Photochemical Ligation Techniques for Carbohydrate Biosensors and Protein Interaction Studies ...

101 Pages·2012·6.43 MB·English

Photochemical Ligation Techniques for
Carbohydrate Biosensors and Protein
Interaction Studies

Oscar Norberg

Doctoral Thesis

Stockholm 2012

Akademisk avhandling som med tillstånd av Kungl Tekniska Högskolan i
Stockholm framlägges till offentlig granskning för avläggande av doktorsexamen i
kemi med inriktning mot organisk kemi fredagen den 30 mars kl 10.00 i sal F3,
KTH, Lindstedtsvägen 26, Stockholm. Avhandlingen försvaras på engelska.
Opponent är Prof. Stéphane Vincent, Facultés Universitaires Notre-Dame de la
Paix, Namur, Belgien
ISBN 978-91-7501-271-1
ISSN 1654-1081
TRITA-CHE Report 2012:10

© Oscar Norberg, 2012
E-print, Stockholm
Oscar Norberg, 2012: “Photochemical Ligation Techniques for Carbohydrate
Biosensors and Protein Interaction Studies”, KTH Chemical Science and
Engineering, Royal Institute of Technology, SE-100 44 Stockholm, Sweden.

Abstract

This thesis concerns the development of surface ligation techniques for the
preparation of carbohydrate biosensors. Several methodologies were developed
based on efficient photochemical insertion reactions which quickly functionalize
polymeric materials, with either carbohydrates or functional groups such as alkynes
or alkenes. The alkyne/alkene surfaces were then treated with carbohydrate azides or
thiols and reacted under chemoselective Cu-catalyzed azide-alkyne cycloaddition
(CuAAC) or photo-radical thiol-ene/yne click chemistry, thus creating a range of
carbohydrate biosensor surfaces under ambient conditions. The methodologies were
evaluated by quartz crystal microbalance (QCM) and surface plasmon resonance
(SPR) flow through instrumentations with recurring injections of a range of lectins,
allowing for real-time analysis of the surface interactions. The developed methods
were proven robust and versatile, and the generated carbohydrate biosensors showed
high specificities and good capacities for lectin binding. The methods were then
used to investigate how varying the glycan linker length and/or a sulfur-linkage
affect the subsequent protein binding. The survey was further explored by
investigating the impact of sulfur in glycosidic linkages on protein binding, through
competition assays with various O/S-linked disaccharides in solution interactions
with lectins.

Keywords: Carbohydrates, Biosensors, CuAAC, Photochemistry, Thiol-ene/yne,
Perfluorophenylazide (PFPA), Lectins, Molecular recognition, Quartz crystal
microbalance (QCM), Surface plasmon resonance (SPR)

III
List of Publications
This thesis is based on the following papers, referred to in the text by their Roman
numerals I-VI:

I. Photo-Click Immobilization of Carbohydrates on Polymeric Surfaces - A
Quick Method to Functionalize Surfaces for Biomolecular Recognition
Studies
Oscar Norberg, Lingquan Deng, Mingdi Yan and Olof Ramström
Bioconjugate Chem., 2009, 20, 2364-2370

II. Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for
Selective Carbohydrate-Protein Interaction Analyses
Oscar Norberg, Lingquan Deng, Teodor Aastrup, Mingdi Yan and Olof
Ramström
Anal. Chem., 2011, 83, 1000-1007

III. Stereoselective Synthesis of Light-Activatable Perfluorophenylazide-
Conjugated Carbohydrates for Glycoarray Fabrication and Evaluation
of Structural Effects on Protein Binding by SPR Imaging
Lingquan Deng, Oscar Norberg, Suji Uppalapati, Mingdi Yan and Olof
Ramström
Org. Biomol. Chem., 2011, 9, 3188-3198

IV. Photogenerated Lectin Sensors Produced by Thiol-Ene/Yne Photo-Click
Chemistry in Aqueous Solution
Oscar Norberg, Irene H. Lee, Teodor Aastrup, Mingdi Yan and Olof
Ramström
Biosens. Bioelectron., 2012, in press

V. Stereocontrolled 1-S-Glycosylation and Comparative Binding Studies of
Photoprobe-Thiosaccharide Conjugates with Their O-Linked Analogs
Lingquan Deng, Xin Wang, Suji Uppalapati, Oscar Norberg, Hai Dong,
Adrien Joliton, Mingdi Yan and Olof Ramström
Submitted for publication

IV
VI. Synthesis and Binding Affinity Analysis of α1-2- and α1-6-O/S-linked
Dimannosides for the Elucidation of Sulfur in Glycosidic Bonds using
Quartz Crystal Microbalance Sensors
Oscar Norberg, Germain Fauquet, Ann-Kathrin Saur, Teodor Aastrup,
Mingdi Yan and Olof Ramström
Preliminary manuscript

Book chapters not included in this thesis:

VII. Dynamic Combinatorial Chemistry – Ligands for Biomolecules
Olof Ramström, Luis Amorim, Rémi Caraballo and Oscar Norberg
In: Dynamic Combinatorial Chemistry (Eds.: J. Reek, S. Otto)
Wiley-VCH Verlag GmbH & Co. 2010, 109-150

VIII. Synthesis of Glyconanomaterials via Photo-Initiated Coupling Chemistry
Xin Wang, Oscar Norberg, Lingquan Deng, Olof Ramström and Mingdi Yan
In: ACS Symp. Ser., 2011, 1091, 49-67

V
Author’s Contribution
The following is a description of my contribution to Publications I to VI, as
requested by KTH.

Paper I: I contributed to the formulation of the research problems, performed the
experimental work and wrote the manuscript.
Paper II: I contributed to the formulation of the research problems, performed the
experimental work and wrote the manuscript.
Paper III: I contributed to the formulation of the research problems, performed part
of the experimental work and wrote part of the manuscript.
Paper IV: I contributed to the formulation of the research problems, performed the
majority of the experimental work and wrote the manuscript.
Paper V: I contributed to the formulation of the research problems and performed
part of the experimental work.
Paper VI: I contributed to the formulation of the research problems, performed part
of the experimental work and wrote the manuscript. Part of the synthesis was
performed by Diploma worker Germain Fauquet and Erasmus student Ann-Kathrin
Saur under my supervision.

VI
Abbreviations
Ac Acetyl
AcOH Acetic acid
AcSH Thioacetic acid
ACVA 4,4-Azobis(4-cyanovaleric acid)
AgOTf Silver trifluoromethanesulfonate
aq. Aqueous
Bn Benzyl
BSA Bovine serum albumin
BS-II Griffonia (Bandeiraea) simplicifolia lectin II
Bz Benzoyl
CCD Charge-coupled device
CI Confidence interval
Con A Concanavalin A
Cu(OTf) Copper(II) trifluoromethanesulfonate
2
CuAAC Copper(I)-catalyzed azide-alkyne cycloaddition
DCM Dichloromethane
DIBAL-H Diisobutylaluminum hydride
DIPEA N,N-Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DMPU 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol ((2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol)
EC The half maximal effective concentration
50
EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
ELISA Enzyme-linked immunosorbent assay
ESI-MS Electrospray ionization mass spectrometry
VII
EtOAc Ethyl acetate
Gal β-D-Galactose
GlcNAc N-Acetyl-β-D-glucosamine
h Hour
Hex Hexane
HIV Human immunodeficiency virus
HMPA Hexamethylphosphoramide
HSA Human serum albumin
IC The half maximal inhibitory concentration
50
ITC Isothermal titration calorimetry
K Association constant
a
K Dissociation constant
d
l Liquid
LAH Lithium aluminum hydride
Man α-D-Mannose
MS Mass spectrometry
M Molecular weight
w
NBS N-Bromosuccinimide (1-Bromo-2,5-pyrrolidinedione)
NHS N-Hydroxysuccinimide
NIS N-Iodosuccinimide
NMR Nuclear magnetic resonance
NOE Nuclear overhauser effect
OEG Oligoethylene glycol
PAAm Polyacrylamide
PBS Phosphate buffered saline
PEG Polyethylene glycol
PEO Polyethylene oxide
PEOX Poly(2-ethyl-2-oxazoline)
PFPA Perfluorophenylazide
PhCHO Benzaldehyde
VIII
pI Isoelectric point
PNA Peanut agglutinin (Arachis hypogaea)
PP Polypropylene
PS Polystyrene
PSA Pisum sativum agglutinin
QCM Quartz crystal microbalance
quant. Quantitative
R2 Coefficient of determination
RCA-I Ricinus communis agglutinin I
rt Room temperature
s.m. Starting material
sat. Saturated
SAM Self assembled monolayer
SD Standard deviation
SEM Standard error of the mean
SPR Surface plasmon resonance
SPRi Surface plasmon resonance imaging
STD Saturation transfer difference
TBABr Tetra-N-butylammonium bromide
TBAI Tetra-N-butylammonium iodide
TBANO Tetra-N-butylammonium nitrite
2
TBASAc Tetra-N-butylammonium thioacetate
TEA Triethylamine
THF Tetrahydrofuran
Thiol-ene Thiol-alkene
Thiol-yne Thiol-alkyne
TMSOTf Trimethylsilyl trifluoromethanesulfonate
Ts p-Toluenesulfonyl
UV Ultraviolet
WGA Wheat germ agglutinin (Triticum vulgaris)
IX
Table of Contents

Abstract ......................................................................................................................... III 
List of Publications ....................................................................................................... IV 
Abbreviations ............................................................................................................... VII 
Table of Contents ........................................................................................................... X 
1.  Introduction ............................................................................................. 1 
1.1.  Carbohydrates .................................................................................................... 1 
1.1.1.  Chemical Aspects of Carbohydrates ......................................................... 2 
1.1.2.  Carbohydrate Synthesis ............................................................................ 3 
1.2.  Carbohydrates in Glycobiology ......................................................................... 4 
1.3.  Lectins in Biology and Technology ................................................................... 5 
1.4.  Methods to Study Protein Interactions with Glycans ........................................ 7 
1.4.1.  Isothermal Titration Calorimetry ............................................................. 7 
1.4.2.  Quartz Crystal Microbalance ................................................................... 9 
1.4.3.  Surface Plasmon Resonance ................................................................... 11 
1.5.  Glycan Surface Ligation Techniques .............................................................. 13 
1.5.1.  Surface Functionalization Chemistry using Unmodified Glycans .......... 13 
1.5.2.  Surface Functionalization Chemistry using Tagged Glycans ................. 15 
1.6.  Glycan Analogues in Drug Design .................................................................. 17 
1.7.  Aim of This Thesis .......................................................................................... 21 
2.  CuAAC Photo-Click Immobilization ................................................... 23 
2.1.  Introduction ..................................................................................................... 23 
2.2.  Perfluorophenylazides (PFPAs) ...................................................................... 23 
2.3.  Synthesis of Photo-probes and Linkers ........................................................... 24 
2.4.  Synthesis of Carbohydrate Azides for Click Chemistry Surfaces ................... 26 
2.5.  Method Design and Development ................................................................... 27 
2.6.  Method Evaluation .......................................................................................... 30 
2.7.  Method Expansion ........................................................................................... 32 
2.8.  Conclusions ..................................................................................................... 37 
X