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Combined Chemoenzymatic Synthesis of N-Glycoprotein Building Blocks.

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Combined Chemoenzymatic Synthesis
of N-Glycoprotein Building Blocks **
ical synthesis of unprotected heterooligosaccharides is laborious and affords only low yields. Despite several modern
and efficient glycosylation procedures, a practical alternative
is often the use of enzymes.
With the goal of synthesizing partial structures of N-glycoproteins, we have used galactosyltransferase to form the
Gal+( 1-4)-GlcNAc bond in the asparagine-linked oligosaccharides 6 and 10 (Scheme 1). The substrates for this enzymatic reaction (5 and 9, respectively) were synthesized via
a chemical route (Scheme 2). The oligosaccharides 4 and 8,
By Joachim Thiem* and Torsten Wiemann
N-Glycoproteins play a central role in processes of biological recognition"". b1 such as oncogenesis,['"] bacterial and
viral infection,['d1 and uptake of various macromolecular
substances. Furthermore, they govern blood-group typeIIeJ
and are responsible for cell-cell recognition, cell growth, and
cell differentiation.["]
HO%
HO
0-P-0-P-0
1
UDP-galactose
Cepimerase
UDP
galactosyl-
UDP
2
substrates
products
HO&
o
)
OH
HO
NHAc OH
HO- HO
%
Ho
4
OH
3
5
HO
HO
OH
HO
7
OH
NHAc
8
10
9
Scheme 1. Galactosylation catalyzed by galactosyltransferase
The ready availability of reference substances for investigations of these highly specific phenomena is therefore of
great importance. The molecular basis of such recognition
processes derives from the extremely diverse ways of coupling monosaccharides; however, this also means that chem-
[*I
[**I
80
Prof. Dr. J. Thiem, Dip1.-Chem. T. Wiemann
Organisch-chemisches Institut der Universitat
Orleansring 23, D-4400 Miinster (FRG)
This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
0 VCH
Verlugsgesellsrhaft mbH. 0-6940 Weinheim.1990
which are not linked to an amino acid, were also synthesized
enzymatically.
The galactosylation was accomplished by a standard procedure. The activated form of galactose required for the
transferase reaction, namely, uridine-5'-diphosphate galactose (UDP-Gal, 2) is very expensive and was therefore generated in situ from UDP-glucose (1) in a reaction catalyzed
by UDP-galactose 4-epimerase. The galactose transfer from
UDP-galactose to the respective acceptor thus Occurs with
release of UDP.
0570-0833/90/0101-0080$02.50/0
Angew. Chem. I n t . Ed. Engl. 29 (1990) No. I
H HO
O
a nn
NHA~---
3
RO
&
AcO
[nBu4N
NaN,IHSO.
ANH
RO
AcO
Pd/C
Na
NHA~
ACNH~
NHAC
CI
*yq+c+
1So.b
120.b
140.b
1
AcNH
OAc
Ac
11
AC-ASP(OH)-OME
EEDP
,OAc
HZSO,
AczO
1
R0-E
AcO
Chitin
-
5:
R
9:
R = HoHO
NHAc
150.b
,OH
H
nz:C
-3%
b
1 0
NHA~
R
Ac
AcO&
AcO
NHAc
Scheme 2. Synthesis of galactosyl acceptors 5 and 9
This two-step galactosylation has also been carried out
with immobilized enzymes and applied to other substrates in
a synthetic cycle involving cofactor regeneration.I2I The
combination of transferase and epimerase in soluble form
offers a good compromise between cost on the one hand and
preparation time and experimental facility on the other.
Galactosylation of N-acetylglucosamine (GlcNAc, 3;
280 mg, 0.43 mmol) and of N,N-diacetylchitobiose (7;
10 mg, 24 pmol) according to the procedure described here
affords N-acetyllactosamine (4; 46 mg, 28 %),[3a1 the central
disaccharide unit of numerous glycoproteins, and trisaccharide 8 (8 mg, 60%),[3b1respectively. Both substances may
also be synthesized starting from UDP-Gal;14] in the case of
N-acetyllactosamine (4), a synthetic cycle involving cofactor
regeneration['] is also possible. The older chemical synthesis
of 4 is much more laborious in our opinion, because of the
large number of steps.[51
The glycoconjugate P-D-galactopyranosyl-(1-+4)-2-acetamido-1 -[(N-acetyl-I -methyl-4-~-aspartyl)amino]-2-deoxyP-D-glucopyranose (6) is present in the urine of patients suffering from aspartylglucosaminyluria.[61 The acceptor 5 required for the enzymatic galactosylation step was synthesized chemically by a procedure similar to that of Hough et
al.['] (Scheme 2). First, GlcNAc (3, 25 g, 113 mmol) was allowed to react with acetyl chloride to give the peracetylated
a-chloride 12a. Its substitution to afford P-azide 13a was
accomplished under phase-transfer catalysis in chloroform/
water. Compound 13a was then reduced to P-amine 14a,
which was coupled with a-methyl N-acetyl-L-aspartate in a
reaction promoted by ethyl 2-ethoxy-I ,2-dihydroquinoline1-carboxylate (EEDQ). The de-0-acetylation with sodium
methoxide affords the aspartylglucosamine 5 (20 % yield
over five steps, based on 3). Before the galactosylation step,
5 has to be chromatographically purified on Bio-Gel P2. The
standard method was again used in the enzymatic galactosylation to 6, which was obtained in 35% yield (0.1-mmol
scale). The expected P(1-4) linkage in 6 was confirmed by
'H NMR
An earlier chemical synthesis of
Angeiv. Chiw~./ti/. Ed. Engl. 29 11990) N o . 1
$3
the completely deprotected form of 6 was accomplished by
Jeanloz et al. in sixteen steps.[*]
Glycopeptide 10 is probably the receptor site for Robinia
lectin on the surface of erythrocyte^.[^] The synthesis of the
asparagine-linked trisaccharide requires, once again, an initial chemical synthesis; galactosyl acceptor 9 was prepared in
analogy to the work of Jeanloz et al.['O1 and Kunz et al.["l
Acetolysis of chitin affords a mixture of oligomers, from
which a-octaacetylchitobiose (11, 10%) can be isolated by
flash chromatography. Octaacetate 11 (4.5 g, 6.7 mmol) was
converted into a-chloride 12 b, which, in analogy to the synthesis of 5, was transformed into a-azide 13 b. Compound
13b was reduced to 14b, which, in turn, was coupled with
cr-methyl N-acetyl-L-aspartate in an EEDQ-promoted reaction to give 15 b. After de-0-acetylation, the free disaccharide 9 (295 mg, 18% over five steps, based on 11) was purified on Bio-Gel P2 and then used in the enzymatic galactosylation. The synthesis of the asparagine-linked trisaccharide
10 was accomplished in 28 % yield (50-pmol scale).[3d1
Experimental Procedure
Enzymatic galactosylation of 3, 5, 7, and 9: The reaction was carried out in
sodium cacodylate buffer (25 mM, pH 7.7) in the presence of manganese chloride (5 mM). After addition of UDP-glucose (2.13 mM), acceptor (13 mM), and
bovine serum albumin (1 mgmL-I), the solution was degassed with nitrogen.
Gakdctosyitransferase (EC 2.4.1.22, 1 U) and UDP-galactose 4-epimerase
(EC 5.1.3.2, 10 U) were then added. After two days at 3 7 T , the reaction mixture was worked up by anion exchange and the products 4,6, 8, and 10 were
purified by gel chromatography (Bio-Gel P2).
Received: July 13, 1989 [Z 3434 IE]
German version: Angew. Ckem. 102 (1990) 78
CAS Registry numbers:
3, 7512-17-6; 4, 32181-59-2; 5, 123883-46-5; 7, 35061-50-8; 8. 72358-28-2; 9,
123883-47-6; 10, 123883-48-7, 11, 7284-18-6; 12a. 3068-34-6; 12b. 7531-49-9;
13a, 6205-69-2; 13b. 29625-70-5; 14a, 4515-24-6; 14b, 29673-51-6; 15a.
123883-49-8; 15b, 123883-50-1; Ac-Asp-OMe, 4910-47-8; chitin, 1398-61-4.
VCH Verlugsgesellschafr m h H , 0-6940 Weinheim. 1990
0570-0833~90l0101-0081$02.5010
81
a) H. Schachter, Clm. Biochem. 17 (1984) 3; b) N. Sharon, H. Lis, Chem.
Erie. News 5 9 / 1 3 ) (19x1) 21: C) T. Feizi. Nature i t o n d o n ) 314 (1985)
53:
,
,
d) J. A. Wilson. J. J.'Shekel, D.C. Wiley. ihid. 289 (1981) 373; e) V. Ginsburg, Ad!,. Enwnol. 36 (1972) 131 ;f) G. E. Edelman, Sci. Am. 1984 (4) 80;
Spekrrum W m . 1984 16) 62.
a) S . L. Haynie, C -H. Wong, G . M. Whitesides. .
I
Org. Chem. 47 (1982)
5416, b) C. Auge, S . David, C. Mathieu, C. Gautheron, Terruhedron terr.
25 (1984) 1467: c) J. Thiem, W. Treder, Angew Chem. 98 (1986) 1100;
Angew. Chem. Inr. Ed. Engl. 25 (1986) 1096.
Selected ' H NMR data (300 MHz, D,O): a) 4. b = 5.19 (d, 1 H, H-1,
Jl,?= 2.2 Hz. a form), 4.71 (d, 1 H, H-1, J,,2= 7.5 Hz, b-form), 4.46 (d,
lH,H-l'.Jr.2 =7.5H~),203(S,3H.NA~);b)8:6=5.2O(d,lH.H-l,
J,., = 2.7 Hz. z form), 4.71 (d. 1 H, H-1, J,,2= 8.1 Hz, p form), 4.62 (d,
3 H, H-1'. J, , 2 , = 7.9 Hz), 4.48 (d, 1 H, H-1". J,..,,.. = 7.8 Hz), 2.08 and
2.05 (each S. each 3H, each NAc): C) 6 : 6 = 5.06 (d, l H , H-I,
J3,,= 9.4 Hz). 4.49 (d, 1 H, H-1', J, , 2 , = 7.6 Hz). 2.95 (dd, 1 H, P-Asn,
J,,p = 5.6 Hz, Ju,D,
= 17 1 Hz), 2.82 (dd, 1 H, F-Asn, J,,u= 7.6 Hz); d)
10:b = 5.09(d, 1 H,H-1, J,.2 = 9.6 Hz),4.62(d. 1 H,H-l'.J,.,, = 8.1 Hz).
4.47 (d. 1 H, H-1", J ,-,,. = 7 7 Hz), 2.06,2.02,2.01 (each s. each 3H. each
NAc).
H. A. Nunez, R. Barker, B,ochemi.strj 21 (1982) 1421
R. T. Lee, Y. C . Lee, Curhohydr. Res. 77 (1979) 270.
R. J. Pollitt, K. M. Pretty, Biochem. .l141 (1974) 141.
D. E. Cowley. L. Hough, C . M. Peach, Curhohydr. Res. 19 (1971) 231.
M. A. E. Shaban, R. W. Jeanloz, Bull. Chem. Six. Jpn. 54 (1981) 3570.
A. M Leseney. R. Bourrillon. S . Kornfeld. Arch. Biocbem. Binphjs. 153
(1972) 831.
M. Spinola, R W. Jeanloz. J Biol. Chem. 245 (1970) 4158.
H. Kunz, H. Waldmann, Angel?. Chem. 97 (1985) 885; Angeu-. Chem. Int.
Ed. Engl. 24 (1985) 883.
shells around their redox centers, (such as cytochrome c,
myoglobin, ferredoxin and phycocyanin) have also shown
that, even though these proteins are directly electrooxidized/
reduced on electrodes, their rates of electron transfer can be
enhanced by adsorbed "promoters" (e.g. bipyridine, methyl
viologen) that bind and orient the proteins, but d o not participate in the redox processes.[41We now find that pyrolytic
carbon and graphite electrode surfaces can be modified with
adsorbed polycationic redox polymers that complex glucose
oxidase. In these structures, electrons are vectorially transferred from enzyme-bound glucose to the electrode via the
redox centers of the enzyme and those of the redox polymer.
Strong adsorption of the redox polymer 1 on graphite is
seen in the cyclic voltammetry of the terpolymer of poly(N-
0
\
N0
N
\
I
I
~ s z o " oCH,
I
NH2
(bpy),C1
1, n = 0.14, rn = 0.81, p
=
0.05
Scheme 1. Structure of the qudternized redox terpolymer 1
Direct Electrical Communication between
Graphite Electrodes and Surface Adsorbed
Glucose Oxidase/Redox Polymer Complexes
By Michael K Pishko, Ioanis Katakis, Sten-Eric Lindquist,
Ling Ye, Brian A . Gregg, and Adam Heller*
The redox centers of glucose oxidase, like those of most
oxidoreductases, are electrically insulated by a protein (glycoprotein) shell. Because of this shell, the enzyme cannot be
oxidized or reduced at an electrode at any potential. In an
earlier communication it was shown that in homogeneous
solutions, water-soluble polycationic poly(viny1pyridine)
complexes of [O~(bpy),Cl]~@
bind to and accept electrons
from reduced glucose oxidase, a polyanion.['l Here we show
that the same polycationic redox polymers are strongly adsorbed on graphite; that these modified surfaces adsorb
strongly the enzyme; and that the surface adsorbed enzyme/
polymer complexes communicate electrically with the electrodes. Electrodes containing these adsorbed complexes are
faster and easier to construct than commercially available
glucose electrodes.
Poly(4-vinylpyridine) (PVP), as well as N-methylated
PVP, were shown earlier to be strongly adsorbed on pyrolytic graphite and to yield, with diffusing redox species, longlived reproducible electrodes. [', 31 Their electrochemistry is
consistent with the physisorption of segments of the macromolecules providing a three-dimensional network into and
out of which ions diffuse. Earlier studies of the electrochemistry of small redox proteins that d o not have thick insulating
[*] Prof. Dr. A. Heller, M. V. Pishko, 1. Katakis, Dr. S.-E. Lindquist, L. Ye,
Dr. B. A. Gregg
Department of Chemical Engineering, University of Texas at Austin
Austin, TX 78712 (USA)
[**I We wish to thank Yinon Degunr for his valuable advice o n the synthesis of
the polymers and Gorun Svensk for the molar mass determination of the
Osmium-containing redox-polymer 1. This work was supported in part by
the Office of Naval Research and the Robert A Welch Foundation.
methyl-4-vinyl pyridinium chloride), 4-aminostyrene, and
the PVP complex of [Os(bpy),CI,] (see Scheme 1); at scan
rates from 2 to 200 mVs-', the peak separation is 30 mV o r
less. Integration of the cyclic voltammograms at low scan
rates (2-5 rnVs-') shows that approximately 1.0 x
moles cm
of 1 are electroactive. The polymer does not
desorb from rotating disk electrodes even at high angular
velocities (2000 rpm), and coulometry shows that less than
10 YOof the polymer is desorbed from the electrodes after 30
days of storage in a stirred water bath. The polycationic
adsorbed polymer I strongly binds glucose oxidase. Cyclic
voltammograms for the oxidation of glucose by the enzymepolymer complex are shown in Figure 1. The current re~
UIVl
0.2
0.1
1
40
30
lo
c
1
"
'
~
-
0.3
'
"
0.4
'
I
t
Fig. 1. Cyclic voltammogram of the l/glucose oxidase complex in 60 mM glucose, 9.9 units catalase mL- I, 0.1 5 M sodium (4-(2-hydroxymethyl)-l-piperazineethanesulfonate (NaHEPES) at pH 7. Scan rate: 5 mVs-' a) no glucose.
b) 60 mM glucose. Potential C1 vs. SCE: glucose oxidase/l/graphite electrode.
sponse at a constant potential of 0.45 V (vs. SCE) persists
for more than 10 hours when electrodes are rotated at
20 rpm in physiological salt solutions that d o not contain
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