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Sialic Acid Analogues and Their Biological
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SYNTHETIC ANALOGUES OF SIALIC ACID AND

THEIR BIOLOGICAL APPLICATION

by

BAO—JEN SHYON G

A THESIS
submitted to
MICHIGAN STATE UNIVERSITY
in partial fulfillment of the requiremrnts
for the degree of
MASTER OF SCIENCE
DEPARTMENT OF BIOCHEMISTRY

1993

ABSTRACT

SYNTHETIC ANALOGUES OF SIALIC ACID AND THEIR BIOLOGICAL
APPLICATION

by

BAO-JEN SHYONG

2,3-dehydro-2-deoxy-N—acetylneuraminic acid (NeuSAcZen) has been synthesized
from N-acetylneuraminic acid (NeuSAc) by a simple, quantitative, one-step conversion.
NeuSAc was dehydrated by peracetylation with trifluoroacetic anhydride (TFAA) in the
presence of a catalytic amount of N ,N—dimethyl-4-aminopyridine (DMAP). 2 ,3-Dehydro-
2-deoxy-neuraminic acid (Neu2en) was also obtained when the ratio of TFAA/DMAP
was changed to 4:3. Seven other sialic acid derivatives were prepared incorporating the
half-chair conformation of NeuSAcZen such as 2,3-epoxy-2-deoxy-N—acety1neuraminic
acid, N —azidoacetyl neuraminic acid, and a substitution of N-acyl or 9—hydroxyl on
Neu5Ac2en.

These derivatives were investigated for their ability to inhibit Vibrio cholerae
sialidase using 2’-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (4MU-Neu5 Ac)
as the substrate. In a kinetic study, seven of the eight analogues of NeuSAcZene were
shown to be competitive inhibitors with K, values ranging from 103 M to 10‘5 M. No
inhibition was observed for the epoxidc—NeuSAcZene compound .

An affinity chromatography method has been developed for separating V. cholerae

neuraminidase from physiological protein mixtures. The affinity column was made by

coupling 2-deoxy-2,3—dehydro-N-deacetyl neuraminic acid (Neu2ene) to Bio-Rio Affi-Gel
10 activated agarose gel. Bound neuraminidase selectivly absorbed from a mixture of
proteins was eluted from the column with 10% neuraminic acid in H20. The total
activity of the recovered neuraminidase from the affinity column was evaluated using
the 4MU-Neu5Ac assay, and the peroxidase-amplified assay. As judged by activity, 80-
10096 of the total activity applied to the column was recovered. The physical properties
of the neuraminidase was examined by electrophoresis. Enzyme samples analyzed before
and after the affinity chromatography appeared as 68 kDa and 16 kDa polypeptide bands
while the molecular weight of V. cholerae neuraminidase has been previously reported

as 24.3 kDa or 90 kDa polypeptides.

ii

ACKNOWLEDGEMENT

I would like to express my sincere gratitude to both my aeademic advisors Dr.
Chuck C. Sweeley and Dr. Rawle I. Hollingsworth, for their guidance and support
during my graduate studies. I am grateful to my committee members, Dr. Bill Wells,
Dr. Shelagh Ferguson-Miller, Dr. Estelle McGroarty, and Dr. Susan E. Conrad, for their
inspiration, valued advice, and help in my research.

Iamthankfultomembersofthemassspectrometry facilityinMSU (Dr. Gage,
Dr. Huang, Bev Chamberlain, Michael Davenport, Melvin Micke, and Kate Noon) for
allowing me to unlimitedly use instruments and all their help. Special thanks go to Dr.
Huang, you have shown a lots of toleration during our work and rebuilted my
confidence. I appreciate your encouragement, and I have learned so much from you in
science and life.

It is with great pleasure that I thank all my colleagues from the laboratory of Dr.
Hollingsworth and Dr. Sweeley have supported, helped, and challenged me during these
years; Doug Wiesner, Dr. Lyla Melkerson-Watson, Amanda Poxon, Kiyoshi and Misa
Ogura, Dr. Bill Hare, Dr. Deborah Lill-Elghanian, Dr. Maria Baconi-Baker, Dr. Zhang
Yuanda, Chuck Campbell, Kim Kyung-II, Seunho Jung, Robert Cedergren, Luc Berube,
Bernard Hummel, and Yin. To my friends beside labmates, Beta Borer, Dr. Christopher
Meyer, Dr. Taha Taha, Barbara Hamel, Lars Peereboom, Susan Leavitt and Carol
McCutcheon, thanks for your friendship, conversation, emergent support, and joy. A

special thank to Chuck Campbell; he is helpful in proofreading of this manuscript.

iii

Finally, I would like to thank my family for their understanding; without it, this

would not have been possible.

iv

TABLE OF CONTENTS

ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
CHAPTER 1: LITERATURE REVIEW
A. Intrroduction
B. Structural Characteristics of Sialic Acids
C. Distribution and Biological Functions of Sialic Acids
D. Synthetic Sialic Acid Derivatives
E. Distribution and Character of Sialidases
F. The Postulated Mechanism of Sialidases
G. Activatiors and Inhibitors of Sialidas
H. References
CHAPTER 2: SYNTHESIS OF SIALIC ACID ANALOGUES: A novel,
one-step conversion of N-acetylneuraminic acid to 2,3-dehydro-2-deoxyl—N—
acetylneuraminic acid and their derivatives.
Abstract
Introduction
Materials and Methods

Materials

11

16

20

24

28‘

37

38

38

40

4O

Methods A 42
Synthesis of 2,3-dehydro-2-deoxy—N—acetylneuraminic acid 42
Synthesis of 2,3-dehydro-2—deoxy-neuraminic acid 43
Synthesis of 5-acetamino—2,3-epoxide—3,5-dideoxy-B—glcero—
D-galacto—nonulopyranosylonate 44
Synthesis of 2,3-dehydro-2-deoxy-N—chloroacetylneuraminic

acid 45
Synthesis of 2,3-dehydro-2-deoxy—N-bromoacetylneuraminic

acid 45
Synthesis of 2,3-dehydro-2-deoxy-N-azidoacetylneuraminic acid 46
Small-scale synthesis of 7 ,9-m-nitrobenzylidene-2,3-dehydro—
2-deoxy-N-acetylneuraminic acid 46
Synthesis of 9-O-m—aminobenzy1—2,3-dehydro-2-deoxy-N—acetyl
neuraminic acid 47
Large-scale synthesis of 9-O-m-aminobenzyl-N-acetylneuraminic
acid , 48

Synthesis of 8,9—isopropylidene-2,3-dehydro—2-deoxy—N-acetyl

neuraminic acid 49

Results and Discussions 49

References 57
CHAPTER 3: INHIBITION STUDIES FOR SYNETHETIC SIALIC ACID

ANALOGUE

Abstract
Introduction
Materials and Methods
Materials
Methods
The preparation of enzyme assay
Experiments for sialidase inhibition
Results and Discussions
References
CHAPTER 4: AN AFFINITY CHROMATOGRAPHY METHOD - An
utilization of sialic acid serivative
Abstract
Introduction
Materials and Methods
Materials
Instrument
Preparation of immobilized sialic acid analogue on Bio-Rad
Affi G-lO gel
Preparation of a fetuin—agarose affinity column chromatography
The activity assay
4MU-Neu5Ac Assay

Peroxidase-amplified assay

vii

61
61
63
63
63

63

9

75

76
77
77
79
79

79

80

82

82

83

SDS-Polyacylamide gel electrophoresis
Results
Affinity separation of V. cholerae neuraminidase from a protein
mixtures
The bound neuraminidase of fetuin-agarose column chromatography
Discussions
References
CHAPTER 5: DISCUSSIONS
1. Synthesis of neuraminic acid analogues
A. Develop a chemical process for converting sialic acid to
NeuSAc2en
B. Develop a photo-activatable sialidase inhibitor
II. Develop an affinity chromatography for isolation sialidase from
physiological complexes

References

viii

83

85

85
89
91
94
96

96

96

100

102

104

LIST OF TABLES

CHAPTER 1 1
Table l. The influence in the activity 0f sialidase is contributed with
N -, O—substitution 6
Table 2. A summary of the inhibtion data about sialidases from various
sources 26
CHAPTER 2 37
Table 1. Relevant 1H-NMR and TLC data for synthetic sialic acid
derivatives in this study 51
CHAPTER 3 60
Table 1: The remaining activity of V. cholerae sialidase with
synthesized Neu5Ac2en derivatives 67
Table 2. Inhibition of Vibrio cholerae neuraminidase by Neu5Ac2en
and synthetic analogues 69
CHAPTER 4 76
Table l. The recovered activity of Vibn'o cholerae sialidase from
Bio-Rad Affi-GIO affinity chromatogaphy 87

ix

LIST OF FIGURES

CHAPTER 1
Figure 1. The structure of N-acetylneuraminic acid (Neu5Ac2en) and
2,3-dehydro-2-deoxyl-N-acetyl neuraminic acid (Neu5Ac2en)
Figure 2. The structure of N -, O-substituents of sialic acids
Figure 3. Reaction scheme for synthesis of 3H-9-PANP-Neu5Ac2en
Figure 4. Reaction scheme for synthesis of ASA-Neu5Ac2en and
[‘BHIASA-NuSAcZen
Figure 5. The postulated mechanism for the binding of substrates and
transition-state analogues to A. sailophiluse sialidase
CHAPTER 2
Figure l. The structure of NeuSAc and the half-chair conformation of
sialyloglycoconjugates
Figure 2. The potential inhibitors of sialidase(s) synthesized for
this study
Figure 3. 300MHz ‘HNMR Spectra of Neu5Ac2en and Neu2en
Figure 4. 300MHz lHNMR Spectra of Neu5N3Ac2en
Figure 5. The whole range of infra-red absorbance spectrum of
Neu5N3Ac2en
CHAPTER 3

Figure l.Thestandardcurveof4MUfrom0thoSOnM

15

17

37

41

50

53

54

55

65

Figure 2. Inhibition kinetics of Neu5Ac2en(S) to sialidase from
V. cholerae using 4MU-Neu5Ac as substrate

Figure 3. Inhibition kinetics of Neu2en to sialidase from
V. cholerae using 4MU-Neu5Ac as substrate

Figure 4. Inhibition kinetics of Neu5N,Ac2en to sialidase from
V. cholerae using 4MU-Neu5Ac as substrate

Figure 5: Inhibition kinetics of AB-NeuAc2en to sialidase from
V. cholerae using 4MU-Neu5Ac as substrate

CHAPTER 4

Figure 1. The common applied affinity chromatography

Figure 2. Gm standard curve for sialidase activity by peroxidase-
amplified assay

Figure 3. Elution profiles of Neu2en Bio-Rad Affi G-lO affinity
chromatography

Figure 4. SDS-PAGE Analysis of V. cholera sialidase with
an affinity column

Figure 5. Elution profiles of fetuin-agarose affinity chromatography

CHAPTER 5

Figure l. The synthesis scheme of Neu5Ac2en developed by Meindl
er. a1. and from this laboratory

Figure 2: Reaction scheme for the synthesis of Neu5Ac2en derivatives

with a modification on its glycerol side chain

70

71

72

74

76

81

86

87

9O

96

97

Figure 3: Reaction scheme for the synthesis of photoaffinity compound 101

CHAPTER]

LITERATURE REVIEW

A. INTRODUCTION

Sialic acids (neuraminic acids) are important components of the carbohydrate
chains of glycoproteins and glycolipids, where they usually occur on the non-reducing
termini of these molecules. The sialic acids are complex acidic sugars derived from
neuraminic acid, a A5-amino-3,5-dideoxy-D-glycero—D-galacto-nonulosonic acid. The
most common member of the sialic acid family is N-acetylneuraminic acid (Neu5Ac)
(Fig. 1A). Because sialic acids occur on cell-surface glycoproteins and acidic
glycosphingolipids (gangliosides) and contain a net negative charge, they have a
pronounced effect on the surface charge and biological function of the cells in which they
occufi‘”). They are thought to be involved in the regulation of cell division by their
incorporation into surface molecules by sialyltransferases or removal by sialidases
(neuraminidases)“-5’.

Sialidase (neuraminidase, EC 3.2.1.18), designated as a N-acetylneuraminosyl—
glycohydrolase, hydrolyzes the terminal a-ketosidic linkage of sialic acid residues from
various sialylated glycoconj ugates‘”). Sialidases have been detected in a variety of
sources including human organs and microorganisms. Animals possess sialidases for the
turnover of their sialoglycoconj ugates“-”. The occurrence of these enzymes in
microorganisms is often associated with pathogenicity“"’-". The role of sialidases in viral

infections has been the subject of many anti-infection studies“). The sialidase activity in

l

2
the cell culture medium of human foreskin fibroblasts is distinctly different between the

sparse density and the confluent density cultures“).

This study utilizes transition-state analogues of neuraminic acid to develop novel
compounds for isolation and determination of sialidase from biological sources. One
approach is to develop efficient inhibitors of these enzymes which requires that such
molecules resemble the transition state for transformation of the substrates to products.
A convenient transition state analogue for utilization in studies of sialidases is 2,3-
dehydro-Z-deoxy-N-acetylneuraminic acid (Neu5Ac2en) (Fig. 1B). This material is
extremely expensive and difficult to synthesize from sialic acid (”M-m. These factors
severely limit the application of Neu5Ac2en in the development of affinity matrices
where considerable amounts of material are required. The most important step in this
project was, therefore, to develop a simple, efficient chemical process for the conversion
of sialic acid to Neu5Ac2en. The specific goals of this study were as follows:

(1)Develop a chemical process for converting sialic acid to Neu5Ac2en;

(2)Develop a method of coupling Neu5Ac2en to a solid support for use as

an affinity matrix without affecting its inhibitory properties. This would

i make it possible to isolate sialidases from complex physiological mixtures

such as spent cell-culture media;

(3) Develop a photoactivatable sialidase inhibitor derived from

Neu5Ac2en which can be prepared in a radiolabeled form for use as a3

photoaffinity probe for radiolabeling sialidases in complex systemsm-“J”;

and

(A)

 

NeuSAc

(B)

 

NéuSAcZene

Figure 1: (A)The structure of free N-aoetylneuraminic acid(Neu5Ac2en). .
(B)The structure of 2,3-dehydro-2-deoxyl—N-acetyl neuraminic acid (Neu5Ac2en).

4
(4) Validate the efficiency of the affinity matrix developed in (2) and the

inhibitory power of the photoaffinity-labeled derivative developed in (3).

B. STRUCTURAL CHARACTERISTICS OF SIALIC ACIDS

Sialic acid is the trivial name for a series of neuraminic acid derivatives (IUPAC
system name: 5-amino—3,5-dideoxy-D—glycero-D-galacto—nonulosonic acids; Neu5Ac),
which are acidic amino sugars. In a recent report“), the number of sialic acids identified
from natural sources was around 25. Neuraminic acid, with a carboxyl group at C-1 , '
provides a negative charge on the carbohydrate components of glycolipids, glycoproteins,
and other glycoconj ugates. In addition to the earboxyl group, sialic acids have other
functional groups such as N-acyl, C7-C8-C9 glycerol side chain, and multiple hydroxyl
groups (Fig. 2, and Table 1). Two kinds of N-substituted sialic acids are widely
encountered in nature, that is, N -acetyl (Neu5Ac) and N-glycolyl (NeuSGc). In addition,
the hydroxyl moieties at C-1, C-4, C-7, C-8, and C-9 can be methylated or esterified
with acetyl, glycolyl, lactyl, methyl, sulphate, or phosphate groups"). In some
glycoconjugates sialic acids with more than one substituent are found. Furthermore,
unsaturated N-acetylneuraminic acid (Neu5Ac2en) has been found as a natural product
in Kamerling et. 01. study‘"). So far, no unsubstituted neuraminic acid has been isolated
from natural sources.

In nature, sialic acids are usually a-ketosidically linked to other carbohydrates in
glycoconjugates while free sich acid exists almost completely as the fi-anomer. The

only fl-ketosidic linkage involving sialic acid known to occur in nature is the activated

 

 

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nucleotide CMPB(5-2)Neu5Ac (cytidine-S’-monophosphate—2-B-N-acetylneuraminic

acid)‘“-"’. Only a low level of free sialic acid has been detected in normal
organismsm-“m’. .

Sialic acids occupy both terminal and internal positions in carbohydrate chains of
glycoconj ugates. Terminal sialic acids are usually linked to galactose (Gal), N-acetyl-D-
galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and glucose (Glc). The
I most common linkages found are a(2-3)Gal, a(2—6)Gal or a(2-6)GalNAc and less
commonly a(2-4)Gal and a(2—4)GlcNAc(’3-2‘). The branched-terminal sialic acids found
in colon mucus glycoproteins of rat were reported to have an a(2-6) linkage to Gal and
GalNAc‘”~"’. Ganglioside Gm is an example of a glycoconjugate containing internal
sialic acids, which are attached either to other sialic acids or to an oligosaccharide
core“. The Neu5Aca(2-8)Neu5Ac—unit and Neu5Aca(2-3)-oligosaccharide core are
common components in sialylpolysaccharides and oligosialylglycoconjugatesm-zm’). The
Neu5Aca(2-9)Neu5Ac, Neu5Aca(2-8)Neu5Ac, and/or Neu5Aca(2-6)Gal linked unit are
present in bacterial polymers. For example, E. coli strain K92 (Bos 12) contains a
mixture of a(2-9) and a(2—8) linkage; the polymers of N. meningitis Serotype C are made

from the repeating unit a(2-9) linkage‘”-"-”"3-"’.

C. DISTRIBUTION AND BIOLOGICAL FUNCTIONS OF SIALIC ACIDS
The variety of substituents and linkage types of sialic acids provides structural
diversity to oligosaccharides of glycoconj ugates. Furthermore, the distribution of sialic

acids also exhibits species— and tissue-specificity. For example, mammalian systems

8
contain N-acetyl, N-glycolyl, O-acyl, sulphate, and phosphate-subsumed neuraminic acids

while Hemichordata (Dolichoglossus kowalevskii) have only N-acetyl neuraminic
acidm-“L Bovine breast muscle tissue has a higher NeuSGc level (45 %) than that of fetal
bovine tissue (0%)”. Gangliosides, consisting of ceramide and sialyloligosaccharide
moieties, are sialic acid-containing glycolipids. In the brain of horse, cow, pig, and
sheep, the level of NeuSGc-containing gangliosides was less than 2 % of total sialic
acids“) while the proportion of that in the erythrocytes of horse and pig reach 95 —
100%°’-‘°’. No mammalian species has been found without sialic acid-containing
glycoconj ugates under normal conditions““’.

The distribution of N-acetyl, N—glycolyl, and O-acyl sialic acids in gangliosides
was shown to correlate with developmental stages in various organisms‘W-m. Bovine
fetal tissue was shown to have a higher level of sialic acids than adult tissues; however,
adult tissues contained a higher percentage of NeuSGc in the total sialic acids than fetal
tissues. It was suggeSted that 9-O-lactyl-Neu5Ac was a differentiation marker in bovine
tissues beeause it was only found in fetal brain tissues but not in the adult”). The
percentage of NeuSGc in gangliosides of rat intestine was shown to rapidly increase after
the first 21 days of life. Similarly, the relative level of Neu5,9Ac, was shown to increase
with maturation of erythrocytes in adult chickens and of colon in rat").

Evidence indicates that Neu5Ac is associated with changes in cell properties. The
location of sialic acid-containing glycoconjugates on the plasma membrane of the cell was
examined in order to correlate the specific functions on certain membrane areas‘“). The

results indicated that the modification of protein-bound sialic acid occurred during the

9 r
mitosis of normal cells. It was also reported that the addition of extra Neu5Ac to growth
media could stimulate DNA synthesis and increase cell density in a fibroblast cell line
(GM03468A) in vitro‘”. This suggested that negatively charged Neu5Ac on
glycoconj ugates might be involved in cell surface regulation events such as cell
recognition, masking of antigenic determinants, contact inhibition, and cell
migration(“v“-‘7v“v").

Total sich acid (TSA) and lipid-bound sialic acid (LSA) content was measured
for normal and transformed cell lines in tissue culture‘mm). An increased level of sialic
acid (0.6 pg) released per mg by sialidase was observed for a transformed baby hamster
kidney cell line (BI-IK-21) as compared to normal cells”). The level of TSA in the
serum of eancer patients with benign tumors was elevated from 1.74 mmol/L to 2.16-
4.15 mmol/l‘” A higher value of TSA was also observed in patients with malignant
tumors‘“'”). The amount of NeuSGc in total lipid-bound sialic. acid of colon cancer
patients is 1% while it is less than 0.01% in the healthy individuals“).

It has been observed that the negative charge on sialic acids can influence the
micro- and macro-structure of glycoproteins on the cell membrane. The conformation
of some glycoproteins with sich acid residues removed has been shown to change with
a corresponding loss of activity‘57'5'5”. Treatment of rat dermal fibroblasts with sialidase
to expose the non-reducing-end saccharides of glycoproteins caused an increase in
deformation of the cell membrane‘“"”. Intrinsic factor, a sialoglycoprotein, appeared to
lose proteolytic resistance and binding ability to vitamin B12 after removal of the terminal

sialic acidsmv‘”. In pathological studies with B. clostridium, infection was shown to

10
occur after a bacterial sialidase cleaved sialic acid from cell surface glycoproteins on the

host‘“-“-“’. These tissues were then defenseless to clostridial proteases‘m. Desialylation
of plasminogen (Pg) in human plasma by sialidase treatment alters its physiological
functions, including an increase of amidolytic and fibrinolytic activity and a decrease in
the binding ability to U937 cells”). However, Chinese hamster ovary (CHO) cells and
mouse 3T3 cells treated with neuraminidase did not demonstrate a change in their binding
ability on glass walls‘“-7°’.

N- and O-acylated sialic acids in glycoconjugates have been described as antigenic
determinants in various mammalian and microbial systems‘“””. For example, Neu5Ac
is the key sugar distinguishing the MN blood group substancesmv”-"". Cholinergic-
specific antigen (Chol-la) from rat brain was found to be a ttisialogangliosidef’s’. Cold
agglutinins, which are antibodies raised against the terminal parts of N- or O-glycosidic
oligosaccharide chains of glycoproteins in the membrane of erythrocytes, were able to
bind Neu5Ac-containing antigensm-m. Waldenstrom macroglobulins also showed specific
binding to Neu5Ac antigens‘m'”). N-glycolylneuraminic acid antigen was found in the
blood group substances of East-Asian dogs. Hanganutziu-Deicher (HD) antibodies,
discovered in sera of patients with several diseases, bound the NeuSGc-antigen of
gangliosides and glycoproteins“"”. Four antigenic gangliosides containing NeuSGc were
isolated from human colon cancer“). Evidence has also indicated that O-acetylation of
sialic acids influences the immunogenicity of glycoconjugates in bacterial polysaccharides
and human melanoma cells“"”’. Colominic acid of E. coli K1, a homopolymer of

Neu5Ac with O-acetylation at 0-9 position, is more immunogenic than non-O-acetylated

ll
homopolysaccharides‘”). NeuS ,9Acz. found in the melanoma-assiciatcd ganglioside

(Gm), is considered to be a tumor-specific antigenm‘”.

D. SYNTHETIC SIALIC ACID DERIVATIVES

Studies of synthetic sialic acid derivatives provide a path to understanding the
biologieal functions of sialic acids. They can be used as substrates and inhibitors of
sialidases, sialyltransferases and other converting enzymes, and to study the mechanism
of sialidase. The following discussion is limited to synthetic transition state analogues
of sialidases. Synthetic sialo—oligosaccharide compounds have been reviewed by Van
der V1eugel‘“’.

2-Deoxy-2,3-dehydro-N-acctylneuraminic acid (Neu5Ac2en) has been known as
a competitive inhibitor for various neuraminidases. The conformation of Neu5Ac2en is
, a half-chair form similar to the intermediate of hydrolysis reaction by sialidase.
Neu5Ac2en has been found as a side product from the synthesis of a-linked
sialodisaccharides‘”'“t"). Neu5Ac2en is synthesized from various halogenated neuraminic
acids. For example, it is obtained by the treatment of 4,7 ,8,9—tetra-O-acetyl-2-chloro-2-,
deoxy-B-N-acctylneuraminic acid with an HCl-eliminating agent such as triethylamine (10
min, 20 °C) or silver carbonate (60—90 min, 80-90 °C), in dioxane or acetone, followed
by O-deacetylation “2'7". Another halogenated neuraminic acid, 4,7 ,8,9-tetra-O—p—
nitrobenzoyl-Z-bromo—Z-deoxy—N—acetylneuraminic acid methyl ester, was converted
quantitatively to Neu5Ac2en by using triethylamine or by treatment of molecular sieves

4A in dichloromethanc. Neu5Ac2en was obtained in 73 96 yield by the full protection

12
bromo sugar after deacetylation‘m. In another scheme, Neu5Ac2en was prepared by

prolonged heating (5hr, 90 °C) of 2,4,7 ,8,9-penta-O—acetyl-N-acetylneuraminic acid in
dioxane, followed by O—deacetylation‘m. Other unsaturated sich acid derivatives with
different N-acyl groups are obtained from a similar process using different N-
acylneuraminic acids as reactants”).

The other common series of unsaturated siafic acids, 2-deoxy-2,3-
dehydroneuraminic acid (Neu2en), are prepared from 2-deoxy-2,3-dehydro—N—
benzyloxyearbonylneuraminic acid by hydrogenolytic cleavage with Pd/BaSOJH2 ('9’.
The yield of this procedure has been reported to be around 73%. In addition, the
benzhydryl ester of Neu2en is prepared with diphenyldiazomethane in methanol. A
series of N -acyl neuraminic acid derivatives and their methyl esters are synthesized from
Neu2en by various N—acyl transferring procedures“°-"’. Neu2en and its N -acyl
derivatives allow us to study the effect of N-substituents on sialidase activity.

Synthetic analogues of sialic acid altered at the C-5 position were prepared on a
microgram scale by Schreiner et. 059°). 2-Deoxy-2,3-dehydro—N-trifluoroacetyl
neuraminic acid (Neu5CF32en) was synthesized by hydrazinolysis of Neu5Ac2en in
hydrazinium hydroxide (72hrs, 85 °C), followed by N-trifluoroacetylation of Neu2en"".
This protocol provided a stereochemieally defined procedure for the preparation of the
N-substituted molecule compared with the traditional Kuhn-Baschang procedure” even
though the yield of the final product (Neu5CF32en) was less than 25 % . 2,3-Didehydro—
2,3-dideoxy-D-glycero-D-galacto-Z-nonulopyranosonic acid (Kdn2en), 5-azido-2,3-

didehydro-2,3,5-trideoxy-D-glycero-D—galacto-2-nonulopyranosonic acid (5-azido-5-

13
deoxy—Kdn2en) were formed from the respective peracetylated methyl esters via

dehydration and Zemphen saponifieation. These C-5 modified analogues of Neu5Ac2en
were used to determine the interaction between the amino, carbonyl groups at C-5
position and the binding site of V. cholerae sialidase.

Synthetic analogues with alternations in the glycerol side chain of sich acid have
been investigated by Erich et. 01‘”). 7-, 8-, 9-Deoxy-Neu5Ac2en was synthesized from
the corresponding iodononulosonic acid. methyl esters by xanthogenation, hydrolysis,
peracetylation, and oxazolino-dehydrationm"). In this series of procedures, the 4,5—
oxazolino derivative was a minor product when l M trimethylsilyl
trifluoromethanesulfonate CF,SO,Si(CI-I,)3 in acetonitrile was used. The major products,
peracetylated methyl esters of 9-deoxy-Neu5Ac2en derivatives, were prepared with only
a eatalytic amount of CF,SO,Si(CH,),, followed by alkaline hydrolysis to give
corresponding to deoxy-Neu5Ac2en compounds in 33-60% yield.

Synthetic analogues of sich acid modified at C-4 were prepared to determine the
structural requirements for sialidase activity. The C4 hydroxyl group of sialic acids has
been converted to azido, amino, N-formyl, N ~acetyl, 4-epi-hydroxy, and 4-epi-azido
substituents via the intermediate 4-epi-Neu5Ac2en (”’7'”). The peracetylated methyl ester
of Neu5Ac was treated with CF,so,Si(CH,), in acetonitrile (2m, 50 °C) to give an 4,5-
oxazoline compound, then with trifluoroacetic acid (TFA) in tetrahydrofuran/water to
cleave the oxazolino ring, followed by deacetylation to form 4-epi-Neu5Ac2en. In
another study, 4—epi-Neu5Ac2en was obtained by acetolysis in sulfuric acid and acetic

anhydride at 40-50 °c for 71mm, The azido substituents of Neu5Ac2en derivatives

14

were prepared from this epimeric derivative by reaction with
triphenylphosphane/ammonia/diethylazodicarboxylate (P(Ph),/NI-I,/DEAC). Other C-4
replacements were obtained by treatment of free 4-azido, or 4-epi-4-azido Neu5Ac with
P(Ph),/'I'HF/I-I,O and hydrolysis.

The above mentioned analogues were synthesized to further understand the
structural requirements for the activity of sialidases in vitro. Another approach to obtain
information about sialidases in vivo is to synthesize photoreactive analogues of sialic
acids. The requirement of a photoactivable probe includes the specific binding to the
active site of sialidase without azido interference. In Wamer’s studies‘13v1‘v‘”, a series
of photoreactable analogues were synthesized through preacetylated 9-O—tosyl-Neu5Ac2en
methyl ester (Fig. 3). 9-Azido—Neu5Ac2en was obtained by treatment of the tosyl
analogue with NaN3 in dry dimethylsulfoxide under N2 at 50 °C overnight. Next, 9-S-(4-
azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9-thio-D-glycero—D-
glacto-non-Z-enonic acid (9-PANP-Neu5Ac2en) was prepared by introducing a
photoactive compound, 4-fluoro—3-nitrophenylazidc, to the C-9 position via a thio
linkage. The 9-thioacetyl—Neu5Ac2en was formed by replacement of the tosyl group with
potassium thioacetate in DMF (80 min, 60 °C). The photoactive product was obtained
after 9-S—acetyl-Neu5Ac2en reacted with 4-fluoro-3-nitrophenylazide/ sodium methoxide
in methanol, followed by peracetylation and base hydrolysis. Also, the radioactive
photoprobe, 9-S-(4-azido-3,53H-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-
tetradeoxy-9-thio-D-glycero-D-glacto—non-2-enonic acid (‘H—9-PANP—Neu5Ac2en) was

prepared by using radioactive 4—fluoro—3-(2,6’H) nitrophenylazide as the starting material.

15

 

 

 

 

CH 3
OH
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Figure 3: Reaction scheme for synthesis of 3H-9-PANP-Neu5Ac2en, which is adapted
from Warner, T. G.(l985).

16
These thio-Neu5Ac2en derivatives were shown to have K, values (1.0 x 10" M) similar

to Neu5Ac2en with lysosomal neuraminidases. In another study‘”), a photoactivable
heterobifunctional reagent, N—hydroxysuccinimide ester of 4-azidosalicylic acid (NHS-
ASA), was incorporated into the 09 position of 9-amino-Neu5Ac2en under slightly basic
conditions (at pH > 7, the half-life time was less than 2hrs.) (Fig. 4). Radioactive
['25IJASA-Neu5Ac2en was also synthesized by coupling ”51 to the photoactive

heterobifunctional reagent according to Hunter’s methodW”.

E. The CHARACTERISTIC OF SIALIDASE

Sialidases (neuraminidases, EC 3.2.1.18), designated as a number of N-
acctylneuraminosyl-glycohydrolases, cleave the terminal a-ketosidic linkage of sich acid
residues from various sialylated g1ycoconjugatcs“°’-‘°’-‘°‘-‘°5’. Sialidases have been isolated
from a variety of sources including human organs and microorganisms. Sialidases found
in bacterial systems are soluble exoenzymes while these enzymes in viruses compose a
part of the envelope membrane to incorporate with hemagglutinin during infection”).
In mammals, sialidase activity is detected in lysosomcs, the Golgi apparatus, the plasma
membrane and the cytosol by using gangliosides, sialylactoses, and 4-methylumbelliferyl-
a—D-N-acetylneuraminic acid (4MU-NANA) as substrates ““5"”.

The optimal pH of sialidase depends on substrate specificity and source.
Sialidases found in bacteria and viruses have an optimal pH of 4.5 to 5.5, depending on
the substrate“°‘-‘“-"’-"3v‘W. In mammals, the optimum pH of lysosomal, Golgi, and

plasma-membrane associated sialidases is 4.0 while that of soluble and extracellular

17

on
HZN

COO
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OH

0
II
1.0.5MNIHCO, ”36—S—O-N: 7
/ °

 

 

a mom Ethyl Acetate 0
i Nils-ASA
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5 H 0
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NflkNufiAdhn
Imfinafion
on "
age... on
5 H o
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[mum-Noam

Figure 4: Reaction scheme for synthesis of ASA-NeuSACZen and [IZSUIASA-
Nu5Ac2en, which is adapted from Horst et. al.(l990).

18
sialidases is reported at around pH 6—6.5“-‘°5"‘5’.

A unique aspect of many types of sialidases is their wide substrate
specificity“-‘°‘-‘°‘-“°"‘3). The activity of various sialidases is studied by using (a(2-
3)sialyllactose (a(2-3)IP-Neu5AcLac) as a reference substrate. Bacterial and viral
sialidases are shown to be relatively non-specific, attacking substrates such as
gangliosides, sialyllactose, sialyloligosaccharides, and sialylglycoproteins. In addition,
Gm, a(2-3)IP-Neu5AcI.acCer, is a better substrate for Vibrio cholerae and Clostridum
pem'ingens sialidase while a(2-6)sialyllactose (a(2-6)II‘ Neu5AcI.ac) is favored by
Arthrobacter ureafaciens sialidase. Mammalian sialidases, on the other hand, are highly
specific. Sialidases of rat liver Golgi hydrolyze the sialyllactoscs, a(2-3)IP Neu5AcI.ac
and a(2-6)II‘Neu5AcLac, but not gangliosides. Other factors, such as the N- and O-
substitution, the length of side-chain, and linkage position in oligosaccharides, can also
affect the activity of sialidases. Studies relating to these factors published before 1980
have been reviewed and summarized by Schauer‘m.

More than one sialidase ean occur in a given tissue. Using 4MU-Neu5Ac as a
substrate, two different sialidases in human leucocytes have been isolated based on their
concanavalin A (Con A) binding abilityum. One is a Con .4 binding form coupled with
B—galactosidasc activity, primarily found in lymphocytes (> 80%). The other is a non-
Con A binding form which is mainly located in granulocytes. Both are classified as
lysosomal sialidases. Two membrane-associated sialidases in rat brain are distinguished
rby a combination of biochemical and immunological methods‘m’. Gangliosides are the

substrates for one of the sialidases while the other had a wider range, including 4MU-

l9
Neu5Ac, gangliosides, sialyloglycoproteins and sialyloligosaccharides. Additionally,

antibodies raised against each sialidase did not cross react. Substrates for a cytosolic
sialidase in rat liver included sialylated oligosaccharides, glycoproteins, glycopeptides,
and gangliosides with an optimal pH of 6.0“"). Lysosomal sialidases in rat liver only
hydrolyzed low-molecular weight oligosaccharides and ganglosides under acidic pH
conditions‘"”.

The activities of extracellular sialidases in the culture medium of human foreskin
fibroblasts were detected by using [’me as substrate“-“"m’. According to the pH
profile, two forms of Gm ganglioside sialidase were found that had maximal activities at
pH near 4.5 and 6.5. The activity level of both sialidases in the culture medium was
also highly dependent on the cell density. The level of sialidase activity at pH 4.5 from
the culture medium increased from 4.1 pmol/hour/ ml to 39 pmol/hour/ml while the cell
density increases from sparse (l x 103 cells/cm?) to confluent density (6 x 10‘ cells/cm’).
In contrast, the activity of the extracellular sialidase with pH 6.5 maximum decreased
from 9 pmol/hour/ ml to a non-detectable level after the cell density reached confluence.
The same experiment has also been performed on plasma-membrane bound sialidases“?
No neutral sialidase activity was detected on the plasma-membrane bound fraction while
the acidic sialidase activity was shown to be 10 times higher than that of culture medium.
On a per cell basis, the acidic form of sialidase activity at confluency stage decreased to
40% of activity of sparse stage. Another study from Yogeeswaran‘m has demonstrated
‘ a similar event. Membrane-bound sialidase activity, which hydrolyzes gangliosides,

decreased to around 50% at the touching stage (preconfluency density) of cell-cell contact

20
in mouse fibroblasts 3T3 cells. However, the sialidase activity of transformed cells

showed no significant change at any stage of cell-contact.

A more sensitive assay of sialidase activity has been developed by Ogura et al.
("5) to analyze low levels of extracellular sialidases in the culture medium. This assay
employed ganglioside (Gm) as the substrate and measured the absorbance of the product
(Gm) coupled with cholera toxin B subunit-horse radish peroxidase at 490 nm. The
detection limit of this assay for ganglioside sialidase was as low as 3 fmol/min. Using
this assay, it has been reported that the culture medium of human fibroblast cells at pH
6.5 contained two sialidases. One of two sialidase activities (47-69 kDa) reached a
maximum by addition of 1% Triton CF-54 while the other sialidase activity (16 kDa) was
not affected by addition of the detergent. The relationship between these two forms of

sialidases remains an open question.

F. The POSTULATED MECHANISM OF SIALIDASES

From studies on the hydrolysis of the glycosidic bond with egg-white lysozyme
and other glycohydrolases, it has been suggested that the transition-state of substrates of
sialidases were likely to be oxo—carbonium ions‘mv‘”). The mechanism of transition-state
binding of sialidase from the soil bacterium Arthrobacter Sialophilus is postulated using
Neu5Ac2en and other sialidase analogues as competitive inhibitors (Fig. 5)“). The Ki
value of Neu5Ac2en and its methyl ester are 1.4 x lO‘M and 4.8 x 10‘ M, respectively,
while the K. value of N-acetylneuraminlactose is 1.0 x 10’ M. However, Neu5Ac-a-

methyl ketoside methyl ester, Neu5Ac-B-methyl ketoside, and its methyl ester at 1.0 x

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22
10'3 M are neither substrates nor inhibitors of this sialidase. This indicated that the free

carboxyl group of sialic acid is essential for the substrate binding to sialidase but it is not
necessary for transition-state analogue binding. The 2C, conformation of the sialic acid
moiety on sialoglycoconj ugates is converted to a half-chair conformation with a planar
geometry at O-Q-C, of the pyranose ring forming the oxo-carbonium cation ion
intermediate. The half-chair conformation of Neu5Ac2en and its a-glycoside methyl
ester fit into the active site of transition-stage of sialidases without any bond bending‘m).

The protein sequence of neuraminidases from influenza A and B varies up to
70%; however, the active site of these sialidases has 24 amino acid residues which are
highly conservedamz). The mechanism of sialidase eatalysis in influenza Altokyo/3/67
(N2) strain has been studied by using site-directed mutagenesis‘m). Seven of 14
mutations were shown to possess no activity with the correct folding structure while two
others were without the correct folding structure. One of two mutations, Asn-l46 to Ser,
is not located in the active site pocket. It has been known that Asn-l46 is one anchor
site for four N -linked carbohydrate side chain of salidases. While the activity of mutant
Asn-l46 vanished, it was suggested that the attachment of the earbohydrates could be
important to form a proper folding structure and to remain active. Other mutations were
located in the active site of sialidase. The catalytic mechanism of sialidase was deduced
from these observations on the activity and the immunoprecipitation. In this model, His-
274 is an indirect proton donor under the physiologieal pH, which eauses a raise in the
pl(. of Glu-276. Then the un-ionized earboxyl of Glu-276 breaks the glycosidic linkage

by a protonation on the oxygen of the pyranose ring, the subsequent cation intermediate

23
might be stabilized by an acidic group (Glu—277), and a water molecule provides a proton

to His-274 and a hydroxyl to the intermediate, leading to the release of the terminal sialic
acid. Other conserved residues were involved in the substrate binding at the active site.
Other strains of influenza A and B also displayed a similar eatalytic model with a slight
difference in the substrate binding residues‘m'mt‘m. A similar mechanism for lysozyme
glycohydrolases and sialidases was postulated by Stryer‘m’. The active site pocket
involved two acidic amino acid residues (Glu-35 and Asp-52) located on the opposite side
of the glycosidic bond cleaved by catalytic reaction.

To facilitate the characterization of the active site of sialidases, synthetic
photoreactive radiolabelling probes provide a useful tool. For example, 1251-ASA-
Neu5Ac2en was used to label the active site of c. pedfingerls sialidase“). Photoaffinity-
labeled sialidase (72 kDa) was cleaved with CNBr to generate 5 peptides with molecular
weights of 27.2, 13.3, 11.4, 10.4 and 7.7 kDa. The 27.2 and 13.3 kDa peptides of 72
kDa sialidase are labelled with the radioionated molecule. This indicated that these two
peptide chains should contain the active sites of C. perfiingens sialidase. No detailed
protein sequence was mentioned in this report. It is worth noting that the specificity of
the photOprobe in mammalian sialidases was low. The active site of lysosomal
neuraminidases in human placenta has been examined with a photolabelling transition?
» state analogue, 3H-9-PANP-Neu5Ac2en‘W. After photolysis, the autoradiogram of the
denatured enzyme complex exhibited two intense bands at 61 kDa and 46 kDa. The first
20 amino acids at the N—terminal of 46 kDa protein was shown to possess 85% identity

with the cloned cDNA sequence of a-galactosidase B from human placenta and 68 %

'24
homology with the sequence of a-galactosidase A from human lung. It was suggested
that the 46 kDa protein, associated with the photoprobe by non-specific hydrophobic
interaction, was not a sialidase. The photoprobe was bound specifically with the 61 kDa
protein since addition Neu5Ac2en decreased the intensity. However, the protein
sequence of 61 kDa band could not be directly determined by automated Edman
degradation due to an unknown blocking effect.

The lysosomal sialidase from human placenta has been reconstituted in vitro by
Verheijen et al."’-‘”’. They indicate that the full activity of lysosomal sialidase was
present in a complex with B—galactosidase (64 kDa), and a protective protein (32 kDa).
The inactive sialidase (66 kDa) isolated from human placenta includes a 38 kDa protein
_ core with 7-14 kDa N -1inked oligosaccharide chain. The active site of sialidase complex
was located on this enzyme. The protective protein, however, was essential for full
activity of the sialidase and the B-galactosidase‘m. The stoichiometry of this complex
was not clear although the binding sites of fl-galactosidase and sialidase on the protective
protein were known to be on different domains. It was suggested that the protective
protein is a processing enzyme because its protein sequence is highly homologous to
yeast protease carboxypeptidase Y (CPY) and the yeast KEXl gene product‘ml. The
activity and stability of sialidase significantly increased after addition of B-galactosidase.
However, no effect occured after addition ofihe protective protein to inactive sialidase.
G. ACTIVATORS AND INHIBITORS OF SIALIDASE

An activator protein, B—glucosidase-stimulating protein, appears to stimulate the

activity of oligosaccharide sialidases in fibroblasts from galactosialidosis and sialidosis

25
patients“”’. The activity of sialidase remained the same after desialylation of the

activator. While all hydrolases are stimulated by fl-glucosidase-stimulating protein, the
. mechanism of activation of the stimulator with sialidases is not yet clear.

It has been reported that some bacterial (V. cholerae and D. pneumoniae) and
viral (some influenza virus strains) sialidases require divalent eation for activity, usually
Ca+’(‘-“). In V. cholerae sialidase, the Ca”2 ion is required for stability of the enzyme
but not essential for the substrate binding‘m-“m. It has been suggested that a salt bridge
is formed between substrate and enzyme. In some mammalian sialidases, mono- and
divalent cations are required for activity. A general effect of monovalent ions is
increased ionic strength, which influences the pH value. A membrane—bound sialidase
detected in Golgi apparatus from rat liver is stimulated by preincubating with 1 mM Ca+2
and Zn+2 0‘”. The lysosomal sialidase of mouse liver is stabilized in the presence of
phenylmethyl sulfonyl fluoride, Ca“, Mg”, Mn”, and Zn+2 (“2). It has been reported
that one of membrane-bound ganglioside sialidases from human liver is activated by
cholate‘w'). The sialidase activity of human promyelocytic leukemia cell line (KL-60) is
raised by incubation with 1 pM retinoic acid or 1.3 % DMSO““’.

Inhibitors of sialidases have been classified into four groups according to source

and character; they are naturally occurring compounds with high molecular weight and
I with low molecular weight, synthetic sialidase inhibitors, inorganic ions and other
compounds. The influence of inhibitors in vivo is summarized in Table 2“"). In general,
the naturally occurring compounds with high molecular weight such as polyanionic

polymers, are non-specific inhibitors due to the interaction between ions. The

26

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27
effectiveness of inhibition of this group toward various sialidases is viruses > bacterial

> mammalian. The next group of inhibitors with low molecular weight are shown to
be more specific. The hydrolysis product of sialidase, Neu5Ac, at high concentration
(up to 10-25 mM) inhibits the activity““’. The inhibitor, Neu5Ac2en, was discovered
in normal urine, serum and in saliva of human”. The level of Neu5Ac2en in sialuria
patients is 10,000 times higher than that of normalmtm’. The normal level of
Neu5Ac2en in humans might cause strong inhibition of sialidases from microorganisms.
Sialyla(2-6)lactose, a substrate of lysosomal sialidases in human, is a competitive
inhibitor for viral sialidases from newcastle disease‘m).

Investigations with synthetic sialidase inhibitors having structural similarity to
sialic acids have been discussed. Other inhibitors with no connection to sialic acid
structure were studied in the l970’s““"‘°’. These results suggest that these compounds
mainly inhibited the activity of bacterial and viral sialidases (Ki value was 4-5 x 10'1
mM). Inhibition by HgC12, HgSO,, Hg(NO3)2, Hg(OOCCH3)2, and KIO, in V. Cholerae
and CI. perfiingens sialidases in vitro was reported by Cabczas“‘°’. Other inorganic ions
such as Cu”, Fe+3 also inhibit the activity of sialidases from all sources. It is suggested
that the inhibition is non-specific and may be due to the interaction between ions and the

thio group of enzyme.

REFERENCES

l. Corfield, A. P., Schauer, R. (1982) In Siaalic Acids: Chemistry, Metabolism and
Function (Cell Biology Momographys, vol. 10) eds. by Schauer, R. , Springer, Wien,
pp5-50.

2. Rosenberg, A. , and Schengrund, C.-L. (eds.) (1976) Biological Roles of Sialic Acid.
Plenum Press, New York, pp

3. Schauer, R. (1985) TIBS, 10, pp 357-360.

4. Usuki, S., Hoop, P.,and Sweeley, C. C. (1988) J. of Biol. Chem. 263, pp10595-

10599.

. Ogura, K., and Sweeley, C. C. (1992) Experimental Cell Research, 199, ppl69-173.

. Muller, H. E. (1974) Behring Inst. Mitt. 55, pp34-56.

. Krasemann, C., and Muller, H. E. (11975) Zbl. Bakt. Hyg. I. A. 231, pp206-2l3.

. Retter, W., Kottgen, E., Bauer, C., and Gerok, W. (1982) In Sialic Acids:

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CHAPTERZ

SYNTHESIS OF SIALIC ACID ANALOGUES: A NOVEL, ONE-STEP
CONVERSION OF N-ACETYLNEURAIVIINIC ACID AND THEIR

DERIVATIVES

Running Title: Synthetic Analogues of Sialic Acid and Their Biological
Application

37

38
ABSTRACT

2,3-Didehydro-2—deoxy-N-acetylncuraminic acid (Neu5Ac2en) has been
synthesized from N-acetylneuraminic acid (Neu5Ac) by a simple, quantitative, one-step
conversion. Neu5Ac was dehydrated by peracylation with trifluoroacetic anhydride
(TFAA) in the presence of N,N-dimethyl-4-aminopyridine (DMAP) as base. 2,3-
Didehydro-2-deoxy-neuraminic acid (Neu2en) was also obtained when the ratio of
TFAA/DMAP was changed to 4:3. 2,3—Epoxy-2-deoxy-N-acetylneuraminic acid (2,3-
epoxy-NeuSAc) was prepared from Neu5Ac2en via epoxidation of the a,B-unsatura_ted
acid with hydrogen peroxide-sodium tungstate. 8,9-Isopropylidene-2,3-didehydro-2-
deoxy-N-acetylneuraminic acid was synthesized from Neu5Ac2en by using copper sulfate
as a eatalyst in acetone. A photoreactable compound, N -azidoacety1 neuraminic acid was
obtained from Neu2en via a N -haloacetyl intermediate. Nine synthetic potential
inhibitors of sialidase(s) have been prepared in this study; four of nine compounds have

not been reported by other laboratories.

INTRODUCTION

5—N -Acety1-5-amino-3,5—dideoxy-D-glycero-D-galacto-nonulosonic acid (N -
acetylneuraminic acid, sich acid, Neu5Ac) .is a naturally-occurring acidic
monosaccharide. Sialic acids are present in tissues from mammals, vertebrates, some
bacteria, and the influenza viruesm. The occurrence of sich acids in nature has been
reviewed‘2'3v4v5'5)- The different substituents found on sialic acid in nature include N-

acetyl, N-glycolyl, O-acetyl, O-methyl, O-phosphate, and O-sulphateu'7'8). Sialic acids

39
are a part of sialylosaccharide chains in glycoconjugates. They are located on terminal

positons as well as internal positions in oligosaccharides via O—ketosidic a-2,3-, a-2,4-,
a-2,6- and a-2,8- linkages. Free sialic acid has also been detected at low levels in
human urine, serum, and saliva(9'1°). The variety of substituents and linkage types and
locations of sich acids affect the rate of catalysis of sialoglycoconjugate hydrolysis by
sialidases" .11.12.l3.14). Many biological functions have been attributed to sialic acid and
its sialoglycoconjugates such as cell recognition and cell adhesionas’m’”).

Sialidases (EC 3.2.1.18, N-acylneuraminosyl glycohydrolases) catalyze the
hydrolytic cleavage of sialic acid residues from sialylglycoconjugates. It has been
reported that sialidase participates in a number of physiological processes such as the
regulation of cell proliferation( 18), clearance of plasma proteins”), the degradation of
gangliosides and glycoproteins‘zo), and neurotransmissionmtzz). In pathogenic
microorganisms, sialidases play an important role in the mechanism by which host cells
are infectedm). Extracellular sialidase activities have been detected at low levels in the
culture medium of human fibroblastsusrz“). Furthermore, it is known that several inherited
metabolic diseases in humans result from sialidase deficiency‘25’26). The wide distribution of
sialidases in cellular organelles and the diversity of biological functions has led to interest
by several groups in the relationship between sich acid structure and the activity of

It has been reported that almost all mammalian, bacterial, and viral sialidases are
inhibited by a transition state analogue, 5-N-acetyl-5-amino—2,3,5-trideoxy-D-glycero-D-

galacto-nonulosonic acid (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid,

40

Neu5Ac2en)(27'28). The conformation of the analogue is very similar to the oxonium ion
intermediate formed during the hydrolysis reaction catalyzed by sialidases (Fig. l)(29).
Thus, this analogue is an excellent probe to determine the active site(s) of sialidases.
The synthesis of Neu5Ac2en in high yield has been a difficult problem due to its acid
lability. Neu5Ac2en has been synthesized from 4,7,8,9-tetra-O-acetyl-2—deoxy-2-halo-
neuraminic acid, by dehydrohalogenation with triethylamine or silver nitrate as catalyst
in two laboratoriesC’O’3 1). One or more steps of protection and deprotection were usually
required during the preparation of Neu5Ac2en and the yield of this procedure was less
than 50 % due to degradation. It has been suggested that sich acid derivatives with
affinity labeling reagents could be useful for characterizing cellular sialidases and other
sialic acid binding proteins‘32t33v34). Three photoactivatable transition state analogues
of sialidase have been synthesized by introducing an aryl azide group in the C-9 position
of Neu5Ac2en via a thio or an amide linkage‘32t33v34). In the present study, we provide
a high-yield, one-step conversion to Neu5Ac2en by introducing and eliminating the
trifluoracetyl group. Several other sialic acid derivatives were also prepared '
incorporating the half-chair conformation of Neu5Ac2en. Furthermore, a
photoactivatable compound was obtained by incorporating an azide group at the N -5

position of Neu5Ac2en.

MATERIALS AND METHODS

AM
N-acetylneuraminic acid was a gift from the MECT Corporation, ToKyo. All

41

(A)

 

Nell SAC

(B)

COOH

   
  

O
H \ R (glycoside)

Figure. 1: (a) The structure of Neu5Ac. (b) The half-chair conformation of
sialyloglycoconjugates for the hydrolysis of sialidase.

42
other reagents were analytical grade and purchased from Sigma, J .T. Baker, or Aldrich.

Thin-layer chromatography (TLC) was performed on pre-coated glass plates of siliea gel
60 F254 (Merck Co.) with l-propanol-water-30% ammonium hydroxide (6:3: 1) as the
developing solvent. Spots were detected by a carbohydrate-spray reagent containing
0.2% orcinol and 5 % H2804 in methanol/water (3/ 1) and heating on a hot plate for 10
min. Orcinol reagent was used for colorimetric analysis of sich acid derivatives within
purification process“). HPLC was carried out using reversed-phase C8 columns
(Partisil 10 ODS-3, 22 x 250 mm, Whatman Co.) with a WATERS-600 instrument
attached to Spectroflow 783 programmable UV detector at 232 nm and a WATERS-740
data processor. Compounds were eluted with a linear gradient of acetonitrile in water
from 5% to 30% over 35 min at a flow rate of 10 rill/min. Size exclusion
chromatography On a Bio-Rad P-2 (10 x 700 mm) column was performed with water as
the mobile phase. IR spectra (CaC12 cell) were recorded with a Nicolet 710 FF-IR
spectrometer. 1H and proton-decoupled 13C NMR spectra were measured in D20 at
300 MHz by using a Varian VXR spectrometer. Negative FAB-MS spectra were
obtained on a JEOL JMS HX-110 double focusing instrument. The samples were

suspended in a matrix of triethanolamine.

Methyl];
Synthesis of 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (Neu5Ac2en, B1)
N-acetyl-neuraminic acid (10 mg, 0.032 mmole) was added to a solution of N ,N -

dimethyl-aminopyridine (80 mg, 0.258 mmole) in dry acetonitrile (0.5 ml). The mixture

43
was cooled to -70 °C and then trifluoracetic anhydride (30 pl, 0.096 mmole) was added.

The mixture was stirred for 1 hr. at 4 °C, and then heated in a teflon-lined screw capped
vial for 6 hours at 70 °C. At the end of this period, the mixture was concentrated to
dryness under a stream of nitrogen. The residue was dissolved in 1 ml H20, then the
pH was adjusted to 11 with 30% ammonium hydroxide. The mixture was extracted with
chloroform (x 3) to remove excess DMAP. The aqueous portion was applied to a Bio-
Gel P-2 column for further purification. After solvent was removed by lyophilization, 8
mg (84%) of Neu5Ac2en was obtained. Large-scale synthesis of Neu5Ac2en has also
been performed with 100 mg of starting material. Other reagents were scaled up with
the same proportions as the small-scale. However, HPLC was required for further
purificaton of Neu5Ac2en from the large-seale preparation. The recovery was 57% (54
mg). Rf: 0.643 (the Rf of Neu5Ac in this mobile phase was 0.473); 1H-NMR (300
MHz, D20): 6 2.091 (s, 3H, NCOQH3), 3.601 (dd, 1H, H-7), 3.646 (dd, 1H, H-9’),
3.885 (dd, 1H, H-9), 3.936 (ddd, 1H, H-8), 4.051 (dd, lH,H-5), 4.213 (dd, 1H, H-6),
4.470 (dd, 1H, H-4), 5.692 (d, 1H, H-3); negative FAB-MS: m/z= 312[M+Na-H]',
290[M-H]', 272[M-H20-H]'.
Synthesis of 2,3-dehydro-2-deoxy-neuraminic acid (Neu2en, B2)

Neu2en was prepared in the same manner as Neu5Ac2en except that the ratio of
N ,N-dimethyl-aminopyridime (40 mg, 0.128 mmole) to trifluoracetic anhydride (30 pl,
0.096 mmole) was changed to 4:3. Yield: 79% ( 6.4 mg). Rf: 0.707 ; 1H-NMR (300
MHz, D20): 6 3.544 (dd, 1H, H-7), 3.602 (dd, 1H, H-9’), 3.855 (dd, 1H, H-9), 3.922

(ddd, 1H, H-8), 4.167 (dd, lH,H-S), 4.345 (dd, 1H, H—6), 4.545 (dd, 1H, H-4), 5.681

44
(d, 1H, H-3); negative FAB-MS: m/z= 248[M-H]‘.

Synthesis of 5-acetamido-2,3-epoxide-3,5-dideoxy-B-glcero-D-galacto-nonulo-
pyranosylonate (2,3-epoxide-Neu5Ac, B3)

The epoxidation of N-acetyl-2,3-dehydro—neuraminic acid was accomplished using
a modification of the Kirshenbaum and Sharpless procedure-“’37). Neu5Ac2en (5 .59
mg, 0.019 mmole) and 0.1 equivalent of Na2WO4'H20 (6.7 mg, 0.0019 mmole) were
added to 0.6 ml H20 in a teflon-lined screw-capped vial. The mixture was stirred, and
the pH of the solution was adjusted to around 6.2 with the addition of 0.1N NaOH
and/or 1N H2SO4. After an additional 10 minutes stirring at room temperature, 1.2
equivalents of aqueous H202 (30% (w/v) solution, 8. 84M, 6.7 pl) was added to the
mixture. The pH of the reaction mixture was kept between 5.8 and 6.2. The progress
of the reaction was followed by TLC. After 2 hours, the excess H202 was evaporated
from the residue under a stream of nitrogen. The resulting mixture was purified by
chromatography on a P-2 column. After the solvent was removed, the residue consisted
of the 2,3-epoxy-Neu5Ac (Rf: 0.507) and the decomposed compound (2,3-diol; Rf:
0.489). According to NMR data, the conversion of epoxide compound from Neu5Ac2en
was near 100% (6.01 mg). However, complete purification of the mixture was not
successful. lH-NMR (300 MHz, D20): 5 2.004 (s, 3H, NCO§H3 of epoxide); 1.978
(s, 3H, NCOQH3 of diol), 3.444 (dd, 1H, H-7), 3.575 (dd, 1H, H-9’), 3.801 (dd, 1H,
H-9), 3.930 (ddd, 1H, H-8), 3.973 (d, 1H, H-3), 4.052 (dd, 1H, H-4),4.164 (dd, 1H,
H-5), 4.274 (dd, 1H, H-6); negative FAB-MS: m/z= 306[M-H]' for epoxy-Neu5Ac and

324[M-H]‘ for 2,3—diol-Neu5Ac.

45
Synthesis of 2-deoxy-2,3-dehydro-N—chloracetyl neuraminic acid (Neu5ClAc2en, B4)

Aqueous triethylamine (20% (v/v)) was prepared with a pH around 11-12.
Neu2en (5 mg, 0.0200 mmole) was added to 750 pl 1120/CH3CN solution (2/1 (v/v)).
The pH of the aqueous solution was adjusted to 8 with 1 M NaHCO3. Excess
chloroacetic anhydride (35 mg, 0.20 mmole) was dissolved in 1 ml acetonitrile. The
chloroacetic anhydride solution was added dropwise to the mixture with stirring at room
temperature over 5 minutes. The pH of the reaction mixture was maintained in 7 to 8
range with l M NaHCO:,. The progress of the reaction was monitored by TLC. After
2 hours the reaction was complete, the resulting mixture was evaporated to dryness,
resuspended in 1 ml H20, and passed through size exclusion column (Bio-Rad P-2
column) for further purification. The fractions containing the sich acid derivatives were
combinated and lyophilzed. Yield: 45%. Rf: 0.59; 1H-NMR (300 MHz, D20): 5 3.985
(s, 2H, NCOCHZCI), 3.491 (dd, 1H, H-7), 3.565 (dd, 1H, H-9’), 3.827 (dd, 1H, H—9),
3.904 (ddd, 1H, H-8), 4.173 (dd, 1H, H-5), 4.313 (dd, 1H, H-6), 4.496 (dd, 1H, H—4),
5.659 (d, 1H, H-3); nagative FAB-MS m/z= 324[M-I-1]', 326 [M-HT.
Synthesis of2-deoxy-2,3-dehydro-N-bromoacetylneuraminic acid (Neu5BrAc2en, 35)

2-Deoxy-2,3-dehydro-N-bromoacetyl neuraminic acid (Neu5BrAc2en) was
obtained in a similar manner as N-ClAcNeu2en except that the bromoacetic anhydride
was replaced with chloroacetic anhydride. Yield: 45%. Rf: 0.60; 1H-NMR (300 MHz,
D20): 6 3.689 (s, 2H, NCOQHzBr), 3.389 (dd, 1H, H-7), 3.526 (dd, 1H, H-9’), 3.777
(dd, 1H, H-9), 3.927 (ddd, 1H, H-8), 4.102 (dd, 1H, H-5), 4.248 (dd, 1H, H-6), 4.410

(dd, 1H, H-4), 5.578 (d, 1H, H-3); nagative FAB-MS mlz= 368[M-H]‘, 370 [M-I-1]'.

46
Synthesis of 2-deoxy-2,3-dehydro-N-azidoacetyl neuraminic acid (Neu5N3Ac2en, B6)

2-Deoxy-2,3-dehydro—N-azidoacetyl neuraminic acid was prepared from 2-deoxy-
2,3—dehydro—N-aminoacetyl neuraminic acid with excess sodium azide (NaN3) at 50 °C
in methanol overnight. 2-Dcoxy-2,3-dehydro-N-aminoacetyl neuraminic acid was
obtained by the treatment of 2-deoxy-2,3-dehydro—N -chloracety1 neuraminic acid with 0.1
N aqueous NH3 for 1 hour. Alternatively, N-azidoAcNeuZen could be directly
prepared from N-haloacetyl Neu2en by addition of NaN3 in methanol at 50 °C for 24
hours. A P-2 column was used for further purifieaton. All operations with the
azidoacetyl were carried out in darkness to prevent decomposition of the product.
Recovery: 80%. IR (Am): 2114 cm’1 ; 1H-NMR (300 MHz, D20): 6 3.814 (s, 2H,
NCOCHZN3), 3.494 (dd, 1H, H-7), 3.529(dd, 1H, H-9’), 3.721 (dd, 1H, H-9), 3.908
(ddd, 1H, H-8), 3.916 (dd, 1H, H-5), 4.458 (dd, 1H, H-6), 4.581 (dd, 1H, H-4), 5.722
(d, 1H, H-3); negative FAB-MS: mlz= 331[M-H]', 303l'M-N2-HT.

Small-scale synthesis of 7 ,9-O-m-nitrobenzylidiene 2,3-dihydro—N-acetyl neuraminic
acid (NB-NeuSAcZen, B7)

Neu5Ac2en and m-nitrobenzylidiene dimethylacetatal were dried in a desiccator
overnight. Neu5Ac2en (5 mg, 0.017 mmole) was dissolved in 200 pl of 0.01% TFA
acetonitrile solution. Dimethylacetyl m-nitrobenzylidene (13.396 mg, 0.068 mmole) was
added to the mixture and stirred overnight. The mixture was neutralized with .
triethylamine (1 drop) and reduced to dryness under N2. The mixture was desalted by
passing through a C18 SepPAK cartridge with the following sequence of solvents: H20

(2ml), HZO/MeOH (1/3; 2ml), MeOH (2ml). The product was eluted with the

47
HZO/MeOH (1/3) fraction. Rf: 0.455; Yield: 78%; 1H-NMR (300 MHz, D20): 6

1.978(s, 3H, NCOQH3), 3.209(dd, 1H, H-7), 3.559(dd, 1H, H-9’), 3.763(dd, 1H, H-9),
3.903(ddd, 1H, H-8), 4.182(dd, 1H, H-5), 4.344(dd, 1H, H-6), 4.505(dd, 1H, H-4),
5.617(d, 1H, H-3), 5.805(s, 1H, OCOC6H4NOZH); 5.927(s, 1H, OCOC6H4N02H);
7.195-7.241(m, 1H, Ph), 7.527-7.594(m, 1H, Ph), 7.732-7.749(m, 1H, Ph), 8.08-
8.209(m, 1H, Ph); negative FAB-MS: m/z 423[M-H]‘. ‘
Synthesis of 9-O-m-aminobenzyl-2,3-dehydro-N-acetyl neuraminic acid (AB-
Neu5Ac2en, B8)

9-O-m-Aminobenzyl-2,3-dehydro-N-acetylneuraminic acid was prepared by the
reduction of 9-O-m-nitrobenzyl—2,3-dehydro-N-acetylneuraminic acid with palladium-poly
(ethylenimine) (Pd-PEI) as a catalyst in HZOIMeOH (5/ 1) in a sealed vial under a
constant H2 gas presure at room temperature overrlight(33). After the catalyst was
removed, the filtrate was evaporated to dryness under N2. Chromatography on a P-2
column was used for further purification. 9-O—m-Aminobenzyl-2,3-dehydro-N-
acetylneuraminic acid was obtained by reducation of 7 ,9-m-nitrobenzylidene-2,3-dehydro—
N-acetylneuraminic acid with sodium cyanoborohydride (NaBH3CN) and hydrogen
chloride gas (in ether) in THF for 2 hours. The Rf: 0.55; lH-NMR (300 MHz, D20):
6 1.978(s, 3H, NCOQH3), 3.209(dd, 1H, H-7), 3.559(dd, 1H, H-9’), 3.763(dd, 1H, H-
9), 3.903 (ddd, 1H, H-8), 4.182(dd, 1H, H-5), 4.344(dd, 1H, H-6), 4.505(dd, 1H, H-4),
5.617(d, 1H, H-3), 5.851(d, 1H, OCOC6H4N0237); 5.947(d, 1H, OCOC6H4NOZH7),
7.523-7.631(m, 1H, Ph), 7.723-7.80(m, 1H, Ph), 8.138-8.289(m, 1H, Ph); negative

FAB-MS: mlz=425[M-H]'.

48
Large-scale synthesis of 9-O-m-aminobenzylidene-N—acetylneuraminic acid (AB-

NeuSAc, B9)

Neu5Ac and m—nitrobenzylidene dimethylacetal were dried in a desiccator
overnight. Neu5Ac (100mg, 0.3236 mmole) was dissloved in 1 ml freshly distilled N ,N—
dimethyl formamide. 1.2 equivilents of m—nitrobenzylidene dimethylacetal (76.50 mg) and
a catalytic amount of toluenesulfonic acid were added to the mixture. The heterogeneous
mixture was allowed to react in a water bath at 70-85 °C until the reactants were
completely dissolved, and then allowed to stand for an additional 15 min. The mixture
was neutralized with triethylamine (1 drop) and evaporated to dryness. The mixture was
desalted by passing down a C8 reverse phase column (1.5 x 7.0 cm). The following
solvent system was employed; H20 (20 ml), H20:MeOH(1 : l; 20 ml), MeOH(20 ml),
MeOH:CHC13(1 :2; 20 ml), and CHC13(20 ml). The product, 7,9-m-nitrobenzylidene-N—
acetylneuraminic acid (NB-Neu5Ac), was eluted with H20:MeOH(1: l). The reduction
of NB-NeuSAc was performed asthat of NB-Neu5Ac2en. Rf: 0.455 ; Yield: 78%. 1H-
NMR (300 MHz, D20): 6 1.978(s, 3H, NCOCH3), 3.209(dd, 1H, H-7), 3.559(dd, 1H,
H-9’), 3.763(dd, 1H, H-9), 3.903 (ddd, 1H, H-8), 4.182(dd, 1H, H-5), 4.344(dd, 1H,
H-6), 4.505(dd, 1H, H-4), 5.617(d, 1H, H-3), 5.851(d, 1H, OCOC6H4N02H7);
5.947(d, 1H, OCOC6H4NOZH,), 7.437-7.550(m, 1H, Ph), 7.662-7.760(m, 1H, Ph),

8.112-8.267(m, 1H, Ph) ; negative FAB-MS: mlz=441[M-H]', 17l[CH(OH)2C6H4N02]'

49
Synthesis of 8,9-isopropylidene-2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid (IP-

Neu5Ac2en, B10)

Neu5Ac2en (27.3 mg, 0.094 mmole) was dissolved in 6 ml of 0.01% TFA in
acetone. A catalytic amount of copper sulfate was added to the mixture. The mixture
was refluxed at 50 °C in an oil bath for 4 hours. The reaction was quenched by the
addition of Na2C03 (20 mg, 0.188 mmole) and the mixture was filtered to remove
solids. The filtrate was evaporated under N2, resuspended in 1 ml MeOH/HZO (1/5),
and applied to a eation exchange column (CM-Sephadex, 10 x 25 mm) to remove copper
sulfate. H20 was used as an eluent (2 ml). Yield: 100% (30 mg); Rf: 0.514; 1H-NMR
(300 MHz, D20): 6 1.329 (s, 6H, CH3OCOQH3), 2.023 (s, 3H, NCOQH3), 3.522 (dd,
1H, H-7), 3.590 (dd, 1H, H-9’), 3.851 (dd, 1H, H-9), 3.911 (ddd, 1H, H-8), 4.172 (dd,
1H, H-5), 4.328 (dd, 1H, H-6), 4.507 (dd, 1H, H—4), 5.664 (d, 1H, H-3); negative

FAB-MS: m/z= 302[M-H]‘

Results and Discussions

The structures of the potential inhibitors of sialidases synthesized for this study
are shown in Fig. 2. The structures of the Neu5Ac2en derivatives were determined by
1H NMR spectroscopy (Table 1). In Neu5Ac2en, the signal of the vinyl proton at C-3
position appeared as a doublet at 6 5.692 ppm. While the H-3ax and H-3ml of Neu5Ac .
gave signals at 6 1.621 and 2.730 ppm, respectively, they vanished after the conversion
was completed. The purity of the product was also shown on the 13‘C NMR (D20, 20

°C) spectrum. The signal for C-2 and C-3 of Neu5Ac2en was present at 6 148.1 ppm

50

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52
and 6 107.8 ppm, respectively; those of C-2 and G3 in Neu5Ac are 6 98.42 and 41.92

ppm, respectively. Other features of the spectra were essentially the same as those
reported for the anticipated product. Thus, it is indicated that no epimerisations or
further dehydrations occurred.

The spectral pattern of Neu2en was similar to that of Neu5Ac2en (Fig. 3, Table
1). However, the signal of acetamino proton at 2.091 ppm in Neu2en disappeared. The
chemical shift for the vinyl proton at C-3 position of Neu2en was observed as a doublet
at 5.832 ppm while the amino group was protonated. In 2,3-epoxy-Neu5Ac, the signal
of the proton at the C-3 position was shifted from 5.692 ppm to 3.978 ppm. The B—
effects for the H—4 of the epoxide group was an upfield shift of about 0.5 ppm.
Chloroacetyl substitution at the N5 position moved the signal to 3.985 ppm. The long
distance effects in H-4, H-5, and H-6 resulted in a downfield shift of about 0.1 ppm. -
The chemical shift for the endo and exo benzylidene proton on IPNB-NeuSAcZen were
present at 5.851 and 5.947 ppm, respectively. The fl-effect of substitution on the H—7
and H-9 protons caused an upfield shift of about 0.2—0.4 ppm. The nitro reduction
caused the chemical shift of aromatic protons to move downfield about 0.3 ppm. In IP-
Neu5Ac2en, the chemical shift of methyl protons appeared at 1.329 ppm. A similar B-
effect was also observed for H-8, H-9 protons.

The 1I-INMR spectral data of N-azidoaeetyl Neu2en are presented in Fig. 4 and
Table 1. The FT-IR spectra of the photoreactable compound are shown in Fig. 5 (see
experimental for detail). The substitution of an azido group on the acetyl group (3. 86

ppm) provided less deshielding than that of the more electronegative chloride atom on

53

 

 

 

 

 

 

 

 

 

 

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the acetyl group present at 3.985 ppm. The results have demonstrated that the

compound has been modified at N—5 position with an azidoacetyl although a
photodeoomposition experiment was not preformed to examine the photolysis products.

The conversion of Neu5Ac to Neu5Ac2en is a dehydration process. However, the
acid lability of Neu5Ac eaused rapid degradation during dehydration either at mild
temperature or with mild acid. To overcome these disadvantages, a protecting group was
required to activate the C-2 hydroxyl group of Neu5Ac. One or more deprotection steps
are also required before elimination. In this work, we effected protection of the primary
and secondary hydroxyl groups and activation of the C-2 tertiary hydroxyl group for
elimination by preacylating with trifluoracetic anhydride in presence of N ,N-dimethyl-4-
aminopyridine. The trifluoroacetyl group not only provides a good leaving group to ‘
activate the hydroxy group at C-2 but also deactivates other hydroxyl groups during the
elimination reaction. The trifluoracetates at other pyranose ring positions are able to
withdraw electron density and to prevent the formation of the electron-deficient species
during dehydration. The role of the N,N-dimethy1-4-aminopyridine in this reaction is
critical in promoting the acylation of the sterically-hindered tertiary hydroxyl group under

mild conditions.

REFERENCES

1, Corfield, A. P., and Schauer, R. (1982) In: Sialic acids chemistry metabolism and
function (Cell Biological Momographys.) vol. 10, eds. by Schauer, R. , Wien,
Springer, pp5-55 .

2. Gottschalk, A. (1960) The chemistrry and Biology of sialic acids and related
substances, Cambridge Universsity Press, Cambridge.

3. Blix, G., aand Jeanloz, R. W. (1969) In: The amino sugars, eds. by Jeanloz, R. W.,
and Balazs, E. A., Academic Press, New York, pp213-265.

4. Tuppy, H. , and Gottschalk, A. ( 1972) In: Glycoproteins, their composition, structure
and function 2nd. ed., eds. by Gottschalk, A., Elserier, Amsterdam, pp403-449.

5. Schauer, R. (1973) Angew. Chem. Int. Edn. 12, pp127-140. ,

6. Na, 8., S., and Dain, J. (1976) In: Biological roles of sialic acid, eds. by Rosenberg,
A., and Schengtrund, C.-L., Plenum Press, New York, pp59-102.

7. Schauer, R. (1985) TIBS, 10, pp357-360.

8. Schauer, R. (1982) Adv. Carbohydr. Chem. Biochem. 254, pp13l-234.

9. Kamerling, J. P., Vliegenthart, J. F. G., Schauer, R., Strecher, G., Montreuil, J.,
(1975) Eur. J. Biochem. 56, pp253-258.

10. Haverkamp, J., Schauer, R., Wember, M., Farriaux, J.-P., Kamerling, J. P.,
Versluis, C., Vliegenthart, J. F. G. (1976) Hoppe-Seyler’s Z. Physiol. Chem. 357,
pp1699-11705.

ll. Corfield, A. P., Michalski, J.-C., Schauer, R. (1981) In: Sialidase and Sialidoses,
perspectives in inherited metabolic diseases, eds. by Tettarmanti, G., Duraand, P.,
and DiDonato, S., vol. 4, Edi Ermes, Milano, pp3-70.

12. Corfield, A. P., Veh, R. W., Wember, M., Michalski, J.-C., Schauer, R. (1981)
Biochem. J. 197, pp293-299. ,

13. Caimi, L., Lombardo, A., Preti, A., Wiesmann, U., and Tettamanti, G. (1979)
Biochim. Biophys. Acta, 571, pp137-146.

14. Sagawa, J., Miyagi, T., and Tsuik, s. (199011. Biochem. 107, pp452-456.

57

15.
16.
17.
18.

19.
20.

21.
22.

23.

24.

25.
26.
27.
28.
29.

30.
31.

32.
33.

58

Matsuda, R., Tanihata, S. (1992) Folia. Pharmacol. Japon. 99, pp363-372.

Bassi, R., and Sonnino, S. (1992) Chem. and Phys. of Lipids , 62, ppl-9.
Yogeeswaran, G., and Hakomori, Sal. (1975) Biochemistry, 14, pp2151-2156.
Usuki, S., Lyu, S.-C., and Sweeley, C. C. (1988) J. of Biol. Chem. 263, pp6847-
6853.

Ashwell, G., and Morel], A. (1974) Adv. enzymol., pp99-128.

Usuki, S., Hoops, P., and Sweeley, C. C. (1988) J. of Biol. Chem. 263, pp10595-
10599.

Harding, 8. B., and Halliday, J. (1980) Nature, 286, pp819-821.

Weis, W. Brown, J. H., Cusack, S., Paaulson, J. L., Skehel, J. J., and Wiley, D.
C. (1988) Nature, 321, pp426—431.

Reutter, W., Kottgen, B., Bauer, C., and Gerok, W. (1982) In: Sialic acids
chemistry metabolism and function (Cell Biologieal Momographys.) vol. 10, eds.
by Schauer, R., Wien, Springer, pp263-305.

Ogura, K., Ogura, M., Anderson, R. L., and Sweeley, C. C. (1992) Anal.
Biochem. 200, pp52-57.

Lowden, J., and O’Brien, J. S. (1979) Am. J. Hum. Genet. 31, ppl-18.

Warner, T. G., and O’Brien, J. S. (1984) Annuaal Rev. Genetics, 17, pp295-341.
Meindl, P., and Tuppy, H. (1969) Z. Physiol. Chem. 350, pp1088-1092.

Meindl, P., and Tuppy, H. (1970) Monatsh. Chem. 101, pp639-647.

Miller, G. A., Wang, P., and Flashner, M. (1978) Biochem. Biophys. Res. Comm.
83, pp1479-1487.

Meindl, P., and Tuppy, H. (1969) Monatsh. Chem. 100, pp1295-1306.

Beau, J. M., Schauer, R., Haverkamp, J., Kammerling, J. P., Dorland, L., and
Vliegenthart, J. F. G. (1980) Eur. J. Biochem. 106, pp531-540.

Warner, T. G. and Lee, L. A. (1988) Carbohydr. Res. 176, pp211-218.

Warner, T. G. and Lee, L. A. (1988) Biochem. Biophys. Res. Commun. 148,
pp1323-l329.

59

34. Horst, G. T. J., Mancini, G. M. S., Brossmer, M., Ross, U. and Verheijen, F. W.
(1990) J. Biol. Chem. 265, pp10801-10804.

35. Schauer, R. (1978) In: Methods in Enzymology, vol. L eds. by Ginsburg, V., New
York, Academic Press, pp64-89.

36. (1981) J. Org. Prep. Proced. Int. 13, ppl37-139.

37. Beau, J. M., Schauer, R., Haverkamp, J., Kammerling, J. P., Dorland, L., and
Vliegenthart, J. F. G. (1984) Eur. J. Biochem. 140, pp203-212.

38. Coleman, D. R., Royer, G. P. (1980) J Org. Chem. 45, pp2268-2269.

CHAPTER3

INHIBITION STUDIES FOR SYNTHETIC SIALIC ACID ANALOGUES

Running Title:Synthetic Analogues of Sialic Acid and 'IIreir Biological Application

61
ABSTRACT

Eight derivatives of 2-deoxy-2,3-dehydro-N-acety1neuraminic acid (Neu5Ac2en),
described in the previous chapter, were investigated for their ability to inhibit Vibrio
cholerae neuraminidase using 2’-(4-methylumbelliferyl)-a-D—N-acetylneuraminic acid
(4MU-Neu5Ac) as the substrate. In a kinetic study, seven of the eight analogues of
Neu5Ac2en were shown to be competitive inhibitors with Ki values ranging from 104

M to 10'5 M. No inhibition was observed for the epoxide-Neu5Ac2en compound.

INTRODUCTION

Neuraminidase (sialidase) is a class of enzyrne found in bacterial, viral and
mammalian origin which selectively hydrolyzes the terminal sialic acid moieties from
sialoglycoconj ugates. Bacterial neuraminidases have been widely used to evaluate the
neuraminidase inhibitors since large amounts of those enzymes were available. Vibrio
cholerae neuraminidase was chosen for this study. This enzyme has maximum activity
at pH 4.6, approximate Km of 1.5 x 10'3 M with 4MU-Neu5Ac as the substrates, and
was activated by 4 mM Cac120). Inhibitors of neuraminidase have been divided into
two groups according to whether their structures were related to sialic acid or note). To
specifically block the activity of bacterial neuraminidases, the inhibitors were required
to have the structure related to the substrate or the product of the enzyme. 2—Deoxy-2,3-
dehydro—N-acetylneuraminic acid (Neu5Ac2en) and its methyl ester, transition state .
analogues of neuraminidase“), have been shown to be competitive inhibitors of various

neuraminidase with Ki values between 5x10'3 and 5x10'6 M depending on the enzyme

62
sources“). Additionally, it has been reported that naturally occurring substrates with

various substitutions of Neu5Ac affect the cleavage rate of sialic acid by neuraminidase
in viva (5,6). It has been concluded that Neu5Ac2en and its derivatives are good tools
to study the enzyme properties beeause Neu5Ac2en is able to inhibit all neuraminidase(s)
from viral, mammalian, and bacterial sources"). With regard to analogues of
Neu5Ac2en, the degree of inhibition by compounds with different N -5 acyl groups has
been reported by Meindle and Tuppy“). No inhibitory activity was observed for an
acidic or basic substitution of N-acyl such as aminoacetyl or earboxyacetyl group. On
the other hand, the N-acyl substitutents with a neutral group such as chloroacetyl or
fluoroacetyl appeared to inhibit the enzyme to a certain degree“). Increasing the length
of N-acyl substitution lowered the inhibitory effect in V. Cholera neuraminidase. For
example, the inhibitory effect for N-butyryl substituted Neu2en was 10 times less than
that of Neu5Ac2en. When the N-acyl group was replaced with a benzoyl group, the Ki
value was 3.0r10'2 M (I(rn=1x10'3 M for 4MU-Neu5Ac as substrate). The activity of
bacterial and mammalian neuraminidases was completely blocked by acetylation of 4-
hydroxyl group of Neu5Ac2en(8'9), while the inhibition constants of 4-epi-Neu5Ac2en
was 2.7):10'4 M (comparison Km=5 mM for V. cholerae sialidase)(1°). The
replacement of 9—hydroxyl with photolabelling compounds has been used to characterize
C. perfringens neuraminidase“ 1’12). These derivatives appeared to have Ki value similar
to Neu5Ac2en (1.5x10'5 M). However, no report has been published about the
inhibitory effect of 9—hydroxy in V. cholerae neuraminidase. The most potential

synthetic inhibitor to date is 2-deoxy-2,3-dehydro-N—tlifluoroacetyl neuraminic acid

63
(Neu5CF3Ac2en) with a Kivalue about 2.5m6 M(13).

In this paper, we describe and compare the inhibitory properties of Neu5Ac2en
derivatives against V aioleme neuraminidase in firm. These Neu5Ac2en derivatives
synthesized in this laboratory could be classified into two groups differing in the position

of substitution N-acyl or 9-hydroxyl.

Materials and Methods

Mater-ink. The following materials were purchased from commercial sources: 4MU-
NeuSAc, 4-methylumbelliferone (4MU), Neu5Ac, Neu5Ac2en and V. Cholerae
neuraminidase (type IV, 0.7 U ml'l) from Sigma Chemical Company (St. Louis, MO).
The inhibitors were prepared as described in a previous chapter. Commercial
Neu5Ac2en was used as the reference for the inhibition assay against V. Cholerae
neuraminidase. All other chemieals and solvents were reagent grade or better. Aminco
fluoro-colorimeter was obtained from Amerieaan Instrument Co. (Silver Spring, MD).
The preparation of enzyme assay. Neuraminidase activity was determined with a
modifieation of the Poticr et al. method“). The activity of V. Cholerae neuraminidase
solutions purchased from commerieal sources (Sigma) were diluted to 4 pU ul'l and used
for these assayes. One unit (U) of neuraminidase activity was defined as 1 umole 4MU-
Neu5Ac hydrolysed per minute. Unless otherwise mentioned, a fluorogenic substrate
(4MU-Neu5Ac) was used as the artificial substrate of neuraminidase. V. Cholerae
neuraminidase activity was measured at 37 °C in a 0.2 ml total volume containing 0.1

M sodium acetate buffer (pH 5.0), 4 mM CaClz and varied concentration of 4MU-

64
Neu5Ac and inhibitors for its optimum activity. For the blank sample, the acetate buffer

was used to replace the enzyme. The hydrolysis reaction was terminated by the addition
of 0.2 M glycine-sodium hydroxide solution (1 ml, pH 10.7) after 45-60 minutes of
incubation. Free 4MU, the hydrolysis product, was measured by fluorescence emission
at 450 nm and excitation at 365 nm with an Aminco fluoro—colorimeter. The fluorimeter
was ealibrated with 25 and 50 nM 4MU and referred as 50% and 100% of relative
fluorescence intensity (RFI), respectively (Fig. 1). The relative fluorescence intensity
of samples was determinated in an aliquot (200 pl) in a total volume 2.2 ml stopper
solution containing 0.2 M glycine-sodium hydroxide (pH 10.7).

Experiments for Sialidase inhibition. For the determination of Km values, 4MU-
Neu5Ac was hydrolysed at the final concentration of 1, 0.5, 0.25, 0.1 mM under the
experimental condition. The concentration of inhibitors are tested at 0.01 mM, 0.1 mM,
and 1 mM in preliminary inhibition experiments. For the determination of Ki value,
Neu5Ac2en and its analogues were investigated at 0.01, 0.05, and 0.1 mM using the
substrate concentration described above. Two replications were prepared at each set of
concentration. The kinetic models of these synthetic inhibitors were determined by the
Lineweaver-Burk double reciproieal plots. The constants ('Km and Ki values) were also
calculated by fitting the data with a nonlinear regression program of Segal to the variants
of the Michaclis-Menten equation for competitive, noncompetitive, uncompetitive and

mixed inhibitionu“).

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Results and Discussions

The inhibition effect of these transition state analogues for V. cholerae
neuraminidase activity has been evaluated by the inhibition assay with 1 mM substrate.
The results summarized ill Table 1 indicate that Neu5Ac2en and its derivatives varied at
N-acyl and 9-hydroxyl positions are inhibitors of V. cholerae sialidase in vitro. The V.
cholerae sialidase activity was inhibited from 90% to 20% varied with the concentration
and substitution of inhibitor. For example, the remaining activity with the inhibitor
(Neu5NClAc2en) at 1 mM, 0,1 mM, and 0.01 mM using 1 mM 4MU-Neu5Ac as
substrate condition are 4%, 13%, and 24%, respectively, which has shown to be the
strongest inhibitor in this study with a Ki value about 2.1x10‘5 M. On the other hand,
the remaining activity with AB-Neu5Ac2en at 1 mM, 0,1 mM, and 0.01 mM under 1
mM substrate condition are 23% , 58% , and 74% , respectively, which is the weakest
inhibitor in this study with a Ki value about 1.1x10'4 M. Comparing the significance
of inhibitory between commerieal Neu5Ac2en (Neu5Ac2en(C)) and synthesized
Neu5A02en (Neu5Ac2en(S)), the activity measured with 1 mM substrate is inhibited up
to 82%, and 88% aq=3.5 x 105 M), respectively. It suggested that no difference is
observed in the inhibition effect for V. cholerae sialidase activity with these two
Neu5Ac2en sources. The interesting analogue, epoxy-Neu5Ac, containing some degree
of 2,3-diol-Neu5Ac has shown no inhibition effect for sialidase from V. cholerae.

The value of Km determined from Lineweaver-Burk plot is about 3.86x10’3 M
with 4MU-Neu5Ac as substrate forV. cholerae sialidase. Apparent Km value similar to

the result from Schreiner et. al. study (3.01x10'3 M)(13), but it has 2-3 times difference

67

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68
comparing the reports from Potier at. al. (1.5x10'3 MW). Based on Lineweaver-Burk

plots of the inhibition kinetics, these analogues substitued on the N -5 or 9-hydroxyl have
been shown to be competitive inhibitors versus the substrate 4MU-Neu5Ac. The
inhibition constant (Ki) of these compound are in the range 1.1x10'4 M to 2.1x10'5 M
and summarized on Table 2. Inhibtion effect of V. cholerae sialidase by Neu5Ac2en(S)
is present in Fig. 2 with a Ki of 3.5x10'5 M, which is 1.5 times higher than the previous
report (2.5):10'5 M)(15). For Neu2en and Neu5N3Ac2en, the inhibition efficacy are
shown in Fig.3 and 4, respectively. The inhibition constant (Ki=4.4x10’5 M) of Neu2en
is found to be nearly equal to that of Neu5Ac2en (3.5x10'5 M) in our study, which is
three orders of nragnitude higher than that of Zbiral er. al. study (2.9x10'2M)“5). This
result could be explained by the neutral amine group on the 05 position instead of a
positive charge of the quaternary ammonium group. The inhibition by Neu5N3Ac2en
is competitive with an apparent Ki (6.2x10'5 M). This finding is encouraged and will
allow the analogue to use for synthesis phtotoactivtable probe(s). The Ki value of other
N-acyl substituted analogues such as Neu5NClAc2en, Neu5NBrAc2en, and N -
arninoacetyl Neu2en are derived in the same manner as described in the methods (see
Table 2), and are similar to the results from Meindl et. al. (4).

The kinetic results demonstrate that NeuSACZen derivatives with substitution on
the 9-hydroxyl position are competitive inhibitors of sialidase from V. cholerae. The
order of decreasing inhibition of this series compound is Neu5Ac2en, IP-NeuSAcZen,
NB-NeuSAcZen, and AB-NeuSAc2en. The inhibition constant of each inhibitor is shown

in Table 2. Apparent Ki value of IP-NeuSAc2en (3.9x10'5 M), which formed an

69

Table 1: Inhibition of Vibrio cholerae neuraminidase by Neu5Ac2en and synthetic
analogues

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inhibitor Ki [mM]tSD
Neu5Ac2en (C) 3.5x10'2i 6.7x10’3
Neu5Ac2en (S) 3.5x10'21:5.1x10'3

Neu2en 4.4x10'2tl.2x10'2
NeuSNClAcZen 2.1x10'219.4x10'3
Neu5NBrAc2en 5.7x 10'2 t 1.4x 10'2
Neu5N3Ac2en harm-Zinnia"2
IP-NeuSAcZen , 3.9x10'2t1.7x10'2
NB-NeuSAc2en 7.9x10'2r_2.9n10'2
AB-NeuSAcZen 1.1x10'115.1x10'2
Epoxyl-NeuSAc 9.93:4

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73
isopropylidene linkage between the 8- and 9-hydroxyl, is likely the same as that of

Neu5Ac2en (3.5x10-5 M). A similar behavior is observed for the inhibition of NB-
Neu5Ae2en (Ki =7.9x10‘5 M). The inhibitory effect by AB-NeuSAcZen for v, cholerae
sialidase (Ki=l.09x10‘4 M) is 20 fold less than that of Neu5Ac2en (Figure 5); this
exception could not be a reasonably explained. Other inhibitors with the C-9 substituted
Neu5Ac2en reported from Horst et. al. and Warner were shown to be like
Neu5Ac2en(l 1'12). The sialidase activity influence by deoxyl side chain of Neu5Ac2en
analogues has been reported and the Ki range is from 1.1x10‘5 M to 9x10‘5 MOW).
The phenomenon indicates that the major force for the inhibition of sialidase causes from
the half-chair configuration of sialidase analogue, and the N—acyl residue. The structure
and conformation of the side chain of N eu5 Ac2en derivatives has lesser influence for
their inhibition on sialidase(s). However, a lone-pair electron element on the side chain

of Neu5Ac2en congener(s) play a role in the recognition of sialidase.

74

 

 

 

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REFERENCES

1. Potier, M., Mameli, L., Belisle, M., Dallaire, L., and Melancon, S. B. (1979) Anal.
Biochem. 94, pp287-296.

2. Schauer, R., and Corfield, A. P. (1981) In: Medicine Chemistry Advances (eds.
Heras, F. G. and Vega, S), Pergamon Press, pp423-434.

3. Meindl, P., and Tuppy, H. (1969) Hoppe-Seyler’s Z. Physiol. Chem. Bd.350,
pp1088-1092.

4. Meindl, P., Bodo, G., Palose, P., Schulman, J., and Tuppy, H. (1974) Virology, 58,
pp457-463.

5. Schauer, R., Vliegenthart, J. F. G. (1982) In: Sialic Acids: Chemistry, Matabolism
and Function (Cell Biology Monography, eds. Schauer, R.), 10, p4-49.

6. Schauer, R. (1985) TIBS, 10, pp357-360.

7. Miller, C. A., Wang, P., and Flashner, M. (1978) Biochem. Biophys. Res.
Commun. 83, ppl4‘79-1487.

8. Schauer, R., Failad, H. (1968) Hoppe-Seyler’s Z. Physiol. Chem. Bd.349, pp961-
970.
9. Corfield. A. P., Sander-Wewer, M., Veh, R. W., Wember, M., and Schauer, R.
(1986) Hoppe-Seyler’s Z. Physiol. Chem. Bd.367, pp433-442. .
10. Kumar, V. Kessler, J., Scott, M. B., Patwardhan, B. H., Taanenbaum, S. W., and
Flashner, M. (1981) Carbohydrate Res. 94, pp123-130.

11. Warner, T. G. (1987) Biochem. Biophys. Res. Commun. 148, pp1323-1329.

12. Horst, G. T. J., Mancini, G. M. S., Brossmer, R., Ross, U., and Verheijen, F. W.
(1990) J. Biol. Chem. 265, pp10801-10804.

13. Schreiner, B., and Zbiral, E. (1991) Carbohydr. Res. 216, pp61—66.

14. Perrella, F. W. (1988) Anal. Biochem. 174, pp437-447.

15. Schreiner, B., and Zbiral, E. Kleineidam, R. G. and Schauer, R. (1991) Liebigs.
Ann. Chem. pp126-134.

16. Zbiral, B., Schreiner, 13., Christian, R., Kleineidam, R. G. and Schauer, R. (1989)
Liebigs. Ann. Chem. pp159—l65.

75

CHAPTER4

AN AFFINITY CHROMATOGRAPHY METHOD - AN UTILIZATION OF
SIALIC ACID DFRIVATIVE

Running Title: Synthetic Sialic Acid Analogues and Their Biological Application

76

ABSTRACT

An affinity chromatography method has been developed for separating
neuraminidases from physiological protein mixtures. The affinity column was made by
coupling 2-deoxy-2,3-didehydro-N-deacety1neuraminic acid (Neu2en) to Bio-Rad Affi-
Gel 10 activated agarose gel. Bound neuraminidase was eluted from the column with
10% neuraminic acid in water. The totaal activity of the recovered neuraminidase from
the affinity column was evaluated using the 4MU-Neu5Ac assay and the peroxidase-
amplified assayutls). As judged by activity, 80-100% of the total activity applied to the
column was recovered. The physical properties of the recoved neuraminidase was
examined by electrophoresis. Enzyme samples analyzed before and after affinity
chromatography appeared as 68 kDa, and 16 kDa polypeptide bands while the molecular

weight of V. cholerae neuraminidase has been previously reported as 24.3 kDa or 90 kDa

polypeptides.

INTRODUCTION

Neuraminidase (N—acylneuraminosyl glycohydrolases; EC 3.2.1.18) has been
found in a wide range of organisms from bacteria to humans. Procedures for purification
of neuraminidase(s) with high specific activity from microorganisms have been well
developed(2v3t4). The activity of most animal neuraminidases has been reported but
complete purification has been hampered by the fact that these enzymes are
predominantly membrane bound having strong hydrophobic interaction(5t5t7). In addition

their lability, and low concentrations, as well as interference by other factors have added

78
to the difficulty of their isolation. Neuraminidase activity has also been detected in the

cell-conditioned media of human fibroblast cell lines (FS-4 and GM03468A)(1»3t9). The
physical and biochemical properties of this neuraminidase differ from that of the
membrane-bound neuranrinidases0'9' 10).

The interaction of (:10st perfringens neuraminidase with immobilized
ligands such as sialic acid derivatives, and sialyl-glycoconj ugates have been studied by
Corfield er. aI.<l 1). In their study, adipic acid dihydrazido-Sepharose 4B (AD-Sepharose)
and/or polymethylacrylic hydrazido-Sepharose 4B (PAH-Sepharose) were used as solid
supports. Their results indicated that neuraminidase was strongly bound to the amino-
linked adsorbents, could not be readily eluted with substrate or free sialic acid and
recoveries were low (40%). On the other hand, binding of neuraminidase to non-
substituted, blocked supports such as alkane chains from two carbons to ten carbons, or
4-nitrophenyloxamic acid were low. Neuraminidase adsorbed onto decyl-agarose support
was recovered from the support in 80% yield and it was suggested that hydrophobic
interactions between ligands and enzyme were involved. The same authors also reported
a similar behavior for neuraminidases from V. cholerae and influenza virus strain
2AlAichi/2/ 68.

A simple affinity chromatography method for the separation of neuraminidase(s)
from other proteins would be a useful tool to extend neuraminidase research. previous
studies with immobilized ligands and neuraminidase(s) indicated that two factors should
be considered in preparation of an affinity column. One is the specific binding of ligand

with neuraminidase through a neuraminic acid analogue having a free C-l carboxylic

79
group‘m. Next is the non-specific hydrophobic interactions between neuraminidase and

the column support‘13'“). This paper reports the preparation of an affinity column
which selectively absorbs neuraminidase using 2-deoxy-2,3-didehydro—N-deacetyl

neuraminic acid (NeuZen) linked with agarose gel (Bio-Rad Affi G-lO).

Methods and Materlak

Materials: Bio-Rad Affi G-lO gel and poly-styrene EIA/RIA plates (96 wells) were
obtained from Bio-Rad laboratories (Richmond, CA). The following substances were
purchased from Sigma Chemical (ST Louis, MO): glucose oxidase type V, a-
galactosidase, fetuin-agarose type III-A, V. cholerae neuraminidase type III (1 .U/ml),
4MU-Neu5Ac, o—phenylenediamine tablet, bovine serum albumin (BSA), and GM, and
Gm, gangliosides. V. alolerae neuraminidase (500 U/ml) was obtained from Koch-
Light laboratory (Colnbrook Bucks, England). Cholera toxin B subunit-horseradish
peroxidase conjugate was purchased from LIST Biological Laboratories (Campbell, CA).
AMICON centricon 10 filter was obtained from Amicon Division (Beverly, MA).
Synthesis of Neu2en has been described in the previous chapter. Other solvents and
reagents used were analytical grade or higher.

Instrument: An Aminco Fluoro-Colorimeter from American Instrument Co. (Siliver
Spring, MD) was used to measure the relative fluorescence intensity (RFI) of free 4MU
for 4MU-Neu5Ac assay at 365 nm for excitation and at 450 nm for emission, which was
calibrated with 25 and 50 nM 4MU and their RFI was so and 100%, respectively. The

sensitivity range of the instrument was fixed at 100 with a vemier control. A microtiter

80
plate reader from Bio-TEX Instruments Inc. (EIA Reader EL-307) was used to obtained

the absorbance of the peroxidase-amplified assay at 490 nm.
Preparation of immobilized sialic acid analogue on Bio-Rad Affi G-lo gel

The activated affinity supports (Bio-Rad Affi G-10 gel) had a ten-carbon spacer
arm. The immobilized ligand on Bio-Rad Affi G-10 gel was Neu2en (Figure 1C), which
is a sialic acid derivative with a free amino group at the N-5 position. The coupling was
performed in aqueous conditions following the product instl'uctions(15), which was briefly
described as the following. After the gel (1 ml) was washed with cold water (x 4
volumes) and drained through a Buchner funnel, the moist gel cake was transferred to
a test tube (15 ml) and the cold ligand solution (20 mmoles/ ml of gel) in 100 mM
HEPES buffer (pH 9-10) was added. The gel slurry was agitated on a shaker at 4 °C
overnight. Ethanolamine HCl (1 M, pH 8, 0.1 ml/per ml gel) was used to block any
remaining active esters in the gel, and it was rocked at 4 °C for 1 hour. The gel was
transferred to a poly-prep chromatography column and washed with water to remove the
uncoupled ligand. Subsequently, the column was equilibrated with 100 mM sodium
acetate buffer (pH 5.0). V. Cholerae neuraminidase (250 m0), glucose oxidase type V
(1.25 U), and a-galactosidase (5 U) were mixed in 100 mM sodium acetate buffer (pH
5.0, 1 ml) and applied to the column. Glucose oxidase type V and a-galactosidase were
eluted with 100 mM sodium acetate (pH 5.0, 40 ml), washed with H20 (100 ml), and
V. cholerae neuraminidase was subsequently eluted with 10% sich acid (5 ml). The
protein fractions were determined by the measurement of the UV absorbance at 280 nm

and confirmed with Lowry’s protein assay. Each protein peak was to concentrated to 0.5

81

(A) - ; coon

OCHZCHZNHz-Sepharose

 

Neu5Ac via a-g‘lycoaida linkage
COOH

OCH2CONHCH2CH2NH-Sephorase

 

Neu5Ac via arnlda linkage

(B)

Neu-fl-Ma via amide linkage

Norman vla anllda linkage

(0.)

New Bio-Rad All! G-lo

 

Figure 1: The common applied affinity chromatography (A) Used sialic acid as ligand
(B) Used sialic acid, its analogues, and sialylglycoproteins as ligands (C) Used Neu2en
as ligand

82
ml in an AMICON centricon 10 cartridge before the activity assays and electrophoresis.

The column was stored at 4 °C with 0.2% sodium azide.

Preparation of a fetuin-agarose affinity column chromatography

A control experiment was performed by preparing a fetuin-agarose column.
Fetuin is a sialic acid containing glycoprotein, which should bind neuraminidase. Fetuin-
agarose gel (200-300 mg/per ml, containing 2% free neuraminic acid) activated with
cyanogen bromide was commercially available. The fetuin-agarose gel (1 ml) was
transfered to a poly-prep chromatography column and washed with 100 mM sodium
acetate buffer (pH 5.0) at 4 °C. The column was pre-saturated with 1% BSA solution
to avoid non-specific binding of V. cholerae neuraminidase. Bound neuraminidase was
eluted with 50 mM sodium phosphate buffer containing 1 M NaCl (pH 6.5, 10
ml)(11'13'16'17). The column was stored at 4 0c: with 0.2% sodium azide.
The activity assay
mamm~

The assay of Potier et. al. was utilized with some modificationsm). In this study,
the activity of V. Cholera neuraminidase was determined before and after affinity
chromatography. The starting enzyme activity was 1 U/ ml, which was used as the
reference. Fractions from the affinity chromatography were diluted to 1 ml with buffer
solution to compare with the reference. The assay mixtures contained the following
components: 100 pl of 200 mM sodium acetate buffer containing 8 mM CaC12 (pH 5.0),
50 pl of 4 mM 4MU-Neu5Ac (the artificial substrate), and 50 pl of the enzyme solution.

After 45 minutes incubation at 37 °C, the enzymatic hydrolysis was quenched by addition

83
of 1 ml of 0.2 M glycine-sodium hydroxide solution (pH 10.7). An aliquot (100 pl) of

eluted neuraminidase was analyzed in a total volume 2.1 ml of 0.2 M glycine-NaOH
solution (pH 10.7), and the RFI value for fluorescence emission at 450 nm was
measured. Two sets of samples were prepared for each assay. Three readings were taken
for each sample. Several batch dilutions of enzyme solution were required to obtain RFI
readings in the liner range.

R 'l m l

V. Cholerae neuraminidase activity was assayed according to the method of Ogura
et. al.“). A Gm standard curve was linear up to 1 pmol (Figure 2). A 10 pmol sample
of GD“ was used as the substrate for the assay. No enzyme was added in the blank
sample. The absorbance for hydrolysis of GNI in the assay was subtracted from the blank
to limit the background interference from Go“. The fractions assaycd were treated in the
same manner as they were for the 4MU-Neu5Ac assay. Two sets of samples were
prepared for each assay. Five readings were taken for each sample.

SDS-El l 'I GIEI l .

The physical properties of enzymes eluted from the affinity column were analyzed
using the Bio-Rad Mini 1D Protean H Dual slab gel system. The concentrated column
fractions were lyophilizcd, suspended in 100 ill SDS sample buffer of Laemmlim),
reduced with 25 mM 2-mercaptoethanol, and applied to SDS PAGE on 12% separating
gels with a 8 % stacking gel. After electrophoresis, the gel was pre-fixed in methyl
alcohol containing 6.75 % acetic acid for 30 minutes, soaked in double distiation water

overnight, and stained with silver nitrate as described by Giuliun et. 01.0031). A middle

g4
Absorbance at 490 nm

 

 

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85
molecular-weight protein standard marker (Sigma Co.) was used to define the

approximate molecular weights of bands on the gel.

 

Conditions for optimal recovery of V. cholerae neuraminidase from an affinity
column were determined by varying the sialic acid concentration in the eluent. Under
the conditions of 4MU-Neu5Ac assay”), a high concentration (Ki =25 mM) of sialic
acid inhibited neuraminidase activity. The interference of sialic acid in peroxidase-
amplified assay has not been previously reported. Therefore, both activity assays were
performed after sialic acid was removed by size exclusion chromatography and with an
ultrafilter PM-lO.

The elution profile and electrophoresis pattern of fractions from the affinity Bio-
Rad G-10 column are shown in Fig. 3,. V. Cholerae neuraminidase (1U, and 250 mU)
was desorbed from the affinity column with 10% . sialic acid (5 ml). The total activity
of the recovered enzyme was examined using with both the 4MU-Neu5Ac and the
peroxidase-amplified assay and approximated 1.1 U and 200 mU, respectively (Table 1).
V. Cholerae neuraminidase isolated from the column migrated as a 68 kDa, and a 16 kDa
polypeptides on a 12% SDS PAGE as did the starting material (Figure 4). The activity
of the neuraminidase was not affected by the column according to the results of 4MU-

Neu5Ac assay and peroxidase-amplified assay.

 

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Sialyl-glycoconj ugates have commonly been employed as immobile ligands in
column chromatography to adsorb neuraminidase from various biological
sourccsm’”'24'25). The recovery of neuraminidase from an immobilized sialyl-
glycoconjugate column is influenced by the nature of the immobilized sialyl-
glycoconjugate and the amount of sialic acid in the sialyl-glycoconj ugate. Fetuin is a
plasma glycoprotein and a good substrate for neuraminidase, but is rapidly hydrolyzed
by the enzyme(1 1). Column degradation is a common problem in neuraminidase
purification from all immobilized sialyl-glycoconjugate column<11v13’15»l7). After
degradation, the resulting terminal saccharides of glycoconjugates may bind with other
glycosidases and cause contamination during the process.

V. Cholerae neuraminidase (l U) bound to a fetuin-agarose column is eluted with
50 mM sodium phosphate buffer containing 1 M NaCl (pH 6.0, 20 ml)(“). The elution
profile and electrophoresis pattern of the fetuin-agarose column is shown in Fig. 5 .
Under optimal conditions, the recovery of neuraminidase activity from the fetuin-agarose
column was less than 10 % . To increase the yield of eluted neuraminidase, the salt
concentration in the acetate buffer was raised to 2 M NaCl. However, no significant
improvement in the recovery of neuraminidase was shown (Fig. 5). Bound neuramidase
could be released from fetuin-agarose column while the column was placed at room

temperature for 48 hrs.

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91
chussion

Sialic acid derivative(s) immobilized on a solid support through the amide (N —
acetylneruaminamide) or the amino group of a-glycoside (N-acetylneuraminic acid) have
been used for affinity chromoatography of C perfringens and V. cholerae neuraminidases
(Fig. 0923537). These studies demonstrated that the N -acetyl neruaminamide-Sepharose
did not signifieatly adsorb C perfiingens and V. cholerae neuraminidases. These results .
were in agreement with the results of Miller et. al.(m, which suggested that the free
carboxyl group of the sialic acid moiety on the substrates was essential for substrate
binding to neuramidase within the catalytic stage.

The hydrophobic interactions between neuraminidase and various ligands have
been studied using the enzyme from bacterial, viral and bovine kidney
sourcesu3’23'29’3o). The binding of neuraminidases to alkyl agarose (range from 02 to
C 10) improved with increasing alkyl chain length. The neuraminidase could be desorbed
from alkyl agarose with 1M NaCl or by shifting the pH from 4.5 to 6. This suggested
that neuraminidase contained a hydrophobic site(s) which assist in the binding of
neuraminidase to alkyl columnsul). However, this interaction was shown to be a non-
specific beeause a similar phenomenon was observed with other glycosidases treated in
this manner‘ 13). The most selective binding of C. perfiingens neuraminidase was
observed on columns of Neu-B-Me and Neu2en linked to AD- or PAH-Sepharose via
amide linkages. However, the enzyme was only recovered from these affinity columns
after treatment with 0.05 M sodium acetate buffer (pH 5.3) at 20 °C and the recovery

was low(1 1). The bound neuraminidase on PAH-Sepharose failed to be eluted with

92
various substrates, products, or inhibitors such as sialyllactose (10 mM), Neu5Ac (100

mM), Neu-B-Me (100 mM), and Neu5Ac2en (10 mM)(2'11’27). Further, hydrolysis of
sialyllactose by neuraminidase bound to these columns demonstrated that the active site
of the enzyme was not occupied by the bound ligand and was still available for enzymatic
activity. This suggested that the spacer linkage from ligand to support was an important
factor and that hydrophobic interactions might again be the main force for the
quantitative binding of neuraminidase.

In our study, a transition state analogue (Neu2en) was coupled to the Bio-Gel
Affi-lO affinity column. The results indicate that V. cholerae neuraminidase was not
only adsorbed to the column specifically but was also easily eluted with 10% neuraminic
acid (pH < 4.0). This is an improvement over the results of Corfield et. all“), in which
the immobilization of Neu2en on PAH-Sepharose was shown to tightly bind two
neuraminidases which could not be eluted with 100 mM Neu5Ac. The improvenment
in recovery probably results from a reduction in hydrophobic interactions in the Neu2en
Bio-Gel Affi-lO column by introducing a 10 earbon spacer arm with polar substituents
rather than an alkyl spacer group as in the study of Corfield et. all“). The specificity
of-neuraminidase binding to the Neu2en Affi-IO column was confirmed by the separation
of V. cholerae neuraminidase from two other proteins in a mixture. The specificity
probably arises from the interactions with the transition state analogue. The bound
neuraminidase was successfully eluted with 10% neuraminic acid (pH < 4.0) illustrating

that the Neu2en Affi-lO column is a true affinity chromatography.

93
The physieal properties of V. cholerae neuraminidase have been determined with

electrophoresis before and after affinity chromatography. Both samples appeared to be
approximately 68 kDa, and 16 kDa polypeptides. However, the molecular weight of V.
cholerae neuraminidase has been estimated by ultr'acentrifugation(32), and reported to be
10-20 kDa. The same enzyme was analyzed by gel electrophesis and reported to be 90
mac”) or to be 24-25 kDa(15). The variation in the molecular weight reported for V.
cholerae neuraminidase from different groups might arise from several different factors
such as the ligand interactions, pH condition, glycolation migration, and partial
degradation.

V. Cholerae neuraminidase was tightly bound to the fetuin-agaroses at 4 0C and
barely desorbed from the column with high salt concentration in the buffer. Eventually,
free sich acid and bound neuraminidase were released from fetuin-agarose column after
prolonged standing at the room temperature (48 hours). These observations are in partial
agreement with the study by Corfield et. all1 1). The large quantity (250 mU) of
neuraminidase employed might cause an increase in the hydrophobic interactions and lead
to poor recovery. Although fetuin is an affinity adsorbant for sialidase(l3’15’17), it is not
useful for chromatography because of the low recovery from the column and its
degradation.

REFERENCES

p—s

. Ogura, K., Ogura, M., Anderson, R. L., and Sweeley, C. C. (1992) Anal. Biochem.
200, pp52—57.

2. Holmquist , L. (1974) Acta Chemica Scandinavica, B 28, pp1065-1068.

. Mohr, B., and Schramm, G. Z. (1960) Naturforsh, B 15, pp568-570.

4. Holmquist, L. , and Brossmer, R. ( 1972) Hoppe-Seyler’s Z. Physiol. Chem. 353,

pp1346-1352.
5. Corfield, A. P., Michalski, J.-C., and Schauer, R. (1981) In: Perspectives in Inherited
Metabolic Diseases, vol. 4, eds. Tettamanti, G., Durand, P., Didonato, S., Edi
Ermes, Milano, pp3-70.
. Miyagi, T., Tsuiki, S. (1984) Eur. J. Biochem. 141, pp75-81.
. I-Iiraiwa, M., Uda, Y., Nishizawa, M., and Miyatake, T. (1987) J. Biochem. 101,
pp1273-1279.
8. Ogura, K., and Sweeley, C. C. (1992) Experimental Cell Research, 199, pp169-l73.
9. Usuki, S., Lyu, S.-C., and Sweeley, C. C. (1988) J. of Biol. Chem. 263, pp6847-
6853.

10. Usuki, S., and Sweeley, C. C. (1988) Indian J. of Biochem. Biophys. 25, pp102-
105.

ll. Corfield, A. P., Corfield, C. A., Wember, M., and Schauer, R. (1985)
Glycoconjugate J. 2, pp45-60.

12. Miller, G. A., Wang, P., and Flashner, M. (1978) Biochem. Biophys. Res. Comm.
83, pp1479-l487.

13. Ziegler, D., Keilich, G., and Brossmer, R. (1980) Fresenius Z. Anal. Chem. 301,

pp99-100.

14. Ziegler, D., Keilich, G., and Brossmer, R. (1982) Fresenius Z. Anal. Chem. 311,

pp384-385.

15. The Bulltin of Activated Immunoaffinity Supports by Bio-Rad Lab. Co.

16. Winkelhake, J.L., and Nicolosson, R. (1980) Anal. Biochem. 71, pp281-289.

0.)

\IO‘

94

95

17. Kabayo, J. P., and Hutchinsson D. W., (1977) FEBS Lett. 78, pp221-224.

18. Potier, M., Mameli, L., Belisle, M., Dallarie, L., and Melancon, S. B. (1979) Anal.
Biochem. 94, pp287-296.

l9. Laemmli, U. K. (1970) Nature (London), 227, pp680-685.

20. Schagger, H., Link, T. A., Engel, W. D., and Von Jagow, G. (1986) In: Methods
in Enzymology (eds. by Fleischer, S., and Fleischer, B.), vol. 126, Academic Press,
Orlando, pp224-237.

21. Giulian, (1983) Anal. Biochem. 129, pp2‘77-287.

22. Corfield , A. P., Parker, T. L., and Schauer, R. (1979) Anal. Biochem. 100, pp221-
232.

23. Michalski, J.-C., Corfield, A. P., and Schauer, R. (1982) Hoppe-Seyler’s Z.
Physiol. Chem. 363, pp1097-1102.

24. Schauer, R., Wember, M., and Tschesche, H. (1984) Hoppe-Seyler’s Z. Physiol.
Chem. 365, pp419-426.

25. Scheid, M., and Choppin, P. W. (1974) Virology, 62, pp125-133.

26. Holmquist, L., Ostman, B. (1975) FEBS Lett. 60, pp327-330.

27. Holmquist, L., Nilsson, G. (1979) Acta Pat. Microbiol. Scand., B 87, pp129-135.

28. Brossmer, R., Ziegler, D., and Keilich, G. (1977) Hoppe-Seyler’s Z. Physiol.
Chem. 358, pp397-400.

29. Crampen, M., Von nicolai, H., and Zilliken, F. (1979) Hoppe-Seyler’s Z. Physiol.
Chem. 360, ppl703-1712.

30. Brossmer, R., Schmotzer, C. H., Keilich, G., Pech, H., and Ziegler, D. (1978)
Hoppe—Seyler’s Z. Physiol. Chem. 359, pp1065-1069.

31. Hatton, M. W. C., and Regoeczi, E. (1973) Biochem, Biophys. Acta, 327, pp114-
120.

32. Schramm, G., and Mohr, E. (1959) Nature (London), 183, ppl677-l678.

33. Pye, J., and Curtain, C. C. (1961) J. Gen. Microbiol. 24, pp423-425.

CHAPTERS

DICU SSION S

I. SYNTHFSIS OF NEURAMINIC ACID ANALOGUFS

 

During the last twenty years, several methods for the syntheses of Neu5Ac2en
have been developed and are summarized on Fig. 1A. Usually, Neu5Ac2en is prepared
from the fully protected 2-deoxy-2-halo sugar by dehydrohalogenation with base or silver
carbonate‘l’m). The alternative process is to treat N—acetylneuraminic acid methyl ester
with acetic anhydride in presence of a catalytic amount of sulfuric acid at 50 °C,
followed by deacetylation with NaOMe/MeOH(4). Under these conditions, a mixture
containing Neu5Ac2en and the 4-epimer Neu5Ac2en was obtained. 2-Deoxy-2,3-
dehydroneuraminic acid (Neu2en) was synthesized by hydrogenolytic cleavage of 2-
deoxy1-2,3-dehydro-N-benzyloxycarbonyl—neuraminic acid, which was formed by reaction
of siafic acid with diphenyldiazomethane‘s).

, In this work, a method for the synthesis of the transition state analogue of
neuraminic acid (Neu5Ac2en and Neu2en) has been developed (Fig. 1B). This procedure
has advantages over conventional methods in that it is a quantitative, simple one-step
conversion from neuraminic acid to Neu5Ac2en, or Neu2en. In this reaction,
epimerization at the C-4 position, which often accompanies the dehydration process, is
prevented by using the base dimethylamino pyridine. A comparison between this method

and the standard method for Neu2en synthesis, was made by varying ratios of

96

97
(A) OH

AcO .'

AczOIHCIO‘ or “07/

Ac 20’ ”'0'“?

   

R'NN

 

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DHAP/TFM (8:3)

"’0" C 6hrs .

   

Neu SAC

NeuSAcZene (84%)

UMP/WM (4/3)
70° C 6111's.

 

Neu2en (798)

Figure l: (A) The synthesis scheme of Neu5Ac2en was developed by Meindl and
Tuppy“»2v3). (B) The synthesis scheme of Neu5Ac2en developed in this laboratory.

98
trifluoroacetic anhydride and dimethylamino pyridine. A series of derivatives at the N—S

position of Neu2en has been obtained using methods similar to previous studies“). These
derivatives were assayed for their ability to competitively inhibit V. cholerae sialidase
using 4MU-Neu5Ac as a substrate. This results were consistant with previous reports(6).

Three analogues of Neu5Ac2en with varying substituents at the 9-hydroxyl group
were prepared in this study (Fig. 2A). The starting material, Neu5Ac2en, was
selectively protected at the 7-hydroxyl and 9-hydroxy with m-nitrobenzylidene
dimethylacetal and subsequently reduced with NaBH3CN and hydrogen chloride gas in
THF. In a kinetic study, the 9-substituted analogues of Neu5Ac2en were shown to be
competitive inhibitors with Ki values ranging from 110 to 80 pM, which is about 3 times
less than that of Neu5Ac2en. These results are not in agreement with those obtained by
Zibiral er. al. (7), who prepared 7-deoxy, 8-deoxy, or 9-deoxy—Neu5Ac2en from the
corresponding peracetylated Neu5Ac methyl ester derivatives by treatment with
trimethylsilyl trifluoromethanesulfonate (CF3SO3Si(CH3)3) (Fig. 23). 9-Deoxy-
NeuSAcZen was shown to be a better inhibitor than Neu5Ac2en for sialidase from V.
cholerae. To determine the contribution of the glycerol side-chain to inhibition, 1,2-O-
m—nitrobenzenzlidene-3—hydroxyl glycerol was prepared and assayed in the same manner.
No inhibition was observed. These results suggested that the binding site(s) of sialidase
did not directly interact with by the side-chain of Neu5Ac2en. One drawback of the
method for preparation of these 9-hydroxyl substituted compounds in this study is that

the reduction regent (NaBH3CN) is difficult to remove completely.

(A)

 

   

 

 

 

 

   

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Figure 2: Reaction scheme for the synthesis of Neu5Ac2en derivatives with a
modification on its glycerol side chain.(A) The synthesis scheme of AB-NeuSAc2en
developed in this laboratory.(B) 9-Deoxyl-Neu5Ac2en was prepared by Zbiral er.
al. (7)

100
ED 1 l |° ll 'l'l °l'l'

Two photoreactive, potential inhibitors of sialidase(s) have been prepared and
analyzed by Warner and Horst et. al. (Fig. 3A)(3t9). These photoactivatable groups were
introduced into the C-9 position via a thio- or an amide- linkage. The starting material
for both compounds was Neu5Ac2en methyl ester. Another photoreactive analogue of
Neu5Ac2en, 5-azido-5-deoxy-Kdn2en, has been prepared by Schreiner et. al. by
treatment of peracetylated S-azide sialic acid methyl ester with CF3SO3Si(CH3)3 in
presence of acetonitrile at 0 °C. However, this compound was not an inhibitor of
sialidase from v. cholerae (Ki=2.5 mM)(1°>.

In this study, a photoactivatable compound, Neu5N3Ac2en, was derived from
Neu2en by incorporating an azide group to a haloacetyl group at the N-S position (Fig.
3B). The photoreactable compound (Neu5N3Ac2en) was shown to be a competitive
inhibitor for v. cholerae sialidase (Ki= 6.1 x 102 mM), which could also be prepared
in a radiolabeled form for use as a photoaffinity probe. To utilize a photoaffinity
compound in physiological extracts, it is necessary to introduce a radiolabel
photoreactable inhibitor without sacrificing its inhibition ability. We did not perform the
radiolabeling synthesis because the procedure involved multiple enzymatic reactions to
incorporate the 1“c into the pyranose ring of sialic acid. In accordance with the
mechanism of catalysis of sialidase, Neu5Ac2en methyl ester has shown no influence on
its inhibitory effectiveness. Therefore, it could be one candidate position to introduce
a radiolabel. In a recent study, 14C labeling at the C—2 position of Neu5Ac was

prepared from N—acyl-2—amino-2—deoxyl-[l-14C] glucose as precursors by a series of

(A)

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Figure 3: Reaction scheme for the synthesis ”of photoaffinity compound. (A) 3H-9-

PANP-NeuSAcZen was prepared by Warner“); (B) Neu5N3Ac2en was prepared in
this laboratory.

102
enzymatic reactions(“).

11. AFFINITY CHROMATOGRAPHY FOR ISOLATION OF SIALIDASE FROM A
PHYSIOLOGICAL COMPLEXES

It has been demonstrated that affinity chromatography is an efficient method to
separate a particular enzyme from a phyiiological mixturemtutl‘b 15). Sialic acid
derivatives immobilized on to various solid supports via a—glycosides have been
employed in affinity chromatography systems for sialidase(s) purificationuztnt 14). It
has also been reported that Neu2en and B—glycosides of sialic acid methyl ester (Neu-B-
Me) were coupled to PAH-Sepharose matrix via the activation of acyl-hydrazido group
with 1010205, 16). Coupling of sialic acid analogues having an aldehyde side-chain via
reduction at the C-7 to soild supports was performed by reductive amination( 12).
Sialoglycoconj ugates have been immobilized on Sepharose and cellulose matrices“, 17).
Among those applications, the a-glycoside of Neu5Ac-Sepharose and related derivatives
have been used to sucessfully purify sialidases from various sources because they can
significantly adsorb sialidase in the presence of sodium acetate buffer (pH 5.5) and the
enzyme can be desorbed from the column with the addition of the benzyl a-ketoside of
NeuSACm). For the Neu2en-PAH-sepharose clomn, the binding of sialidase was
strong, unfortunately there was no straightforward process to elute the bound sialidase.
The main concern when using sialoglycoconjugates in affinity chromatography is that the
column will be degraded by the enzyme.

In this study, Neu2ar was immobilized on Bio-Rad Affi-Gel 10 via its free amino

group. The following improvements over the Neu2en—PAH-sepharose column were

103
observed. First, the affinity column could selectively adsorb V. cholerae sialidase(s)

from artificial protein mixtures (See details in chapter 4). The bound V. cholerae
sialidase(s) could be quantitatively eluted with 10% aqueous sialic acid (Neu5Ac) at 4
°C. The activity of the recovered sialidase(s), as determined by the 4MU-Neu5Ac and
the peroxidase-amplified assay, was retained. Overall, the Neu2en-Affi-10 column has
advantages over a-glycoside of Neu5Ac-Sepharose and Neu2en-PAH-Sepharose
chromatographies.

This study provided two useful tools for the handling of sialidase(s) in complex
physiological mixtures. One is a series of sich acid analogues with inhibitory
characteristics and the other is an efficient affinity chromatography column. It will be
interesting to examine the behavior of the affinity column when physiological extracts are
applied. Problems might include the modification of the elution system, and the variety

of the specificities within the families of sialidase(s).

REFERENCES

. Meindl, P., and Tuppy. H. (1967) Monatschefte fur chemie, 98, pp53-60.

. Meindl, P., and Tuppy. H. (1969) Monatschefte fur chemie, 100, pp1295-1306.

. Meindl, P., and Tuppy. H. (1970) Monatschefte fur chemie, 101, pp639-647.

. Kumar, V., Kessler, J., Scott, M. B., Patwardhan, B. H., Taanenbaum, S. W., and

Flaskner, M. (1981) Carbohydr. Res. 94, pp123-l30.

. Meindl, P., and Puppy, H. (1973) Monatschefte fur chemie, 104, pp402-4l4.

6. Meindl, P., Bodo, G., Palese, P., Schulman, J., and Tuppy, H. (1974) Virology,
58, pp457-463.

7. Zbiral, B., Schreeiner, B., Christian, R., Kleineidam, R. G., and Schauer, R. (1989)
Liebigs. Ann. Chem. pp 159-165.

8. Warner, T. G. (1987) Biocchem. Biophys. Res. ccommun. 148, ppl323-l329.

9. Horst, G. T. J., Maancini, G. M. S., Brossmer, R., Rose, U. and Verheijen, F. W.
(1990) 265, pp10801-10804.

10. Schreiner, B., and Zbiral, E. (1991) Carbohydr. Res. 216, pp611-66.

ll. Kayser, H., Zeitler, R., Hoppe, B., and Reutter, W. (1992) J. Labelled Compound
and Radioopharmacenticals, 31, pp7ll-715.

l2. Holmquist, L. (1974) Acta. chem. Scand. B28, pp1065-1068.

l3. Holmquist, L., and Ostrnan, B. (1975) FEBS Lett, 60, pp327-330.

l4. Holmquist, L., and Nilsson, G. (1979) Acta Path. Microbiol. Scand. B87, pp129-
135.

15. Corfield, A. P., Parker, T. L., Schauer, R. (1979) anal. Biochem. 100, pp221-252.

16. Corfield, A. P., Corfield, C. A., Wember, M., and Schauer, R. (1985)
Glycoconjugate J. 2, pp45-60.

l7. Winkelhake, J. L., and Nicolson, G. L. (1976) Anal. Biochem. 71, pp281-289.

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104

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