Versatile Functionalization of Polylysine
J. Am. Chem. Soc., Vol. 121, No. 25, 1999 5921
Scheme 1
process is of pharmaceutical interest because chronic expression
occurs in certain inflammatory conditions, such as psoriasis and
rheumatoid arthritis. Acute disorders such as reperfusion injury
or asthma caused by excessive leukocyte recruitment represent
1
9
another important target. Several recent reports suggest that
multivalency plays an important role in the inflammatory
2
0
response. To probe this, we required a reliable access to
x
multivalent sLe ligands. We were also interested in well-
x
a
characterized glycopolymers containing sLe or sLe and biotin
as a functional group for competitive selectin binding assays,
allowing the determination of IC50 values of potential selectin
antagonists.
reaction times and elevated temperatures were applied. More-
over, partial hydrolysis to unwanted carboxylic acid functions
in the polymer cannot be avoided completely. Polylysine has
been functionalized directly. Michael addition to a carbohydrate
containing an acrylamide residue gave a highly charged gly-
In this paper, we present a novel, reliable, and reproducible
access to glycopolymers. Commercially available polylysine 3
2
1
is converted into its chloroacetamide 4. This new, DMF-
soluble and reactive homopolymer can easily be transformed
into water-soluble homopolymers by treatment with hydrophilic
thiols. Glycopolymers with predeterminable carbohydrate con-
tent are available by reaction with substoichiometric amounts
of saccharides containing a mercapto substituent, followed by
treatment with an excess of thioglycerol to cap the remaining
1
6
copolymer with more than 80% of free amino functions.
Neither capping nor further functionalization of the amino
groups was reported. Coupling of polylysine and carbohydrates
with carboxylic acid functions has also been reported, but the
carbohydrate incorporation was incomplete.17
22
Our interest in such glycopolymers arose from our involve-
chloroacetamide groups. The scope of the method is explored
1
8
x
a
ment in studying the selectin oligosaccharide recognition. E-
and P-selectins are expressed on endothelial cells upon stimula-
tion and mediate the recruitment of leukocytes to sites of injury
by preparing sLe - and sLe -glycopolymers with up to four
different components. The appropriate characterization of the
products is described in detail. Furthermore, the use of such
conjugates as multivalent receptor blockers and multivalent
ligands for assay development is discussed.
x
x
or infection, recognizing sialyl Lewis (sLe )-related carbohy-
drate epitopes on the leukocyte surface.19 Control over this
(
14) Acryl polymer 2 was obtained from Synthesome, Gesellschaft fuer
Results and Discussion
medizinische Biochemie mbH, Heimdall Str. 4, D-81739 Muenchen,
Germany. H NMR spectroscopy (500 MHz, D2O, 60 °C) showed that the
1
Acylation of Polylysine Hydrobromide. Polylysine of
different well-characterized, narrow molecular weight fractions
is commercially available in the L, D/L, and D forms. This
polymer is nonimmunogenic and biodegradable and has already
integral per proton of the ethanolamine side chain has approximately 90%
of the value per proton of the polymer backbone, indicating 90%
incorporation of ethanolamine. Accordingly, compound 2 contains 10% of
carboxylic acid functions. Additional evidence for the presence of unwanted
carboxylic acid functions in compound 2 was obtained as follows:
Compound 2 was dissolved in 0.01 M HCl to transform carboxylates into
the free carboxylic acid functions, followed by repeated ultrafiltration until
the eluted solution became neutral. An ecxess of N,N-dimethylaminopyridine
23
been explored as a drug carrier. The polylysine hydrobromides
used in this work were purchased from Sigma and had a
3
molecular weight Mw of approximately 50 000 (Table 2). We
intended to transform poly-L-lysine hydrobromide (L-3)24 into
the per-N-chloroacetyl derivative L-4 in order to subsequently
modify this reactive homopolymer by thiol substitution (Scheme
(
DMAP, 5.0 equiv) was added to form the corresponding carboxylic acid
salt. The solution was resubjected to ultrafiltration until the elute became
neutral. Following lyophilization, the compound was analyzed by H NMR
1
(400 MHz, D2O). A singlet at 3.10 ppm (6 H of DMAP) and two doublets
at 6.80 and 7.95 ppm (2 H of DMAP each) indicated 8% DMAP with respect
to the polymer backbone. Accordingly, 2 contains at least 8% of carboxylic
acid functions. Within the experimental error,the sum of the integrals per
proton of the ethanolamine side chain and of DMAP equaled the integral
per proton of the polymer backbone. Similarly, commercially available
glycopolymers are prepared by converting polyacrylate 2 first with
substoichiometric amounts of carbohydrates (DMF, NEt3, 40 °C, 24 h),
followed by treatment with ethanolamine (see ref 12a). We have evidence
that such compounds can contain even more carboxylic acid residues
2
). A suspension of L-3 in a 3:1 mixture of DMF and 2,6-
lutidine was treated with chloroacetic anhydride. Formation of
a clear solution indicated the consumption of L-3. The product
L-4 was isolated in 98% yield by precipitation. The compound
is insoluble in water but readily dissolves in DMF and DMSO.
(20) (a) Berg, E. L.; Robinson, M. K.; Mansson, O.; Butcher, E. C.;
Magnani, J. L. J. Biol. Chem. 1991, 266, 14869. (b) Welply, J. K.; Abbas,
S. T.; Scudder, P.; Keene, J. L.; Broschat, K.; Casnocha, S.; Gorka, C.;
Steininger, C.; Howard, S. C.; Schmuke, J. J.; Graneto, M.; Rotsaert, J.
M.; Manger, I. D.; Jacob, G. S. Glycobiology 1994, 4, 259. (c) Spevak,
W.; Foxall, C.; Charych, D. H.; Dasgupta, F.; Nagy, J. O. J. Med. Chem.
1996, 39, 1018. (d) Lin, C.-C.; Kimura, T.; Wu, S.-H.; Weitz-Schmidt, G.;
Wong, C.-H. Bioorg. Med. Chem. Lett. 1996, 6, 2755. (e) Stahn, R.;
Schaefer, H.; Kernchen, F.; Schreiber, J. Glycobiology 1998, 8, 311.
(21) Chloroacetylation of lysine dendrimers and subsequent functional-
ization with cystein-containing peptides has been described earlier. The
resulting dendrimers contain only one type of active residue (up to eight
end groups). Zhang, L.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363.
(22) In a preliminary communication, we have described the preparation
(
>25%).
(
15) (a) Stahl, W.; Ahlers, M.; Walch, A,; Bartnik, E.; Kretzschmar, G.;
Grabley, S.; Schleyerbach, R. Eur. Pat. Appl. 0601417A2, 1992. (b) Thoma,
G.; Ernst, B.; Schwarzenbach, F.; Duthaler, R. O. Bioorg. Chem. Med. Lett.
1
997, 7, 1705.
16) Romanowska, A.; Meunier, S. J.; Tropper, F. D.; Laferri e` re, C. A.;
Roy, R. Methods Enzymol. 1994, 242, 90.
(
(
17) (a) Monsigny, M.; Roche, A.-C.; Midoux, P.; Mayer, R. AdV. Drug
DeliVery ReV. 1994, 14, 1 and references therein. (b) Mahato, R. I.;
Takemura, S.; Akamatsu, K.; Nishikawa, M.; Takakura, Y.; Hashida, M.
Biochem. Pharmacol. 1997, 53, 887.
(18) (a) Scheffler, K.; Ernst, B.; Katopodis, A.; Magnani, J. L.; Wang,
W, T.; Weisemann, R.; Peters, T. Angew. Chem., Int. Ed. Engl. 1995, 34,
a
1841. (b) Thoma, G.; Schwarzenbach, F.; Duthaler, R. O. J. Org. Chem.
1996, 61, 514. (c) Baisch, G.; Oehrlein, R. Angew. Chem., Int. Ed. Engl.
1996, 35, 1812. (d) Jahnke, W.; Kolb, H. C.; Blommers, M. J. J.; Magnani,
of a sialyl Lewis polymer: Thoma, G.; Magnani, J. L.; Oehrlein, R.; Ernst,
B.; Schwarzenbach, F., Duthaler, R. O. J. Am. Chem. Soc. 1997, 119, 7414.
(23) Acylated poly-L-lysine derivatives have been used as drug carriers.
Acylation was necessary to avoid both toxicity and nonspecific binding to
mammalian cells caused by the polycationic properties. (a) N e` gre, E.;
Monsigny, M.; Mayer, R. Tetrahedron 1993, 49, 6991. (b) Gonsho, A.;
Irie, K.; Susaki, H.; Iwasawa, H. Biol. Pharm. Bull. 1994, 17, 275.
(24) In this paper, poly-L-lysine derivatives are designated as L, poly-
D/L-lysine derivatives are designated as D/L, and poly-D-lysine derivatives
are designated as D. Polymer identification numbers without an extension
do not specify the configuration.
J. L.; Ernst, B. Angew. Chem., Int. Ed. Engl. 1997, 36, 2603. (e) Norman,
K.; Andersson, G. P.; Kolb, H. C.; Ley, K.; Ernst, B. Blood 1998, 91, 475.
(
19) (a) Welply, J. K.; Keene, J. L.; Schmuke, J. L.; Howard, S. C.
Biochim. Biophys. Acta 1994, 1197, 215. (b) Springer, T. A. Cell 1994, 76,
01. (c) Kansas, G. S. Blood 1996, 88, 3259. (d) Cines, D. B.; Pollak, E.
3
S.; Buck, C. A.; Loscalzo, J.; Zimmermann, G. A.; McEver, R. P.; Pober,
J. S.; Wick, T. M.; Konkle, B. A.; Schwartz, B. S.; Barnathan, E. S.; McCrae,
K. R.; Hug, B. A.; Schmidt, A.-M.; Stern, D. M. Blood 1998, 91, 3527.