A MODEL FOR THE BINDING OF Cl DERIVATTZED GLUCOSE
COMPOUNDS TO CONCANAVALIN A
Dawn Patricia Cherico
B.A., Central Methodist College
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
This thesis for the Master of Science
Dawn Patricia Cherico
has been approved
Cherico, Dawn Patricia (M.S., Chemistry)
A Model For The Binding Of Cl Derivatized Glucose Compounds To
Thesis directed by Professor Douglas F. Dyckes
The synthesis of 1 -benzamido-1 -deoxy-P-D-glucopyranose was
successfully accomplished. The first step in the synthesis involved the
0- acetylation of glucose by acetyl bromide while at the same time introducing the
bromine atom on the anomeric carbon. The bromine was then displaced by an
azide ion in the (3 position, followed by catalytic hydrogenation of the azide
producing a primary amine. The amine was then benzoylated. Finally, the last step
involved a base-driven deprotection of the hydroxyl groups of the pyranose ring.
The compounds produced in each step of the synthesis were identified by *H and
13C Nuclear Magnetic Resonance (NMR) spectroscopy.
The binding of glucose and various derivatives to concanavalin A (Con A)
is already known, but this research wanted to investigate whether glucose
derivatized with a substantially larger substituent at the Cl position on the
pyranose ring, for example, 1-benzamido-1-deoxy-P-D-glucopyranose, would still
bind to Con A. By using a benzamido group, a chromophore is introduced to the
pyranose ring allowing for the binding to Con A to be measured
spectrophotometrically. A preliminary binding study indicated that 1-benzamido-
1- deoxy-P-D-glucopyranose bound to Con A, with an average binding constant
(Kas) of 1.7 x 104 M1. This value is comparable to binding constants for
saccharides that have a high affinity for Con A. Unfortunately, the binding study
experiment could not be reproduced in order to confirm results.
This abstract accurately represents the content of
recommend its publication.
I wish to express my sincere thanks to Dr. Douglas F. Dyckes for his
support, understanding, and patience throughout this project.
I would like to also thank Dr. Robert Damrauer and Dr. Donald Zapien for
their friendship and support during the completion of my degree.
Finally, I would like to thank all those who contributed in any way by their
moral support, patience, and kind words, especially Robert Nelson and all of my
List of Abbreviations...................................................x
1. Introduction....................................................... 1
1.2 Concanavalin A...................................................1
1.3 Synthesis Strategy...............................................4
1.4 Binding Study....................................................6
2.2 General Methods....................................................7
2.2.1 Thin Layer Chromatography (TLC)..................................7
2.2.2 Nuclear Magnetic Resonance (NMR).................................8
2.2.3 Melting Points...................................................9
2.2.4 Ultraviolet (UV) Spectroscopy....................................9
2.3 Synthetic Procedures...............................................9
2.3.1 Synthesis of Acetobromoglucose (2,3,4,6-tetra-Oacetyl-a-D-
glucopyranosyl bromide) (Compound I).............................9
2.3.2 Synthesis of 2,3,4,6-tetra-0-acetyl-(3-D-ghicopyranosyl azide
2.3.3 Synthesis of 2,3,4,6-tetra-O-acetyl-P-D-glucopyranosyl amine
2.3.4 Synthesis of 2,3,4,6-tetra-O-acetyl- 1-benzamido- 1-deoxy-P-D-
ghicopyranose (Compound IV).....................................14
2.3.5 Synthesis of 1-benzamido- 1-deoxy-P-D-glucopyranose
2.4 Spectrophotometric Study of the Binding of Compound V to
3. Results and Discussion..............................................19
3.1 Compound 1......................................................19
3.2 Compound Q......................................................26
3.3 Compound IQ.....................................................31
3.4 Compound IV.....................................................36
3.5 Compound V......................................................41
3.7 Binding Study Results...........................................51
3.9 Suggestions for Further Studies.................................54
2.1 Con A/l-benzamido-1 -deoxy- |3-D-glucopyrano se (Compound V)
binding study scheme. 18
3.1 HETCOR Coirelations. 49
3.2 *H and 13C chemical shift assignments. 50
3.3 Binding study data for 1-benzamido- 1-deoxy-P-D-glucopyranose
to concanavalin A. 52
1.1 Makelas Classification of the of Four Groups of Pyranoses. 2
1. 2 Schematic Drawing of the Synthesis of 1-benzamido- 1-deoxy- 5
3.1 *H NMR at 200 MHz of Acetobromoglucose (2,3,4,6-tetra-
O-acetyl-a-D-glucopyranosyl bromide) (Compound I) in CDCI3. 21
3.2 13C NMR at 200 MHz of Acetobromoglucose (2,3,4,6-tetra-
O-acetyl-a-D-ghicopyranosyl bromide) (Compound I) in CDCI3. 22
3.3 COSY NMR at 200 MHz of Acetobromoglucose (2,3,4,6-tetra-
O-acetyl-a-D-glucopyranosyl bromide) (Confound I) in CDCI3. 23
3.4 *H NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-glucopyranosyl
azide (Confound II) in CDC13. 27
3.5 13C NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-glucopyranosyl
azide (Compound II) in CDCI3. 28
3.6 COSY NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-
glucopyranosyl azide (Confound II) in CDCI3. 29
3.7 *H NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-glucopyranosyl
amine (Compound III) in CDC13. 33
3.8 13C NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-glucopyranosyl
amine (Compound HI) in CDCI3. 34
3.9 , COSY NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl-P-D-
ghicopyranosyl amine (Confound HI) in CDC13. 35
3.10 ^NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl- 1-benzamido-1-
deoxy-P-D-glucopyranose (Compound IV) in CDCI3. 37
3.11 COSY NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl- 1-benzamido-
1-deoxy-P-D-glucopyranose (Confound IV) in CDCI3. 38
3.12 13C NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl- 1-benzamido-1-
deoxy-P-D-glucopyranose (Compound IV) in CDCI3. 40
3.13 *H NMR at 200 MHz of 1-benzamido- 1-deoxy-P-D-glucopyranose
(Compound V) in D20. 42
3.14 COSY NMR at 200 MHz of 1-benzamido- 1-deoxy-P-D-
glucopyranose (Compound V) in D20. 43
3.15 13C NMR at 200 MHz of 1-benzamido- 1-deoxy-P-D-
glucopyranose (Compound V) in D20. 45
3.16 HETCOR NMR at 200 MHz of 2,3,4,6-tetra-O-acetyl- 1-benzamido-
1-deoxy-P-D-glucopyranose (Compound IV) in CDC13. 47
3.17 HETCOR NMR at 200 MHz of 1-benzamido- 1-deoxy-P-D-
glucopyranose Compound V) in D20. 48
LIST OF ABBREVIATIONS
benzamido- 1-deoxy- 3-
Concanavalin A Con A
T etramethylsilane TMS
Homonuclear Correlation Spectroscopy COSY
Heteronuclear Correlation HETCOR
The ultimate goal of this research was to determine whether concanavalin
A can be used as a purification method for carbohydrate labeled synthetic peptides.
The first step in accomplishing this goal was to synthesize a substituted glucose
labeled with a large substituent at the Cl pyranose ring position. The substituent
chosen was a benzoyl functional group. Following the synthesis of the compound,
it was used to determine if glucose derivatives of this type bind to concanavalin A
1.2 Concanavalin A
The lectin concanavalin A (Con A), a protein from the Jack bean
(Canavalis ensiformis), was first isolated by Sumner in 1919, crystallized and
identified as a hemagglutinin by Sumner and Howell in 1936. (1) Con A is
classified as an a-D-glucopyranosyl- and a-D-mannopyranosyl-binding lectin
because it binds saccharides which have the same hydroxyl configuration as group
three of Makelas structures, Fig. 1.1.(2) Makela classified saccharides based on
the configuration of the 3- and 4-hydroxyl groups on the pyranose ring .(3)
Saccharides that bind to Con A must have the proper orientations of the hydroxyl
groups at the C-3 and C-4 position, as well as a D-configuration at the C-5
position, in order to maximize hydrogen bonding of the saccharide to the active
site of Con A. (2) (4) The C-2 hydroxyl group does not hydrogen-bond to the
saccharide binding site and therefore is not required. (4)
Fig. 1.1: Makelas Classification of the of Four Groups ofPyranoses/3)
Con A has been shown to consist of aggregates of polypeptide subunits
with each having a molecular weight of approximately 27,000. Con A aggregates
forming dimers measured at molecular weight of approximately 52,000 at pH 5.6
and below, and tetramers with a measured molecular weight of about 110,000 in
solutions above pH 5.6.(3)(5) Each subunit has a binding site (SI) for one
saccharide(6) and two anion binding sites (S2), one of which binds a transition
metal ion (Zn2+, Co2+, Mn2+, or Mg2+) and one of which binds a calcium ion
(Ca2+). (7) Legume lectins use Ca2+ and the transition metals ions for stabilization
of the SI site by holding the surface amino acids in the appropriate position for
hydrogen bonding to the hydroxyl groups of the saccharides.^
Specificity of various saccharides for Con A has been examined over the
years. A study by Poretz and Goldstein examined binding of C-1 and C-2
derivatives of glucose and mannose to Con A in order to explore the
stereochemistry of the protein binding site. (9) Their experiments utilized the
quantitative hapten inhibition technique to demonstrate if their derivatives would
inhibit dextran from binding to Con A. (10) Their study indicated that many
different types of a- and J3- D-mannose and D-ghicose derivatives have inhibiting
power, and thus a binding affinity for Con A. Their study also concluded that even
though the hydrogen of the C-2 hydroxyl does not hydrogen bond in the binding
site, the oxygen atom provides a polar binding site for interaction of the saccharide
to Con A. The Cl oxygen may also provide a polar binding site, although data
suggest that some C-l derivatives appear to be good inhibitors of the binding of
dextran to Con A.(9)
1.3 Synthesis Strategy
It was hoped that it would be possible to synthesize a glucose molecule
with a large substituent at the Cl position which would still bind Con A. For this
research, the benzoyl group was chosen as the functional group because it has a
large extinction coefficient, which would be useful for spectrophotometric studies
when examining binding to Con A. The amide linkage was chosen because it
would be a better functional group for attaching peptides to the Cl position of
saccharides. The synthetic strategy for 1-benzamido- l-deoxy-(3-D-ghicopyranose
is schematically drawn in Fig. 1.2. (11) The first step in the synthesis requires
protection of the hydroxyl groups on glucose by acetylation. This can be done by
the addition of acetyl bromide to glucose. After acetylation of the hydroxyl
groups, the acetate group on the anomeric carbon becomes a good leaving group
and is nucleophilically displaced by bromide. Subsequently, bromide can be easily
displaced by nucleophilic substitution (Sn2) using an azide ion, which then places
an azide group at the (3 position. The azide then can be reduced by catalytic
hydrogenation producing a |3 primary amine. The amine is then benzoylated
leading to the incorporation of the chromophore onto the pyranose ring. Finally,
the pyranose ring can be deprotected by a base-driven de-O-acetylation, leaving
the hydroxyl groups free to hydrogen bond with Con A.
Fig. 1. 2 Schematic Drawing of the Synthesis of 1 -b enzamido-1 deoxy- (3-D-
1.4 Binding Study
After the synthesis of 1-benzamido- l-deoxy-(3-D-glucopyranose, it can be
used in a binding study to determine if it binds to Con A. The binding study
consists of combining solutions of known concentrations of Con A and 1-
benzamido- 1-deoxy-P-D-glucopyranose and allowing equilibrium between the free
and bound species to be reached. The equilibrium is depicted as follows:
Con A + D . ConA*D
1^= [Con A*D]/[Con A][D]
where Con A, D, Con A*D, and represent concanavalin A, 1-benzamido-1-
deoxy-P-D-glucopyranose, Con A/l-benzamido- 1-deoxy-P-D-glucopyranose
complex, and association binding constant, respectively. (12-15) After equilibrium
is achieved, the solutions are centrifuged through filter units. The absorbances of
unbound 1-benzamido-1-deoxy-P-D-glucopyranose in the filtrates are measured
spectrophotometrically at 228 nm and converted to concentrations after correcting
for any contributions from Con A control absorbances. The concentrations of
bound and free Con A can be calculated from the concentrations of 1-benzamido-
1-deoxy-P-D-ghicopyranose in the filtrates. After all the concentrations are
determined the association binding constants can be calculated as well
Acetyl bromide (99%) and a-D-glucose were purchased from Aldrich.
Benzoyl Chloride and Silver Nitrate (ACS grade, 99.8%) were purchased from
Adams Catalyst (Platinum Oxide) was purchased from Matheson Coleman &
Sodium azide was purchased from Sigma Chemicals.
2.2 General Methods
2.2.1 Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) was performed using aluminum plates
pre-coated with 0.2 mm layer of silica gel 60 (F254) (EM Science). Samples were
spotted on the plates using a 1 pL micropipet and the chromatograms were
developed in one of the following solvent systems:
system A: ethyl ether/petroleum ether (2/1)
system B: ethyl acetate/isopropyl alcohol/ILO (9/4/2)
The identification of the components was determined by one or more of the
following detection methods:
detection 1: Plate was sprayed with a solution containing 2% ninhydrin
in acetone, and then heated for 10 minutes at 110C.
detection 2: Plate was sprayed with a solution containing 3% phenol
and 5% sulfuric acid in 95% ethanol, and then heated for
10 minutes at 110C.
detection 3: Plate observed under ultraviolet light (254 nm).
2.2.2 Nuclear Magnetic Resonance (NMR)
Nuclear magnetic resonance analyses (NMR) were performed using a
Gemini 200 Broad Band spectrometer. Chemical shifts, 5, are reported in ppm.
Solutions were prepared in CDC13 containing TMS (8 = 0.00) as internal reference
or in D2O using HOD (5 = 4.60) as internal reference. Pre-programmed two
dimensional heteronuclear shift correlation experiments, HETCOR, in which one
dimension contains the 13C spectrum and the other coupled spectrum, and two
dimensional *H homonuclear correlation experiments, COSY, were also performed
to assist in the identification of compounds synthesized.
2.2.3 Melting Points
Melting points were determined using a Mel-Temp Laboratory Devices
melting point apparatus. All melting points reported are given without correction.
2.2.4 Ultraviolet (UV) Spectroscopy
UV spectra were recorded on Hewlett Packard 8452A Diode Array
Spectrophotometer. All spectra were obtained at room temperature using 1 cm
2.3 Synthetic Procedures
2.3.1 Synthesis of Acetobromoglucose (2,3,4,6-tetra-O-acetyl-a-D-
glucopyranosyl bromide) (Compound I)
Compound I was synthesized according to modified procedures described
by Koenigs and Knorr,(7# Charante/77) and Dale.(18) Acetyl bromide (14 mL,
0.19 mol) was added to a 100 mL round-bottomed flask fitted with a reflux
condenser and a calcium chloride drying tube and containing a-D-glucose (5.4 g,
0.030 mol).. The mixture was stirred with a Teflon stir bar at room temperature
for about 20 minutes until the colorless mixture changed to a dull straw yellow
solution. The apparatus was then put into a ice bath and the mixture was stirred
overnight. The mixture became thick and difficult to stir. The reaction was halted
by the addition of chloroform (75 mL) to the flask and stirred until the product
dissolved. The chloroform mixture was then transferred to a 250 mL separatory
funnel and washed with water (3X50 mL), sodium bicarbonate solution (4X50
mL), and one final water wash (50 mL). The chloroform layer was then dried over
calcium chloride and rotary evaporated. The thick syrupy residue was dissolved in
10 mL ethyl ether and transferred to a beaker with the aid of an additional 10 mL
ether. Compound I was readily precipitated as a white solid by the addition of
petroleum ether (20 mL) with vigorous stirring in an ice bath. The precipitate was
collected on a Buchner funnel Yield: 6.7g (55%); m.p.: 88-89C (lit. m.p.: 88-
89C)(76-7$); TLC Rf= 0.6 (system A and detection 2). 1HNMR(CDC13) 5
(ppm) 2.04-2.11 (12H, -OAc), 4.11-4.38 (m, 3H, H-5 and H-6 ), 4.84 (dd, 1H, H-
2), 5.17 (t, 1H, H-4), 5.57 (t, 1H, H-3), 6.62 (d, 1H, H-l). 13C NMR (CDC13) 8
(ppm) 20.59 and 20.70 (-CH3), 61.04 (C6), 67.27 (C4), 70.26 (C2), 70.70 (C3),
72.25 (C5), 86.68 (Cl), 169.67, 170.00, 170.05, and 170.72 (C=0).
2.3.2 Synthesis of 2,3,4,6-tetra-0-acetyI-(3-D-glucopyranosyl azide
Compound II was synthesized according to procedures described by
Bolton and Jeanloz with some modifications. (19) Two separate 20 mL solutions
were freshly prepared, one containing silver nitrate (3.4 g, 0.021 mol) and the
other containing sodium azide (1.4 g, 0.020 mol). The silver nitrate solution was
added to the sodium azide solution and stirred with a magnetic stirrer for about 5
minutes. Immediately there was a formation of a white precipitate of silver azide
in the solution. The precipitate was washed by decantation with water (40 mL),
95% ethanol (3 X 40 mL), ethyl ether (3 X 40 mL), and finally with chloroform (4
X 40 mL). The precipitate was then resuspended in chloroform (15 mL).
A sample of compound I (3.0 g, 3.3 mmol) was dissolved in chloroform
(10 mL) and transferred with the aid of a small amount of chloroform to a round-
bottomed flask containing the silver azide suspension. The mixture was refluxed
for about 45 minutes at 60-65C with stirring. As the reaction proceeded the
white AgN3 turned a greenish, silver color. The mixture was filtered and the
filtrate was rotary evaporated. The residue was dissolved in chloroform (15 mL),
pentane (20 mL) was added, and the solution was put into the refrigerator
overnight to crystallize. The colorless needle-like crystals were collected on a
Buchner funnel. Yield: 1.4 g (52%). ILC Rf = 0.5 (system A and detection 2);
mp.: 120.5-124.5C (lit. mp.: 127C)(20); H NMR (CDC13) 5 (ppm) 2.01-2.11
(12H, -OAc), 3.79-3.84 (m, 1H, H-5), 4.20-4.27 (m, 2H, H-6), 4.66 (d, 1H, H-l),
4.97 (t, 1H, H-2), 5.11 (t, 1H, H-4), 5.23 (d, 1H, H-3). 13C NMR (CDC13) 8 (ppm)
20.59 and 20.74 (-CH3), 61.74 (C6), 67.97 (C4), 70.73 (C2), 72.71 (C3), 74.13
(C5), 88.03 (Cl), 169.43, 169.53, 170.35, and 170.84 (C=0).
2.3.3 Synthesis of 2,3,4,6-tetra-0-acetyI-(3-D-glucopyranosyl amine
Compound IH was synthesized according to the procedure described by
Bolton and Jeanloz.(79) Compound II (1.2 g, 3.23 mmol) was suspended in 95%
ethanol (30 mL) in a 100 mL round-bottomed flask and Adams Catalyst,
platinium oxide, (0.13 g) was added. A hydrogenation apparatus was set up
which was flushed with nitrogen prior to the addition of hydrogen gas. Hydrogen
gas was bubbled into the azide mixture with stirring for about two hours. The
solution cleared and there were no visible signs of any undissolved starting
material TLC analysis using ninhydrin (detection 1) indicated that a primary
amine had formed. The catalyst was removed by filtration and the filtrate was
evaporated by rotary evaporation, resulting in a thick syrupy residue with
suspended crystals. Ethyl ether (20 mL) and water (10 mL) were added to the
residue, which only partially dissolved. The mixture was transferred to a
separatory iiumel using additional water (10 mL). The water layer was extracted
three more times with 20 mL aliquots of ethyl ether. The ether layers were
combined and concentrated to a thick syrupy residue by rotary evaporation. The
amine derivative crystallized as needles after the addition of 30 mL ethyl ether
which was added to dissolve the syrupy residue. The needle-like crystals were
collected by vacuum filtration. To ensure the best yield of the product, the ether
filtrate was again concentrated to a thick residue as previously described. The
residue was dissolved in ethyl acetate (20 mL), pentane (40 mL) was added to the
mixture, and it was allowed to sit overnight in the refrigerator. Additional needle-
like crystals formed. Both sets of crystals were analyzed by TLC. The results
indicated that they were the same product, Rf = 0.08 for both crystals (system A
and detection 1) and approximately the same purity. Identity was also confirmed
by *11 NMR. Combined yield: 0.4 g (38%), mp.: 120-122C. *11 NMR (CDC13)
8 (ppm) 1.97 (s, 2H, -NH2), 2.02-2.10 (12H, -OAc), 3.67-3.74 (m, 1H, H-5),
4.07-4.29 (m, 3H, H-l and H-6), 4.84 (t, 1H, H-2), 5.04 (t, 1H, H-4), 5.25 (d, 1H,
H-3). 13C NMR (CDC13) 8 (ppm) 2.65 and 2.83 (-CH3), 62.38 (C6), 68.88 (C4),
72.15 (C2), 72.84 (C3), 74.27 (C5), 85.13 (Cl), 169.76, 170.43, and 170.90
2.3.4 Synthesis of 2,3,4,6-tetra-0-acetyl-l-benzamido-l-deoxy-P-D-
glucopyranose (Compound IV)
Compound IV was synthesized with some modifications to procedures
described by Bolton and Jeanloz.(79) A solution of compound HI (0.27 g, 0.78
mmol) was prepared in 1.6 mL pyridine. Benzoyl chloride (0.16 mL, 1.37 mmol)
was carefiilly added to the solution in an ice bath. The reaction flask was removed
from the ice bath, fitted with a calcium chloride drying tube, and stirred at room
temperature for about 30 hours. One drop of ice water was then added, forming a
cloudy mixture. After standing for about five minutes the mixture cleared, forming
a light brown/yellow solution. An additional 40 mL of water was added, file
mixture was transferred to a 250 mL separatory funnel and was extracted with
chloroform (2 X 40 mL). The chloroform layer was washed with cold dilute HC1
(2 X 25 mL), 0.1 M cadmium chloride (2 X 25 mL), 0.5 M sodium bicarbonate (4
X 25 mL), and with water (3 X 25 mL). The chloroform layer was then dried over
anhydrous sodium sulfate, filtered, and concentrated to a syrupy residue on a
rotary evaporator. The syrupy residue was dissolved in 2 mL ethyl acetate and
transferred to a 50 mL beaker with the aid of an additional 6 mL ethyl acetate.
Pentane (16 mL) was added and the solution was allowed to stand in a refrigerator
until crystals formed. The white feathery clumps of crystals which formed after
two days were collected by vacuum filtration. Yield: 0.23 g (65%); m.p.:>270C,
decomposes. rH NMR (CDC13) 5 (ppm) 2.05-2.08 (12H, -OAc), 3.88-3.95 (m,
1H, H-5), 4.07-4.41 (m, 2H, H-6), 5.02-5.17 (m, 2H, H-2 and H-4), 5.36-5.50 (m,
2H, H-l and H-3), 7.04 (d, 1H, NH), 7.42-7.55 (m, 3H, H-4 and H-5), 7.74-7.79
(m, 2H, H-3). 13C NMR (CDC13) 8 (ppm) 20.63 and 2.76 (-CH3), 61.71 (C6),
68.33 (C4), 70.91 (C2), 72.71 (C3), 73.75 (C5), 79.06 (Cl), 127.39-132.92
(aromatic C), 167.32 (N-C=0), 169.81, 170.10, 170.84, and 171.79 (C=0).
2.3.5 Synthesis of 1-benzamido-l-deoxy-fi-D-glucopyranose
The synthesis of compound V involved the de-O-acetylation of compound
IV. This procedure was a modification of the de-O-acetylation procedure of
Marshall and Neuberger/27) Compound IV (0.16 g, 0.78 mmol) was added to
0.6 N LiOH in a small round bottomed flask. Initially the compound only partially
dissolved. The mixture was stirred at room temperature for about 12 hours by
which time most of the starting compound appeared to have been dissolved. The
mixture was then neutralized by passing it through a 8.5 X 1.0 cm Amberlite IR-
120 (If) column (Mallinckrodt) which had been pre-washed with 5 volumes of
water. Five mL fractions were collected at a flow rate of about 2.5 mL/min. TLC
using system B and detection C indicated a primary spot at Rf= 0.6 for fractions
1-9. These fractions were pooled and concentrated to dryness by rotary
evaporation forming a white solid. Yield = 0.088 g (87%); mp.: not performed. XH
NMR(D20), 5 (ppm) 3.31-3.69 (m, 6H, H2-6), 4.99 (d, 1H, H-l), 7.30-7.48 (m,
3H, H-4 and H-5), 7.60-7.65 (m, 2H, H-3). 13C NMR (D20) 5 (ppm) 60.50,
69.21, 71.74, 76.53, 77.65 (C2 C6), 79.90 (Cl), 127.46 (C3), 128.79 (C45),
132.76 (C2 and C5), 171.96 (Cl).
2.4 Spectrophotometric Study of the Binding of Compound V to
All binding studies were performed in microcentrifuge tubes. Binding
studies were performed in 20 mM Tris-HCl at pH 7.2, containing 1 mM Mn2+, 1
mM Ca2+, 0.5 M NaCL Two separate stock solutions containing Con A, 23.3
mM, (Sigma Chemical Company, Type V, lyophilized) and compound V, 94.1
mM, were prepared. The concentration of Con A was determined
spectrophotometrically at 280 nm using A1701 cm =11.4. (13) The molecular weight
of the Con A monomer, 27,000, was used for calculating protein (binding site)
molarity. Binding studies were done using 1000 pL solutions containing varying
amounts of Con A and compound V, see Table 2.1, and were incubated at room
temperature for approximately 48 hours. Solutions were filtered and centrifuged
on Eppendorf centrifuge at 6000 X g through Millipore ULTRAFREE-MC 10,000
NMWL filter units. The filtrates were collected and the absorbances were
measured at 228 nm in 1.0 cm quartz microcuvettes.
Table 2.1: Con A/l-benzamido- 1-deoxy-P-D-glucopyranose
(Compound V) binding study scheme
Solution ID Amount of 23.3 mM Stock Con A (pL) Amount of Buffer (m-L) Amount of 94.1 mM stock Derivative (pL)
A 500 500
B 500 500
C 500 500
D 250 750
E 250 250 500
F 500 250 250
G 750 250
3. Results and Discussion
3.1 Compound I
The first step in the synthesis of 1-benzamido-l-deoxy-P-D-glucopyranose
(Compound V) involved the acetylation of the hydroxyl groups of glucose
followed by the replacement of the acetate group on the anomeric carbon by
bromine through a nucleophilic reaction, producing compound I. The first
attempts to synthesize this compound followed a procedure in which acetyl
bromide was added to glucose and heated at about 60C, until a clear straw yellow
syrupy mixture was formed. When crystallizing acetobromoglucose from the
syrupy residue produced by this procedure, a yellow impurity contaminated the
white compound. Whenever the yellow color was seen, degradation appeared to
occur more rapidly. Compound I is very unstable at room temperature and
degrades rapidly in the presence of moisture. Hie white powder will slowly turn
brown and finally to a thick black liquid. When the yellow color was present, TLC
detected several additional spots not corresponding to the primary spot at
Rf = 0.6 (system A and detection 2). These additional compounds made
identification of products in subsequent synthesis steps very difficult.
In subsequent syntheses an alternate procedure was found by Charante,(77J
in which the same starting compounds were stirred in an ice bath, instead of
heating, until a thick straw syrupy residue formed as described in Synthetic
Procedures, Section 2.3.1. Compound I crystallized readily from the syrupy
residue and degradation did not occur as rapidly as previously. It is nevertheless
essential to store the compound in an evacuated dessicator in the freezer to help
maintain its stability. TLC also indicated a reduction in the number and intensity of
spots other than the primary spot corresponding to compound I when using this
procedure. The observed melting point range, 88-89C, also agreed with literature
values. (16-18) The identity of compound I was confirmed by H, 13C, and COSY
NMR spectra, Figures 3.1, 3.2, and 3.3, which were compared to spectra
published in The Aldrich Library of13C and H FT NMR Spectra. (22)
The interpretation of the 'H spectrum was supported by the two-
dimensional homonuclear Correlation Spectroscopy (COSY) spectrum Starting
with the XH spectrum, there is a multiplet upfield between 2.04 and 2.11 ppm
integrating to 12 protons, which corresponds to the -CH3 protons of the acetyl
groups. The region from 4.00 to 7.00 ppm corresponds to the C-H protons of the
pyranose ring. There is only one doublet in that region integrating to one
Fig. 3.1 lH NMR Spectrum at 200 MHz of Acetobromoglucose (Compound I) in CDC13.
Fig. 3.2 ,3C NMR Spectrum at 200 MHz of Acetobromoglucose (Compound I) in CDC13.
Fig. 3.3 COSY NMR Spectrum at 200 MHz of Acetobromoglucose (Compound I) in CDC13.
proton, at 6.62 ppm. This resonance is assigned to the proton attached to the
anomeric carbon, Cl, because it is the only proton with only one neighbor.
Additionally, protons on carbons next to electronegative atoms, such as bromine
and oxygen, are deshielded by the electron-withdrawing effect. Cl has both
bromine and oxygen atoms attached, making it the most electronegative ring
carbon. In the COSY spectrum, the Cl proton at 6.62 ppm correlates with a
doublet of doublets at 4.84 ppm, integrating to one proton, which can therefore be
assigned to the proton on C2, being split by two non-equivalent neighbors. The
C2 proton correlates to a triplet at 5.57 ppm, integrating to one proton, which is
assigned to the proton on C3. The C3 triplet correlates to another apparent triplet
integrating to one proton, at 5.17 ppm, which can be assigned to the proton on C4.
Neither the C3 nor the C4 proton signal is expected to be a pure triplet because
protons on C2, C3, C4, and C5 are nonequivalent. The coupling constants for
each neighboring pair are close, however, leading to the apparent triplets for C3
and C4. In the latter case, the non-equivalence explains the broadening of the
resonances at the baseline. Assignment of the C5 and C6 protons is not as
obvious. There is a multiplet between 4.11 and 4.38 ppm integrating to three
protons. The C4 resonance correlates to downfield density of the multiplet
assigning the C5 protons within the multiplet. The C5 proton strongly correlates
with the remaining region of the multiplet, corresponding to the C6 protons. The
singlet seen at 7.27 ppm corresponds to the solvent, CDC13.
The assignments of the 13C spectrum will be discussed more in detail in
Section 3.6. The spectrum itself corresponds favorably to a published
spectrum. (22) The spectral regions will be discussed now because they can be
easily identified. The resonances at 20.59 and 20.70 ppm correspond to methyl
carbons. The resonances at 61.04, 67.27, 70.26, 70.70, 72.25, 86.68 ppm
correspond to the five pyranose ring carbons and the C6 carbon. The resonances
at 169.67, 170.00, 170.05, 170.72 ppm are identified as carbonyl carbons. The
TMS can be seen at 0.00 ppm and the solvent is seen as three large resonances
centered at 77.12 ppm.
3.2 Compound 11
The synthesis of compound n, described in Section 2.3.2, involved the
displacement of the bromide ion on the anomeric carbon of compound I by the
highly reactive nucleophilic azide ion. The azide ion was introduced into the
reaction by a suspension of silver azide. It was determined experimentally that in
order for this reaction to proceed properly, it was essential to eliminate the water
from the silver azide before the addition of acetobromoglucose to the suspension.
Reaction progress was monitored by TLC and if the silver azide was not washed
free of water it did not react completely with compound I. Compound IIs identity
was confirmed by *11 NMR, 13C NMR and COSY spectra which compared
favorably to published NMR data.(20) Figures 3.4, 3.5, and 3.6, respectively,
provide spectra for the purified azide compound.
Comparing the NMR for compound I andJl, the most significant
difference is for the resonance for the proton on Cl which is more upfield at 4.66
ppm for compound n, compared to 6.62 ppm for compound I. The increased
shielding is due to the replacement of the bromine ion by the azide ion, which
appears to be less electron-withdrawing than the bromine ion. The resonances for
the protons on C2, C4, and C3 remain in the same order in the spectrum, 4.97,
5.11, and 5.23 ppm, confirmed by the COSY spectrum, but have moved in closer
proximity to each other. The resonances for C5 and C6 now can be distinguished
Fig. 3.4 'H NMR spectrum at 200 MHz oi'2,3,4,6-tetra-0-acetyl-B-D-glucopyranosyl azide (Compound II) in CDC13.
1 BO 1dO
"I 1 1 T
Fig. 3.5 13C NMR spectrum at 200 MHz ot'2,3,4,6-tetra-0-acetyl-J3-D-glucopyranosyl azide (Compound II) in CDCI3.
Fig. 3.6 COSY NMR spectrum at 200 MHz of 2,3,4,6-tetra-O-acetyl-B-D-glucopyranosyl azide (Compound II) in CDCI3.
The multiplet seen between 4.20-4.27 ppm which integrates to two protons
corresponds to the protons on C6. The proton on C5 can be assigned to the
multiplet integrating to one proton between 3.79-3.84 ppm. These assignments
are confirmed from the correlations identified by the boxes drawn on the COSY
spectrum, Fig 3.6.
The 13C spectrum obtained for compound II corresponds to published
chemical shifts by Sabesan/2QJ The resonances for compound II are assigned as
follows: 20.59 and 20.74 ppm (-CH3), 61.74 ppm (C6), 67.97 ppm (C4), 70.73
ppm (C2), 72.71 ppm(C3), 74.13 ppm(C5), 88.03 ppm (Cl), 169.43,169.53,
170.35, and 170.84 ppm (C=0).
3.3 Compound HI
The hydrogenation of compound II, described in Section 2.3.3, proceeded
fairly rapidly producing the amine derivative, compound m. The reduction of the
azide to the primary amine was monitored by TLC using ninhydiin (detection A) as
the indicator. After filtration of the catalyst and removal of the ethanol by rotary
evaporation, an attempt was made to dissolve the residue in ether and water. The
product did not completely dissolve at first however, during the extraction the
suspended matter slowly appeared to dissolve and distribute between the layers.
The ether layer was collected and concentrated by rotary evaporation. A problem
was then encountered when an attempt was made to redissolve the residue in
ether. Needle-like crystals immediately formed with the addition of ether. The
crystals were collected by vacuum filtration. The crystals and the ether filtrate
were tested by TLC to determine if the crystals were indeed the amine derivative,
or if it remained in the filtrate. TLC indicated that the crystals were the amine
product, producing a primary ninhydiin positive spot at
Rf = 0.08 (system A). The filtrate also produced a major spot corresponding to the
same Rf, indicating that not all of compound HI had crystallized. The ether filtrate
again was concentrated on the rotary evaporator. The syrupy residue was
dissolved in 40 mL ethyl acetate and allowed to crystallize overnight in the
refrigerator after the addition of 40 mL pentane, producing needle-like crystals.
The crystals were collected and analyzed for identity. Both sets of crystals were
analyzed by NMR, resulting in similar spectra, and TLC, each producing a primary
spot at Rf = 0.08 (system A), indicating that both crystals were the same product.
The identity of the compound was determined by 'HNMR, 13C NMR and COSY,
Figs. 3.7, 3.8, and 3.9, respectively.
The 'H NMR spectrum for compound El produced resonances for the
protons on the carbons of the pyranose ring in the same order as compound 11,
3.67-3.74 ppm (C5), 4.07-4.29 ppm (C6), 4.84 ppm (C2), 5.04 ppm (C4), and
5.25 ppm (C3), with the exception of the proton attached to Cl. The Cl proton
moved further upfield resonating in the same region as the C6 protons. The set
appears as a multiplet between 4.07-4.29 ppm, integrating to three protons. An
extra broad resonance appears at 1.97 ppm, integrating to two protons, next to the
region corresponding to the -CH3 protons of the acetyl groups, 2.02-2.10 ppm
These two protons are assigned to the pair of nitrogen protons. The peak is broad
due to the fact that protons on nitrogen atoms can undergo exchange. The 13C
NMR spectrum looks very much like the spectra for the previous compounds aside
from the small changes in the observed chemical shifts. Assignments will be
discussed in Section 3.6.
Fig. 3.7 'H NMR spectrum at 200 MHz of 2,3,4,6-tetra-O-acetyl-B-D-glucopyranosyI amine (Compound III) in CDC13.
Fig. 3.8 13C NMR spectrum at 200 MHz of 2,3,4,6-tetra-O-acetyl-B-D-glucopyranosyl amine (Compound III) in CDCI3.
3.4 Compound IV
The procedure for the benzoylation of compound HI on the nitrogen of Cl,
described in Section 2.3.4, proceeded as expected, attaching the benzoyl group to
the Cl position of the pyranose ring. The addition of the aromatic functional
group to the pyranose ring of glucose, produces some significant differences seen
in the NMR spectra in comparison to the previous derivatives. In the NMR
spectrum, Fig. 3.10, the multiplets for the protons on C6, 4.07-4.41 ppm, and C5,
3.88-3.95 ppm, remain at approximately the same chemical shifts as seen in
compound HI, as do the methyl protons, 2.05-2.08 ppm. The remaining protons
on the pyranose ring now are seen as two multiplets, each integrating to two
protons. The assignments are unclear without examining correlations, drawn as
boxes on the COSY spectrum, Fig. 3.11. The resonance assigned to the C5
proton, in addition to correlating to the C6 resonance, is also coupled to the
multiplet between 5.02-5.17 ppm, integrating to two protons. Thus one proton of
this resonance can be assigned to the C4 proton. The C4 multiplet correlates to
the multiplet, integrating to two protons, between 5.36-5.50 ppm, which assigns
one of the protons, therefore, as the C3 proton. This multiplet correlates to a
doublet at 7.04 ppm, integrating to only one proton. The only proton with one
neighbor is attached to nitrogen, thus the doublet is assigned to the nitrogen
Fig. 3.10 H NMR spectrum at 200 MHz of 2,3,4,6-tetra-O-acctyl-l-benzamido-glucopyranose (Compound IV) in CDCI3.
Fig. 3.11 COSY NMR spectrum at 200 MHzof2,3,4,6-tetra-0-acetyl-l-benzamido-glucopyranose (Compound IV) in CDC13.
(amide) proton which is in the spectral region normally associated with amide
protons. Since the multiplet between 5.36-5.50 ppm integrates to two protons
and the C3 proton is not expected to correlate to the nitrogen proton, the extra
proton density is assigned to the Cl proton. The pyranose ring proton attached to
C2, is assigned to the multiplet between 5.36-5.50 ppm, accounting for the
remaining unassigned proton density. The aromatic protons are seen downfield as
two multiplets between 7.42-7.55 ppm and 7.74-7.79 ppm. Assignments will be
discussed in Section 3.6.
The 13C NMR spectrum, Fig 3.12, still remains similar to the previous
compound with the exception of the additional resonances from the benzoyl group.
The aromatic carbons are seen in the region between 127.39-132.92 ppm and the
new carbonyl carbon is seen at 167.32 ppm. The identity of the pyranose carbons
and aromatic carbons will be discussed in Section 3.6.
Fig. 3.12 l3C NMR spectrum at 200 MHzof2,3,4,6-tetra-0-acetyl-l-benzamido-glucopyranose (Compound IV) in CDC13.
3.5 Compound V
The final step in the synthesis of 1-benzamido- 1-deoxy-P-D-glucopyranose,
compound V, involved a base driven deprotection of the hydroxyl groups of the
pyranose ring of compound IV. In the attempts to recrystallize compound V, the
product was dissolved in 2-propanol, and allowed to sit in the freezer for several
days. It was determined that compound V would not crystallize in this solvent and
no crystallization procedure was determined. An attempt was made to remove the
2-propanol by rotary evaporation which resulted in the formation of a white
compound. The identity of the compound was determined by NMR. In JH NMR
spectrum, Fig. 3.13, a doublet integrating to one proton is seen at 4.99 ppm. This
resonance is assigned to the proton on Cl. This doublet correlates to the multiplet
between 3.31-3.69 ppm, see COSY spectrum, Fig. 3.14. This multiplet integrates
to six protons which corresponds to the protons attached C2-C6. The pyranose
ring protons resonances, except the proton on Cl, have moved upfield compared
to the previous compounds. This is because the acetyl groups have been the
removed thus leaving less electron withdrawing hydroxyl groups attached to the
carbons. The aromatic protons are observed as two multiplets between 7.30-7.48
ppm, integrating to three protons, and 7.60-7.65 ppm, integrating to two protons.
There is no resonance seen
Fig. 3.13 H NMR spectrum at 200 MHz of 1-benzamido-l-deoxy-B-D-glucopyranose (Compound V) in D20.
Fig. 3.14 COSY NMR spectrum at 200 MHz of 1-benzamido-l-deoxy-B-D-glucopyranose (Compound V) in D20.
for the proton attached to the nitrogen at the Cl position because is has
exchanged with the solvent, D20, in addition to the exchange of the hydroxyl
protons. The proton spectrum also shows evidence of residual 2-propanol
resulting in a large doublet at 0.95 ppm and a multiplet at approximately 3.80 ppm
The assignments of the aromatic protons will be identified in Section 3.6.
The 13C NMR spectrum, Fig 3.15, still remains similar the spectrum for
compound IV, except the acetyl carbons are no longer present. The carbon
resonance assignments will be identified in Section 3.6.
Fig. 3.15 13C NMR spectrum at 200 MHz of l-benzamido-l-deoxy-li-D-glucopyranose (Compound V) in D20.
The combination of the two HETCOR spectra, Figs. 3.16 and 3.17
obtained for compound IV and compound V, respectively, and the published shift
assignments for compound II(20) make it possible to assign the pyranose ring
resonances in the 13C NMR spectrum for all the compounds. The HETCOR
spectrum for compound V helps to assign the multiplets corresponding to aromatic
protons. The HETCOR spectrum for compound IV did not show correlations for
the aromatic protons, therefore these assignments were not identified. The
HETCOR correlations are drawn on both spectra and described in Table 3.1.
Table 3.2 summarizes all the carbon and aromatic proton assignments that were
Fig. 3.16 HETCOR NMR spectrum at 200 MHz of 2,3,4,6-tetra-O-acetyl-l-benzamido-glucopyranose (Compound IV) in CDC13.
Fig. 3.17 HETCOR NMR spectrum at 200 MHz of 1-benzamido-l-deoxy-B-D-glucopyranose (Compound V) in D20.
Table 3.1 HETCOR Correlations.
*H Shift (ppm) *H Shift 13C Shift 13C Shift
Assignment Correlation Assignment
Compound IV 3.88-3.95 H-5 73.75 C5
4.07-4.41 H-6 61.71 C6
5.02-5.17 H-2 70.911 C2
H-4 68.33 1 C4
5.36-5.50 H-l 79.06 Cl
H-3 72.75 C3
Compound V 4.99 H-l 79.90 Cl
7.60-7.65 H-3 127.46 C3
7.30-7.45 H-4 128.79 C4
H-5 132.71 C5
xCarbon assignment concluded from published data for compound 11.(20)
Table 3.2 'H and l3C Chemical Shift Assignments.1
C1 C2 C3 C4 C5 C6 C1' C2' C3' C4 C5' H-3' H-4' H-5'
I 86.68 70.26 70.70 67.27 72.25 61.04
II 88.03 70.73 72.71 67.97 74.13 61.74
III 85.13 72.15 72.84 68.88 74.27 62.38
IV1 2 79.06 70.91 72.71 68.33 73.75 61.71
V 79.90 71.74 76.53 69.21 77.65 60.50 171.96 132.76 127.46 128.79 132.76 7.60 7.30 7.30
-7.65 -7.48 -7.48
1AII shifts are in ppm.
2Aromatic carbons and protons not assigned.
3.7 Binding Study Results
After solutions containing varying amounts of Con A and compound V
were allowed to equilibrate at room temperature, they were centrifuged through
10,000 NMWL centrifuge units. The filtrates were collected and the absorbances
were measured at 228 nm Absorbances were corrected for contributions from
Con A in the filtrates determined from the measured absorbances of filtrates of
solutions containing only Con A. The data for the experiment are summarized in
Table 3.3. The binding experiment indicated that 1-benzamido-l-deoxy-(3-D-
glucopyranose did bind to Con A with an average association binding constant,
Kas, determined to be 1.6 X104 M*1. The binding constant is comparable to
published binding constants, for example, methyl a-D-mannopyranoside of
7.6 X 103 and 0.82 X 104 Wl.(12)(14) Unfortunately, this experiment was not
reproducible. Two additional attempts were made, one in which a phosphate
buffer, pH 6.8, was used and the other attempt used Tris-HCl buffer, pH 7.2, with
a shorter incubation time of about 24 hours instead of 48 hours. Neither of the
additional experiments indicated that compound V was bound to Con A because
the absorbances of the filtrates determined the concentrations of the unbound
derivative to be the same as that of the controls which contained no Con A.
Table 3.3 Binding study data for 1-benzamido-l-deoxy-B-D-glucopyranose to concanavalin A.
Con A Cone. (mM) rCon A1 Compound V Cone. (mM) [D1 Average1 Absorbance at 228 nm Corrected Absorbance at 228 nm Absorbance Difference (Bound ComDound \n Cone, of Compound V Bound T1 (mM) KaS M'1
0.0474 ***** 0.172672 ***** ***** ***** *****
0.0474 0.0471 0.59782 0.42515 0.22869 0.0165 1.7 X 104
***** 0.0471 0.65384 ***** ***** *****
0.0237 ***** 0.09416 ***** ***** *****
0.0237 0.0471 0.60462 0.51045 0.14338 0.0103 2.1 X 104
0.0474 0.0235 0.41211 0.23944 0.09227 0.00654 0.9 X104
***** 0.0235 0.33171 ***** ***** ***** *****
Average absorbance of duplicate samples.
2Absorbance for one sample.
This research investigated the synthesis of 1-benzamido- 1-deoxy-P-D-
glucopyranose. Synthetic procedures for the incorporation of a benzoyl functional
group to the Cl position of the pyranose ring of glucose were successfully
developed. Confirmation of the structures of all synthetic compounds were
performed by NMR.
The preliminary binding study experiment indicated that 1-benzamido-1-
deoxy-p-D-glucopyranose binds to Con A. Subsequent studies were unable to
confirm the binding results, however. The average binding constant of 1.6 X 104
M"1 for the first experiment was a surprise because it compares favorably to
published binding constants for a-methyl-D-mannopyranoside which has a strong
affinity for Con A and is stronger than any known binding constants for glucose
derivatives. (12) (14) The inconsistancies in the results of subsequent experiments
indicates that 1-benzamido- 1-deoxy-P-D-glucopyranose may not actually bind to
3.9 Suggestions for Further Studies
Additional binding experiments will need to be performed in order to
confirm the binding results of 1-benzamido-1 -deoxy-P-D-glucopyranose to Con A
To confirm the results in future experiments, alternate procedures other than the
one described in this research should be performed. For example, if larger
amounts of the compound can be synthesized, equilibrium dialysis experiments
might be an alternative procedure to use to determine if 1-benzamido-l-deoxy-(3-
D-glucopyranose binds to Con A (23) Additionally, another alternate method
described by Bessler could be used, in which a spectrophotometric study
determined the binding constants of various carbohydrates by the displacement of a
chromogenic ligand, /7-nitrophenyl a-D-mannopyranoside, from Con A. (13)
In conclusion, if binding of 1-benzamido- 1-deoxy-P-D-glucopyranose to
Con A can be repeated, additional glucose derivatives will need to be synthesized.
These derivatives will involve substituting various small peptides at the Cl position
of the pyranose ring via an amide linkage. The derivatives will then need to be
analyzed to determine if binding to Con A remains. The synthetic procedures used
for this research may prove initially to be too time consuming and alternative
methods may prove to be more productive in order to determine quickly if various
derivatives bind to Con A A method by Nimura(2^ describing a procedure for
derivatizing enantiomeric amino acids using 2,3,4,6-tetra-O-acetyl-P-D-
glucopyranosyl isothiocyanate appears to be a more efficient method for attaching
glucose to the N-terminal end of peptides. The compound, 2,3,4,6-tetra-O-acetyl-
(3-D-glucopyranosyl isothiocyanate, is more costly than other procedures, but
eliminates a number of the steps described in Section 2.3, and thus the time
required for synthesizing derivatives is reduced.
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