Macro-Glycoligands: Methods and Protocols

228 Pages·2016·8.873 MB·English

by  Xue-Long Sun (eds.)

Methods in
Molecular Biology 1367
Xue-Long Sun Editor
Macro-
Glycoligands
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Macro-Glycoligands
Methods and Protocols
Edited by
Xue-Long Sun
Cleveland State University, Cleveland, OH, USA
Editor
Xue-Long S un
Cleveland State University
Cleveland, OH, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3129-3 ISBN 978-1-4939-3130-9 (eBook)
DOI 10.1007/978-1-4939-3130-9
Library of Congress Control Number: 2015951795
Springer New York Heidelberg Dordrecht London
© Springer Science+Business Media New York 2 016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com)
Prefa ce
Carbohydrate recognition is a crucial event in many biological processes, such as cell-cell
signaling, immune recognition events, pathogen/host interactions, tumor metastasis, tis-
sue growth and repair, etc. Therefore, carbohydrate recognition has come to the forefront
of biological scientifi c research aiming to uncover the molecular mechanisms of many phys-
iological and pathological processes and discover potential therapeutic targets or diagnostic
mechanisms for various diseases. Cell surface carbohydrates, existing as glycoproteins, gly-
colipids, or proteoglycans, are often involved in these biological processes. Consequently,
reconstitution of cell surface carbohydrate epitopes to mimic authentic compositions and
presentations has become the key research in either studying carbohydrate recognition or
developing therapeutic and diagnostic tools.
It has been known that multivalent interactions facilitate both specifi city and affi nity in
carbohydrate–protein interactions, referred to as the “cluster glycosidic effect.” In the past
decades, glycopolymers, namely, polymers with carbohydrate pendant groups, have been
extensively explored as multivalent carbohydrate ligands for studying on carbohydrate–pro-
tein interactions and for important biomedical applications. For example, glycopolymers
can act as agonists or antagonists for understanding the molecular mechanisms of many
biological processes and also provide tremendous opportunities for therapeutic applica-
tions. In addition, glycopolymers can serve as potential receptors for biochip/biosensor
development, which can be used for understanding carbohydrate–protein interaction, sub-
strate specifi city of carbohydrate-processing enzymes, antibody profi ling, biomarkers, and
pathogen and toxin identifi cation applications. Therefore, there is a high demand for devel-
oping facile methods and protocols for synthesizing and characterizing glycopolymers of
different interests. This book aims to give the reader detailed research methods and proto-
cols for the synthesis, characterization, and biomedical applications of glycopolymer-based
macro-glycoligands.
In this book M acro -G lycoligands: M ethods and Protocols , we have received excellent
contributions from experts in the fi eld. Altogether 17 book chapters cover the recent
advances in carbohydrate chemistry and polymer chemistry, and glycobiology aimed at
understanding and controlling the outcomes of carbohydrate recognition with particular
emphasis on glycopolymer-based macro-glycoligand. The book content is divided into
three parts: (I) Synthesis and characterization of glycopolymers; (II) Glycopolymer-
nanoparticle conjugates; (III) Surface immobilized glycopolymers. The compilation of
these book chapters provides a comprehensive and practical methods and protocols and
timely reference to the state of the art in recent glycopolymer research and applications.
There are seven chapters (Chapters 1 – 7 ) in P art I , which covers recent advances in the
synthesis and characterization of glycopolymers and their biomedical applications as well.
Glycopolymers carrying pendant sugar moieties can be synthesized by either direct polym-
erization of carbohydrate-containing monomers with protection group or without protec-
tion group, by the postpolymerization conjugation of glycans and synthetic polymers, or by
grafting of carbohydrate ligand to polymeric materials. Over the past decades, a variety of
direct polymerization methods have been developed, including cyanoxyl-mediated free-
radical polymerization (CMFRP), reversible addition-fragmentation chain transfer (RAFT)
v
vi Preface
polymerization, ring opening metathesis polymerization (ROMP), cationic ring-opening
polymerization (CROP), ring-opening polymerization (CROP), and atom transfer radical
polymerization (ATRP). Chapter 1 provides a straightforward synthesis of chain-end
functionalized glycopolymers via CMFRP, in which no protection/deprotection is needed.
In Chapter 2 , a protecting-free synthesis of well-defi ned glycopolymers via RAFT is fully
demonstrated. Chapter 3 provides a unique method for generating end-labeled amino ter-
minated monotelechelic glycopolymers by ROMP. Chapter 4 provides a facile protecting-
group-free synthetic approach to glycopolymers bearing large biologically relevant
oligosaccharides having sialic acids via reversible addition-fragmentation chain transfer
polymerization (RAFCTP). Chapter 5 presents a detailed methodology to functionalize
poly(2-ethyl-2-oxazoline) in a stereoselective manner with a range of carbohydrates that
can serve as biological targeting units. Chapter 6 presents a method for the in situ function-
alization and (co-) polymerization of allylglycine N -carboxyanhydride in a facile one-pot
procedure, combining radical thiol-ene photochemistry and nucleophilic ring-opening
polymerization techniques, to yield well-defi ned heterofunctional glycopolypeptides.
Finally, Chapter 7 describes a method for preparation of graft copolymers with glycosami-
noglycan side chains, which mimic the structure and composition of proteoglycans.
Part II (Chapters 8 – 14 ) covers recent advances in the synthesis and characterization of
glycopolymer-nanoparticle conjugates and their biomedical applications as well.
Nanomaterials are a promising tools for biomedical research and applications, as it is pre-
dicted to be benefi cial in tackling clinical problems. Glycopolymer-based nanostructures are
invaluable tools to both study biological phenomena and design future targeted drug deliv-
ery systems. On the other hand glyconanoparticles (GNPs), such as sugar-coated gold, iron
oxide, or semiconductor nanoparticles, have magnetic or fl uorescence properties, making
this multivalent glyco-scaffold suitable for carrying out studies on carbohydrate-mediated
interactions and applications in molecular imaging and targeted drug delivery applications.
In this book, Chapter 8 describes the methods to prepare well-defi ned glycopolymer-
containing diblock copolymers via RAFT, to self-assemble these macromolecules and to
start assessing the in vitro interactions of the self-assembled structures with live cells.
Recently, high luminescence , single excitation narrow emission, low photobleaching prop-
erties and low toxicity of high quality water-soluble Quantum Dots (QDs) have attracted
attention for in vivo labeling/imaging of cells. Chapter 9 describes a synthetic approach to
biotinylated glycopolymer functionalized quantum dots, with special emphasis on the
development of high quality water-soluble and bioactive QDs with low toxicity for fl uores-
cent probes in biomedical applications. Both ATRP and RAFT polymerization allow for
facile surface modifi cation and eventual surface polymerization of monomers exhibiting
biological mimicking capabilities. In Chapter 10 , ATRP was carried out in a “grafting
from” approach to obtain well-defi ned polypentafl uorostyrene modifi ed polymeric micro-
spheres. These fl uorinated materials were then converted to fl uorinated glycopolymers
using a thioglucose salt and thiol-halogen “click” chemistry without the need for any fur-
ther deprotection chemistry. Further, Chapter 11 describes the synthesis of glycopolymer-
grafted polymer particles using two types of surface-initiated living radical polymerization:
the fi rst is ATRP, and the other is photoiniferter polymerization. In Chapter 12 , a synthetic
approach to prepare nonspherical glycopolymer-coated iron oxide nanoparticles is pro-
vided, by combining the convenience of inorganic shape control, catecholic chemistry, and
thiol-ene reaction. On the other hand, Chapter 13 describes a synthetic approach to
glycopolymer- gold(I) nanoparticle conjugate for cancer therapy via three-step strategies to
Preface vii
incorporate thiol and dithiocarbamate functionality for the stabilization of gold nanoparticles
and the cancer drug for therapeutic application via RAFT polymerization method. Finally,
in Chapter 14 , a polymer-stabilized glycosylated gold nanoparticle platform was demon-
strated with precisely engineered heterotelechelic poly N -hydroxyethyl acrylamide poly-
mers bearing a carbohydrate moiety at one end for lectin interaction and a thiol at the other
for gold particle attachment.
Part III (Chapters 1 5 – 17 ) covers recent advances in surface immobilization of glyco-
polymers and their biochip/biosensor development and applications. The presentation of
carbohydrates on an array can provide a means to model (mimic) oligosaccharides found
on cell surfaces. Tuning the structural features of such carbohydrate arrays can therefore
be used to help elucidate the molecular mechanisms of protein-carbohydrate recognition
on cell surfaces. Chapter 1 5 presents a strategy to directly correlate the molecular and
structural features of ligands presented on a surface with the kinetics and affi nity of carbo-
hydrate–lectin binding. Both Surface Plasmon Resonance (SPR) spectroscopy and atomic
force microscopy (AFM) confi rmed the spatial distribution of carbohydrate ligands within
the surface grafted polymer layer and their lectin binding features. In Chapter 16 , a che-
moenzymatic synthesis of O -cyanate chain-end functionalized sialyllactose-c ontaining gly-
copolymers and their oriented sialyloligo-macroligand formation for glycoarray and
glyco-biosensor applications are demonstrated in detail. This oriented sialyloligo-macroli-
gand platforms are expected to facilitate both affi nity and specifi city of protein binding and
thus provide a versatile tool for profi ling glycan recognition via glycoarray and SPR-based
glyco-biosensor. The cellular glycocalyx controls many of the crucial signaling pathways
involved in cellular development. Synthetic materials that can mimic the multivalency and
three-dimensional architecture of native glycans serve as important tools for deciphering
and exploiting the roles of these glycans. Chapter 1 7 describes an approach for remodeling
cell surface glycocalyx with glycopolymer-based proteoglycan mimetics that binds FGF2
as a cell-surface engineering strategy to infl uence stem cell specifi cation.
In this book, we provide a detailed methods and protocols for the synthesis and char-
acterization of glycopolymers and their biomedical applications. Various controlled radical
polymerization techniques have been successfully employed for the synthesis of chain-end
functionalized glycopolymers with narrow polydispersity. The two signifi cant features of
the chain-end functionalized glycopolymers are multivalency, which can help increase the
affi nity and specifi city of bimolecular recognition, and chain-end functional group, which
can facilitate direct one-to-one attachment and oriented immobilization of glycopolymers
onto solid surfaces for mimicking cell surface carbohydrates. These chain-end functional-
ized glycopolymers were covalently or noncovalently attached to proteins, nanoparticles,
and glass slides in a site-specifi c fashion and lead to oriented glycopolymer presentation that
will fi nd important biomedical applications. Particularly, oriented glycopolymer-based gly-
can microarrays have exhibited high potential as a high-throughput analytical tool for inves-
tigating biological processes engaged with carbohydrates.
As an editor of this M ethods in Molecular Biology Series , I am very grateful to the S eries
Editor John M. Walker for this opportunity, and I am greatly indebted to all authors, who
responded with great enthusiasm to my initial proposal by contributing manuscripts. Also,
I would extend my gratitude to Springer for support of this special issue. With respect to
the readers, I hope that this compilation of chapters will provide not only practical methods
and protocols but also a timely overview and reference to Carbohydrate Recognition and
viii Preface
Application. Further, it will stimulate new ideas for hypothesis-driven research in this cer-
tainly fascinating area of glycoscience. Finally, I do hope this Macro -G lycoligands: Methods
and Protocols book will contribute to the transformation of the discipline of glycoscience
from highly specialized research domain to the mainstream biology.
Cleveland, OH, USA Xue-Long S un
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
PART I SYNTHESIS AND CHARACTERIZATION OF GLYCOPOLYMERS
1 Synthesis of Chain-End Functionalized Glycopolymers
via Cyanoxyl-Mediated Free Radical Polymerization (CMFRP). . . . . . . . . . . . 3
Valentinas G ruzdys , J inshan T ang , Elliot C haikof , and Xue-Long S un
2 P rotecting-Group-Free Synthesis of Well-Defined Glycopolymers
Featuring Negatively Charged Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . 13
Luca Albertin
3 G lycopolymers Prepared by Ring-Opening Metathesis
Polymerization Followed by Glycoconjugation Using a Triazole-Forming
“Click” Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Ronald Okoth and A mit Basu
4 P rotecting-Group-Free Synthesis of Glycopolymers and Their Binding
Assay with Lectin and Influenza Virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Tomonari T anaka , T adanobu T akahashi , and Takashi S uzuki
5 C arbohydrate-Based Initiators for the Cationic Ring-O pening
Polymerization of 2-Ethyl-2-Oxazoline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Christine Weber , M ichael G ottschaldt , R ichard H oogenboom ,
and Ulrich S. S chubert
6 Heterofunctional Glycopolypeptides by Combination
of Thiol-Ene Chemistry and NCA Polymerization. . . . . . . . . . . . . . . . . . . . . . 61
Kai-Steffen Krannig and Helmut S chlaad
7 P reparation of Proteoglycan Mimetic Graft Copolymers . . . . . . . . . . . . . . . . . 6 9
Matt J . K ipper and Laura W . P lace
PART II GLYCOPOLYMER NANOPARTICLE CONJUGATES
8 Galactosylated Polymer Nano-objects by Polymerization-I nduced
Self-Assembly, Potential Drug Nanocarriers. . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Mona S emsarilar , Irene C anton , and Vincent L admiral
9 S ynthetic Approach to Biotinylated Glyco-Functionalized Quantum Dots:
A New Fluorescent Probes for Biomedical Applications. . . . . . . . . . . . . . . . . . 109
Christian K. A dokoh , J ames D arkwa , and Ravin Narain
10 Surface Modification of Polydivinylbenzene Microspheres
with a Fluorinated Glycopolymer Using Thiol-Halogen Click Chemistry. . . . . 123
Wentao S ong and Anthony M . Granville
11 G lycopolymer-Grafted Polymer Particles for Lectin Recognition . . . . . . . . . . . 137
Michinari Kohri , T atsuo T aniguchi , and Keiki Kishikawa
ix