Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase

Article abstract of DOI:10.1016/j.carres.2004.05.028

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannos

Full text of DOI:10.1016/j.carres.2004.05.028

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2091–2100
Accessibility of N-acyl-
D
-mannosamines to N-acetyl-
acid aldolase
D
-neuraminic
Yanbin Pan, Tiffany Ayani, Janos Nadas, Shouming Wen and Zhongwu Guo*
Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH44106, USA
Received 15 January 2004; accepted 31 May 2004
Abstract—N-Acetyl-
NeuNAc using chemically modified
tives of -mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propi-
onyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl- -mannosamines proved to be as good substrates as, or even
better than, the natural N-acetyl- -mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was pro-
posed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of -mannosamine
derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-
butanoyl-, and N-phenylacetyl- -mannosamines may be employed to bioengineer NeuNAc on cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
D
-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface
D
-mannosamines. To explore the optimal substrates for this application, eight N-acyl deriva-
D
D
D
D
D
Keywords: N-Acyl-
D
-mannosamine; N-Acetyl-
D
-mannosamine; N-Acetyl-
D
-neuraminic acid; Sialic acid; Aldolase
1. Introduction
by Reutter12–14 and Bertozzi15–17 and their co-workers in
the bioengineering of NeuNAc on cell surfaces. Based
upon this pioneering work, we recently developed a new
strategy to overcome the problem of immunotolerance to
tumor-associated carbohydrate antigens (TACAs) for
cancer vaccine development.18 The underlying principle
is that after a cancer patient or an animal is immunized
with a synthetic, artificial TACA bearing modified sialic
acid(s) to establish a specific immune response, the pa-
tient or animal is treated with a correspondingly modified
biosynthetic precursor of sialic acid to initiate the
expression of the artificial TACA on tumor cells. The pre-
stimulated immune system will then wipe out the marked
tumors.
As the terminal and thereby the most exposed sugar unit
of a majority of oligosaccharides of natural glycopro-
teins and glycolipids on cell surfaces, N-acetyl-D-neur-
aminic acid (NeuNAc, 1, also known as N-acetylsialic
acid) plays a critical role in various biological events.1–3
For example, the overexpression of NeuNAc is closely
correlated with many phenotypes of tumors, so sialyl-
ated carbohydrate antigens on tumor cells are important
molecular templates and targets for cancer vaccine
design and other biological investigations.4–10
The biosynthesis of NeuNAc, and therefore sialoglyco-
conjugates, utilizes N-acetyl-D-mannosamine (ManNAc,
2) as a key substrate.11 It is well recognized that this
biosynthetic pathway can tolerate unnatural substrates,
especially ManNAc derivatives with modified N-acyl
groups, and the phenomenon was extensively exploited
Our preliminary studies utilizing N-propionyl-D-
mannosamine (ManNPr 3) as the glycoengineering
precursor and polysialic acid on cancer cells as the target
antigen18 gave encouraging results with regard to the
glycoengineering and selective immunotargeting of
cancer. However, the initial design was not necessarily
the optimal one, because more distinctive modifications
of NeuNAc may produce more effective vaccines and
* Corresponding author. Tel.: +1-216-368-3736; fax: +1-216-368-3006;
e-mail: zxg5@case.edu
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.05.028

2092
Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
make the glycoengineered tumors more distinguishable.
In this context, the caveat is that the modified -man-
(ManNTFP, 9) derivatives, were prepared from com-
mercial -mannosamine (10) by reaction with the cor-
D
D
nosamine as precursor must be available to the biosyn-
thetic pathways of sialic acid and sialoglycoconjugates.
Recently Bertozzi and her co-workers19 identified that
the bottleneck for the biosynthetic machinery of sialic
responding acyl anhydrides or, for ManNTFP, by
reaction with 1-hydroxybenzotriazole (HOBt) ester of
trifloropropionic acid in methanol (Scheme 2). The ex-
pected products were obtained in 68–75% yield after
silica gel column chromatography and subsequent
recrystallization from mixtures of ethanol and ethyl
acetate.
Standard samples of N-acylsialic acids (11–17) were
prepared according to Scheme 3. Direct hydrolysis of
NeuNAc under either acidic or basic conditions caused
its decomposition, probably due to the involvement of
its keto a-hydrogens in the open-chain form. Therefore,
NeuNAc (1) was first converted into the benzyl glyco-
side 2028–30 that could be hydrolyzed under basic con-
ditions smoothly to afford 21. Selective acylation of the
free amino group of 21 using various acyl anhydrides or
the active ester of trifluoropropionic acid was followed
by catalytic debenzylation to give N-acylsialic acids 11–
18. All synthetic intermediates and final products in
Schemes 2 and 3 were positively identified by NMR
spectroscopy and by mass spectrometry.
acid to use N-acyl-D-mannosamines was its 6-O-phos-
phorylation, the first biosynthetic step, while the en-
zymes involved in the remaining steps were more
permissive. The efficient incorporation of various N-
acyl-
D
-mannosamines into cell-surface sialoglycoconju-
-mannosamine
gates12;19 suggests that the modified
D
precursors can somehowbypass the substrate-specific
step. It was proposed that the conversion of unnatural
D
-mannosamines to sialic acids took advantage of Neu
NAc aldolase,19 an enzyme that usually catalyzes the
degeneration of sialic acid (Scheme 1). However, since
this reaction is completely reversible, NeuNAc aldolase
is also involved in the biosynthesis of NeuNAc.20
Moreover, it has been observed that NeuNAc aldolase
can tolerate a variety of modifications of
D-mannos-
amine. Thus, NeuNAc aldolase has been used to syn-
thesize a variety of NeuNAc derivatives or analogs by
an enzymatic method.19–27
To find out the optical substrates for the bioengineer-
ing of NeuNAc on tumor cells, and other cells as well, by
utilizing the permissibility of NeuNAc aldolase, it is
necessary to understand the reactions of various N-acyl-
Commercial NeuNAc aldolase purchased from
CALBIOCHEMꢁ was utilized to investigate the enzy-
matic accessibility of N-acyl-D-mannosamines. The
reactions were carried out at 37 ꢂC in a buffer (pH 7.4,
I ¼ 0.1) containing 0.05 M potassium phosphate and
7.5% (v/v) dithiothreitol.23;24 Pyruvic acid was used in
large excess (10 equiv), and its concentration could be
considered as a constant during the treatment of reac-
tion kinetics. The sialic acid products of the reactions
were assayed with the modified Svennerholm (resor-
cinol) method.31
D
-mannosamines with the enzyme. For this purpose, the
D-
accessibility of a number of N-acyl derivatives of
mannosamine to NeuNAc aldolase was investigated.
2. Results and discussion
The UV–vis absorption spectra of the Svennerholm
reaction products of N-acylsialic acids 1 and 11–17 were
analyzed first. As shown in Figure 1, although they had
nearly identical kmax (587 nm), their molar extinction
coefficients (e) were different. Since the structure of
Svennerholm product is not well defined,31 howthe acyl
groups affected the e was not clear. Nevertheless, it
seemed that bulky (Piv) and electron deficient (Bz,
PhAc, and TFP) acyl groups resulted in a decrease in the
e values of the Svennerholm reaction products. More-
over, the product of NeuNBz showed a much stronger
absorption band at ca. 425 nm than all other derivatives.
Due to the differences of the UV–vis spectra, the indi-
Eight N-acyl-D-mannosamines, including N-acetyl
(ManNAc, 2), N-propionyl (ManNPr, 3), N-butanoyl
(ManNBu, 4), N-iso-butanoyl (ManNi-Bu, 5), N-piva-
loyl (ManNPiv, 6), N-phenylacetyl (ManNPhAc, 7), N-
benzoyl (ManNBz, 8), and N-trifluoropropionyl
OH
OH
HO
NeuNAc
aldolase
O
NHAc
O
HO
HO
HO
O
+
COOH
1 (NeuNAc)
Scheme 1. NeuNAc aldolase-catalyzed reaction.
AcNH
OH
CO2H
HO
HO
2 (ManNAc)
2 R = CH3 (ManNAc)
3 R = CH2CH3 (ManNPr)
4 R = CH2CH2CH3 (ManNBu)
5 R = CH(CH3)2 (ManNiBu)
6 R = C(CH3)3 (ManNPiv)
7 R = CH2Ph (ManNPhAc)
8 R = Ph (ManNBz)
O
NH2 HCl
O
HO
HO
HO
1. NaOH, MeOH
2. RCOOR', MeOH
HO HN
HO
HO
R
OH
O
OH
10
9 R = CH2CF3 (ManNTFP)
Scheme 2. Preparation of N-acyl-D-mannosamines (2–9).

Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
2093
1. MeOH,H+
2.Ac2O, Pyr.
OAc
OAc
OAc
OAc
AgOTf,
BnOH
HCl,
COOCH3
OAc
Cl
COOCH3
O
OBn
AcO
AcO
AcHN
OAc
AcO
AcHN
OAc
AcOH
O
O
COOCH3
1
AcHN
87%
>99%
93%
AcO
18
AcO
AcO
20
19
2N NaOH, heat
OH
OH
OH
OH
OH
COOH
OBn
COOH
OH
H2, Pd/C HO
RCONH
HO
RCONH
COOH
OBn
RCOOR'
72-95%
HO
OH
O
O
O
84-94%
H2N
HO
HO
11 R = CH2CH3 (NeuNPr)
HO
21
22 R = CH2CH3
23 R = CH2CH2CH3
24 R = CH(CH3)2
25 R = C(CH3)3
26 R = CH2Ph
27 R = Ph
12 R = CH2CH2CH3 (NeuNBu)
13 R = CH(CH3)2 (NeuNiBu)
14 R = C(CH3)3 (NeuNPiv)
15 R = CH2Ph (NeuNPhAc)
16 R = Ph (NeuNBz)
28 R = CH2CF3
17 R = CH2CF3 (NeuNTFP)
Scheme 3. Preparation of N-acyl-D-neuraminic acids (11–17).
Figure 1. UV–vis spectra of the Svennerholm reaction products of: (a) NeuNAc, (b) NeuNPr, (c) NeuNBu and NeuNi-Bu, (d) NeuNPhAc, (e)
NeuNTFP, (f) NeuNPiv, and (g) NeuNBz.
vidual calibration curves of 1 and 11–17 with regard to
their absorbance at 580 nm were obtained using sialic
acid concentrations ranging from 0 to 0.2 mM, all of
which gave excellent linear correlations. It was also
examined by Svennerholm method.31 The results are
summarized in Figure 2a and b. The reactions had two
phases (Fig. 2a). During the first 5 h, they exhibited the
pseudo zero-order kinetics (Fig. 2b). Thereafter, most
reactions leveled off and eventually reached a plateau
within 24 h. The reaction of ManNTFP was the slowest
of all eight derivatives tested, but it reached a similar
plateau ca. 50 h later.
It is well established that aldolase-catalyzed reactions
are reversible (Eq. 1),21;22 and the reaction rate can then
be expressed by Eq. 2.32 At the initial stage, the con-
centration of NeuNR was rather low, so the reverse
reaction (kÀ1[NeuNR][Enz]) could be neglected, and
Eq. 2 can be simplified to Eq. 3. Meanwhile, pyruvic
acid was used in large excess and the enzyme remained
unchanged. Thus, the concentrations of enzyme [Enz]
and pyruvic acid [PA] could also be taken as con-
stants. Although the substrate concentration [ManNR]
proved that N-acyl-D-mannosamines, pyruvic acid or
their mixture had no influence on the sialic acid analysis
under the Svennerholm conditions. Therefore, the
method could be safely used to assay the enzymatic
reactions.
Next, we established the suitable concentrations of N-
acyl-D-mannosamine (5–50 mM) and aldolase (0.9 unit/
mL) for the convenient and accurate analysis of sialic acid
products by the Svennerholm method. The transforma-
tion of N-acyl-
thus studied with 18.2 mM of N-acyl-
D
-mannosamines to N-acylsialic acids was
-mannosamine,
D
0.9 unit/mL of aldolase and 182 mM of pyruvic acid.
For the reaction analysis, exact aliquots of the reac-
tion mixtures were withdrawn at various time points and

2094
Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
12
d½NeuNRŠ=dt ¼ k1½ManNRŠ½PAŠ½EnzŠ
ð3Þ
ð4Þ
ð5Þ
d½NeuNRŠ=dt ¼ k0
½NeuNRŠ ¼ k0t
On the other hand, with the accumulation of NeuNR,
the term kÀ1[NeuNR][Enz] of Eq. 2 would become more
and more significant, while the consumption of ManNR
reduced k1[ManNR][PA][Enz]. As a result, the reactions
slowed down and gradually reached an equilibrium (the
plateau).
10
8
6
4
The initial reaction rates of various N-acyl-D-man-
nosamine derivatives were different (Fig. 2b), even
though their conversion rates at the equilibriums were
similar (69% 1.8%). Compared to ManNAc
(k0 ¼ 0:0114), the reactions of ManNPr (k0 ¼ 0:0131),
ManNi-Bu (k0 ¼ 0:0140), ManNPiv (k0 ¼ 0:0137) and
ManNPhAc (k0 ¼ 0:0159) were slightly faster, but the
reactions of ManNBz (k0 ¼ 0:0093) and ManNTFP
(k0 ¼ 0:0034) were obviously slower. Since 5–7 that bear
rather bulky N-acyl groups such as i-Bu, Piv, and PhAc
were as good substrates as ManNAc, it seems that the
steric effect is not critical for determining the enzymatic
accessibility of these substrates. On the contrary,
ManNTFP and ManNBz with strongly electron-defi-
cient acyl groups were poor substrates, which may
indicate that the electronic effect probably has a signif-
2
0
0
500
1000
1500
Time (min)
2000
2500
3000
ManNAc
ManNPiv
ManNTFP
ManNPr
ManNBz
ManNiBu
ManNPhAc
(a)
7
C = 0.0159t, R2 = 0.9946 (PhAc)
C = 0.0140t, R2 = 0.9994 (iBu)
C = 0.0137t, R2 = 0.9984 (Piv)
C = 0.0131t, R2 = 0.9998 (Pr)
6
5
4
3
2
1
0
C = 0.0114t, R2 = 0.9949 (Ac)
C = 0.0093t, R2 = 0.9921 (Bz)
icant influence on the accessibility of N-acyl-D-man-
nosamines to the aldolase, and the highly electron-
deficient acyl groups retard the reaction. However,
ManNPhAc with a mildly electron-deficient acyl group
is a good substrate, which suggests that, besides elec-
tronic effect, other factors such as hydrophobic inter-
actions, may also affect the substrate–aldolase binding.
ManNPhAc was especially interesting to us, as it was
the best substrate for NeuNAc aldolase and thus pos-
sibly the most efficient precursor for engineering cell-
surface sialic acid. Meanwhile, since a phenylacetyl
group is distinctly different from an acetyl group, the
phenylacetyl derivatives may form effective vaccines.
The reaction of ManNPhAc was then studied in detail.
As shown in Figure 3a, with pyruvic acid in excess, the
reaction rate increased with the concentration of
ManNPhAc from 1.82 to 182 mM. At lowconcentra-
tions, the reaction rate (k0) had a linear correlation with
[ManNPhAc] (Figure 2b), which can be explained by
Eq. 3. However, the reaction rate leveled off at higher
[ManNPhAc]. The results fit a Lineweaver–Burk plot
(Fig. 2c).32 Because cellular levels of mannosamine are
usually much lower than the tested concentrations, the
NeuNAc aldolase-catalyzed reaction rate will probably
observe a linear correlation with the concentration of
ManNPhAc in the biological system. Therefore, any
measures that can improve the cellular concentrations of
ManNPhAc would be able to increase the biosynthesis
of PhAc-modified sialic acid by cells and thereby im-
C = 0.0034t, R2 = 0.9979 (FPr)
0
100
200
300
Time (min)
400
500
600
ManNAc
ManNBz
ManNPr
ManNPhAc
ManNiBu
ManNTFP
ManNPiv
(b)
Figure 2. The reaction kinetics of N-acyl-D-mannosamines (18.2 mM)
with pyruvic acid (182 mM) and NeuNAc aldolase (0.9 unit/mL):
37 ꢂC, 0.05 M potassium phosphate buffer (pH 7.4, I ¼ 0:1) containing
7.5% (v/v) dithiothreitol.
changed with the proceeding of reactions, at the initial
stage this change was relatively small, and [ManNR]
could be considered as a consistent. Eq. 3 can thus be
simplified to Eqs. 4 and 5, showing the zero-order kinetics.
(k1)
K
ð1Þ
ManNR + PA + Enz
NeuNR+Enz
(k-1)
reaction rateðmÞ ¼ d½NeuNRŠ=dt
¼ k1½ManNRŠ½PAŠ½EnzŠ
À k ½NeuNRŠ½EnzŠ
ð2Þ
À1

Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
2095
0.04
6
5
4
3
2
1
0
C = 0.0331t
R
2 = 0.9982
R2 = 0.9997
C = 0.0274t
2 = 0.9792
R
0.03
0.02
0.01
C = 0.0158t
2 = 0.9951
R
C = 0.0075t
R2 = 0.9932
C = 0.0028t
R2 = 0.9972
C = 0.0013t
R2 = 0.9958
0
0
50
100
Time (min)
150
200
0
50
100
150
200
(a)
C ([ManNPhAc], mM)
(b)
1.8 mM
3.6 mM
9.1 mM
18 mM
800
91 mM
182 mM
1/k ' = 1368.5/C + 2.1657
R2 = 0.9951
600
400
200
0
0
0.2
0.4
0.6
(c)
1/C
Figure 3. The impact of ManNPhAc concentrations (1.8, 3.6, 9.1, 18, 91, and 182 mM) on its reaction with pyruvic acid (182 mM) and NeuNAc
aldolase (0.9 unit/mL): 37 ꢂC, 0.05 M potassium phosphate buffer (pH 7.4, I ¼ 0:1) containing 7.5% (v/v) dithiothreitol.
prove the efficiency of cell-surface sialic acid engineer-
ing.
In conclusion, a number of N-acyl derivatives of
precursors identified, ManNi-Bu and ManNPhAc are
especially interesting to us, because our initial studies
also indicated that N-i-Bu and N-PhAc modified sialic
acids could induce robust IgG antibodies desirable for
cancer immunotherapy.36 Therefore, we are currently
investigating in detail the bioavailability of ManNi-Bu
and ManNPhAc to cells, as well as their efficiency to
glycoengineer tumors in vitro and in vivo.
D
-mannosamine were prepared, and their accessibility to
NeuNAc aldolase was studied. It was demonstrated that
NeuNAc aldolase could tolerate a range of N-modifi-
cations of D-mannosamine, and as substrates of Neu-
NAc aldolase, ManNPr, ManNBu, ManNi-Bu,
ManNPiv, and ManNPhAc were as good as or even
better than the natural ManNAc. These results suggest
that ManNPr, ManNBu, ManNi-Bu, ManNPiv, and
ManNPhAc may be used as effective biosynthetic pre-
cursors to engineer cell-surface sialic acid. Coupled with
the fact that artificially modified sialooligosaccharides
are highly immunogenic,33–35 these results should be
valuable for the development of newcancer immuno-
therapies. Among the potentially useful biosynthetic
3. Experimental
3.1. General methods
NMR spectra were recorded on a Gemini-300 FT NMR
spectrometer. Proton chemical shifts are reported in
ppm (d) downfield from tetramethylsilane (TMS).

2096
Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
Coupling constants (J) are reported in Hertz (Hz). The
fast-atom-bombardment mass spectra (FABMS) were
obtained with a Kratos MS-25RFA spectrometer.
Optical rotations were determined with a Perkin–Elmer
241 polarimeter, and thin-layer chromatography (TLC)
was performed on silica gel GF254 plates with detection
by charring with phosphomolibdic acid–EtOH or 5%
H2SO4–EtOH. Anhydrous solvents and other reagents
were purchased from vendors and used without further
purification.
isomer) d 5.07 (d, 1H, J1;2 1.3 Hz, H-1), 4.28 (dd, 1H, J2;3
4.8 Hz, H-2), 4.02 (dd, 1H, J3;4 9.7 Hz, H-3), 3.75–3.84
(m, 3H, H-5,6,60), 3.58 (dd, 1H, J4;5 10.8 Hz, H-4), 2.22
(t, 2H, J 7.6 Hz, Bu), 1.47–1.60 (m, 2H, Bu), 0.86 (t, 3H,
J 7.5 Hz, Bu); (b-isomer) d 5.0 (d, 1H, J1;2 1.7 Hz, H-1),
4.41 (d, 1H, J2;3 4.3 Hz, H-2), 3.74–3.84 (m, 3H, H-
3,6,60), 3.48 (dd, 1H, J 9.8, 9.3 Hz, H-4), 3.32–3.45 (m,
1H, H-5), 2.25 (t, 2H, J 7.3 Hz, Bu), 1.47–1.68 (m, 2H,
Bu), 0.89 (t, 3H, J 7.4 Hz, Bu); FABMS: m=z 232.2
[M+Hþ)H2O], 250.3 [M+Hþ]; HRFABMS: Calcd for
C10H19NO6 [M+Hþ] m=z 250.1285; found 250.1293.
3.2. N-Acyl derivatives (2–8) of
D-mannosamine
3.2.4. N-iso-Butanoyl-D-mannosamine (5). 252 mg (73%);
1
20
D
To solution of -mannosamineÆHCl (300 mg,
a
D
½aŠ +10 (c 1.0, H2O); H NMR (D2O, 300 MHz): (a-
1.39 mmol) in 5 mL MeOH and 0.5 mL of 3 N NaOH
was added dropwise 1.0 mL of an acyl anhydride while
cooling the mixture in an ice-water bath. After the
reaction was finished (within 3 h as indicated by TLC),
the mixture was neutralized with 2 N HCl and then
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (eluent:
MeOH–CH2Cl2) to afford N-acylmannosamine that was
further purified by passing through a Bio-Gel P2 column
using distillated water as eluent. The fractions contain-
ing the expected product were combined, freeze-dried,
and finally recrystallized from EtOH and EtOAc to give
a white crystalline product.
isomer) d 5.11 (d, 1H, J1;2 1.2 Hz, H-1), 4.32 (dd, 1H, J2;3
4.7 Hz, H-2), 4.07 (dd, 1H, J3;4 9.7 Hz, H-3), 3.77–3.92
(m, 3H, H-5,6,60), 3.63(t, 1H, J 9.6 Hz, H-4), 2.54–2.70
(m, 1H, i-Bu), 1.10 (d, 6H, J 6.9 Hz, i-Bu); (b-isomer) d
5.04 (d, 1H, J1;2 1.3 Hz, H-1), 4.45 (dd, 1H, J2;3 3.9 Hz,
H-2), 3.77–3.92 (m, 3H, H-3,6,60), 3.52 (dd, 1H, J 9.9,
9.6 Hz, H-4), 3.38–3.44 (m, 1H, H-5), 2.54–2.70 (m, 1H,
i-Bu), 1.11 (d, 6H, J 6.9 Hz, i-Bu); FABMS: m=z 250.1
[M+Hþ], 272.1 [M+Naþ], 232.1 [M+Hþ)H2O];
HRFABMS: Calcd for C10H19NO6 [M+Hþ] m=z
250.1285; found 250.1273. Anal. Calcd for (C10H18NO6)
C, 48.19; H, 7.68; N, 5.62. Found: C, 47.79; H, 7.73; N,
5.44.
20
D
20
D
3.2.1. N-Acetyl-
D
-mannosamine (2). 210 mg (70%); ½aŠ
3.2.5. N-Pivaloyl-
D
-mannosamine (6). 273 mg (76%); ½aŠ
+11 (c 1.0, H2O) (lit.37+9.7); 1H NMR (D2O, 300 MHz):
(a-isomer) d 5.10 (d, 1H, J1;2 1.3 Hz, H-1), 4.30 (dd, 1H,
J2;3 4.6 Hz, H-2), 4.02 (dd, 1H, J3;4 9.7 Hz, H-3), 3.81–
3.83 (m, 2H, H-6,60), 3.80 (m, 1H, H-5), 3.59 (dd, 1H,
J4;5 9.0 Hz, H-4), 2.03 (s, 3H, Ac); (b-isomer) 5.00 (d,
1H, J1;2 1.6 Hz, H-1), 4.42 (dd, 1H, J2;3 4.4 Hz, H-2), 3.81
(m, 1H, H-3), 3.80–3.85 (m, 2H, H-6,60), 3.50 (dd, 1H, J
9.9, 9.6 Hz, H-4), 3.41–3.43 (m, 1H, H-5), 2.07 (s, 3H,
Ac); FABMS: m=z 204.0 [M+Hþ)H2O], 221.1 [Mþ],
222.1 [M+Hþ], 244.1 [M+Naþ]; HRFABMS: Calcd for
C8H15NO6 [M+Hþ] m=z 222.0972; found 222.1016.
+10 (c 1.0, H2O); 1H NMR (D2O, 300 MHz): (a-isomer)
d 5.13 (s, 1H, H-1), 4.33 (dd, 1H, J1;2 1.6 Hz, J2;3 4.6 Hz,
H-2), 4.09 (dd, 1H, J3;4 9.6 Hz, H-3), 3.80–3.92 (m, 3H,
H-5,6,60),3.63 (t, 1H, J 9.1 Hz, H-4), 1.21 (s, 9H, Me);
(b-isomer) d 5.08 (d, 1H, J1;2 1.6 Hz, H-1), 4.47 (dd, 1H,
J
2;3 4.1 Hz, H-2), 3.8–3.9 (m, 3H, H-5,6,60), 3.47 (t, 1H, J
9.4 Hz, H-4), 3.40–3.46 (m, 1H, H-5),1.23 (s, 9H, Me);
FABMS: m=z 246.1 [M+Hþ)H2O], 264.1 [M+Hþ];
HRFABMS: Calcd for C11H21NO6 [M+Hþ] m=z
264.1442; found 264.1455. Anal. Calcd for (C11H20NO6)
C, 50.18; H, 8.04; N, 5.32. Found: C, 50.39; H, 8.12: N,
5.22.
3.2.2. N-Propionyl-
D
-mannosamine (3).38 215 mg (68%);
½aŠ +7.0 (c 1.0, H2O); H NMR (D2O, 300 MHz): (a-
20
D
1
3.2.6. N-Phenylacetyl-D-mannosamine (7). 300 mg (73%);
1
20
D
isomer) d 5.08 (d, 1H, J1;2 1.6 Hz, H-1), 4.29 (dd, 1H, J2;3
4.8 Hz, H-2), 4.02 (dd, 1H, J3;4 9.7 Hz, H-3), 3.75–3.86
(m, 3H, H-5,6,60), 3.58 (t, 1H, J 9.5, H-4), 2.29 (q, 2H, J
7.6 Hz, Pr), 1.07 (t, 3H, J 7.6 Hz, Pr); (b-isomer) d 4.99
(d, 1H, J1;2 1.7 Hz, H-1), 4.42 (dd, 1H, J2;3 4.4 Hz, H-2),
3.75–3.86 (m, 3H, H-3,6,60), 3.48 (t, 1H, J 9.5, H-4),
3.33–3.43 (m, 1H, H-5), 2.33 (q, 2H, J 7.7 Hz, Pr), 1.09
(t, 3H, J 7.7 Hz, Pr); FABMS: m=z 218.1 [M+Hþ)H2O],
236.1 [M+Hþ], 258.1 [M+Naþ]; HRFABMS: Calcd for
C9H17NO6 [M+Hþ] m=z 236.1129; found 236.1133.
½aŠ )10 (c 1.0, H2O); H NMR (D2O, 300 MHz): (a-
isomer) d 7.33–7.45 (m, 5H, H–Ar), 5.11 (d, 1H, J1;2
1.5 Hz, H-1), 4.33 (dd, 1H, J2;3 4.7 Hz, H-2), 4.08 (dd,
1H, J3;4 9.9 Hz, H-3), 3.80–3.87 (m, 3H, H-5,6,60), 3.63
(dd, 1H, J4;5 9.5, Hz, H-4), 3.69, 3.65 (2d, 2H, J 15.0 Hz,
Bn); (b-isomer) d 7.33–7.45 (m, 5H, H–Ar), 5.04 (d, 1H,
J1;2 1.5 Hz, H-1), 4.46 (dd, 1H, J2;3 4.4 Hz, H-2), 3.80–
3.87 (m, 2H, H-3,6), 3.77 (dd, 1H, J 12.0, 5.4 Hz, H-60),
3.75 (s, 2H, Bn), 3.51 (dd, 1H, J 9.7, 9.5 Hz, H-4), 3.40
(ddd, 1H, J 9.7, 4.8, 2.3 Hz, H-5); FABMS: m=z 280.1
[M+Hþ)H2O], 298.1 [M+Hþ], 320.1 [M+Naþ], 336.1
[M+Kþ]; HRFABMS: Calcd for C14H19NO6 [M+Hþ]
m=z 298.1285; found 298.1348. Anal. Calcd for
3.2.3. N-Butanoyl-
D
-mannosamine (4).39 264 mg (75%);
½aŠ +9.0 (c 1.0, H2O); H NMR (D2O, 300 MHz): (a-
20
D
1

Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
2097
(C14H18NO6) C, 56.56; H, 6.44; N, 4.71. Found: C,
56.16; H, 6.39: N, 4.46.
4.53 (2d, 2H, J 10.9 Hz, CH2Ph), 4.15 (dd, 1H, J 10.2,
1.9 Hz, H-6), 3.81–3.94 (m, 4H), 3.76 (dd, 1H, J 12.6,
6.1 Hz, H-9), 3.31 (t, 1H, J 10.3 Hz, H-5), 2.85 (dd, 1H,
J, 12.4, 4.6 Hz, H-3e), 1.79 (t, 1H, J 12.4 Hz, H-3a).
20
3.2.7. N-Benzoyl-
D
-mannosamine (8). 300 mg (74%); ½aŠ
D
)17 (c 1.0, H2O); 1H NMR (D2O, 300 MHz): (a-isomer)
d 7.5–7.8 (m, 5H, H–Ar), 5.26 (s, 1H, J1;2 1.1 Hz, H-1),
4.58 (dd, 1H, J2;3 4.7 Hz, H-2), 4.17 (dd, 1H, J3;4 9.8 Hz,
H-3), 3.8–3.9 (m, 3H, H-5,6,60), 3.65–3.78 (t, 1H, J2;3
9.7 Hz, H-4); (b-isomer) d 7.5–7.8 (m, 5H, H–Ar), 5.14
(s, 1H, H-1), 4.72–4.73 (m, 1H, H-2), 3.8–3.9 (m, 3H, H-
3,6,60), 3.64 (t, 1H, J 9.8 Hz, H-4), 3.44–3.50 (m, 1H, H-
5); FABMS: m=z 266.3 [M+Hþ)H2O], 284.3 [M+Hþ];
HRFABMS: Calcd for C13H17NO6 [M+Hþ] m=z
284.1129; found 284.1131.
3.5. Benzyl glycosides of N-acyl-D-neuraminic acid (22–
27)
To a solution of 21 (150 mg, 0.42 mmol) in MeOH
(4 mL) was added an anhydride (1.26 mmol) at 0 ꢂC
slowly. The mixture was stirred at 0 ꢂC for 1 h and then
at rt until TLC showed that the reaction had gone to
completion. Workup as shown in Section 3.2 and col-
umn chromatography (1:4 to 1:1 MeOH–EtOAc) of the
product afforded 22–27.
3.3. N-Trifluoropropionyl-D-mannosamine (9)
3.5.1. Benzyl glycoside of N-propionyl-D-neuraminic acid
To the solution of 1-hydroxybenzotriazole (297 mg) and
N,N0-dicyclohexylcarbodiimide (454 mg) in 5 mL of
DMF was added 194 lL of 3,3,3-trifluoropropionic acid
(2.2 mmol). After the reaction mixture was stirred at
(22). 165 mg (95%); Rf 0.59 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): d 7.40
(m, 5H, H–Ar), 4.80, 4.52 (2d, 2H, J 11.3 Hz, Bn), 3.96–
3.60 (m, 6H), 3.58 (d, 1H, J 9.0 Hz, H-7), 2.88 (dd, 1H, J
4.4 Hz, 12.2 Hz, H-3e), 2.34 (m, 2H, COCH2), 1.71 (t,
1H, J 11.8 Hz, H-3a), 1.10 (t, 3H, J 7.5 Hz, CH3);
HRFABMS: Calcd for C19H27NO9 [M+Naþ] m=z
436.1584; found 436.1584.
0 ꢂC for 1 h and at rt for additional 3 h,
D-mannosa-
mineÆHCl (300 mg, 1.39 mmol) was added. The reaction
mixture was stirred overnight, and the precipitate was
then filtered off. The filtrate was condensed under re-
duced pressure, and the residue was purified by silica gel
column chromatography (eluent: MeOH–CH2Cl2) to
afford 9 that was further purified by a Bio-Gel P2 col-
3.5.2. Benzyl glycoside of N-butanoyl-D-neuraminic acid
(23). 167 mg (93%); Rf 0.65 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): d 7.40
(m, 5H, H–Ar), 4.77, 4.53 (2d, 2H, J 11.3 Hz, Bn), 3.96–
3.60 (m, 7H), 2.80 (dd, 1H, J 4.4 Hz, 13.2 Hz, H-3e),
2.30 (t, 2H, J 7.2 Hz, COCH2), 1.72 (t, 1H, J 11.8 Hz, H-
3a), 1.64 (q, 2H, J 7.2 Hz, CH3CH2), 0.94 (t, 3H, J
7.2 Hz, CH3); HRFABMS: Calcd for C20H29NO9
[M+Naþ] m=z 450.1740; found 450.1740.
umn and finally recrystallized from EtOH and EtOAc to
20
D
give white crystalline 9 (329 mg, 82%). ½aŠ +3.0 (c 0.5,
1
H2O); H NMR (D2O, 300 MHz): (a-isomer) d 5.11 (d,
1H, J1;2 1.6 Hz, H-1), 4.35 (dd, 1H, J2;3 4.6 Hz, H-2), 4.05
(dd, 1H, J3;4 9.7 Hz, H-3), 3.78–3.86 (m, 3H, H-5,6,60),
3.58 (dd, 1H, J 9.9, 9.7 Hz, H-4), 3.32 (q, 2H, JHF 10.7,
CH2CF3); (b-isomer) d 5.02 (d, 1H, J1;2 1.6 Hz, H-1),
4.48 (dd, 1H, J2;3 4.4 Hz, H-2), 3.78–3.86 (m, 3H, H-
3,6,60), 3.48 (t, 1H, J 9.9 Hz, H-4), 3.38–3.42 (m, 1H, H-
5), 3.36 (q, 2H, JHF 10.7, CH2CF3); FABMS: m=z 272.3
[M+Hþ)H2O], 290.1 [M+Hþ], 312.1 [M+Naþ];
HRFABMS: Calcd for C9H14NO6F3 [M+Hþ] m=z
290.0846; found 290.0856. Anal. Calcd for
(C9H13NO6F3): C, 37.38; H, 4.88: N, 4.84. Found: C,
37.01; H, 4.70: N, 5.09.
3.5.3. Benzyl glycoside of N-iso-butanoyl-D-neuraminic
acid (24). 51 mg (84%); Rf 0.80 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): d 7.40
(s, 5H, H–Ar), 4.75, 4.52 (2d, 2H, J 10.8, CH2Ph), 3.89–
3.55 (m, 6H), 3.56 (d, 1H, J 8.6 Hz, H-7), 2.81 (dd, 1H, J
4.4, 13.2 Hz, H-3e), 2.55 (m, 1H, COCH), 1.71 (t, 1H, J
12.8, H-3a), 1.15 (2 d, 6H,J 7.0 Hz, 2CH3); HRFABMS:
Calcd for C20H29NO9 [M+Naþ] m=z 450.1740; found
450.1747.
3.4. Benzyl glycoside of D-neuraminic acid (21)
A solution of compound 2030 (2.4 g, 4.1 mmol) refluxed
in 2 N NaOH (50 mL) for 6 h was cooled to rt, and
concd HCl was added to neutralize it to pH 6. The
mixture was then concentrated under reduced pressure,
and the residue was extracted with MeOH to remove
NaCl. The extracts were combined, concentrated, and
3.5.4. Benzyl glycoside of N-trimethyl-D-neuraminic acid
(25). 176 mg (95%); Rf 0.64 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 200 MHz): d 7.37
(m, 5H, H–Ar), 4.70, 4.48 (2d, 2H, J 10.8, CH2Ph), 4.05
(d, 1H, J 10.3, H-6), 3.85–3.73 (m, 4 H), 3.64–3.53 (m,
1H), 3.47 (m, 1H), 2.82 (dd, 1H, J 3.6, 12.4 Hz, H-3e),
1.64 (t, 1H, J 12.1 Hz, H-3a), 1.14 (s, 9H, 3CH3);
HRFABMS: Calcd for C19H27NO9 [M+Naþ] m=z
436.1584; found 436.1584.
purified by column chromatography (1:6 MeOH–
20
D
EtOAc) to give 21 (1.25 g, 85%). ½aŠ )40 (c 0.5, H2O);
Rf 0.59 (eluent: 1:1:1:1 BuOH–HOAc–EtOAc–H2O); 1H
NMR (D2O, 300 MHz): d 7.45 (m, 5H, H–Ar), 4.70,

2098
Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
3.5.5. Benzyl glycoside of N-phenylacetyl-
D
-neuraminic
isomer) d 4.07–3.98 (m, 3H), 3.85–3.83 (m, 2H), 3.66
(dd, 1H, J 12.5, 7.2 Hz, H-9), 3.59 (d, 1H, J 8.1 Hz, H-
7), 2.34 (q, 2H, J 7.9 Hz, COCH2), 2.26 (dd, 1H, J 4.2,
13.0 Hz, H-3e), 1.86 (t, 1H, J 12.4 Hz, H-3a), 1.15 (t, 3H,
J 7.6 Hz,CH3). HRFABMS: Calcd for C12H21NO9 m=z
[M+Naþ] 346.1114; found 346.1117.
acid (26). 181 mg (91%); Rf 0.78 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 200 MHz): d 7.3–
7.5 (m, 10H, H–Ar), 4.67, 4.52 (2d, 2H, J 9.5 Hz,
CH2Ph), 3.75–3.68 (m, 6H), 3.60 (s, 2H, CH2Ph), 3.49
(dd, 1H, J 12.3, 7.1 Hz, H-9), 2.74 (dd, 1H, J 4.0,
12.4 Hz, H-3e), 1.64 (t, 1H,
J
12.2 Hz, H-3a);
HRFABMS: Calcd for C24H29NO9 [M+Naþ] m=z
498.1740; found 498.1740.
3.7.2. N-Butanoyl-D-neuraminic acid (12). 30 mg (90%);
20
½aŠ )27 (c 1.0, H2O); Rf 0.31 (eluent: 1:1:1:1 BuOH–
D
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): (b-
isomer) d 4.02–3.98 (m, 2H), 3.95 (d, 1H, J 9.6 Hz, H-9),
3.85–3.74 (m, 2H), 3.61 (dd, 1H, J 12.0, 6.4 Hz, H-90),
3.55 (d, 1H, J 9.0 Hz, H-7), 2.29 (t, 2H, J 7.2 Hz,
COCH2), 2.26 (dd, 1H, J 4.2, 13.0 Hz, H-3e), 1.86 (t,
1H, J 12.4 Hz, H-3a), 1.63 (q, 2H, J 7.2 Hz, CH3CH2),
0.95 (t, 3H, J 7.2 Hz, CH3). HRFABMS: Calcd for
C13H23NO9 [M+Naþ] m=z 360.1271; found 360.1247.
3.5.6. Benzyl glycoside of N-benzoyl-D-neuraminic acid
(27). 174 mg (90%); Rf 0.59 (eluent: 1:1:1:1 BuOH–
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): d
8.01–7.43 (m, 10H, H–Ar), 4.70, 4.54 (2d, 2H, J 10.0 Hz,
CH2Ph), 4.05 (d, 1H, J 10.3, H-6), 4.2–3.6 (m, 6H), 2.83
(dd, 1H, J 4.8, 12.5 Hz, H-3e), 1.77 (t, 1H, J 12.3 Hz, H-
3a); HRFABMS: Calcd for C23H27NO9 [M+Naþ] m=z
484.1584; found 484.1570.
3.7.3. N-iso-Butanoyl-D-neuraminic acid (13). 31 mg
20
D
3.6. Benzyl glycoside of N-trifluoropropionyl-D-neurami-
nic acid (28)
(93%); ½aŠ )28 (c 1.0, H2O); Rf 0.38 (eluent: 1:1:1:1
BuOH–HOAc–EtOAc–H2O);
1H
NMR
(D2O,
300 MHz): (b-isomer) d 4.08–3.78 (m, 4H), 3.62 (dd, 1H,
J 12.9, 6.6 Hz, H-9), 3.54 (d, 1H, J 8.8 Hz, H-7), 2.58 (m,
1H, COCH), 2.26 (dd, 1H, J 5.2, 12.8 Hz, H-3e), 1.85 (t,
1H, J 12.5 Hz, H-3a), 1.15 (2d, 2 · 3H, J 7.0 Hz, 2 CH3).
HRFABMS: Calcd for C13H23NO9 [M-H+2Na]þ m=z
382.1089; found 382.1084.
To a solution of 1-hydroxybenzotriazole (149 mg) and
N,N0-dicyclohexylcarbodiimide (227 mg) in 5 mL of
DMF was added 97 lL of 3,3,3-triflouropropionic acid
(1.1 mmol). The reaction mixture was stirred at 0 ꢂC for
1 h, then at rt for 3 h and 21 (150 mg, 0.42 mmol) was
added to it. The reaction mixture was then stirred
overnight, and the resulting precipitate was filtered off.
The filtrate was concentrated under reduced pressure,
and the residue was purified by silica gel column chro-
matography to afford 28 (140 mg, 72%). Rf 0.79 (eluent:
1:1:1:1 BuOH–HOAc–EtOAc–H2O); 1H NMR (D2O,
300 MHz): d 7.40 (br, 5H, H–Ar), 4.74, 4.54 (2d, 2H, J
10.2 Hz, CH2Ph), 3.30 (q, 2H, J 10.9 Hz, CH2CF3), 2.79
(dd, 1H, J 12.5, 4.6 Hz, H-3e), 1.86 (t, 1H, J 12.5 Hz, H-
3a); HRFABMS: Calcd for C19H24)F3NO9 m=z
[M+Naþ] 490.1301; found 490.1354.
3.7.4. N-Trimethylacetyl-D-neuraminic acid (14). 32 mg
20
D
(93%); ½aŠ )31 (c 1.0, H2O); Rf 0.40 (eluent: 1:1:1:1
BuOH–HOAc–EtOAc–H2O);
1H
NMR
(D2O,
600 MHz): (b-isomer) d 4.08–3.78 (m, 4H), 3.60 (dd, 1H,
J 12.0, 6.0 Hz, H-9), 3.45 (d, 1H, J 9.0 Hz, H-7), 2.25
(dd, 1H, J 4.8, 12.6 Hz, H-3e), 1.84 (t, 1H, J 12.6 Hz, H-
3a), 1.06 (s, 9H, 3CH3). HRFABMS: Calcd for
C14H25NO9 [M+Naþ] m=z 374.1427; found 374.1482.
3.7.5. N-Phenylacetyl-D-neuraminic acid (15). 35 mg
20
D
(91%); ½aŠ )14 (c 1.0, H2O); Rf ¼ 0:40 (eluent: 1:1:1:1
3.7. N-Acyl-D-neuraminic acids 11–17
BuOH–HOAc–EtOAc–H2O);
1H
NMR
(D2O,
300 MHz): (b-isomer) d 7.42 (m, 5H, H–Ar), 4.08–3.96
(m, 3H), 3.77 (m, 1H), 3.71–3.66 (m, 1H), 3.68 (s, 2H,
CH2Ph), 3.54 (dd, 1H, J 11.7, 6.4 Hz, H-9), 3.49 (d, 1H,
J 7.8 Hz, H-7), 2.32 (dd, 1H, J 4.9, 13.1 Hz, H-3e), 1.86
(t, 1H, J 13.0 Hz, H-3a). HRFABMS: Calcd for
C17H23NO9 [M+Naþ] m=z 408.1270; found 408.1271.
A mixture of 22–28 (0.1 mmol) and 10% palladium-on-
carbon (0.01 mol) in MeOH (5 mL) was stirred vigor-
ously at rt under a hydrogen atmosphere until TLC
showed the disappearance of the starting material. The
catalyst was filtered off, and the filtrate was concentrated
under reduced pressure. The residue was purified by
silica gel column chromatography (eluent: 20:80:1 to
50:50:1 MeOH–EtOAc–AcOH) to give 11–17 that were
further purified by a Bio-Gel P-2 column using distilled
water as eluent. Recrystallization from 1:2 EtOH–
EtOAc afforded pure products.
3.7.6. N-Benzoyl-D-neuraminic acid (16). 31 mg (84%);
20
½aŠ )15 (c 1.0, H2O); Rf 0.39 (eluent: 1:1:1:1 BuOH–
D
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): (b-
isomer) d 7.64–7.43 (m, 5H, H–Ar), 4.15 (s, 3H), 3.83–
3.73 (m, 2H), 3.65–3.50 (m, 2H), 2.26 (dd, 1H, J 4.2,
3.7.1. N-Propionyl-
D
-neuraminic acid (11). 30 mg (94%);
12.5 Hz, H-3e), 1.86 (t, 1H,
J
11.6 Hz, H-3a).
20
D
½aŠ )21 (c 1.0, H2O); Rf 0.28 (eluent: 1:1:1:1 BuOH–
HRFABMS: Calcd for C16H21NO9 [M+Naþ] m=z
394.1114; found 394.1140.
HOAc–EtOAc–H2O); 1H NMR (D2O, 300 MHz): (b-

Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
2099
Metabolism and Function in Particular During Viral
Infection; Gabius, H.-J., Gabius, S., Eds.; Chapman &
Hall: Weinheim, 1997; pp 245–259.
3.7.7. N-Trifluoropropionyl-D-neuraminic acid (17).
20
34 mg (90%); ½aŠ )16 (c 1.0, H2O); Rf 0.43 (eluent:
D
1:1:1:1 BuOH–HOAc–EtOAc–H2O); 1H NMR (D2O,
300 MHz): (b-isomer) d 4.13–4.03 (m, 3H), 3.86–3.78 (m,
2H), 3.60 (dd, 1H, J 11.4, 6.6 Hz, H-9), 3.55 (d, 1H, J
9.0 Hz, H-7), 3.35 (q, 2H, J 10.5, CF3CH2), 2.32 (dd,
1H, J 4.6, 13.2 Hz, H-3e), 1.86 (t, 1H, J 13.2 Hz, H-3a).
HRFABMS: Calcd for C12H18F3NO9 [M+Hþ] m=z
378.1012; found 378.1013.
3. Varki, A. FASEB J. 1997, 11, 248–255.
4. Schauer, R. Adv. Exp. Med. Biol. 1988, 228, 47–72.
5. Hakomori, S.; Zhang, Y. Chem. Biol. 1997, 3, 97–
104.
6. Kobata, A. In Cancer Cells and Metastasis; Montreuil, J.,
Vliegenthart, J. F. G., Schachter, H., Eds.; Elsevier:
Amsterdam, 1996; pp 183–241.
7. Livingston, P. O. Curr. Opin. Immunol. 1992, 4, 624–
629.
8. Ragupathi, G. Cancer Immunol. Immunother. 1998, 46,
82–87.
3.8. General procedure for the enzymatic reactions
9. Livingston, P. O. Immunol. Rev. 1995, 145, 147–156.
10. Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed.
2000, 39, 837–863.
11. Comb, D. G.; Roseman, S. J. Am. Chem. Soc. 1958, 80,
497–499.
12. Keppler, O. T.; Horstkorte, R.; Pawlita, M.; Schmidt, C.;
Reutter, W. Glycobiology 2001, 11, 11R–18R.
13. Kayser, H.; Geilen, C. C.; Paul, C.; Zeitler, R.; Reutter,
W. FEBS 1992, 301, 137–140.
14. Schmidt, C.; Stehling, P.; Schnitzer, J.; Reutter, W.;
Horstkorte, R. J. Biol. Chem. 1998, 273, 19146–19152.
15. Yarema, K. J.; Mahal, L. K.; Bruehl, R. E.; Rodriguez, E.
C.; Bertozzi, C. R. J. Biol. Chem. 1998, 273, 31168–31179.
16. Mahal, K. L.; Yarema, K. J.; Bertozzi, C. R. Science 1997,
276, 1125–1128.
To a 1.5 mL vial was added ManNR (7.27 lmol) in
130 lL of 0.05 M potassium phosphate buffer (pH 7.4,
I ¼ 0:1), 72.73 lmol pyruvic acid in 100 lL buffer,
2.0 lmol dithiothreitol in 30 lL buffer, and 140 lL di-
luted NeuNAc aldolase solution (0.35 U). The reaction
was kept at 37 ꢂC with occasional shaking. At time
points of 20, 40, 90, 180, 360, 720, and 1440 min, 40-lL
aliquots were taken out and put on dry ice for 30 min.
After that, the samples were directly added into resor-
cinol reagent and analyzed by the modified Svennerholm
method described below.
3.9. Modified Svennerholm method31 for the analysis of
sialic acid products
17. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010.
18. Liu, T.; Guo, Z.; Yang, Q.; Sad, S.; Jennings, H. J. J. Biol.
Chem. 2000, 275, 32832–32836.
19. Jacobs, C. L.; Goon, S.; Yarema, K. J.; Hinderlich, S.;
Hang, H. C.; Chai, D. H.; Bertozzi, C. R. Biochemistry
2001, 40, 12864–12874.
20. Fitz, W.; Schwark, J.-R.; Wong, C.-H. J. Org. Chem.
1995, 60, 3663–3670.
21. Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem.
Rev. 1996, 96, 443–473.
22. Wymer, N.; Toone, E. J. Curr. Opin. Chem. Biol. 2000, 4,
110–119.
23. Fitz, W.; Wong, C.-H. J. Org. Chem. 1994, 59, 8279–8280.
24. Ichikawa, Y.; Liu, J. L. C.; Shen, G. J.; Wong, C.-H. J.
Am. Chem. Soc. 1991, 113, 6300–6302.
25. Lin, C.-C.; Lin, C.-H.; Wong, C.-H. Tetrahedron Lett.
1997, 38, 2649–2652.
26. Sparks, M. A.; Williams, K. W.; Lukacs, C.; Schrell, A.;
Priebe, G.; Spaltenstein, A.; Whitesides, G. M. Tetrahe-
dron 1993, 49, 1–12.
An aliquot (40 lL) of the enzymatic reaction mixture
was added to a 1.5 mL solution of the resorcinol reagent
(0.2% w.t. resorcinol, 80% v/v concd HCl and 0.25% v/v
0.1 M CuSO4, in H2O). After the mixture was heated in
a boiling water bath for 30 min and cooled to rt, to it
was added 3 mL of the extraction solvent (85:15 Bu-
OAc–BuOH). The mixture was vigorously shaken on a
vortex mixer, and after the mixture settled down, the
two layers were carefully separated. The absorbance of
the organic layer was measured at 580 nm with a UV–vis
spectrophotometer using a 1.0-cm quartz cuvette with
the extraction solvent as blank. The calibration curves
of all sialic acid derivatives were obtained by the same
measurement.
27. Halcomb, R. L.; Fitz, W.; Wong, C.-H. Tetrahedron:
Asymmetry 1994, 5, 2437–2442.
28. Roy, R.; Laferriere, C. Can. J. Chem. 1990, 68, 2045–
2054.
Acknowledgements
29. Byramova, N. E.; Tuzikov, A. B.; Bovin, N. V. Carbohydr.
Res. 1992, 237, 161–175.
30. Brossmer, R.; Gross, H. J. Meth. Enzymol. 1994, 247, 153–
176.
31. Svennerholm, L. Biochim. Biophys. Acta 1957, 24, 604–
611.
We thank the financial support for this work by a re-
search grant from NIH/NCI (1R01 CA95142) and a
seed grant from the Ohio Cancer Research Associates.
MS measurements were performed by Mr. J. Faulk.
32. Marangoni, A. G. Enzyme Kinetics: A Modern Approach;
Wiley-Interscience: Hoboken, NJ, 2002.
33. Ritter, G.; Boosfeld, E.; Calves, M. J.; Oettgen, H. F.; Old,
L. J.; Livingston, P. O. Immunobiology 1990, 182,
32.
34. Ritter, G.; Boosfeld, E.; Asluri, S.; Calves, M. J.; Oettgen,
H. F.; Old, L. J.; Livingston, P. O. Int. J. Cancer 1991, 48,
379–385.
References
1. Rosenberg, A. E. Biology of the Sialic Acids; Plenum: New
York, 1995.
2. Reutter, W.; Stasche, R.; Stehling, P.; Baum, O. In The
Biology of Sialic Acids: Insights into Their Structure,

2100
Y. Pan et al. / Carbohydrate Research 339 (2004) 2091–2100
35. Jennings, H. Int. J. Infect. Dis. 1997, 1, 158–164.
36. Chefalo, P.; Pan, Y.; Nagy, N.; Harding, C.; Guo, Z.,
Glycoconjugate J., in press.
38. Keppler, O. T.; Stehling, P.; Herrmann, M.; Kayser, H.;
Grunow, D.; Reutter, W.; Pawlita, M. J. Biol. Chem. 1995,
270, 1308–1314.
37. Spivak, C. T.; Roseman, S. J. Am. Chem. Soc. 1959, 81,
2403–2404.
39. Charter, N. W.; Mahal, L. K.; Koshland, D. E. J.;
Bertozzi, C. R. J. Biol. Chem. 2002, 277, 9255–9261.