No. 10] Proc. Japan Acad. , 73, Ser.

B (1997) 205

Synthesis of Glycopeptide-conjugates via Ring-opening Polymerization of

Sugar-substituted a-Amino Acid N-Carboxyanhydrides (GlycoNCAs)
By Masahiko OKADA,t~ Keigo Aol, and Kaname TSUTSUMIUCHI

Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University

Furo-cho, Chikusa-ku, Nagoya 464-01

(Communicated by Seizo OKAIURA,M.J.A., Dec. 12, 1997)

Abstract : This article briefly reviews our recent investigation on the synthesis and biological functions of
artificial glycopeptide-conjugates via ring-opening polymerization of 1)-glucose- or N-acetyl-D-glucosamine-
substituted L-serine N-carboxyanhydrides (glycoNCAs). Primary amine-initiated polymerization of glycoNCAs
proceeded without side reactions to give linear glycopeptide-conjugates of controlled chain lengths. A variety of
block copolymers and graft copolymers containing glycopeptide segments were synthesized by utilizing the
living nature of the ring-opening polymerization of glycoNCAs. Reaction of primary amine-terminated
poly(amido amine) dendrimers of different generations with a slight excess of glycoNCAs at -30°C gave a new
type of dendrimers whose surfaces were modified with the corresponding mono(glycopeptide) moieties.
Oligomerization of glycoNCAs with poly(amido amine) dendrimer as a multifunctional macroinitiator at 27°C
provided globular macromolecules coated with oligo(glycopeptide) chains. Molecular recognition abilities of
these linear and globular glycopeptide-conjugate macromolecules were evaluated by the hemagglutination
inhibition assay.

Key words : a-Amino acid N-carboxyanhyd rides; glycopeptide; dendrimer; ring-opening polymeriza-
tion; macroinitiator; molecular recognition.

Introduction. Glycoconjugates such as glycopro-
teins and glycolipids are widely distributed in nature, e.g.,
in intra- and extracellular liquids, cell membranes, cell
walls, connective tissues and blood. The glycochain parts
in these glycoconjugates play essential roles in diverse
biological functions such as intercellular communication,
transportation of proteins from one part to another of a
cell, and rendering proteins resistant to enzymatic
degradation but allowing them to be recognized by certain
protein receptor. 1)2)
Synthetic strategies for glycoconjugates, in particular
glycopeptides, are more complex and laborious compared
with the syntheses of oligopeptides or oligosaccharides.
With the advent of precise synthetic methodologies in
glycopeptide chemistry,3~'4~ a number of artificial glyco-
conjugates having functions similar to those of naturally
occurring glycoproteins and glycolipids have been designed
and synthesized so far.5~'h~The purposes of synthesizing
artificial glycoconjugates are not only to prepare structural-
ly well-defined models for biochemical and immunochemi-
t) Correspondence to: M. Okada.
cal studies but also to create polymeric materials
possessing sophisticated properties characteristic of
naturally occurring glycoconjugates.
In this article, we describe briefly our recent
investigation on the synthesis and biologicalfunctions of
linear and globular glycopeptide-conjugate macro-
molecules, based on the ring-opening polymerization of
sugar-substituted a-amino acid N-carboxyanhydrides
(hereafter referred to as glycoNCAs). Our work aims at
developing new functional polymeric materials applicable
for biochemical and biomedical purposes.
Synthesis of linear glycopeptide-conjugates
by ring-opening polymerization of glycoNCAs.
Homopolymerizationof GlycoNCAs. Structurally simple
polypeptides consisting of only one kind of a-amino acid
residues can be conveniently synthesized by anionic ring-
opening polymerizationof NCAs.7~~y~ Althougha methodol-
ogy for synthesizing glycoNCAswas established by Rude
et al. 10)a long time ago, ring-opening polymerization of
glycoNCAs had never been reported until we presented
our first paper on the ring-opening polymerization of a
glucose derivative-substituted L-serine NCA (1, X= OAc)
206 M. OKADA, K.
in 1994.11)
0-glycosylated serine moieties of naturally occurring
glycoproteins plays a significant role in various life
phenomena. For example, an N-acetyl-D-glucosamine
(G1cNAc)-linked serine part appears to be highly dynamic
and responsive to cellular stimuli in a fashion analogous to
phosphorylation.12) This is one of the reasons why we
chose 0-glycosylated L-serine NCAs as monomers for
glycoconjugate macromolecular synthesis by ring-opening
polymerization.
Anionic ring-opening polymerization of glycoNCAs 1
was carried out in dichloromethane or acetonitrile at 25°C
by using n-hexylamine as an initiator (Scheme 1). 11)13)In
contrast to the general tendency that anionic polymeriza-
tion of NCAs carrying a bulky substituent is often
accompanied by side reactions such as hydantoic acid
formation, glycoNCAs 1 undergo anionic polymerization
without side reactions, irrespective of the bulky sugar
moiety, to give the corresponding polypeptides 2 of a
narrow molecular weight distribution. Spectroscopic
analysis of 1 showed a hydrogen bonding between the
hydrogen atom on the ring nitrogen and the oxygen atom of
the acetyl group at either 0(2) or 0(6) of the sugar
residue. It seems, therefore, very likely that such a
hydrogen bonding retards deprotonation from the NCA
ring, thus preventing the occurrence of side reactions. 14)
The deacetylation of 2 with hydrazine in methanol at 0°C
gave poly(L-serine)s (3) with controlled chain lengths
having a 13-D-glucose or GIcNAc unit in each repeating
unit. Neither racemization of the asymmetric carbons nor
the cleavage of the glucosidic linkages occurred during the
deacetylation.
Glycopeptide block copolymers. The living nature of the
primary amine-initiated polymerization of glycoNCAs was
further demonstrated by the AB-type block copolymer
synthesis using a "one pot two-stage feeding" technique.
Thus, after completion of the first-stage polymerization of
glycoNCA 1 (X=OAc), the second monomer, L-alanine
NCA, was added to the reaction mixture. The second-
Aoi, and K. TSUTSUMIucHI [Vol. 73(B),
stage polymerization was initiated by the propagating
amino end group of the A-block of the first monomer to
produce the AB-type block copolymer, which on deacetyla-
tion gave poly(glycopeptide)-polyalanine block copolymer
(4) 11)
Synthetic homopolypeptides obtained by the NCA
method are generally of limited solubility, and hence
difficult to handle, due to strong intrachain or interchain
hydrogen bonds. To enhance their tractability, conjugation
of polypeptides with other synthetic polymers has been
undertaken by block- or graft-copolymerizations.15) We
have synthesized various block and graft copolymers
containing poly(glycopeptide) segments. Some typical
examples are illustrated in Scheme 2.
2-Alkyl- or 2-aryl-substituted-2-oxazolines undergo
cationic ring-opening polymerization to give living poly(2-
alkyl- or 2-aryl-substituted-2-oxazoline)s with an oxazoli-
nium ion at the growing chain end. 16)By utilizing the living
character of the ring-opening polymerizations of both
NCAs and 2-oxazolines, we synthesized a glycopeptide-
containing block copolymer by mutual termination of the
respective living polymerizations of glycoNCA 1(X=OAc)
and 2-methyl-2-oxazoline.1 `) The block copolymer was
deacetylated by hydrazine under mild conditions to yield
the corresponding poly(glycopeptide)-polyoxazoline block
copolymer (5). As expected, the block copolymer is
soluble in methanol, dimethylformamide, and dimethyl
sulfoxide, although poly[0-(1 -D-glucopyranosyl)-L-serine]
(3, X=OH) was insoluble in these solvents.
Alternatively, poly(glycopeptide)-polyoxazoline block
copolymers were synthesized by the polymerization of 1
(X=OAc) using c)-amine-terminated poly(2-methyl-2-ox-
azoline) or poly(2-phenyl-2-oxazoline) macroinitiators.14)
The degrees of polymerization of polypeptide segments of
the resulting block copolymers were close to the
monomerlmacroinitiator feed molar ratios, indicating
nearly quantitative initiation efficiency. 14)
Graft copolymers with glycopeptide branches. Graft
copolymers with controlled glycopeptide branch lengths
No. 10] Glycopeptide Conjugates via
Scheme
were synthesized by the macromonomer method. Gly-
copeptide macromonomers 6 were prepared by the ring-
opening polymerization of glycoNCAs using p-vinylbenzyl
amine as an initiator, followed by deacetylation. 18) The
terminal vinyl group was not affected during the deacetyla-
tion. Water-soluble glycoconjugates 7 having glycopeptide
branches were prepared by copolymerization of the
glycopeptide macromonomer 6 with acrylamide.18) The
same graft copolymer was also synthesized by the
copolymerization of the acetyl-protected macromonomer
with acrylamide, followed by deacetylation of the resulting
copolymer.
Polymerization of glycoNCA 1 with an a-styryl-type
poly(2-methyl-2-oxazoline) macromonomer gave a block
copolymer-type macromonomer consisting of a poly(2-
methyl-2-oxazoline) segment and a sugar-bearing poly(L-
serine) segment. Radical copolymerization of the macro-
monomer with acrylamide gave a graft copolymer having
glycopeptide branches with a poly(2-methyl-2-oxazoline)
spacer.
Synthesis of globular glycopeptide-conju-
gates based on poly(amido amine) dendrimers.
Sugarpersubstituted dendrimers, "Sugar Balls". Naturally
occurring multiantennary oligosaccharides show a much
higher affinity to the multisubunits receptors than simple
monosaccharide-receptor binding. A proper geometric
arrangement of terminal sugar residues of natural
multiantennary oligosaccharides appears to be a prere-
quisite for their highly specific molecular recognition
ability.19)Taking this into consideration, we have proposed
Polymerization of GlycoNCAs 207
2
a new concept of intersugar space-regulated globular
glycoconjugate macromolecular design using a dendrimer
skeleton, 20)
Lactose and maltose derivative-persubstituted
poly(amido amine) dendrimers of the third to fifth
generations (hereafter indicated as G=3.0-5.0) were
successfully synthesized by the reaction of the amine
terminated poly(amido amine) dendrimers of the corres-
ponding generations with an excess amount of unprotected
lactonolactone and maltonolactone in dimethyl sulfoxide at
27 and 40°C, respectively.20) We named these sugar-
persubstituted dendrimers "Sugar Balls", because the
surface of the globular poly(amido amine) dendrimers is
covered with sugar residues covalently bonded to the
dendrimer skeleton. Recently, different synthetic methods
for sugar-modified dendrimers have been proposed by Roy
et al. 21),22)and Stoddart et al. 23),24)
Glycopeptide-type sugar balls. As described in the
previous section, the primary amine-initiated ring-opening
polymerization of glycoNCAs has two important features:
First, the polymerization proceeds without side reactions
to yield polypeptides of controlled chain lengths. Secondly,
the initiation is much faster than the propagation. These
features prompted us to use poly(amido amine) dendrimer
as a multifunctional macroinitiator for the polymerization of
glycoNCAs to construct glycopeptide-type sugar balls.
The ring-opening oligomerization of glycoNCAs 1 was
carried out in chloroform at 27°C by using primary amine-
terminated poly(amido amine) dendrimer (8, ethylene-
diamine core, G=3.0-5.0) as an initiator to give acetyl-
208 M. OKAVA, K. Aol,
Scheme
protected glycopeptide-type sugar balls in quantitative
yields (Scheme 3).25)The MWIMnvalues determined by
the size exclusion chromatography (SEC) were 1.03-1.11,
reflecting a rapid initiation/slow propagation system.
This point was clearly confirmed by the experiment
carried out at -30°C for 15 min with a molar ratio of
glycoNCA 1 (X=NHAc) to the primary amino group of
1.18. The MW/MRvalue was close to that of the dendritic
macroinitiator 8 (G=3.0, MW/Mn=1.02).26)The molecular
weight (1.92x104) determined by vapor pressure
osmometry agreed well with the calculated value
(1.92x104). These results together with spectroscopic
data indicate that each primary amino group on the
dendrimer surface reacted with a molecule of glycoNCA1
to give a mono(glycopeptide)-typesugar ball precursor (9,
G=3.0, n=1.0), and that the initiation of the ring-opening
polymerizationof the glycoNCA 1 is definitely faster than
the propagation.
Deacetylation of the sugar moieties of 9 by hydrazine
monohydrate in methanol at room temperature gave
glycopeptide-type sugar balls 10. The sugar balls 10 are
soluble in dimethyl sulfoxide and water, while linear
oligo(glycopeptide)3 of a similardegree of polymerization
is not soluble in the former solvent.
Biological functions of glycopeptide-conju-
gates. Molecular design of artificialglycoconjugateshas
and K. TsuTsuMIUCHI [Vol. 73(B),
3
been primarily directed toward development of new
polymeric materials exhibiting a strong and specific affinity
to lectins and cells. The binding ability of sugar balls
carrying glucose or galactose residues on the surface with
concanavalin A (Con A) was investigated by quantitative
precipitation experiments and by a competitive binding
method.20> As expected, Con A which specifically
recognizes D-glucose and D-mannose residues, bound only
D-glucose-covered sugar ball. A 1200 fold molar excess of
D-glucose was needed to disrupt this binding interaction.
Wheat germ agglutinin (WGA) is known to have
several sugar binding sites, which specifically recognizes
N-acetyl-D-glucosamine and its oligomers. Extracellular
carbohydrate moieties of erythrocyte interact with the
lectin to make agglutination. We examined inhibition
activities of multivalent glycopeptide-carrying polyacryla-
mide graft copolymer 7 against erythrocyte agglutination
by WGA lectin.18) In the hemagglutination inhibition assay,
N-acetyl-D-glucosamine itself did not inhibit aggregate
formation between erythrocyte and WGA up to the N-
acetyl-D-glucosamine concentration of 2.3 x 10-2 mol/L.
On the other hand, the minimum sugar concentration of the
graft copolymer 7 containing N-acetyl-D-glucosamine units
in its branches to inhibit hemagglutination was 4.9x 10 _ 5
mol/L. The efficient interaction between 7 and WGA is
apparently ascribable to the multivalency of the sugar
No. 10] Glycopeptide Conjugates via Polymerization
moieties of 7, i.e., the so-called cluster effect.
Our recent experiment showed that the minimum
sugar concentrations of the mono(glycopeptide)-type sugar 6)
balls 10 (X=NHAc, n=1) of the third to sixth generations
to inhibit hemagglutination by WGA were as low as
7)
7.6x 10-7-2.0 x 10h mol/L, much lower than those for
the graft copolymer 7 described above. This finding clearly 8)
demonstrates that the proper geometrical arrangement of
the recognition sites on the surface of the sugar balls is 9)
indispensable for highly specific molecular recognition.
Concluding remarks. We have described the
outline of our recent investigation on the synthesis and 10)
biological functions of linear and globular artificial
11)
glycopeptide-conjugates via ring-opening polymerization of
glycoNCAs 1. These glycopeptide-conjugates, particularly 12)
globular ones, are not only of scientific significance but also
of practical importance as functional materials. Poly(amido
amine) dendrimers have hollow cavities inside, which can 13)
accommodate low molecular weight compounds. Since
sugar moieties on the surface of globular macromolecules 14)
act as recognition sites, sugar balls which can encapsulate
15)
drug molecules in their internal cavities may be promising
candidate materials for target-directing drug delivery
systems. Actually, we found that glycopeptide-type sugar 16)
balls of the fifth and sixth generations encapsulated low
molecular weight compounds effectively.27~ Furthermore,
preliminary experiments have shown that glycopeptide- 17)
type sugar balls 10 form ion complexes with DNA,
suggesting that they may be used as a DNA carrier. `8) 18)
These findings encourage us to make further efforts to
19)
create intelligent materials applicable for biochemical and
biomedical purposes from artificial glycopeptide-conju-
20)
gates.
Acknowledgment. The authors sincerely thank 21)
our coworkers, K. Itoh, E. Aoki, A. Yamamoto, and H.
Noda for their continuous collaboration. The present work 22)
was financially supported in part by Grant-in-Aid (Nos. 23)
08246106 and 09450349) from the Ministry of Education,
Science, Sports, and Culture of Japan.
24)
References
1) Lee, Y. C., and Lee, R. T. (1992) In Glycoconjugates (eds. 25)
Allen, H. J., and Kisailus, E. C.). Marcel Dekker, New York,
26) Aoi, K., Tsutsumiuchi, K., Yamamoto, A., and Okada, M.
pp. 121-165.
2) Dwek, R. (1996) Chem. Rev. 96, 683-720.
3) Garg, H. G., von dem Bruch, K., and Kunz, H. (1994) Adv. 27) Tsutsumiuchi, K., Aoi, K., and Okada, M. (1997) Polym.
Carbohydr. Chem. Biochem. 50, 277-310.
4) Wong, C.-H., Halcomb, R. H., Ichikawa, Y., and Kajimoto, T. 28) Aoi, K., Yamamoto, A., Tsutsumiuchi, K., Okada, M., Kato,
(1995) Angew. Chem. Int. Ed. Engl. 34, 412-432 and
521-546.
5) Lee, R. T., and Lee, Y. C. (1994) In Neoglycoconjugates:
of GlycoNCAs 209
Preparation and Applications (eds. Lee. Y. C., and Lee, R.
T.). Academic Press, San Diego, pp. 23-50.
Okada, M. (1996) In Polymeric Materials Encyclopedia (ed.
Salamone, J. C.). CRC Press, Boca Raton, vol. 4, pp.
2834-2840.
Imanishi, Y. (1984) In Ring-Opening Polymerization (eds. Ivin,
K. J., and Saegusa, T.). Elsevier, London, pp. 523-602.
Kricheldorf, H. K. (1987) a-Amino Acid N-Carboxyanhydrides
and Related Heterocycles. Springer, Berlin, pp. 59-209.
Kricheldorf, H. R. (1989) In Comprehensive Polymer Science
(eds. Eastmond, G. C., Ledwith, A., Russo, S., and Sigwalt,
P.). Pergamon, Oxford, vol. 3, pp. 531-551.
Rude, E., Westphal, 0., Hurwitz, E., Fuchs, S., and Sels, M.
(1966) Immunochemistry 3, 137-151.
Aoi, K., Tsutsumiuchi, K., and Okada, M. (1994) Macro-
molecules 27, 875-877.
Haltiwanger, R. S., Kelly, W. G., Roquemore, E. P.,
Blomberg, M. A., Dong, L.-Y. D., Kreppel, L., Chou, T.-Y.,
and Hart, G. W. (1992) Biochem. Soc. Trans. 20, 264-269.
Okada, M., and Aoi, K. (1995) Macromolecular Report A32,
Suppls. 5 and 6, 907-914.
Tsutsumiuchi, K., Aoi, K., and Okada, M. (1997) Macro-
molecules 30, 4013-4017.
Okada, M. (1994) In Macromolecular Design. Concept and
Practice (ed. Mishra, M. K.). Polymer Frontier Internation-
al, New York, pp. 359-389.
Kobayashi, S., and Saegusa, T. (1984)
.In Ring-Opening Poly-
merization (eds. Ivin, K. J., and Saegusa, T.). Elsevier,
London, pp. 761-807.
Tsutsumiuchi, K., Aoi, K., and Okada, M. (1995) Macromol.
Rapid Commun. 16, 749-755.
Aoi, K., Tsutsumiuchi, K., Aoki, E., and Okada, M. (1996)
Macromolecules 29, 4456-4458.
Lee, Y. C., and Lee, R. T. (1995) Acc. Chem. Soc. 28,
321-327.
Aoi, K., Itoh, K., and Okada, M. (1995) Macromolecule 28,
5391-5393.
Zanini, D., Park, W. K. C., and Roy, R. (1995) Tetrahedron
Lett. 36, 7383-7386.
Zanini, D., and Roy. R. (1996) J. Org. Chem. 61, 7348-7354.
Ashton, P. R., Boyd, S. E., Brown, C. L. N., Jayaraman, N.,
Nepogodiev, S. A., and Stoddart, J. F. (1996) Chem. Eur. J.
2, 1115-1128.
Ashton, P. R., Boyd, S. E., Brown, C. L., Nepogodiev, S. A.,
Meijer, E. W., Peerlings, H. W. I., and Stoddart, J. F. (1997)
Chem. Eur. J. 3, 974-984.
Aoi, K., Tsutsumiuchi, K., Yamamoto, A., and Okada, M.
(1997) Tetrahedron 53, 15415-15427.
(1997) Macromol. Rapid Commun. (in press).
Preprint, Japan 46, 2876-2877.
M., and Tsukagoshi, N. (1997) Polym. Preprint, Japan 46,
746.