Citation for published version (APA):
Lempens, E. H. M. (2011). Bioconjugation strategies for multivalent peptide ligands. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR693309
DOI:
10.6100/IR693309
Document status and date: Published: 01/01/2011 Document Version:
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PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Technische Universiteit Eindhoven, op gezag van de rector magnificus, prof.dr.ir. C.J. van Duijn, voor een
commissie aangewezen door het College voor Promoties in het openbaar te verdedigen
op maandag 10 januari 2011 om 16.00 uur
door
Edith Helène Martine Lempens
prof.dr. E.W. Meijer
Copromotor: dr. M. Merkx
Cover design: ICMS Animationstudio, TU/e Printing: Wöhrmann Print Service, Zutphen
A catalogue record is available from the Eindhoven University of Technology Library ISBN: 978-90-386-2406-8
Chapter 1
Well-Defined Multivalent Macromolecules for Biomedical Applications
1.1 Introduction 2
1.2 Multivalent interactions 2
1.3 Multivalent scaffolds for biomedical applications 4
1.4 Well-defined multimodal dendrimers 7
1.5 Bioorthogonal chemistry 12
1.6 Aim and outline of the thesis 15
1.7 References 17
Chapter 2 A Versatile, Modular Platform for Multivalent Ligands Based on a Dendritic Wedge 2.1 Introduction 26
2.2 Synthesis of labeled dendrons 27
2.3 Peptide dendrons 29
2.4 A systematic study on multivalent interactions 31
2.5 Multivalent peptides as CD40L mimetics 33
2.6 Conclusion 36
2.7 Experimental section 36
2.8 References 47
Chapter 3 Oxime Chemistry as a Complementary Bioconjugation Method for the Modification of Dendritic Wedges 3.1 Introduction 52
3.2 Synthesis of dendritic wedges for oxime chemistry 52
3.3 Synthesis of a collagen mimic via oxime chemistry 53
3.4 Protein dendrons 56
3.5 Peptide-protein hybrid dendrons 59
3.6 Conclusion 62
3.7 Experimental section 62
3.8 References 68
Chapter 4 Dendrimer Display of Tumor-Homing Peptides 4.1 Introduction 72
4.2 Selection of tumor-homing peptides 73
4.3 Synthesis of functionalized dendrons 75
4.5 Targeting lymphatic vessels 80
4.6 Discussion and conclusion 83
4.7 Experimental section 84
4.8 References 89
Chapter 5 Efficient and Chemoselective Surface Immobilization of Proteins using Aniline-Catalyzed Oxime Chemistry 5.1 Introduction 94
5.2 Preparation of aminooxy-functionalized surfaces 95
5.3 Immobilization of NaIO4 treated peptides/proteins 96
5.4 Immobilization of PLP treated proteins 98
5.5 Influence of receptor density 100
5.6 Discussion and conclusion 105
5.7 Experimental section 106
5.8 References 110
5.9 Appendix 112
Chapter 6 Engineering of Proteolytically Activatable Antibodies for Dual-Specific Targeting 6.1 Introduction 116
6.2 Covalent intramolecular approach via oxime chemistry 117
6.3 Covalent intramolecular approach via photoaffinity labeling 120
6.4 Non-covalent intermolecular approach 122
6.4.1 Design of bivalent epitopes 122
6.4.2 Anti-HIV1 as model system 124
6.4.3 Synthesis of peptide-DNA conjugates 124
6.4.4 Characterization of antibody complexes 129
6.5 Conclusion 132 6.6 Experimental section 133 6.7 References 135 6.8 Appendix 137 Summary 139 Samenvatting 143 Curriculum Vitae 147 Dankwoord 151
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AbstractIn biological systems the affinity and specificity of binding are often enhanced by combining multiple weak interactions, an effect known as multivalency. Researchers have followed nature’s exquisite example by combining several low affinity ligands e.g. carbohydrates, peptides or proteins onto a synthetic scaffold in order to create high affinity multivalent ligands for specific delivery of therapeutics or imaging probes to diseased tissue. Among the many different scaffolds that have been developed, dendrimers are of particular interest as they have several unique properties that make them attractive for use in biomedical applications. To date multivalent and multimodal dendritic structures have predominantly been synthesized by statistical modification of peripheral groups. However, potential application of such probes in patients demands well-defined and monodisperse materials with unique structures. Progress in the field of chemical biology involving chemoselective ligation methods renders this challenge possible and as a result promising well-defined dendritic structures for use in biomedical applications have been developed in recent years.
Part of this work has been accepted for publication:
1.1 Introduction
Chemical biology is a relatively new scientific field spanning the traditional disciplines of chemistry and biology. It involves the application of synthetic chemistry to study and manipulate biological systems at a molecular level. Organic synthesis allows the construction of molecular assemblies with a wide variety of functional groups and 3-dimensional topologies. Combining synthetic compounds with biological macromolecules such as carbohydrates, proteins or oligonucleotides has resulted in a number of hybrid structures that are promising candidates for use in targeted imaging and drug delivery. The concept of using “magic bullets” that specifically seek, target and destroy diseased cells with minimal damage to normal tissue has been around since Ehrlich postulated more than 100 years ago the existence of specific receptors that bind antigens.1 In 1975,
Ringsdorf was the first to report on a model in which synthetic scaffolds were used to encapsulate low molecular weight drugs.2 He proposed that a combination of desired
properties could be achieved by linking appropriate structural motifs to different domains along a polymer chain. In this design one part was responsible for solubility and body distribution while another was used to link the drug to the polymer chain by a bond which was either stable or labile in vivo. Finally, a unit which enhances preferential uptake into target tissue was conjugated to the carrier molecule. The resulting hybrid compound with a diameter in the nanometer range was expected to lead to long blood circulation times, thereby increasing its efficacy. Ringsdorf’s initial design was soon followed and other scaffolds such as dendrimers, micelles and liposomes were introduced as smart biomaterials. The search for novel technologies in this area continues to be a very active area of research. Although much progress is achieved in recent years, many fundamental problems are still present with a special position for targeting the scaffolds to a specific place. Active targeting is not achieved yet and novel insights are required to conquer this enormous challenge. For many, nature is a rich source of inspiration in order to arrive at active biological targeting.
1.2 Multivalent interactions
Nature often uses multiple low affinity interactions to enhance the overall affinity and specificity of binding.3 This effect is known as multivalency and plays a pivotal role in e.g.
adhesion of viruses or bacteria to cells, immune responses and protein-protein interactions. Inspired by nature’s success, researchers nowadays often combine several low affinity ligands e.g. carbohydrates, peptides or proteins onto a single scaffold in order to create high affinity multivalent nanoparticles for specific delivery of therapeutics or imaging probes.4 An empirical approach to quantify the enhanced affinity of multivalent
interactions uses the term effective molarity (Meff).5 This molarity is defined as the ratio
between the intermolecular and intramolecular dissociation constant and depends on the length and flexibility of the linker. Krishnamurthy et al. elaborated the concept for the dissociation of a bivalent ligand from a bivalent receptor (Figure 1.1).6 The first step is an
intermolecular process with a dissociation constant equal to ¼ of the monovalent dissociation constant (K inter). This factor derives from the four possibilities of binding
(two ligands can bind to two binding sites).7 The second step is intramolecular
with a dissociation constant of 2 times KDintra. The statistical factor of 2
represents the two possible modes of dissociation. The overall dissociation constant of the multivalent complex (KDavidity) thus depends on the
monovalent dissociation constant (KDinter), the effective molarity and a
statistical factor. In practice multivalent binding will only take place when the effective molarity exceeds the dissociation constant of the interaction. The term effective concentration (Ceff) is
conceptually similar to the more
generally used effective molarity but describes the advantage for an intra- over an intermolecular interaction from physical geometries of the complex.8 Effective
concentration represents the altered concentration of ligand experienced by a free binding site of a multivalent receptor after binding has taken place at a neighbouring site.9For flexible linkers that display a random coil behavior, the effective concentration
can be calculated from the probability that the two ends of the linker are at a position that equals the distance between the binding sites.8 C
eff and Meff are closely correlated for
multivalent interactions except when cooperativity is involved. Cooperativity takes place when the binding of a ligand influences the binding strength of the receptor towards subsequent interactions.10 So far this phenomenon has not been assigned for synthetic
multivalent ligands in contrast to biological systems.
The design of multivalent ligands is often complicated by the fact that the position of binding sites in a multivalent target and their mutual spacing are unknown. As a result small statistical effects are frequently observed due to the use of linkers that are either too short or too long for simultaneous binding to take place. For example the interaction shown in Figure 1.2a between a bivalent ligand and a bivalent receptor theoretically increases the overall affinity only by a factor of 4. In contrast, the chelate effect mainly contributes to the enhanced affinity of a multivalent interaction.11 Simultaneous
occupation of multiple binding sites decreases the overall rate of dissociation as rebinding of partially separated complexes is favored. Besides the length also the nature of the linker is important.12 Linkers that are long and flexible are easy to design and prepare
but result in relatively low effective concentrations (Figure 1.2b). In contrast, rigid linkers that exactly span the distance between the binding sites lead to high effective concentrations but are much more difficult to develop (Figure 1.2d).13 If the linker is a
little too short (or too long) the conformation is strained resulting in an unfavourable
KDavidity ¼ KDinter
2 KDintra =
KDavidity = (2 KDintra)(¼ KDinter) = ½ (KDinter)2/Meff
Figure 1.1. Empirical model describing the dissociation of a bivalent ligand from a bivalent receptor.
D C
B A
energy term (Figure 1.2c).3a Especially for multivalent proteins from which no crystal
structure has been determined the choice of linker length is a returning problem.
Figure 1.2. For the design of optimal high-affinity multivalent ligands the length and nature of the linker are important. a) Schematic representation of the statistical effect. The increased local concentration of the multivalent ligand leads to a higher affinity even when only one receptor is engaged. b-d) Schematic representation of the chelate effect. b) Long and flexible linkers result in an unfavorable entropy penalty. c) Rigid linkers that are slightly too long result in an unfavorable strained conformation. d) Perfectly matched rigid linker.
Most examples of synthetic multivalent ligands use carbohydrates as recognition units.14 A paradigm example is the work of Fan et al. who incorporated structural
information of the heat-labile enterotoxin (LT) from E. coli, a member of the AB5 family of
bacterial toxins, into the design of pentavalent inhibitors.15The five B subunits of LT are
arranged symmetrically and can bind to five ganglioside GM1 groups protruding from cells of the gastrointestinal lumen. The distance between the toxin’s nonadjacent binding sites is 45 Å and was effectively spanned by flexible 4,7,10-trioxa-1,13-tridecanediamine linkers that were coupled to a rigid acylated pentacyclen core. D-galactose, the terminal carbohydrate unit of the natural GM1 receptor was coupled to the linker and used as ligand. The pentavalent structure showed a 105-fold increase in affinity compared to
galactose and is therefore a promising drug candidate for treatment of bacterial infections. Several crystal structures of AB5 toxins formed an excellent basis for designing
potent inhibitors through a structure-based approach. However, precise three-dimensional structural information is often unavailable. Therefore most of the current approaches towards designing multivalent ligands are based on the attachment of monovalent ligands to multiple sites of generic backbones, such as polymers, membranes and dendrimers.4, 16 However, the affinity enhancements of these multivalent ligands
vary widely ranging anywhere from zero to up to nine orders of magnitude. 1.3 Multivalent scaffolds for biomedical applications
The architecture of supramolecular scaffolds including shape, orientation of ligands, flexibility, size and valency greatly influences their binding properties.16 For example,
globular structures may not be capable of spanning large distances needed to cluster multiple proteins in a cell membrane, but may effectively occupy multiple binding sites on an oligomeric receptor. To date, a wide variety of scaffolds from many structural classes has been used (Figure 1.3) and some of the many interesting reports are highlighted in this paragraph.
Porphyrins,17 calixarenes18 and carbohydrates19 are examples of low molecular weight
scaffolds that typically possess a rigid (cyclic) core structure to which a few ligands can be conjugated.20 Trauner et al. developed tetraphenylporphyrins bearing cationic side
chains for simultaneous binding towards highly conserved Asp and Glu residues in each of the four subunits of human KV1.3 potassium channels.18c The authors demonstrated a
strong interaction in the nanomolar range and a reduction in current passing through the channel. Kitov and coworkers prepared a multivalent ligand consisting of 10 carbohydrate head groups attached to a single glucose scaffold.19 This inhibitor bound to pentameric
Shiga-like toxins with an activity that is 107 fold higher than that of the monovalent
ligand. Interestingly, the crystal structure revealed a hamburger type of structure with one ligand binding in between two pentameric subunits. A slightly larger scaffold is a cyclic decapeptide introduced by Mutter et al. which has been used as a starting point for the introduction of four peptides that bind to αvβ3 integrins over expressed in
tumor-induced angiogenesis.21 Binding experiments demonstrated only a five-fold increase in
affinity over the monovalent peptide. In this case the distance between neighboring integrins in the cell membrane is clearly too large for the multivalent scaffold to span and as a result only a statistical effect is observed.
Figure 1.3. Synthetic scaffolds used for the multivalent display of ligands include small molecules like porphyrin (a), linear polymers (b), dendrimers (c), micelles (d) and liposomes (e).
Polymers are linear scaffolds composed of a central backbone that allows the presentation of multiple ligands. Early functionalized polymers typically suffered from a broad range of molecular weights thereby obscuring structure-function relationships and information regarding receptor topology. However, modern polymer chemistry, in particular ring-opening metathesis polymerization (ROMP), provides new opportunities for the synthesis of well-defined polymers with narrow polydispersity.22 In a recent
example Kiessling et al. demonstrated the ability of dinitrophenyl functionalized ROMP polymers to cluster B cell antigen receptors onto specific membrane domains and to generate immune responses in vivo.23 Atom transfer radical polymerization (ATRP) has
emerged as a second powerful method that provides polymers with well-defined
A N H N N H N B C D E
molecular weights. Maynard and coworkers showed that ATRP can even be performed in water with proteins.24
Since the late 1970s, dendrimers have evolved into a versatile new class of compounds in between small organic molecules and polymers. Their stepwise synthesis affords highly branched, essentially monodisperse macromolecules with a well-defined number of peripheral groups.25 As a result of their nearly perfect molecular composition,
dendrimers are more likely than other structures to meet the strict regulatory requirements for polymer-based materials intended for use in humans.26
Glycodendrimers have found wide use in the study of cellular events mediated by lectins. Poly(amido amine) dendrimers functionalized with mannose at the exterior were reported by Mangold and Cloninger.27 Efficient bivalent binding towards dimeric Concanavalin A
was demonstrated using isothermal titration calorimetry. Although most research has concentrated on glycodendrimers some other contributions haven been described. For example, polylysine dendrimers displaying 32 naphthalene disulfonate units at their periphery are used as the pharmaceutical ingredient in Vivagel, a topical vaginal microbicide for HIV prevention.28 These dendrimers prevent infection by binding to the
receptor gp120 on the viral coat of HIV-1, which in turn prevents the virus from entering target cells.
Micelles and liposomes are spherical noncovalent assemblies composed of amphiphilic macromolecules that have distinct hydrophobic and hydrophilic domains.29 They can be
prepared in a wide range of sizes but the arrangement and orientation of ligands is difficult to control. Micelles are attractive for medical applications30due to their relatively
small size and the possibility to load them with hydrophobic drugs. This is illustrated by the work of Nasongkla et al. who prepared micelles filled with chemotherapeutic agents and superparamagnetic iron oxide nanoparticles for use in tumor targeting.31 At the
surface various amounts of the well-known cyclic RGD peptides were incorporated. Flow cytometry analysis showed that the uptake into tumor cells was dependent on the density of cyclic RGD with a maximum of 70% uptake for micelles of which 76% of the lipids was functionalized with cyclic RGD. The synthesis and binding behavior of multivalent protein-functionalized micelles has also been reported.32 CNA35, a collagen
binding domain present in an adhesion protein from Staphylococcus aureus, was coupled via native chemical ligation to pegylated phospholipids. The affinity towards collagen increased with increasing protein-to-lipid ratio as demonstrated by strongly decreased dissociation rates in surface plasmon resonance.32c Liposomes differ from micelles in that
they consist of a lipid bilayer with an internal aqueous compartment. In a beautiful example Kane and coworkers functionalized liposomes with an inhibitory peptide for anthrax toxin at different densities and observed a transition in potency at an interpeptide separation that matched the distance between the binding sites.33 These
pattern-matched liposomes bound heptameric anthrax more than 50,000 fold stronger than the corresponding monovalent peptide making it a potent strategy for treatment of viral and bacterial diseases
Several other examples of noncovalently formed multivalent scaffolds have been reported in literature. Well-known are the peptide amphiphiles from Stupp and coworkers that self-assemble into cylindrical micelles.34 In one of their promising studies a short
pentapeptide (IKVAV) was incorporated that promotes neurite sprouting and growth.34c
Densely packed nanofibers induced differentiation of neural progenitor cells into neurons thereby mimicking the network formed by natural laminin. Synthetic supramolecular polymers based on discotic monomers that self-assemble into columnar stacks were prepared by Müller and Brunsveld.35 Strong binding of these polymers consisting of
different ratios of unfunctionalized and mannose-functionalized monomers towards bacterial lectins was demonstrated using fluorescence microscopy. Furthermore, hydrogen-bonded pentaplex assemblies of DNA that form in the presence of Cs+ have
been investigated for binding to homopentameric C-reactive protein (CRP) produced by the liver in response to infection.36 Five strands containing a sequence of at least four
successive isoguanine bases were functionalized with phosphocholine head groups and assembled in a parallel orientation. The binding affinity of the pentaplex scaffold towards CRP was three orders of magnitude higher than that of the functionalized single strand. In contrast to peptide amphiphiles and supramolecular polymers, the structural valency that can be reached with oligonucleotides is limited. The same is true for several other popular noncovalent assemblies that use for example the self-association domains of leucine zippers37 or the strong interaction between biotin and streptavidin.38
In this paragraph, some successful examples were discussed in which structure-based information of the target’s multiple binding sites was included in the design process leading to affinity enhancements of several orders of magnitude. Disappointing affinities originating from only statistical effects are frequently observed as well. Several reports exist in which researchers focus on the design and synthesis of a certain scaffold to which in the end a popular bioactive ligand is conjugated.21 However, the succesfull examples
demonstrate that it is advantegous to work the other way around and to choose a multivalent scaffold based on the geometry of the target protein.
1.4 Well-defined multimodal dendrimers
In order to use the multivalent scaffolds discussed in the previous paragraph as “magic bullets” the introduction of other functionalities (e.g. imaging probes and therapeutics) is required.39 Dendrimers are in principle highly suited for in vivo use due
to their controlled synthesis and monodisperse structure. In the past, numerous groups have reported on the statistical modification of dendrimers with uniform surface groups for applications such as boron neutron capture therapy (BNCT),40 magnetic resonance
imaging (MRI)41 or drug delivery.42 However, statistical modification forfeits the
advantage of monodispersity and the homogeneity of the material. The key to the successful synthesis of well-defined, monodisperse dendritic structures is accurate control over surface functionalities which can be addressed separately. This can be achieved using e. g. orthogonal protected functional groups, chemoselectively
addressable functionalities or a combination of both. Historically, convergent and divergent approaches are used to synthesize dendrimers, both having their pros and cons. The divergent approach was introduced first by the groups of Tomalia and Newkome and is mostly used because of its ease in synthesis.43 These dendrimers
however often lack – certainly at higher generations - full control over purity and hence the exact number of end groups. The convergent approach was introduced by Hawker and Fréchet and is synthetically more demanding.44 It typically yields dendrimers of very
high purity and the synthetic approach involves the production of dendrons. The latter are useful on their own and as building blocks for the synthesis of asymmetric dendrimers. Nowadays, the convergent approach is the more prominent one with the exception of divergent solid-phase approaches used for the synthesis of multiple antigen peptides (MAPs) based on a branched lysine core.45
In 1996, Qualmann and co-workers were the first to report the synthesis of a defined multifunctional dendron based on a lysine core.46 Simultaneous incorporation of eight
carborane clusters at the outer sphere and an antibody fragment together with a fluorescent probe at the focal point make this structure a promising candidate for use in boron neutron capture therapy (Figure 1.4). By using TentaGel PAP resin for the synthesis, an additional PEG-tail was incorporated to increase its solubility in water. This example shows how four different
functionalities can be conjugated in a highly controlled manner via fast and efficient solid phase chemistry. Several years later, 4th-generation
mannosylated poly(lysine) wedges with a fluorescent group at the C-terminus were prepared by Kantchev et al. to mimic the mannose-receptor-mediated capture and uptake of pathogens by dendritic cells.47 Conjugation of these
glycodendrons is a potentially useful strategy to enhance the immunogenicity of e.g. peptides in vaccine design. A similar strategy was recently used by Lusvarghi et al. to assemble various generations of peptide-functionalized poly(lysine) wedges with a biotin group at the focal point. These constructs were studied for their potential use as biosensors in order to detect bacterial spores in air, water or food supplies.48
Direct comparison of the weakly binding monovalent spore-binding peptide with
Figure 1.4. Schematic representation of a second generation poly(lysine) dendritic wedge functionalized with 80 boron atoms (red), an antibody fragment and a fluorescent probe (green).46 N H O O PEG HNSO2 N H N N H H N N H H N O S O O NH2 NH2 O O HN N H H2N O O N H O H2N O NH O NH2 H N O NH2 NH O HN HN O NH N H O H2N O NH2 O NH NH O H2N O NH2 B10 B10 B10 B10 B10 B10 B10 B10 L in ke r Targeting Fluorescence BNCT
the divalent and tetravalent dendrons revealed an increase in affinity of 1 and 2 orders of magnitude, respectively.
Besides the use of poly(lysine) dendritic structures, several groups have reported the functionalization of other types of dendrons. For example, PAMAM wedges have been condensed with DNA after modification of the focal point with a peptide-modified PEG. The peptide (WIFPWIQL) is capable of targeting the construct to cells expressing the glucose-regulated protein-78 kDa (GRP-78) identified in tumors. In vitro experiments showed that the dendron efficiently targeted and transfected prostate carcinoma cells and therefore has the potential to be used in tumor-targeted gene delivery.49
Kostiainen and co-workers also prepared precisely defined dendritic wedges that are biocompatible and show efficient gene transfection. They used first and second generation Newkome-type wedges modified with spermine at the periphery. This naturally occurring polyamine is capable of high-affinity binding to DNA as shown by an ethidium bromide displacement assay. Functionalization of the focal point with a maleimide-group allowed attachment of proteins via their free cysteines. Conjugation of the adhesion protein hydrophobin (HFBI) to a second generation spermine dendron generated a self-assembling amphiphile that promoted efficient gene transfection in vitro.50
The use of multifunctional dendritic wedges for tumor imaging was demonstrated by the synthesis of peptide dendrons bearing a DOTA label. Monomeric, dimeric and tetrameric amino acid dendrons
H N NH O O O NH O O O N N N N NN N H NH NH HN HN O O O O O R G D f N N N N O OH O OHHO O O NH O O O N N N N N N DOTA c(KRGDf) c(KRGDf) c(KRGDf) c(KRGDf) DOTA c(KRGDf)
Figure 1.5. Structure of a DOTA-conjugated (green) tetravalent cyclo[RGDfK] peptide (red) dendrimer.52 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 O O N N N O O O O R2 R2 O HOHO HO O HO N N N O O N O O N N N O 6 R1= R2=
Figure 1.6. Asymmetric dendrimer containing 16 mannose groups and two coumarin fluorophores.54
were made, starting from 3-hydroxy methyl benzoate or 3,5-dihydroxy methyl benzoate and 2-bromoethylamine.51 In the end, a microwave-assisted 1,3-dipolar cycloaddition
was performed to functionalize the dendritic alkynes with N-ε-azido modified c[KRGDf]. Selective binding of these cyclic peptides to αvβ3 integrins is thought to prevent tumor
growth by antagonizing angiogenesis. After connection of the DOTA moiety to the dendritic core, solid phase binding assays and biodistribution studies were performed (Figure 1.5). The tetrameric construct showed enhanced affinity for αvβ3 integrins and
significant higher uptake by tumor cells in athymic mice with a subcutaneously growing SK-RC-52 renal cell carcinoma.52
In another study Wu et al. explored the use of synthetic glycodendrons as a new strategy to coat single-walled carbon nanotubes (SWNT) and to promote their binding to cells. These nanotubes have been employed for imaging, drug delivery and cancer cell targeting, but modifications are required to diminish their cytotoxicity. Bifunctional dendrimers based on the biocompatible building block 2,2-bis(hydroxymethyl)propionic acid (G2 and G3) were functionalized with a variety of peripheral carbohydrate moieties. Conjugation of a pyrene group to the focal point allowed binding to SWNTs through
π-π
interactions.53 In vitro experiments with these biocompatible carbon nanotubes revealedreceptor specific binding and labeling of the cell membrane.
So far, only dendritic wedges with a single modification at the focal point have been discussed. However, several groups have recently reported the preparation of functional dendrimers by merging two dendritic blocks together. Wu et al. used the 2,2-bis(hydroxymethyl)propionic acid building block for the synthesis of dendrons with either an alkyne or an azide functionality at the focal point. Two series of up to the 4th generation were synthesized and coupling of various blocks proceeded smoothly using copper(I)-catalyzed azide-alkyne cycloaddition. Finally, an asymmetrical dendrimer with two 7-diethylaminocoumarin dyes and sixteen mannose groups (Figure 1.6) was prepared, which exhibited a 240-fold greater potency than monomeric mannose in the hemagglutination assay.54
A similar concept was used for the development of multivalent target-specific MRI contrast agents for in vivo imaging of cardiovascular diseases. Two poly(lysine) wedges of which one has a thioester at its focal point and the other a cysteine residue were coupled via native chemical ligation. The chemical structure of the dendrimer consisting of a second generation DTPA-wedge and a first generation peptide-wedge (GRGDS) is shown in Figure 1.7.55 Deguise and co-workers used the azide-alkyne cycloaddition for
the synthesis of well-defined glycodendrimers containing fucoside residues at one side and galactoside residues at the other side. These two carbohydrates inhibit either PA-IL or PA-IIL lectins present in gram-negative bacteria such as Pseudomonas aeruginosa and are therefore interesting therapeutic agents for the prevention of bacterial infections. The repeating unit for the dendron synthesis was an aromatic diazido acid and merging of the two building blocks was based on the formation of amide linkages with a bis-amine function.56 Hetero-bifunctional dendrimers up to eight fucoside and eight galactoside
residues showed binding and clustering of both PA-IL and PA-IIL lectins. The three examples above show that by merging two dendrons the ratio between the number of labels and the number of targeting units can be controlled and optimized. Furthermore, both functionalities are physically separated from each other in distinct clusters allowing optimal presentation of ligands for binding to receptors.
Besides the use of dendritic wedges for the synthesis of multifunctional dendrimers, a few other approaches have been explored. Fuchs et al. prepared a first generation bifunctional polyamidoamine dendrimer by using the orthogonal protected 3,5-bis(3-aminopropyl)benzoic acid as a branching unit.57 They incorporated three dansyl
fluorescence tags for intracellular detection and three oligopeptides (GRFG) that are cleaved in the presence of cathepsin B, a cysteine protease that is often overexpressed in malignant cells. Furthermore a 2,3-D/L-diaminopropionic acid (Dpa) ligand was
introduced to the N-terminus of the peptide for potential complexation of anticancer-active Pt2+. Confocal fluorescence microscopy confirmed the rapid internalization of these
dendrimers in HeLa cells. Coupling of antitumor agents to the cleavable peptides might generate promising constructs for targeted drug delivery.58 Another drug delivery system
based on PEGylated triazine dendrimers derivatized with two groups for radioiodination and 16 anticancer agents, was investigated by Lim and Simanek (Figure 1.8). Their synthetic route is based on stepwise reactions of cyanuric chloride with amine nucleophiles and gives rise to an interesting dendritic structure in which the ratio of three different functional groups is precisely defined.59
H2N O N H NH HS O H N O H N NH O HN Ac-Gly-Arg-Gly-Asp-Ser-Gly-Gly-Cys NH Ac-Gly-Arg-Gly-Asp-Ser-Gly-Gly-Cys
O NH
NH Ac-Gly-Arg-Gly-Asp-Ser-Gly-Gly-Cys
Ac-Gly-Arg-Gly-Asp-Ser-Gly-Gly-Cys
O N H O HN O NH N H O HN H N O HN HN O H N O S R O S R O S S R O N H H N HN HN O HN O O S R O S R H N S R O O S R N O O H N O OH N O N N HOOC HOOC COOH COOH 4 R R= DTPA
Most of the structures presented in this paragraph are based on dendritic wedges bearing multiple ligands at the periphery and a single label (fluorescent dye or contrast agent) at the focal point. However, it is more favorable to have multiple labels as well for visualization during in vivo measurements. This issue is addressed in the few examples of merged dendritic wedges that currently exist. Here, the ratio between number of labels and number of targeting units can be controlled. Further interdisciplinary research between synthetic chemists and biologists will be needed to fully benefit from the advantages of well-defined multivalent and multifunctional dendrimers.
1.5 Bioorthogonal chemistry
Developments in chemical biology have increased the efficiency of bioconjugation methods enabling complex macromolecules to be assembled. Key to this is the use of bioorthogonal reactions whose components react rapidly and selectively with each other under physiological conditions in the presence
of a whole spectrum of functional groups found in natural ligands.60 In contrast to
classical bioconjugation methods that typically target the side chains of lysine or cysteine residues, chemoselective reactions result in a single controlled modification thereby minimizing the chance of inactivation. In addition to the synthesis of complex hybrid structures as shown in the previous paragraph, bioorthogonal chemistry has mainly been employed to prepare synthetic full-length proteins out of unprotected peptide fragments, to label biomolecules for in vivo visualization and to orient biomolecules on surfaces. The most widely used chemoselective approaches are based on the Staudinger, Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition, imine ligation (oxime and hydrazone) and native chemical ligation reactions (Figure 1.9). Although these conjugation methods have already been applied very successfully, chemists and biologists keep developing further strategies for the synthesis of biomolecules bearing chemical reporter groups. In this respect, particular emphasis is placed on bioorthogonality and achievement of mild reaction conditions that are transferrable to in vivo applications. In contrast, for the modification of multivalent scaffolds or surfaces with biomolecules as described in this thesis the easy accessibility of starting materials and fast reaction kinetics are even more
Figure 1.8. Drug delivery system based on triazine dendrimers developed by Lim and Simanek.59 N N N N R1 HN N N N N NH NH N N N N N N N N N N HN NH HN HN N N N N N N N N N N N N R2 R3 R3 R2 R2 R3 R3 R2 N N O OH H N HN Paclitaxel O O N HN PEG N HN HN N N N N N N R2 R3 R3 R2 N HN NH N N N N N N R2 R3 R3 R2 R1= R2= R3=
important. Therefore this section focuses on these characteristics for the four ligation methods mainly used nowadays.
Figure 1.9. Schematic overview of widely used chemoselective reactions for conjugation of biomolecules. a) Staudinger ligation of azides and triarylphosphines. b) Cu(I)-catalyzed [3+2] cycloaddition of azides and alkynes to form triazoles. c) Oxime formation between aldehydes/ketones and aminooxy groups. d) Native chemical ligation between thioesters and cysteine residues.
The Staudinger ligation was introduced as a chemoselective ligation for bioconjugation by Saxon and Bertozzi in 2000.61 The reaction proceeds through the nucleophilic attack of
a phosphine on an azide to give an aza-ylide intermediate, which is trapped in an intramolecular fashion by the methoxycarbonyl group to give an amide bond after hydrolysis.62 Later, a traceless version using phosphinothioesters was reported in which
amide bonds are produced without inclusion of the phosphine oxide moiety.63 The
traceless Staudinger ligation is of particular interest for ligation of peptide fragments as shown successfully by Wong et al. for the synthesis of glycopeptides in high yield.64 In
addition to its use in chemical peptide synthesis, the Staudinger ligation has frequently been explored for the preparation of bioconjugates.65 The research groups of Tirrell and
Bertozzi have described a general strategy for the incorporation of azides into recombinant proteins for subsequent chemoselective modification.66 Thereto,
azidohomoalanine is activated by methionyl-tRNA synthetase of E. coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. The exquisite bioorthogonality of azides has even enabled the use of the Staudinger ligation for probing cell-surface glycans in living animals although the reaction kinetics turned out to be relatively slow.67 R O N H2 O R N O N3 NH O O PPh2 O O Ph2P N3 N N N SR O HN 2 S H O N H O SH O + + + + A C B D
Azides can also participate in Huisgen-type [3+2] cycloadditions, such as the Cu(I)-catalyzed click-reaction described independently by the groups of Meldal and Sharpless.68
This cycloaddition of azides and alkynes yields exclusively the 1,4-substituted triazole isomer, whereas the uncatalyzed version delivers 1,4 and 1,5 isomers in equal amounts. Furthermore, the catalyzed reaction is bioorthogonal, fast, high-yielding and proceeds under mild conditions. The reaction has found various applications in material and polymer science as well as in the specific labeling of biomolecules. However, the use of Cu(I)-catalyzed azido-alkyne 1,3-dipolar cycloadditions (CuAAC) in living systems has been hindered by the toxicity of Cu(I). To improve the biocompatibility of this reaction Bertozzi et al. developed a strain-promoted reaction in which the ring strain of a cyclic substituted cyclooctyne is used as the driving force.69 Fast bioconjugation of functional
labels to azido-functionalized glycoproteins was shown in live cells and zebrafish embryos.70 In addition to azides, also alkynes can be incorporated into recombinant
proteins by replacing methionine in the culture medium by 2-amino-5-hexynoic acid.71
All imine ligations proceed via a common mechanism involving the nucleophilic attack of an amino group on a protonated carbonyl followed by a dehydration step. Although the free carbonyl is favored in water, compounds with α-effect nitrogens can be used to shift the equilibrium towards the imine.72 α-Effect nitrogens such as hydrazides or aminooxy
groups, whose basicities are lowered by neighboring nitrogen or oxygen atoms, react with aldehydes or ketones at acidic pH to form oxime or hydrazone linkages, respectively.73 Oxime ligated products are stable under physiological conditions
illustrated by their use in bioconjugation and labeling studies.74 Hydrazones on the other
hand are more dynamic75 and of special interest for dynamic covalent chemistry.76
Several approaches currently exist to introduce carbonyl groups into peptides and proteins including synthetic, semi-synthetic and genetic procedures. Oxidation of serine or threonine residues using sodium periodate is a fast and easy method to introduce a carbonyl at the N-terminus of a protein.77 Essentially full conversion is achieved but care
has to be taken to prevent oxidation of sensitive residues such as Met, Cys and Trp. In 2006, Francis and coworkers showed that N-terminal ketones can also be introduced site-specifically using oxidation with pyridoxal 5’-phosphate.78 This method can be applied to
many proteins without recombinant modification, but the reaction is not quantitative and the yield depends on the nature of the N-terminal amino acid.79 For proteins in which the
N-terminus is functionally important, recombinant procedures have recently become available that allow site-specific introduction of ketone functionalities at any place in a protein. Carrico and coworkers e.g. showed that the formylglycine generating enzyme present in E. coli recognizes an LCTPSR motif introduced into target proteins and oxidizes the cysteine residue posttranslationally into an aldehyde functionality.80 Despite the
availability of several methods to introduce carbonyl groups, slow reaction rates (especially at physiological pH) have hampered the scope of this approach in the past. Recently, Dirksen et al. explored the use of aniline as a nucleophilic catalyst for oxime and hydrazone ligations.72, 76, 81 Rate enhancements of up to three orders of magnitude
were achieved enabling reactions to be performed even at low concentrations and neutral pH.
Native chemical ligation was developed in 1994 by Dawson and Kent to facilitate the chemical synthesis of proteins from unprotected peptide fragments.82 The reaction
between an N-terminal cysteine and C-terminal thioester proceeds under aqueous conditions and gives a peptide bond at the site of ligation. The amide-forming intramolecular step had been reported before by Wieland and coworkers.83 When applied
in the context of biologically derived proteins native chemical ligation is often called expressed protein ligation.84 Initially, the method was restricted to cysteines but
continued advances, including the use of removable auxiliary groups and desulphurization methods, have significantly broadened the scope of the method.85
Although thioesters are not very stable and therefore not ideal for in vitro or in vivo labeling of biomolecules, native chemical ligation has found wide application in peptide chemistry and in the functionalization of nanoparticles or surfaces with biomolecules.86
The introduction of thioesters into peptides is straightforward in the case of tBoc chemistry87, but requires the use of special resins when Fmoc-protected amino acids are
used.88 In addition, an expression and purification strategy based on protein splicing has
been developed to obtain recombinant proteins with a C-terminal thioester. Muir et al. showed that recombinant proteins can be prepared as a fusion with a C-terminal intein domain and a chitin binding domain.84 After purification using a chitin column, treatment
with thiols induces intein-catalyzed cleavage and elution of the protein of interest with a C-terminal thioester.
1.6 Aim and outline of the thesis
Future challenges in the fields of targeted drug delivery and molecular imaging include the rational design and synthesis of well-defined multivalent ligands with high affinity and specificity for biological targets. More systematic studies on the strength of multivalent interactions are however required in order to fine-tune structure-function relationships concerning the number of functional groups and the influence of length and flexibility of the linker. Generic strategies that allow fast and easy screening of constructs with different linkers or functional groups are attractive especially for multivalent targets of which no structural information about the position of binding sites is available. The aim of this research is to develop novel synthetic strategies for well-defined multivalent ligands and to study their binding behavior using various biochemical techniques. The use of chemo- and regioselective reactions plays a crucial role throughout this thesis in order to ensure homogeneous presentation and activity of multivalent peptides and proteins.
In the first part of this thesis a general methodology is presented for the synthesis of well-defined multivalent and multimodal dendritic wedges. The ability of this type of macromolecule to present multiple groups at the surface, while maintaining a well-defined structure with low polydispersity makes them ideal for use in biomedical applications. In Chapter 2, the quantitative functionalization of asymmetric polyamide
dendrons that possess N-terminal cysteine residues at the periphery with C-terminal thioester peptides using native chemical ligation is described. Conjugation of biologically relevant groups at the focal point resulted in a series of related structures with highly controlled valency (2-5) that can be used directly in a systematic study of the strength of multivalent interactions. Oxime chemistry is complementary to native chemical ligation as it allows functionalization with peptides and proteins via their N-terminus. The applicability of this chemoselective reaction is reported in Chapter 3 using the same polyamide dendrons but this time functionalized with aminooxy groups. Although efficient ligation of peptides was observed steric hindrance prevented the formation of multivalent protein constructs. In collaboration with the University of California in Santa Barbara the tumor homing behavior of our dendrons modified with peptides developed via in vivo phage display was explored. Chapter 4 describes the modification of the dendritic wedge with a short linear peptide that homes to clotted plasma proteins. Although a specific receptor in tumor tissue was recognized, the extravasation was affected by the size of the construct. In contrast, a highly positively charged cyclic peptide with cell penetrating properties was capable of directing the entire dendritic architecture towards a specific receptor in tumor lymphatics. These results emphasize the influence of peptide properties and overall size on the biodistribution of dendritic structures.
The use of aniline-catalyzed oxime chemistry as an efficient bioconjugation method was further explored in Chapter 5 for the site-selective immobilization of proteins on biosensor surfaces. Carboxylate-functionalized SPR chips were modified with aminooxy groups via a newly developed bifunctional linker. Proteins were oxidized under mild conditions resulting in the introduction of an N-terminal ketone and injected over the aminooxy-modified surfaces. The influence of pH and aniline catalysis on the ligation rate were investigated as well as the activity towards binding partners. Our protein-functionalized chip surfaces showed an increased binding capacity compared to surfaces prepared via standard amine coupling. In addition, this novel immobilization strategy was used to investigate how receptor density influences the binding affinity and kinetics of bivalent ligands connected via either a long and flexible or a short and rigid linker.
The development of bivalent ligands connected via long and rigid linkers that bind simultaneously to both binding sites of an antibody is challenging due to the large distances that need to be spanned. In Chapter 6 the investigation of the use of double stranded DNA as a linker is described. DNA is attractive because of its long persistence length and the possibility to easily tune the distance by the number of base pairs. Peptide-DNA conjugates were successfully synthesized using the selective reaction between a thiol and a maleimide group. Using size-exclusion chromatography, the formation of cyclic bivalent 1:1 complexes was demonstrated after association of the functionalized DNA with antibodies. The complexes formed could be applied in a novel dual specific targeting concept based on proteolytic activation of blocked antibodies in order to increase their specificity for tumor tissue.
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