In Situ Preparation of β-D-1-O-Hydroxylamino Carbohydrate Polymers Mediated by Galactose Oxidase

Article abstract of DOI:10.1021/ol0258379

(Equation Presented) Galactose oxidase produced a C-6 aldehyde in various terminal-containing galactose hydroxylamines for the simultaneous in situ generation of an A-B type condensation for the construction of unique oxime polymers. Molecular weights of the corresponding polymers were determined to be in the range of 4200-8900 g/mol, respectively. This indicates that approximately 20-25 sugar units were incorporated in these unique polymers.

Full text of DOI:10.1021/ol0258379

ORGANIC
LETTERS
2
002
Vol. 4, No. 11
863-1866
In Situ Preparation of
1
â-D-1-O-Hydroxylamino Carbohydrate
Polymers Mediated by Galactose
Oxidase
Peter R. Andreana, Wenhua Xie, Huai N. Cheng, Lei Qiao, Dennis J. Murphy,
Qu-Ming Gu, and Peng G. Wang*
Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202-3489
pwang@chem.wayne.edu
Received March 7, 2002
ABSTRACT
Galactose oxidase produced a C-6 aldehyde in various terminal-containing galactose hydroxylamines for the simultaneous in situ generation
of an A−B type condensation for the construction of unique oxime polymers. Molecular weights of the corresponding polymers were determined
to be in the range of 4200−8900 g/mol, respectively. This indicates that approximately 20−25 sugar units were incorporated in these unique
polymers.
Synthetic or modified natural carbohydrate polymers have
organs or drug delivery systems have been developed by
ingeniously taking advantage of the recognition processes
of carbohydrates in vivo. The selective incorporation of
1
a wide variety of practical applications including hydrogels,
adsorbents, biorecognition agents,3 and glycoconjugate
2
7,8
4
assemblies. Carbohydrates conjugated to a variety of materi-
sugars and their derivatives into polymers is taking on
increased importance, particularly in the preparation of
als, including proteins, lipids, and synthetic polymers, have
been utilized in diverse scientific disciplines.5 For instance,
novel techniques of medical treatments such as artificial
,6
9
biocompatible and biodegradable materials. The vast major-
ity of in vitro preparation of sugar-containing polymers have
utilized glycosidases for polysaccharide synthesis involving
(
1) (a) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869-1880,
10
unnatural monosaccharide precursors or lipases, esterases,
and references therein. (b) Kumashiro, Y.; Huh, K. M.; Ooya, T.; Yui, N.
Biomacromolecules 2001, 2, 874-879. (c) Vazquez-Duhalt, R.; Tinoco,
R.; D’Antonio, P.; Topoleski, L. D. T.; Payne, G. F. Bioconjugate Chem.
and proteases for poly(sugar ester, amide, or acrylate)
11
synthesis in organic solvents.
2
001, 12, 301-306. (d) Han, J. H.; Krochta, J. M.; Kurth, M. J.; Hsieh,
The highly selective condensation reactions of ketones or
aldehydes with aminooxy, hydrazide, or thiosemicarbazide
Y.-L. J. Agric. Food Chem. 2000. 48, 5278-5282. (e) Lee, K. Y.; Bouhadir,
K. H.; Mooney, D. J. Macromolecules 2000, 33, 97-101. (f) Eiselt, P.;
Lee, K. Y.; Mooney, D. J. Macromolecules 1999, 32, 5561-5566. (g)
Dorski, C. M.; Doyle, F. J.; Peppas, N. A. Polym. Mater. Sci. Eng. Proc.
1
997, 76, 281.
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4, 33-39.
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(6) Lee, Y. C.; Lee, R. T. in Methods of Enzymology Vol. 247 -
Neoglycoconjugates, Part B: Biomedical Applications; Abelson, J. N.;
Simon, M. I.; Colowick, S. P.; Kaplan, N. O., Eds.; Academic Press: New
York, 1994.
(
2
(
Kopecek, J. Pharm. Res. 1999, 16, 1010-1019. (b) Wroblewski, S.;
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(8) Ouchi, T.; Ohya, Y. In Neoglycoconjugates: Preparation and
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Macromol. Chem. Phys. 2000, 201, 2245-2250.
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5) Lee, Y. C.; Lee, R. T. in Methods of Enzymology Vol. 242 -
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1
0.1021/ol0258379 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/02/2002

groups have been widely used for the conjugation of small
and macromolecules. The reactions proceed in aqueous
conditions, and their high selectivity negates the requirement
for protection of other functional groups on the coupling
partners.12 Galactose oxidase (GO) selectively oxidizes
exposed primary hydroxyl groups in nonreducing, terminal
galactose and N-acetyl galactosamine residues to the corre-
sequential A-B condensations with hydroxylamine moieties.
The result would be novel, high molecular weight oxime
polymers that are relatively stable to hydrolysis. This
methodology would allow enzyme-mediated polymer syn-
thesis under simple and mild conditions and provide products
with a relationship to natural carbohydrate polymers in which
most are connected from the anomeric carbon.
The synthesis of the â-D-1-O-hydroxylamine galactose
(GO substrates 5-8 for in situ polymerization) moieties is
shown in Scheme 2. Previously, â-D-1-O-hydroxylamine
13
sponding aldehyde. The galactose oxidase/NaBH
3
CN method
is well-known and widely used as a technique to label cell-
surface glycoconjugates.14 GO also has broad substrate
specificity for a variety of galactose- and nongalactose-based
1
5
compounds. The native reaction on galactose yields a C-6
aldehyde, which has been shown to undergo spontaneous
Scheme 2. Synthetic Route to â-D-1-O-hydroxylamino
16
Carbohydratesa
Schiff base formation in the presence of amines. Recently,
Dordick and co-workers reported the chemoenzymatic
synthesis of sugar-containing polyamines using GO coupled
with chemical reduction. GO was first used to selectively
2
oxidize C6-CH OH to an aldehyde, and then in a subsequent
reaction, polycondensation of the aldehyde with diamines
via reductive amination, either in an AA-BB or A-B
fashion, afforded the poly(galactose amine)s.17
Our approach to synthesizing nonnatural carbohydrate
polymers takes advantage of an anomeric hydroxylamino
1
8
preparation, first reported by Roy and co-workers. In the
consideration of the fact that chemoselective ligation of an
aldehyde and a hydroxylamine proceeds readily in aqueous
solvents for the formation of oximes which are stable in
water, we envisioned that the GO oxidation of â-D-1-O-
hydroxylamino galactose (Scheme 1) and related compounds
Scheme 1. In-Situ Polymerizationa
a
(a) 33% HBr/AcOH, 6 h. (b) NHS, (1:1, CH
2 2 3
Cl :1 M NaCO ),
2
4 h. (c) NH
2
NH -H O, 12 h.
2
2
sugars prepared in the same manner have been purified by
1
8b
a
preparative HPLC. However, in the present study, an
effective way for purification on a relatively large scale was
desirable. To this end, ion-exchange columns were found to
be inadequate due to stability problems of the products under
1
20 U GO, 1200 U catalase in 10 mL of phosphate buffer (50
mM pH 7.0) containing 3 mg of CuSO
stream of air.
4 2
‚5H O with a continuous
19
such conditions. Recrystallization with a variety of solvent
systems could only reduce the amount of impurities.20
Finally, it was found that purification on gram-scale could
be achieved by utilizing a gel-filtration column.
in aqueous buffer solutions would lead to the formation of
polysaccharides by selective C6-CH OH oxidations and
2
(10) (a) Fort, S.; Boyer, V.; Greffe, L.; Davies, G. J.; Moroz, O.;
Christiansen, L.; Schuelein, M.; Cottaz, S.; Driguez, H. J. Am. Chem. Soc.
2
1
000, 122, 5429-5437. (b) Shoda, S.-I.; Kobayashi, S. Trends Polym. Sci.
997, 5, 109-115.
(13) Suzuki, Y.; Suzuki, K. J. Lipid Res. 1972, 13, 687-690.
(14) (a) Eccleston, E. D.; White, T. W.; Howard, J. B.; Hamilton, D. W.
Mol. Reprod. DeV. 1994, 37, 110-119. (b) Enrich, C.; Gahmberg, C. G.
Biochem. J. 1985, 227, 565-572. (c) Kumarasamy, R.; Blough, H. A. Arch.
Biochem. Biophys 1985, 236, 593-602.
(
11) (a) Li, J.; Xie, W.; Cheng, H. N.; Nickol, P. G.; Wang, P. G.
Macromolecules 1999, 32, 2789-2792. (b) Li, J.; Cheng, H. N.; Nickol,
R. G.; Wang, P. G. Carbohydr. Res. 1999, 316, 133-137. (c) Xie, W.; Li,
J.; Chen, D.-P.; Wang, P. G. Macromolecules 1997, 30, 6997-6998. (d)
Xie, W.; Li, J..; Chen, D.-P.; Wang, P. G. Polym. Prepr. 1997, 38, 279-
(15) Avigad, G.; Amaral, D.; Asensio, C.; Horecker, B. L. J. Biol. Chem.
1962, 237, 2736.
2
80. (e) Andreana, P. R.; Xie, W.; Wang, P. G. In Synthesis and
(16) Yalpani, M.; Hall, L. D. J. Polym. Sci. Polym. Chem. Ed. 1982, 20,
3399-3420.
(17) Liu, X.-C.; Dordick, J. S. J. Am. Chem. Soc. 1999, 121, 466-467.
(18) (a) Cao, S.; Tropper, F. D.; Roy, R. Tetrahedron 1995, 51, 6679-
6686. (b) Rodriguez, E. C.; Marcaurelle, L. A.; Bertozzi, C. R. J. Org.
Chem. 1998, 63, 7134-7135.
(19) Three different types of cation-exchange resin were used; Amberlite
IRP-64, Amberlite IRP-69, and NaFion R NRSO XR ion-exchange resin
from DuPont Company.
(20) H2O/MeOH with varying concentrations; H2O/CH3CN with varying
concentrations.
Modifications of Carbohydrates Through Biotechnology In Frontiers in
Polymer Synthesis/Modification by Biocatalysis; Gross, R., Cheng, H. N.,
Eds; 2001, in press. (f) Patil, D. R.; Dordick, J. S.; Rethwisch, D. G.
Macromolecules 1991, 24, 3462-3463. (g) Wang, Z.-L.; Hiltunen, K.;
Orava, P.; Seppala, J.; Linko, Y.-Y. Pure Appl. Chem. 1996, A33, 599-
6
1
12. (h) Patil, D. R.; Rethwisch, D. G.; Dordick, J. S. Biotechnol. Bioeng.
991, 37, 639-646.
(
12) (a) Muir, T. W. Structure 1995, 3, 649-652. (b) Lemieux, G. A.;
Bertozzi, C. R. Trends Biotechnol. 1998, 16, 506-513. (c) Hang, H. C.;
Bertozzi, C. R. Acc. Chem. Res. 2001, 34, 727-736.
1864
Org. Lett., Vol. 4, No. 11, 2002

GO-catalyzed oxidation reactions of â-D-1-O-hydroxyl-
amino terminal galactose proceeded readily at room tem-
perature. The corresponding rates of reaction for â-D-1-O-
hydroxylamino lactose, melibiose, and â-D-gal(1f3)-D-
arabinose were found to be slower under the same conditions.
As was expected, polymerization took place for all â-D-1-
O-hydroxylamino compounds; the progress was monitored
via TLC. As the generation of the aldehyde proceeded, a
distinctly new spot began to materialize at the base of the
silica gel TLC plate, while the color concentration of the
Table 1. GO-Mediated Polymerization of
â-D-1-O-hydroxylamino Sugars
entry
monomer
yield (%)
Mw
Mw/Mnd
1a
2a
3a
1-ONH2 Gal
1-ONH2 Lac
1-ONH2 Mel
1-ONH2-Gal-Ara
1-ONH2 Gal
1-ONH2 Lac
1-ONH2 Gal
1-ONH2 Lac
62
44
12
47
51
38
60
42
4200
7800
4500
7300
4000
7500
5100
8900
1.7
1.9
2.2
2.4
1.9
1.8
2.0
2.1
4a
5b
6b
7c
starting material faded (solvent system i-PrOH/H
2
O/(28-
1
8c
3
0%)NH OH:7/3/2). For comparison, Figure 1 shows the H
4
a
b
Reactions conducted in phosphate buffer (50 mM, pH 7.0). Reactions
conducted in phosphate buffer (50 mM, pH 5.5). c Resulting polymers from
method a were stirred in 0.20 M acetic acid for 24 h. d Polydispersity where
Mn is the number average molecular weight.
polymer from the reaction of â-D-1-O-hydroxylamino lactose
6
(entry 2) in the phosphate buffer (pH 7.0) has a molecular
weight (MW) of 7800. The corresponding reaction of â-D-
-O-hydroxylamino galactose 5 (entry 1), however, gave a
1
polymer with a lower molecular weight (MW) of 4200. Since
it is known that acidic conditions promote oxime formation,
we performed selective GO reactions in a buffer at pH 5.5
only to obtain lower molecular weight polymers. This was
attributed to the fact that optimal GO oxidation conditions
are conducted in a 50 mM phosphate buffer at pH 7.0.
However, slightly improved results were obtained by stirring
the resulting mixture in an acidic solution (0.2 M HOAc)
for a prolonged time (24 h), entries 7 and 8 (Table 1).
These novel C-6 oxime-linked polymers show a fair
amount of monomeric carbohydrate unit polymerization. For
instance, in the galactose example (entry 1), the resulting
polymer contains a MW of 4200, which corresponds to the
polymerization of 24 galactose units. For lactose (entry 2),
the resulting MW of the polymer is 7800, which translates
to 23 monomeric lactose units. The number of substituted
monomeric units increases when the resulting polymers are
allowed to stir in 0.20 M acetic acid (entries 7 and 8) from
Figure 1. 1H NMR of the starting â-D-1-O-hydroxylamino sugars
galactose and lactose) and the resulting polymers. Note the syn
(
and anti peaks in the 6.0-8.0 ppm region. (a) â-D-1-ONH
2
2
galactose, (b) oxime Gal-polymer, (c) â-D-1-ONH lactose, (d)
oxime lactose-polymer.
NMR spectra of the starting material and isolated products
from polymerization reactions of 5 and 6. The spectra of
the products show significant differences from those of the
starting materials. In addition to completely different display
patterns, all the peaks have been considerably broadened.
The key features of the spectra are the two signals in the
region from 6.90 to 8.00 ppm, which belong to the proton
on the oxime carbon. It is possible to distinguish between
syn and anti isomers since the H-CdN shift is upfield in
the sterically more compressed form, and the more hindered
substitute (syn to the OR) is upfield to the less hindered
2
3 to 26 for both entries. These values indicate the same
number of monomeric units form the oxime-linked polymer
regardless of monosaccharide or disaccharide carbohydrates.
HPLC of the retentate material gave the calculated M /M
w n
(polydispersity) values indicated in Table 1. If polymers of
higher molecular weights are desired, the number of con-
densing chains must be smaller, so that the polymerization
reaction rate is slower and can become similar to the thermal
polymerization rate, thus broadening the polydispersity.
22
Polymers were subjected to decomposition conditions and
were found to be stable at pH 2, 25 °C (1 M HOAc/NaOAc)
(
anti).
The molecular weights of the resulting polymers were
determined by HPLC size exclusion chromatography using
2 4
and at pH 11, 25 °C (1 M Na HPO ).
In summary, we have demonstrated that GO-mediated
selective oxidation of â-D-1-O-hydroxylamino galactose and
other â-D-1-O-hydroxylamino sugars containing a galactose
terminus resulted in the formation of carbohydrate polymers,
which contain oxime linkages between the C-6 of galactose
21
dextran standards. As summarized in Table 1, the resulting
(21) (a) Dubin, P. L. Carbohydr. Polym. 1994, 25, 295-303. (b) Hester,
R. D.; Lundy, C. E. Polym. Mater. Sci. Eng. 1986, 54 190-193. (c) Rollings,
J. E.; Bose, A.; Caruthers, J. M.; Okos, M. R.; Tsao, G. T. Polym. Prepr.
(22) Polymers were heated to 100 °C at pH 2.0 and 11 for 2 h. At pH
1
981, 22, 294-295.
7.0 the polymer did not decompose when heated.
Org. Lett., Vol. 4, No. 11, 2002
1865

and the anomeric carbon, either of the same galactose moiety
or of other sugar units (D-lactose, â-gal-D-arabinose, and
D-melibiose). The GO-mediated polymerization, under en-
vironmentally benign conditions, provides a unique and
convenient way for the synthesis of nonnatural polysaccha-
rides with novel structures, closely related to those found in
nature.
P.G.W. acknowledges a research grant from Hercules
Incorporated.
Supporting Information Available: Experimental details
for the syntheses of compounds in Scheme 2, steps a-c, as
well as the reaction conditions for the GO-mediated polym-
erization. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. P.R.A. acknowledges a summer
2001 graduate fellowship from Wayne State University and
OL0258379
1866
Org. Lett., Vol. 4, No. 11, 2002