Design and synthesis of biotin chain-terminated glycopolymers for surface glycoengineering

Article abstract of DOI:10.1021/ja025788v

Biotin chain-terminated glycopolymers were generated by cyanoxyl-mediated free-radical polymerization using a biotin-derivatized arylamine initiator with high conversion (75%) and low polydispersity (1.30). Streptavidin-biotinylated glycopolymer binding w

Full text of DOI:10.1021/ja025788v

Published on Web 05/31/2002
Design and Synthesis of Biotin Chain-Terminated Glycopolymers for Surface
Glycoengineering
Xue-Long Sun,* Keith M. Faucher, Michelle Houston, Daniel Grande, and Elliot L. Chaikof*
Laboratory for Biomolecular Materials Research, Departments of Surgery and Bioengineering,
Emory UniVersity School of Medicine, Atlanta, Georgia 30322, and School of Chemical Engineering,
Georgia Institute of Technology, Atlanta, Georgia 30332
Received February 1, 2002
Fundamental studies of glycopolymer properties have provided
insight regarding carbohydrate-mediated biomolecular recognition
processes that may be attributed, in part, to a multivalent or cluster
effect.1 Significantly, these efforts hold relevance for both phar-
maceutical and biomaterial applications. For example, recent
investigations have synthesized glycopolymers with surface anchor-
ing groups located along the polymer backbone to generate
glycosurfaces with potential utility in bio- and immunochemical
assays,2 as well as biocapture analysis.3 Nonetheless, glycopolymers
bearing a series of pendant anchoring groups characteristically
demonstrate reduced bioactivity due to steric hindrance, which is
only partially offset through the introduction of a spacer arm
between the anchor and the polymer backbone. Avidin (streptavi-
din)/biotin-based surface engineering has the advantage of being
rapidly completed in a mild aqueous environment, with simple
washing and purification steps. Thus, potential damage to candidate
surface ligand groups due to the conjugation process is limited and
any moiety that can be biotinylated can be immobilized onto an
avidin/streptavidin surface. As such, polymers with biotinylated end
groups have recently been used to generate self-organizing protein-
polymer hybrid amphiphiles,4 as well as molecularly engineered
surfaces.5 Characteristically, these chain-end functionalized poly-
mers are prepared by further modification or conversion in several
steps after initial polymer synthesis.6 In this report, we describe a
straightforward approach to synthesize biotin chain-terminated
glycopolymers of low polydispersity (Figure 1), via use of a biotin-
derivatized arylamine initiator employed in a cyanoxyl-mediated
free radical polymerization scheme.
A representative chain end-functionalized glycopolymer was
designed as shown in Figure 1, in which multivalent lactose units
serve as ligands for lectins and/or antibodies,7 and a single biotin
group provides an anchor by its specific binding activity to avidin
or streptavidin.8 Modulating lactose density as well as polymer
solubility was achieved by using acrylamide as a comonomer and
a spacer arm (X) between biotin and polymer backbone was used
for further optimization of polymer-avidin/streptavidin interaction.
The synthetic strategy relies on our previously developed cyanoxyl-
mediated free-radical polymerization process, in which arylamines
have been identified as candidate initiators.9 Specifically, the biotin
derivatized arylamine was used as an initiator for cyanoxyl-mediated
free radical polymerization of 2-acrylaminoethyl lactoside and
acrylamide so as to provide the desired biotin chain-terminated
glycopolymers for subsequent use in glycosurface engineering.
In the current study, 4-aminobenzyl-biotinamide 1 and 210 were
designed and investigated as initiators for the polymerization of
2-acrylaminoethyl lactoside 711 with acrylamide. Treatment of 1
Figure 1. Structure of biotin chain-terminated glycopolymer.
Scheme 1. Synthesis of Biotin Chain-Terminated Glycopolymer
with HBF4 and NaNO2 in deoxygened H2O-THF (1:1) gave the
arenediazonium cation 3, which upon reaction with NaOCN at 50
°C afforded the biotinyl aryl free radical 5 and the cyanoxyl free
radical (•OCtN) as the initiating system for copolymerization of
glycomonomer 7 and acrylamide. The biotin chain-terminated
glycopolymer 8 was generated in 75% conversion as a white spongy
powder. Similarly, using biotin-cap-arylamide 2 as the initiating
species, glycopolymer 9 was generated with a spacer arm between
biotin and the polymer chain in 70% conversion.The resultant
copolymers were characterized by NMR spectroscopy (Figure 2),
as well as by size-exclusion chromatography (SEC) coupled with
both refractive index and laser-light scattering (LLS) detectors.
Comparison of the integrated signal from the phenyl protons (7.10
ppm, H2′,6′ and 7.18 ppm, H3′,5′) with that due to the anomeric
protons of lactose (4.36 ppm, H1′-Lact and 4.43 ppm, H1-Lact), as
well as that of the polymer backbone methine (2.10 ppm, CH) and
methylene (1.55 ppm CH2), indicated an average polymer composi-
tion of 10 lactose and 70 acrylamide units. Notably, downfield shifts
of H2′, H6′ phenyl protons (7.10 ppm) demonstrated C-C bond
formation between the phenyl group and the polymer backbone.
The actual molar mass (Mn) was 12 kDa with a polydispersity index
(Mw/Mn) of 1.3 from SEC/RI/LLS for 8.
A gel shift assay was performed to verify streptavidin-glyco-
polymer binding in a solution-phase system. Streptavidin-glyco-
polymer complexes were generated as illustrated in lanes E and F
(Figure 3, Gel I), while not observed on incubation with a
comparable glycopolymer lacking a biotin group [lane D]. Since
each streptavidin (60 kDa) contains four identical subunits, a
streptavidin band remains present when mixed with only 2 equiv
* To whom correspondence should be addressed. E-mail: echaiko@emory.edu
or xsun@emory.edu.
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10.1021/ja025788v CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
In conclusion, cyanoxyl-mediated free-radical polymerization
with a biotin-derivatized arylamine initiator provides a straightfor-
ward strategy for generating biotin chain-terminated glycopolymers.
Streptavidin-biotin binding was verified by a SDS-PAGE gel shift
assay and the fabrication of a glycocalyx-mimetic surface achieved.
The present approach will facilitate the production of glycosurface
arrays of varying carbohydrate species type and density.
Acknowledgment. This work was supported by grants from
the NIH. We thank Dr. Jinho Hyun and Dr. A. Chilkoti at Duke
University for the kind gift of streptavidin precoated PET mem-
branes.
Figure 2. 1H NMR spectrum of biotin-terminated glycopolymer (8).
Supporting Information Available: Detail synthetic procedures
and spectral data for intermediates and final compounds, SDS-PAGE
gel shift assay, and glycosurface fabrication and lectin binding
procedures (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
References
Figure 3. Streptavidin-biotin binding SDS-PAGE gel shift assay: (I) 2
equiv of glycopolymer; (II) 20 equiv of glycopolymer. A, marker; C,
Streptavidin alone; D, streptavidin + p-chlorophenyl-glycopolymer (10);
E, streptavidin + biotin-glycopolymer (8); F, streptavidin + biotin-cap-
glycopolymer (9).
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Figure 4. (A) Schematic of lectin binding to a glycopolymer-derivatized
surface; (B) fluorescent image of FITC-lectin bound onto a patterned PET
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(10) The biotin-containing arylamine initiators 1 and 2 were prepared by the
condensation of commercial available p-nitrobenzylamine with N-hydroxy-
succinimidyl-biotin, and N-hydroxy-succinimidyl-biotinamidocaproate fol-
lowed by hydrogenation with Pd-C in methanol in high yield, respec-
tively.
of biotin-glycopolymer (Gel I, Lanes E and F), but disappears in
the presence of an excess of glycopolymer (Gel II, Lanes E and
F). The retarded migration of streptavidin-glycopolymer complexes
may be due to both an increase in molecular weight and a reduction
in the capacity of the apolar portions of streptavidin to interact with
the alkyl moiety of sodium dodecysulfate (SDS).13 In this system,
the presence of a spacer arm did not have a measurable impact on
glycopolymer/streptavidin affinity.
Biotin and streptavidin activation techniques have played an
important role in the development of biofunctionalized surface for
sensor or biomaterial applications.14,15 Glycopolymer-coated sur-
faces were produced by incubating streptavidin-derivatized PET
membranes16 in a glycopolymer solution (1 mg/mL in PBS) for 1
h at room temperature. Membranes were subsequently washed with
PBS and incubated in a solution of a FITC-labeled galactose binding
lectin (1 mg of psophocarpus tetragonolobus/mL in PBS). As
demonstrated in Figure 4, lectin binding was observed in regions
of glycopolymer immobilization, while no such activity was noted
in surface regions not derivatized with streptavidin.
(11) 2-Acrylaminoethyl lactoside 7 was prepared from lactose per-acetate via
four steps including glycosylation, hydrogenation, and acrylation with
acryloyl chloride in 53% total yield.
(12) 4-Chlorophenyl-glycopolymer 10 was prepared in a similar procedure as
described for 8 by using 4-chloroaniline as initiator.
(13) Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406-4412.
(14) Yang, Z.; Belu, A. M.; Liebman-Vinson, A.; Sugg, H.; Chilkoti, A.
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(15) Hyun, J.; Zhu, Y.; Liebman-Vinson, A.; Beebe, T. P.; Chilkoti, A.
Langmuir 2001, 17, 6358-6367.
(16) Biotin-modified poly(etheneterephthate) (PET) surface was patterned with
streptavidin by microcontact print (40 µm streptavidin stripes with 40
µm spacing): ref 14.
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