-(phenylmethylene)-D
-glucopyranoside (3)20
1-Benzyl-
D-glucopyranoside (17 g, 63 mmol) was slurried in
acetonitrile (150 mL) and benzyl-dimethylacetal (40.5 g,

1228
O. M. T. Pearce, A. Varki / Carbohydrate Research 345 (2010) 1225–1229
0.26 mol). (+)-Camphor-10-sulfonic acid (154 mg, 0.67 mmol) was
added and the reaction mixture was stirred at room temperature
for three hours after which TLC (100% EtOAc) showed complete
consumption of the starting material (Rf 0.3) and the formation
of the product (Rf 0.8). The reaction was quenched with Et3N
(1.8 mL, 17.8 mmol) and the reaction mixture was dried under vac-
uum. The crude product was purified by flash silica chromatogra-
phy (Petrol/EtOAc 1:1) to yield the product 3 as a white gum
(18.9 g, 84%).
1H NMR (D2O, 500 MHz) (assigned for the major anomer)
0
d = 3.31 (ddd, 1H, J5,6 2.3 Hz, J5,6 5.7 Hz, J4,5 10.0 Hz, H-5), 3.38
(at, 1H, J 9.5 Hz, H-4), 3.56 (dd, 1H, J1,2 1.4 Hz, J2,3 4.6 Hz, H-2),
0
0
3.60 (dd, 1H, J5,6 5.3 Hz, J6,6 12.3 Hz, H-6), 3.75 (dd, 1H, J5,6
2.3 Hz, J6,6 12.3 Hz, H-60), 3.83 (dd, 1H, J2,3 4.9, J3,4 9.4, H-3), 5.01
(d, 1H, J1,2 1.8 Hz, H-1). m/z (ESI+) 180 (M+H+, 90%). HRMS m/z
(ES+) Calcd for C6H14NO5 (M+H+) 180.0866. Found 180.0865. Spec-
tral data were in agreement with a commercial standard (manno-
samine standard purchased from Sigma).
0
1H NMR (CDCl3, 500 MHz) (assigned for the major anomer; al-
pha) d = 3.49 (at, 1H, J 9.5 Hz, H-4), 3.63 (dd, 1H, J1,2 4.0 Hz, J2,3
9.2 Hz, H-2), 3.71 (at, 1H, J 10.4 Hz, H-60), 3.83 (atd, 1H, J 5.2 Hz,
1.6. 2-Deoxy-2-[(hydroxyacetyl)amino]-
D
-Mannose (6)9j
0
10.0 Hz, H-5), 3.95 (at, 1H, J 9.2, H-3), 4.21 (dd, 1H, J5,6 4.3 Hz,
2-Amino-2-deoxy- -mannopyranoside (1.4 g, 6.5 mmol) was
D
J6,6 10.3 Hz, H-60), 4.56 (d, 1H, Ja,b 11.8 Hz, PhHaHbO), 4.67 (d, 1H,
dissolved in water (60 mL) with sodium bicarbonate (10.8 g,
130 mmol) and cooled in an ice bath. Acetoxyacetyl chloride
(4.2 mL, 39 mmol) was added slowly to the reaction mixture. After
30 min TLC (EtOAc/MeOH 7:3) showed complete consumption of
the starting material (Rf 0.0) and the formation of the product (Rf
0.4). The reaction mixture was neutralized with mixed bed resin
and concentrated under vacuum to yield 6 as a white gum (1.0 g,
94%).
0
Ja,b 11.7 Hz, PhHaHbO), 5.01 (d, 1H, J1,2 4.0 Hz, H-1), 5.51 (s, 1H,
PhCH), 7.30–7.40 (m, 10H, 2 Ph). m/z (ESI+) 359 (M+H+, 70%), 376
(M+NH4þ, 70%). HRMS m/z (ES+) Calcd for C20H22O6 (M+H+)
359.1411. Found 359.1413.
1.4. Benzyl 2-azido-2-deoxy-4,6-O-(phenylmethylene)-D-
mannopyranoside (4)21
1H NMR (D2O, 500 MHz) (assigned for the major anomer)
d = 3.30 (m, 1H, H-5), 3.45 (at, 1H, J 10.0 Hz, H-4), 3.58 (dd, 1H,
Pyridine (8.8 g, 111.6 mmol) was dissolved in dry CH2Cl2
(300 mL) followed by addition of trifluoromethanesulfonic
anhydride (8.6 g, 30.7 mmol) and cooled to ꢀ30 °C. 1-Benzyl,
J5,6 5.2 Hz, J6,6 12.0 Hz, H-6), 3.69–3.73 (m, 2H, H-2, H-60), 3.78
0
(m, 1H, H-3), 4.02 (s, 2H, COCH2OH), 4.91 (d, 1H, J1,2 1.4 Hz, H-1).
m/z (ESI+) 260 (M+Na+, 100%). HRMS m/z (ES+) Calcd for C8H15NO7-
Na (M+Na+) 260.0741. Found 260.0742.
4-6-O-(phenylmethylene)-D-glucopyranoside (10 g, 27.9 mmol)
was dissolved in CH2Cl2 (250 mL) and added dropwise over
30 min under anhydrous conditions to the cooled reaction mixture.
After 2 h TLC (Petrol/EtOAc 4:1) showed almost complete con-
sumption of the starting material (Rf 0.0) and the formation of
the unisolated intermediate (Rf 0.7). The reaction was quenched
with brine and the organic layer was dried with sodium sulfate, fil-
tered and concentrated under vacuum (<30 °C). The crude interme-
diate (oil) was dissolved in dry dimethylformamide (500 mL) and
sodium azide (5.4 g, 83.7 mmol) was added to the solution. The
reaction mixture was heated to 50 °C and stirred under argon for
16 h, after which time TLC (Petrol/EtOAc 10%) showed the forma-
tion of the product (Rf 0.4). The reaction was quenched with excess
water and the product was extracted with diethyl ether, dried over
magnesium sulfate, filtered and concentrated under vacuum. The
crude product was purified by flash silica column chromatography
(Petrol/EtOAc 9:1) to yield the product 4 as a clear/colourless oil
(7.7 g, 72%).
1.7. N-Glycolylneuraminic acid (1)9a
2-Deoxy-2-[(hydroxyacetyl)amino]-
9.68 mmol), sodium pyruvate (24.8 g, 48.34 mmol) and pyruvate
lyase (1553 L, 33.76 mg/mL, from P. multocida (PmNanA), plasmid
D
-mannopyranose
(2.4 g,
l
gifted from Dr. Xi Chen) were dissolved in Tris–HCl (524 mL,
100 mM) and pH was confirmed to be 7.5. The reaction mixture
was incubated at 37 °C with shaking for 20 h. TBA analysis was
used to confirm formation of the product, and predicted a 50% con-
version. The reaction solution was passed through a Dowex-50 col-
umn (diameter = 2.5 cm, height = 15 cm). The column was washed
with water (5 ꢁ 75 mL). The combined eluent and washes were
passed though an AG 1 ꢁ 8 ion exchange column (diame-
ter = 2.5 cm, height = 100 cm). The column was washed with
10 mM formic acid (7 ꢁ 500 mL). The product was eluted from
the column with 1 M formic acid (approx 1 L). The clear/colourless
eluent was concentrated under vacuum to yield the product as a
white gum (1.6 g, 50%).
1H NMR (assigned for the major anomer; alpha) (CDCl3,
500 MHz) d = 3.81 (m, 2H, H-5, H-60), 3.91 (at, 1H, J 9.5 Hz, H-4),
4.02 (dd, 1H, J1,2 1.4 Hz, J2,3 4.0 Hz, H-2), 4.22 (m, 1H, H-6), 4.32
(dd, 1H, J2,3 3.9 Hz, J3,4 9.6 Hz, H-3), 4.50 (d, 1H, Ja,b 11.7 Hz,
PhCHaHbO), 4.70 (d, 1H, Ja,b 11.7 Hz, PhCHaHbO), 4.88 (d, 1H, J1,2
1.3 Hz, H-1), 5.58 (s, 1H, PhCH), 7.30–7.40 (m, 10H, 2 Ph). m/z
(ESI+) 384 (M+H+, 100%), 401 (M+NH4þ, 65%), 406 (M+Na+, 35%).
1H NMR (D2O, 500 MHz) (assigned for the major anomer)
0
0
d = 1.73 (t, 1H, J 11.7 Hz, H-3), 2.17 (dd, 1H, J3 ,4 4.9 Hz, J3,3
13.2 Hz, H-30), 3.38 (t, 1H, J 9.2 Hz, H-7), 3.46 (dd, 1H, J8,9 6.3 Hz,
0
0
J9,9 12.0 Hz, H-9), 3.59–3.62 (m, 1H, H-8), 3.68 (dd, 1H, J8,9
2.5 Hz, J9,9 11.7 Hz, H-90), 3.85 (t, 1H, J 10.3 Hz, H-5), 3.98–4.07
0
(m, 2H, H-4, H-6), 4.00 (s, 2H, COCH2OH). m/z (ESI+) 324 (MꢀH+,
1.5. 2-Amino-2-deoxy-D-mannose hydrochloride (5)
100%).
1-Benzyl-2-azido-2-deoxy-4,6-O-(phenylmethylene)-
D
-manno-
1.8. Neu5Gc/Neu5Ac feeding
pyranoside (3.3 g, 8.6 mmol) was dissolved in MeOH (100 mL) and
2 M HCl (aq) (8.6 mL, 17.2 mmol of HCl) was added. Pd(OH)2/C
(825 mg) was added to the reaction mixture. The reaction mixture
was saturated with H2 (g) and stirred for 48 h under H2 (g) after
which time TLC (EtOAc/MeOH 7:3) showed complete consumption
of the starting material (Rf 1.0) and the formation of the product (Rf
0.3). The reaction mixture was filtered through Celite and washed
with MeOH. The clear/colourless solution was concentrated under
vacuum. The product was dissolved in water (50 mL) and washed
with Et2O (3 ꢁ 50 mL). The aqueous layer was dried under vacuum
to yield the product 5 as a clear/colourless gum (1.6 g, 89%).
THP-1 cells were grown in Dulbecco’s Modified Eagle’s Medium
with 5% human serum. Freshly passaged cells were incubated with
1.9 mM Neu5Gc or Neu5Ac, or just media for three days. Cells were
lifted from culture flasks using 2 mM ethylenediaminetetraacetic
acid (EDTA, 10–15 min, rt) and immediately pelleted (centrifuged
1400 rpm) and the supernatant was removed. The cell pellet was
resuspended in 3% paraformaldehyde (20 min, rt). After fixing, cells
were pelleted, then resuspended in 1% fish gelatin (sigma) in phos-
phate-buffered saline (PBS) (blocking buffer). Once prepared
400,000 cells were used for each staining reaction at 4 °C. The cell

O. M. T. Pearce, A. Varki / Carbohydrate Research 345 (2010) 1225–1229
1229
5. Bardor, M.; Nguyen, D. H.; Diaz, S. L.; Varki, A. J. Biol. Chem. 2005, 280, 4228–
4237.
6. Hedlund, M.; Padler-Karavani, V.; Varki, N.; Varki, A. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 18936–18941.
7. Pham, T.; Gregg, C. J.; Karp, F.; Chow, R.; Padler-Karavani, V.; Cao, H.; Chen, X.;
Witztum, J. L.; Varki, N. M.; Varki, A. Blood 2009, 114, 5225–5235.
8. Commercial sources are limited, and may not continue. Source: Inalco
pharmaceuticals. Website: www.inalcopharm.com.
pellet was resuspended in 100 lL of either affinity-purified chicken
anti-Neu5Gc antibody17 or a control chicken IgY (Jackson Immuno-
Research, West Grove, PA) diluted 1:5000 in blocking buffer and
incubated for 20 min at 4 °C. Cells were pelleted, then washed with
500
l
L of blocking buffer and pelleted. Cells were resuspended in
100
l
L Cy5-conjugated Donkey-anti-chicken IgY (Jackson Immu-
9. Examples of chemical procedures to produce neuraminic acids and derivatives
thereof: (a) Auge, C.; David, S.; Gautheron, C.; Malleron, A.; Cavaye, B. New J.
Chem. 1988, 12, 733–744; (b) Kang, S. H.; Choi, H. W.; Kim, J. S.; Youn, J.-H.
Chem. Commun. 2000, 227–228; (c) Danishefsky, S. J.; DeNinno, M. P. J. Org.
Chem. 1986, 51, 2615–2617; (d) Danishefsky, S. J.; DeNinno, M. P.; Chen, S.-L. J.
Am. Chem. Soc. 1988, 110, 3929–3940; (e) Hartlieb, S.; Gunzel, A.; Gerardy-
Schahn, R.; Munster-Kuhnel, A. K.; Kirschning, A.; Drager, G. Carbohydr. Res.
2008, 343, 2075–2082; (f) Takahashi, T.; Tsukamoto, H.; Kurosaki, M.; Yamada,
H. Synlett 1997, 1065–1066; (g) Li, L.-S.; Wu, Y.-L.; Wu, Y. Org. Lett. 2000, 2,
891–894; (h) Mack, H.; Brossmer, R. Tetrahedron Lett. 1992, 33, 1867–1870; (i)
Hong, Z.; Liu, L.; Hsu, C.-C.; Wong, C.-H. Angew. Chem., Int. Ed. 2006, 45, 7417–
7421; (j) Choi, S. K.; Lee, S.; Whitesides, G. M. J. Org. Chem. 1996, 61, 8739–8745.
10. Examples of enzymatic methods to make neuraminic acids: (a) Wang, T. H.;
Chen, Y. Y.; Pan, H. H.; Wang, F. P.; Cheng, C. H.; Lee, W. C. BMC Biotechnol. 2009,
9, 63; (b) Kuboki, A.; Okazaki, H.; Sugai, T.; Ohta, H. Tetrahedron 1997, 53,
2387–2400; (c) Humphrey, A. J.; Fremann, C.; Critchley, P.; Malykh, Y.; Schauer,
R.; Bugg, T. D. H. Bioorg. Med. Chem. 2002, 10, 3175–3185; (d) Kim, M.-J.;
Hennen, W. J.; Sweers, H. M.; Wong, C.-H. J. Am. Chem. Soc. 1988, 110, 6481–
6486; (e) Huang, S.; Yu, H.; Chen, X. Angew. Chem., Int. Ed. 2007, 46, 2249–2253.
11. Fierfort, N.; Samain, E. J. Biotechnol. 2008, 134, 261–265.
12. Chambers, D. J.; Evans, G. R.; Fairbanks, A. J. Tetrahedron: Asymmetry 2003, 14,
1767–1769.
13. Czech, B. P.; Bartsch, R. A. J. Org. Chem. 1984, 49, 4076–4078.
14. (a) Crossman, A.; Smith, T. K.; Ferguson, M. A. J.; Brimacombe, J. S. Tetrahedron
Lett. 2005, 46, 7419–7421; (b) Liu, X.; Kwon, Y.-U.; Seeberger, P. H. J. Am. Chem.
Soc. 2005, 127, 5004–5005.
15. Okamoto, N.; Kanie, O.; Huang, Y.-Y.; Fujii, R.; Watanabe, H.; Shimamura, M.
Chem. Biol. 2005, 12, 677–683.
16. Li, Y.; Yu, H.; Cao, H.; Lau, K.; Muthana, S.; Tiwari, V. K.; Son, B.; Chen, X. Appl.
Microbiol. Biotechnol. 2008, 79, 963–970. Plasmid kindly gifted from Professor
Xi Chen.
noResearch, West Grove, PA), diluted 1:1000 in blocking buffer,
incubated for 20 min at 4 °C in the dark, then washed as mentioned
above. The stained cells were resuspended in 500 lL PBS, the data
collected on a FACSCalibur (BD Biosciences Immunocytometry Sys-
tems, San Jose, CA) and analyzed with Flowjo software (Tree Star,
Ashlan, OR). Competition assays were done by addition of ‘free’
Neu5Gc during the primary antibody staining step.
Acknowledgements
We thank Sandra L. Diaz and Christopher J. Gregg for useful dis-
cussions and technical help; Professor Xi Chen for the aldolase en-
zyme plasmid; Biswa Choudhury and Natasha Naidu for pulsed
amperometric analysis of compounds; Dr. Katie J. Doores and Dr.
Ieuan G. Davies for critical reading of the manuscript and the Can-
cer Research Institute/Samuel and Ruth Engelberg Fellowship for
funding (O.M.T.P). This work was supported by NIH Grant
R01GM32373 to A.V.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.04.003.
17. Tangvoranuntakul, P.; Gagneux, P.; Diaz, S.; Bardor, M.; Varki, N.; Varki, A.;
Muchmore, E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12045–12050.
18. Diaz, S. L.; Padler-Karavani, V.; Ghaderi, D.; Hurtado-Ziola, N.; Yu, H.; Chen, X.;
Brinkman-Van der Linden, E. C. M.; Varki, A.; Varki, N. M. PLos ONE 2009, 4, 1–
10.
References
1. Varki, A. Nature 2007, 446, 1023–1029.
2. (a) Zhang, M.; Lewis, A. L.; Cho, J. L.; Miller, M.; Varki, A. Blood 2007, 109, 4280–
4287; for a review see, (b) Crocker, P.; Paulson, J. C.; Varki, A. Nat. Rev. Immunol.
2007, 7, 255–266.
19. Yonda, Y.; Krainz, K.; Liebner, F.; Potthast, A.; Rosenau, T.; Karakawa, M.;
Nakatsubo, F. Eur. J. Org. Chem. 2008, 475–484.
3. Varki, A. Trends Mol. Med. 2008, 14, 351–360.
4. Varki, A. Am. J. Phys. Anthropol. 2001, 44, 54–69.
20. Lubineau, A.; Thieffry, A.; Veyrieres, A. Carbohydr. Res. 1976, 46, 143–148.
21. Paulsen, H.; Helpap, B.; Lorentzen, J. P. Liebigs Ann. Chem. 1987, 431–437.