12210
J. Am. Chem. Soc. 1999, 121, 12210-12211
8
Methylated Mono- and Diethyleneglycol
Functionalized Polylysines: Nonionic, r-Helical,
Water-Soluble Polypeptides
poly(L-glutamate esters) (eq 1). Although this aminolysis reaction
Miaoer Yu, Andrew P. Nowak, and Timothy J. Deming*
9
resulted in significant polymer chain cleavage, nearly all the side
Departments of Materials and Chemistry
UniVersity of California, Santa Barbara
Santa Barbara, California 93106
chains could be functionalized and the resulting polymers were
found to be water soluble. However, studies on their helical
structure were limited since these polymers contained substantial
random coil content when dissolved in water. The best example,
poly(N-hydroxybutyl-L-glutamine), PHBG, is ca. 65% helical in
neutral water at 20 °C.8b To date, an entirely helical, nonionic
water-soluble, high molecular weight polypeptide has not been
developed.
Darrin J. Pochan
Department of Materials Science and Engineering
UniVersity of Delaware, Newark, Delaware 19716
There have been many attempts to use poly(N-hydroxyalkyl-
L-glutamines) in biomedical applications; however, they are
recognized as foreign and rapidly degraded in vivo.1 Thus, these
materials are useful only where fast erosion is desired and are
ineffective at imparting protection from biological attack (i.e.
biocompatability). Most biocompatability strategies employ PEG,
which is typically grafted onto other polymers, including polypep-
tides, to improve their properties in vivo. PEG is nonionic, water-
soluble and, most importantly, not recognized by immune
systems.12 It is believed that PEG imparts biocompatability
through formation of a hydrated “steric barrier” at the surface of
a material that cannot be penetrated or recognized by biological
molecules.12 As such, block or graft copolymer drug carriers
containing PEG are able to circulate for long periods in the
bloodstream without degradation.13
ReceiVed October 11, 1999
0
Polypeptides have been studied for use in biomedical applica-
tions for some time. Applications such as drug delivery typically
require water-soluble components to enhance their ability for
circulation in vivo. The problem with common water-soluble
polypeptides (e.g. poly-L-lysine and poly-L-aspartate) is that they
are polyelectrolytes that display pH-dependent solubility and
limited circulation lifetime due to aggregation with oppositely
1
2
11
3
charged biopolymers. Nonionic, water-soluble polypeptides are
desired for biomedical applications since they avoid these
problems, and can also display the stable secondary structures of
proteins that influence biological properties. However, in contrast
4
to short peptides (<25 residues), all nonionic homopolypeptides
derived from naturally occurring amino acids are notoriously
insoluble in water.1,5 We have developed methylated mono- and
We wanted to incorporate the attractive properties of PEG into
14
polypeptides. Incorporation of short ethyleneglycol (EG) repeats
diethyleneglycol-functionalized polylysines that are the first
example of nonionic, water-soluble, high molecular weight
polypeptides, which are completely R-helical in solution. These
exceptionally stable helices are also resistant to proteases similar
to pure poly(ethylene glycol), PEG. These polypeptides are new
rodlike PEG” building blocks that can be used to incorporate
biochemical stability, self-assembly, and water solubility into
polypeptides.
onto amino acid monomers was pursued as opposed to the well-
documented approach of grafting PEG to the ends or side chains
of polypeptides.12 Our strategy avoids the need for expensive
amino- or carboxylato-functionalized PEG molecules necessary
for coupling, which typically must be short (less than 5000 Da)
to ensure high functionalization. Furthermore, the presence of
short EG repeats on every amino acid side chain should result in
a high density of EG around the polymer chain. Whitesides et al.
have shown that surfaces coated with a high density of short
chains contaning as few as two EG repeats are as effective in
“
Once it was discovered that high molecular weight poly(L-
serine) was insoluble in water, there was considerable interest
6
in development of nonionic water-soluble, conformationally
regular polypeptides derived from chemically modified amino
15
passivating the surfaces as high molecular weight PEG. In effect,
our polypeptides would be surrounded by an EG sheath that
should mimic the physical properties of PEG, yet not deleteriously
affect the secondary structure of the polypeptide core. The
molecular weights of these “PEG-mimic” polymers could also
be easily adjusted by controlling the degree of polymerization of
the amino acid.
acids.7,8 These materials were desired to allow fundamental studies
on the R-helical structure in solution in the absence of electrostatic
perturbations. The best materials resulting from this work are the
poly(N-hydroxyalkyl-L-glutamines), derived from aminolysis of
(
1) (a) Sela, M.; Katchalski, E. AdV. Protein Chem. 1959, 14, 391-478.
(
b) Fasman, G. D., Ed. Poly R-Amino Acids; Dekker: New York, 1967. (c)
EG-functionalized lysine monomers and polymers were pre-
pared as shown in Scheme 1. Lysine was chosen as the amino
acid component for the ease of coupling of the side-chain amine
with inexpensive EG-containing carboxylates and for its propen-
sity to form stable R-helical conformations. The formation of EG-
Stahmann, M. A., Ed. Polyamino Acids, Polypeptides, and Proteins; University
of Wisconsin Press: Madison, 1962.
(
2) (a) Kopacek, J. J. Controlled Rel. 1990, 11, 279-290. (b) Langer, R.
S.; Peppas, N. A. ReV. Macromol. Chem. Phys. 1983, C23, 61-126.
3) (a) Gabizon, A.; Papahadjopoulos, D. Proc. Natl. Acad. Sci. U.S.A. 1988,
5, 6949-6953. (b) Tomlinson, E.; Davis, S. S., Eds. Site-Specific Drug
DeliVery; Wiley: Sussex, 1986.
4) Scholtz, J. M.; York, E. J.; Stewart, J. M.; Baldwin, R. L. J. Am. Chem
Soc. 1991, 113, 5102-5104.
5) (a) Bamford, C. H.; Elliot, A.; Hanby, W. E. Synthetic Polypeptides;
(
8
(
(9) Noskov a´ , D.; Kotva, R.; Ryp a´ cek, F. Polymer 1988, 29, 2072-2075.
(10) (a) Ryp a´ cek, F.; Saudek, V.; Pytela, J.; Sˇ karda, V.; Drobn ´ı k, J.
Makromol. Chem. Suppl. 1985, 9, 129-135. (b) Hayashi, T.; Tabata, Y.;
Nakajima, A. Polym. J. 1985, 17, 463-471.
(
Academic Press: New York, 1956. (b) Kulkarni, R. K.; Blout, E. R. J. Am.
Chem Soc. 1962, 84, 3971-3972. (c) Goodman, M.; Felix, A. M. Biochemistry
(11) (a) Pratten, M. K.; Lloyd, J. B.; H o¨ rpel, G.; Ringsdorf, H. Makromol.
Chem. 1985, 186, 725-733. (b) Yokoyama, M.; Kwon, G. S.; Okano, T.;
Sakura, Y.; Seto, T.; Kataoka, K. Bioconj. Chem. 1992, 3, 295-301.
(12) Zalipsky, S.; Lee, C. In Poly(EthyleneGlycol) Chemistry: Biotechnical
and Biomedical Applications; Harris, J. M., Ed.; Plenum: New York, 1992.
(13) Zalipsky. S. AdV. Drug DeliVery ReV. 1995, 16, 157-182.
(14) Nelson, J. C.; Saven, J. G.; Moore, J. S.; Wolynes, P. G. Science 1997,
277, 1793-1796.
1
964, 3, 1529-1534.
(
6) (a) Bohak, Z.; Katchalski, E. Biochemistry 1963, 2, 228-237. (b)
Quadrifoglio, F.; Urry, D. W. J. Am. Chem Soc. 1968, 90, 2760-2765.
7) Thoma, G.; Patton, J. T.; Magnani, J. L.; Ernst, B.; O¨ hrlein, R.; Duthaler,
R. O. J. Am. Chem Soc. 1999, 121, 5919-5929.
(
(
8) (a) Lupu-Lotan, N.; Yaron, A.; Berger, A.; Sela, M. Biopolymers 1965,
3
3
7
, 625-655. (b) Lupu-Lotan, N.; Yaron, A.; Berger, A. Biopolymers 1966, 4,
65-368. (c) Okita, K.; Teramoto, A.; Fujita, H. Biopolymers 1970, 9, 717-
(15) Prime, K. L.; Whitesides, G. M. J. Am. Chem Soc. 1993, 115, 10714-
10721.
38.
1
0.1021/ja993637v CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/11/1999