Glycopolypeptides via living polymerization of glycosylated-l-lysine N-carboxyanhydrides

Article abstract of DOI:10.1021/ja107425f

The preparation of new glycosylated-l-lysine-N-carboxyanhydride (glyco-K NCA) monomers is described. These monomers employ C-linked sugars and amide linkages to lysine for improved stability without sacrificing biochemical properties. Three glyco-K NCAs were synthesized, purified, and found to undergo living polymerization using transition metal initiation. These are the first living polymerizations of glycosylated NCAs and were used to prepare well-defined, high molecular weight glycopolypeptides and block and statistical glycocopolypeptides. This methodology solves many long-standing problems in the direct synthesis of glycopolypeptides from N-carboxyanhydrides relating to monomer synthesis, purification, and polymerization and gives polypeptides with 100% glycosylation. These long chain glycopolypeptides have potential to be good mimics of natural high molecular weight glycoproteins.

Full text of DOI:10.1021/ja107425f

Published on Web 10/05/2010
Glycopolypeptides via Living Polymerization of
Glycosylated-L-lysine N-Carboxyanhydrides
Jessica R. Kramer† and Timothy J. Deming*,†,‡
Department of Chemistry and Biochemistry, and Department of Bioengineering, UniVersity of
California, Los Angeles, California 90095-1600, United States
Received August 17, 2010; E-mail: demingt@seas.ucla.edu
Abstract: The preparation of new glycosylated-L-lysine-N-carboxyanhydride (glyco-K NCA) monomers is
described. These monomers employ C-linked sugars and amide linkages to lysine for improved stability
without sacrificing biochemical properties. Three glyco-K NCAs were synthesized, purified, and found to
undergo living polymerization using transition metal initiation. These are the first living polymerizations of
glycosylated NCAs and were used to prepare well-defined, high molecular weight glycopolypeptides and
block and statistical glycocopolypeptides. This methodology solves many long-standing problems in the
direct synthesis of glycopolypeptides from N-carboxyanhydrides relating to monomer synthesis, purification,
and polymerization and gives polypeptides with 100% glycosylation. These long chain glycopolypeptides
have potential to be good mimics of natural high molecular weight glycoproteins.
the sugar substituents and the NCA rings.12 An improved
synthesis of O- and S-linked glyco-serine as well as glyco-
Introduction
Glycosylated peptides and proteins are ubiquitous in nature
and display a wide range of biological functions including
mediation of recognition events, protection from proteases, and
lubrication in eyes and joints.1 Similarly, synthetic glyco-
polypeptides are also expected to show great potential as
biomedical materials (e.g., scaffolds for tissue repair and drug
carriers), as well as serve as valuable tools for probing
carbohydrate-protein interactions.2,3 Although block and hybrid
copolypeptides capable of forming vesicles,4,5 fibrils,6 and other
structures7,8 are readily prepared by amino acid N-carboxyan-
hydride (NCA) polymerization,9 the synthesis of well-defined
glycopolypeptide materials has been challenging. Even though
O-linked glyco-serine NCAs have been known for some
time,10,11 their synthesis is inefficient, and their polymerization
gives only short, oligomeric products where chain growth is
likely inhibited by steric and H-bonding interactions between
threonine NCAs was recently reported; however, these mono-
mers were not sufficiently purified to allow polymerization.13
To overcome these difficulties in synthesis, purification, and
polymerization of glycosylated NCAs, we have prepared new
glycosylated-L-lysine NCA (glyco-K NCA) monomers that
employ C-linked sugars and amide linkages to lysine14 for
improved stability without sacrificing biochemical properties.
Three different glyco-K NCAs were synthesized, purified, and
found to undergo living polymerization using transition metal
initiation.15 These are the first living polymerizations of gly-
cosylated NCAs allowing preparation of well-defined, high
molecular weight glycopolypeptides and glycopolypeptide
containing block copolymers.
Aside from direct polymerization of glycosylated NCAs, other
strategies to prepare glycopolypeptides rely primarily on the
addition of sugars to existing polypeptides. Glyconamidated
polypeptides were prepared by reaction of D-gluconolactone or
lactobionolactone with poly(L-lysine),16,17 or by coupling of ꢀ-D-
galactosylamine to glutamic acid residues,18 to attach carbohy-
† Department of Chemistry and Biochemistry.
‡ Department of Bioengineering.
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Csako, G.; Herp, A. Mol. Cell. Biochem. 1994, 137, 39–55. (d) Jentoft,
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875–877. (b) Tsutsumiuchi, K.; Aoi, K.; Okada, M. Macromol. Rapid
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10.1021/ja107425f  2010 American Chemical Society

Glycopolypeptides
A R T I C L E S
Figure 1. Synthesis of glyco-K NCA monomers. Reagents and conditions: (a) allyltrimethylsilane, BF3-Et2O, MeCN (55-75% yield); (b) NaIO4, RuCl3 ·3H2O,
MeCN:CCl4:H2O (72-92% yield); (c) NHS, DCC, THF; (d) R-Z-L-Lys-OH, NaHCO3 (75-81% yield for (c) and (d)); (e) Cl2CHOMe, DCM, 50 °C (59-64%
yield). 8a ) R-gal-K NCA (R1, R4 ) H; R2, R3 ) OAc). 8b ) R-glc-K NCA (R1, R3 ) H; R2, R4 ) OAc). 8c ) R-man-K NCA (R1, R4 ) OAc; R2, R3
) H).
drates via amide linkages. Also using polypeptide precursors,
glycopolypeptide synthesis has been reported using either
copper-catalyzed azide-alkyne19,20 or thiol-ene21 click chem-
istry, respectively. While promising, these methods can suffer
from incomplete sugar functionalization,21 presentation of sugars
in non-native forms (i.e., ring-opened),16,17 or incorporation of
triazole groups19,20 that may limit biological uses. Consequently,
we have focused on the glycosylated NCA approach to obtain
well-defined glycopolypeptides with 100% glycosylation, as well
as the ability to incorporate glycosylated residues either as
random or blocky sequences. We designed glycosylated-L-lysine
monomers because many derivatives of lysine NCAs are readily
synthesized and polymerized to high molecular weights (>100 000
Da),22 and because acid and base stable linkages can be readily
incorporated to prevent deglycosylation. We chose the glyco-
sylated-L-lysine structure shown in Figure 1, which has been
used for glycopeptide synthesis14 and employs C-linked sugars
and amide linkages to lysine. Although these are non-native
linkages, it is well-known that C-linked glycopeptides can bind
targets with nearly equal affinity and conformation as native
O-linked analogues23,24 and are widely utilized when stable
glycoprotein mimetics are desired.25
to literature procedures followed by isolation of the pure R
anomers (Figure 1 and Supporting Information).14 Purification
of anomers at this stage in the synthesis is crucial for the ultimate
preparation of optically pure, glyco-K NCA monomers. Mixtures
of anomers would lead to polypeptides containing different sugar
configurations, which could make analysis of their properties
difficult. Both R- and ꢀ-anomers are common in glycopeptides
and glycoproteins.26 Although our methodology allows use of
either anomer, we chose to use the R-anomers here because
they were easily obtained in high purity and natural glycopep-
tides utilize both R- and ꢀ-anomers.27 The allyl sugar derivatives
were oxidized to carboxylic acids, converted to N-hydroxysuc-
cinimide (NHS) esters, and then coupled to NR-carbobenzyloxy-
L-lysine (NR-Z-L-lysine) to give the desired conjugates (Figure
1).14 The preparation of glyco-K NCAs was accomplished using
22c
Cl2CHOCH3 followed by purification by precipitation and
flash chromatography to give NCAs suitable for polymerization.
All three glyco-K NCAs were obtained with acceptable yields
(60-95%) in each step. Furthermore, all three monomers were
found to polymerize efficiently using (PMe3)4Co28 initiator in
THF at room temperature (Figure 2),15 yielding polymers in
excellent yields (Table 1, see Supporting Information).
To check for chain-breaking side reactions during polymer-
izations, experiments to verify molecular weight control and
extension of active chains were performed. Variation of
monomer to initiator ratios for each glyco-K NCA gave
glycopolypeptides whose lengths increased linearly with stoi-
chiometry and which possessed narrow chain length distributions
(Mw/Mn). Data for the R-D-mannose-L-lysine monomer (R-
man-K NCA) are shown in Figure 3a, and data for the other
two monomers are given in the Supporting Information. Good
chain length control was obtained, and soluble homoglyco-
polypeptides could be prepared with degrees of polymerization
(DP) greater than 300 residues, significantly larger than chains
prepared from other glycosylated NCAs (DP < 50).12a As
compared to shorter chains, long chain glycopolypeptides are
expected to be much better mimics of natural high molecular
weight glycoproteins. We found that the polymers had higher
than theoretical molecular weights, calculated from monomer
Results and Discussion
The sugar-lysine conjugates were prepared using ꢀ-D-glucose
(glc), ꢀ-D-mannose (man), and ꢀ-D-galactose (gal). C-Allylation
of glc, man, and gal pentaacetates was accomplished according
(19) (a) Tang, H.; Zhang, D. Biomacromolecules 2010, 11, 1585–1592.
(b) Huang, J.; Habraken, G.; Audouin, F.; Heise, A. Macromolecules
2010, 43, 6050–6057.
(20) Xiao, C.; Zhao, C.; He, P.; Tang, Z.; Chen, X.; Jing, X. Macromol.
Rapid Commun. 2010, 31, 991–997.
(21) Sun, J.; Schlaad, H. Macromolecules 2010, 43, 4445–4448.
(22) (a) Gallot, B.; Fafiotte, M. Macromol. Rapid Commun. 1996, 17, 493–
501. (b) Schaefer, K. E.; Keller, P.; Deming, T. J. Macromolecules
2006, 39, 19–22. (c) Yu, M.; Nowak, A. P.; Pochan, D. P.; Deming,
T. J. J. Am. Chem. Soc. 1999, 121, 12210–12211.
(23) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim, S.; Kishi, Y. J. Am.
Chem. Soc. 1998, 120, 11297–11303.
(24) Wang, J.; Kovac, P.; Sinay, P.; Gluademans, C. P. J. Carbohydr. Res.
1998, 308, 191–193.
(25) (a) Levy, D E.; Tang, C. The Chemistry of C-Glycosides; Pergamon:
Tarrytown, NY, 1995. (b) Postema, M. H. D. C-Glycoside Synthesis;
CRC Press: London, UK, 1995. (c) Bertozzi, C. R.; Bednarski, M. D.
J. Am. Chem. Soc. 1992, 11, 2242–2245, 4. (d) Shao, H.; Zerong Wang,
Z.; Lacroix, E.; Wu, S.-H.; Jennings, H. J.; Zou, W. J. Am. Chem.
Soc. 2002, 124, 2130–2131. (e) Gustafsson, T.; Hedenstrom, M.;
Kihlberg, J. J. Org. Chem. 2006, 71, 1911–1919. (f) Ranoux, A.;
Lemie`gre, L.; Benoit, M.; Gue´gan, J. P.; Benvegnu, T. Eur. J. Org.
Chem. 2010, 1314–1323. (g) Peri, F.; Cipolla, L.; Rescigno, M.; Ferla,
B. L.; Nicotra, F. Bioconjugate Chem. 2001, 12, 325–328.
(26) (a) Taylor, C. M. Tetrahedron 1998, 54, 11317–11362. (b) Vliegent-
hart, J. F. G.; Casset, F. Curr. Opin. Struct. Biol. 1998, 8, 565–571,
5. (c) Haase, C.; Seitz, O. Top. Curr. Chem. 2007, 267, 1–36.
(27) (a) Shibata, S.; Takeda, T.; Natori, Y. J. Biol. Chem. 1988, 263, 12483–
12485. (b) Debeer, T.; Vliegenthart, J. F. G.; Loffler, A.; Hofsteenge,
J. Biochemistry 1995, 34, 11785–11789.
(28) (PMe3)4Co was prepared according to literature procedure. Klein, H. F.;
Karsch, H. H. Chem. Ber. 1975, 108, 944–55.
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A R T I C L E S
Kramer and Deming
Figure 2. Polymerization of glyco-K NCAs and glycopolypeptide deprotection. Reagents and conditions: (a) (PMe3)4Co, THF; (b) (H2N)2 ·H2O, MeOH.
10a ) poly(R-gal-K) (R1, R4 ) H; R2, R3 ) OH). 10b ) poly(R-glc-K) (R1, R3 ) H; R2, R4 ) OH). 10c ) poly(R-man-K) (R1, R4 ) OH; R2, R3 ) H).
Table 1. Synthesis of Glycopolypeptides Using (PMe3)4Co in THF
order of monomer addition. In addition to block copolymers,
equimolar mixtures of the three glyco-K NCAs were also
at 20 °C
monomera
Mnb
Mw/Mnb
DPc
yield (%)d
copolymerized to yield statistical ternary glycocopolypeptides
of controllable chain length (Table 1). Overall, these data show
that the glyco-K NCAs were able to undergo living polymer-
ization when initiated with (PMe3)4Co, similar to conventional
NCAs,15 and this chemistry enabled preparation of the first
examples of glycosylated diblock copolypeptides.
10 R-man-K NCA
25 R-man-K NCA
50 R-man-K NCA
75 R-man-K NCA
100 R-man-K NCA
25 glyco-K NCAse
50 glyco-K NCAse
15 870
36 920
81 340
121 300
158 600
47 470
84 710
1.21
1.14
1.19
1.11
1.14
1.23
1.07
32
74
163
242
317
95
91
95
89
94
92
88
94
169
It is noteworthy that polymerizations of glyco-K NCAs
proceeded efficiently at room temperature to yield polymers with
DP > 150 in less than 3 h, as compared to 3-6 days for
polymerization of O-linked glyco-serine NCAs (DP < 25) using
amine initiators.12a The sluggish polymerization of O-linked
glyco-serine NCAs is thought to be due to either the proximity
of the bulky peracetylated sugar to the NCA ring or the
H-bonding of acetyl carbonyls with NH groups of NCAs.12b,c
Although we also see 1H NMR evidence for H-bonding in our
glyco-K NCAs (see Supporting Information), the increased
tether length between sugar and NCA likely weakens this
interaction and removes steric hindrance allowing efficient
polymerization. However, the influence of H-bonding is not
negligible, because the glyco-K NCAs were found to polymerize
2-5 times more slowly than other lysine NCA derivatives. As
all of the acetyl protected poly(glyco-K)s were found to be
a Number indicates equivalents of monomer per (PMe3)4Co.
b Molecular weight and polydispersity index (as determined by GPC/
LS). c DP ) number average degree of polymerization from GPC/LS.
d Total isolated yield of glycopolypeptide. e Statistical terpolymers
prepared from a 1:1:1 mixture of R-man-K:R-gal-K:R-glc-K NCAs.
to initiator stoichiometry, which is due to the known incomplete
efficiency of (PMe3)4Co initiation in THF.29 In any case,
reproducible and precisely controlled glycopolypeptide lengths
were readily prepared. Chain extension experiments of active
chains to prepare diblock copolymers (Table 2, see Supporting
Information) all proceeded in high conversion to yield predict-
able compositions, and with no evidence of inactive chains by
GPC analysis (Figure 3b). Diblock copolymers were prepared
by combining different glyco-K NCAs, as well as by combina-
tion of glyco-K NCAs with conventional NCAs regardless of
Figure 3. (a) Molecular weight (Mn, [) and polydispersity index (Mw/Mn, 9) of poly(R-man-K) as functions of monomer to initiator ratio ([M]/[I]) using
(PMe3)4Co in THF at 20 °C. (b) GPC chromatograms (normalized LS intensity versus elution time in arbitrary units (au)) of glycopolypeptides after initial
polymerization of R-man-K NCA to give a poly(R-man-K)68 homoglycopolypeptide (A); and after chain extension by polymerization of R-gal-K NCA to
give a poly(R-man-K)68-b-poly(R-gal-K)200 diblock glycopolypeptide (B).
Table 2. Synthesis of Diblock Copolypeptides Using (PMe3)4Co in THF at 20 °C
first segmentb
diblock copolymerc
first monomera
second monomera
Mn
Mw/Mn
DP
Mn
Mw/Mn
DPd
yield (%)e
30 Lys NCA
30 R-man-K NCA
30 R-man-K NCA
90 R-man-K NCA
90 Lys NCA
90 R-gal-K NCA
18 100
34 090
34 090
1.15
1.23
1.23
69
68
68
124 000
86 820
134 200
1.01
1.07
1.12
281
269
268
93
99
91
a First and second monomers added stepwise to the initiator; number indicates equivalents of monomer per (PMe3)4Co. Lys NCA
)
ε-Z-L-lysine-N-carboxyanhydride.30 b Molecular weight and polydispersity index after polymerization of the first monomer (as determined by GPC/LS).
c Molecular weight and polydispersity index after polymerization of the second monomer. d Total degree of polymerization of diblock glycopolypeptide.
e Total isolated yield of diblock glycopolypeptide.
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A R T I C L E S
Figure 4. (a) Circular dichroism spectrum of poly(R-gal-K) at 20 °C, 92% R-helical (10a). (b) Percent of R-helical content (% helicity, calculated using
molar ellipticity at 222 nm) of poly(R-gal-K) as a function of temperature. Mn ) 90 040 Da, 0.25 mg/mL in deionized water, pH 7. Molar ellipticity is
reported in millideg ·cm2 ·dmol-1
.
soluble in THF, it appears that there are no significant interchain
H-bonding interactions in the polymers. Furthermore, all three
homoglycopolypeptides were found to have good water solubil-
ity after deprotection using NH2NH2 ·H2O in MeOH. To
investigate the solution conformations of these samples, circular
dichroism (CD) spectra of the poly(glyco-K)s were measured
in deionized water after purification and removal of all residual
cobalt impurities by extensive dialysis against deionized water.
All three glycopolypeptides were found to be R-helical in water
at room temperature, with characteristic minima at 208 and 222
nm, indicating greater than 88% helicity31 (Figure 4a, see
Supporting Information). Poly-L-lysine inherently prefers to be
R-helical when uncharged,32 and the bulky sugar groups are
positioned far enough away from the backbone to avoid
perturbing this conformation. Measurement of CD spectra from
4 to 90 °C revealed that the R-helical conformation of poly(R-
gal-K) was gradually disrupted as temperature was increased.
This behavior is likely due to disruption of amide H-bonding
by interactions with water molecules.33 These disordered
polypeptides remained water-soluble, and their R-helical con-
formations were completely regained upon cooling, showing
this process is reversible. Ternary glycocopolypeptides contain-
ing randomly placed sugars also were found to be R-helical (see
Supporting Information), indicating that the poly-L-lysine
backbone is directing the chain conformation rather than any
specific interactions between sugars. This result allows the
display of a diverse variety of sugars off of ordered R-helical
polypeptide scaffolds, potentially useful for diagnostic applications.
Conclusion
We have prepared new glycosylated lysine NCAs that
undergo living polymerization to give well-defined, high mo-
lecular weight homoglycopolypeptides and block and statistical
glycocopolypeptides. This system solves many long-standing
problems in the direct synthesis of glycopolypeptides from
NCAs relating to monomer synthesis, purification, and polym-
erization and is advantageous in that polypeptides with 100%
glycosylation are easily obtained. These water-soluble glyco-
polypeptides have potential to impart functionality and improve
biocompatibility in copolypeptide materials, such as hydrogels
for tissue engineering and vesicles for drug delivery, as well as
for the preparation of structurally defined sugar presenting
polymers for glycomics research.
Acknowledgment. This work was supported by the NSF
IGERT: Materials Creation Training Program (MCTP) - DGE-
0654431, the NSF under award No. DMR 0907453, and the
California NanoSystems Institute.
(29) Deming, T. J.; Curtin, S. A. J. Am. Chem. Soc. 2000, 122, 5710–
5717.
Supporting Information Available: Experimental procedures
and spectral data for all new compounds. Polymerization data,
Mn versus [M]/[I] plots, and circular dichroism spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(30) ε-Z-L-Lysine-N-carboxyanhydride was prepared according to literature
procedure. Yamamoto, H.; Hayakawa, T. Biopolymers 1977, 16, 1593–
1607.
(31) Morrow, J. A.; Segal, M. L.; Lund-Katz, S.; Philips, M. C.; Knapp,
M.; Rupp, B.; Weigraber, K. H. Biochemistry 2000, 39, 11657–11666.
(32) Fasman, G. D. Poly R-Amino Acids; Dekker: New York, 1967.
(33) Lupu-Lotan, N.; Yaron, A.; Berger, A.; Sela, M. Biopolymers 1965,
3, 625–655.
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