“This book is, indeed, a very timely account of various aspects of the chemical and biological properties of dendrimers, which make them such a versatile tool both for fundamental research and applied biochemistry.”

“Dendrimers are still in their infancy, but they attract more and more attention of scientists from various disciplines, and this book will be a very helpful tool for those who are already well familiar or only starting to become acquainted with them.”

Overview of the state-of-the-art in a rapidly moving research…..be essential reading for graduates and academics involved in research in the area.

“This book is, indeed, a very timely account of various aspects of the chemical and biological properties of dendrimers, which make them such a versatile tool both for fundamental research and applied biochemistry.”

“Dendrimers are still in their infancy, but they attract more and more attention of scientists from various disciplines, and this book will be a very helpful tool for those who are already well familiar or only starting to become acquainted with them.”

— “The Biochemist Evolution website”

Overview of the state-of-the-art in a rapidly moving research…..be essential reading for graduates and academics involved in research in the area.

— “Chemistry and Industry, 6th November 2006 (David Smith)”

Dendrimers in Medicine and Biotechnology

New Molecular Tools

By U. Boas, J.B. Christensen, P.M.H. Heegaard

The Royal Society of Chemistry

Copyright © 2006 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-852-6

Contents

Chapter 1 Dendrimers: Design, Synthesis and Chemical Properties, 1,
Chapter 2 Properties of Dendrimers in Biological Systems, 28,
Chapter 3 Dendrimers as Drug Delivery Devices, 62,
Chapter 4 Dendrimer Drugs, 90,
Chapter 5 Dendrimers in Diagnostics, 130,
Chapter 6 Dendrimers as Biomimics, 152,
Subject Index, 173,

CHAPTER 1

Dendrimers: Design, Synthesis and Chemical Properties

1.1 Introduction

The dendritic structure is a widespread motif in nature often utilised where a particular function needs to be exposed or enhanced. Above ground, trees use dendritic motifs to enhance the exposure of their leaves to the sunlight, which is crucial to maintain life and growth via the photosynthesis. The shade of the tree crown creates a microenvironment maintaining higher humidity and more stable temperatures throughout the day compared to the surroundings. Also beneath ground, the trees have a maximum need to expose a large functional surface when collecting water from the soil. A large dendritic network of roots provides an excellent motif for that purpose (Figure 1.1).

In the “design” of animals and humans, evolution often ends up creating dendritic solutions to enhance particular properties. When breathing air into our lungs the air passes through a tremendous dendritic network of bronchioles and alveoli in order to give maximum surface for the transfer of oxygen into the bloodstream. Also the arterial network transporting the oxidised blood to the different organs progress into dendritic patterns, before the blood is transported back to the heart via the venous system. The central nervous system and the brain consist of a large amount of cells growing into dendritic structures in order to gain the largest exchange of material (and information) with the surrounding tissue. Microglia cells serving as multifunctional helper cells in the brain, form dendritic strucures when activated during pathological or degenerative states in the brain (Figure 1.2). Also here the dendritic structure ensures maximum delivery of secreted anti-inflammatory interleukins to the diseased brain tissue.

Another striking example of dendritic structures in nature discovered just recently, is the tremendous number of foot-hairs on the Gecko’s feet. These foot-hairs “setae” split up into an impressive dendritic network of tiny foot hairs “spatulae”, enabling the Gecko to “stick” to surfaces through dry adhesion without the need of humidity to create surface tension. Examinations of the Gecko’s foot-hairs have revealed that the structures of the millions of end foot-hairs are so microscopic that the adhesion between the surface and the gecko foot is thought to be achieved by weak attractive quantum chemical forces from molecules in each foot-hair interacting with molecules of the surface, the so-called Van der Waal forces. By applying a dendritic pattern, the enhancement of a certain function can sometimes greatly exceed the sum of single entities carried on the surface, because of the synergy gained by a dendritic presentation of a function. So nature has, indeed, applied dendritic structures throughout evolution with great success.

In synthetic organic chemistry the creation and design of dendritic compounds is a relatively new field. The first successful attempt to create and design dendritic structures by organic synthesis was carried out by Vögtle and co-workers in 1978. These relatively small molecules were initially named “cascade molecules” and already then Vögtle and co-workers saw the perspectives in using these polymers as, e.g. molecular containers for smaller molecules. However, after this first report, several years passed before the field was taken up by Tomalia’s group at Dow Chemicals. They had during the years developed a new class of amide containing cascade polymers, which brought these hitherto quite small molecular motifs into well-defined macromolecular dendritic structures. Tomalia and co-workers baptised this new class of macromolecules “dendrimers” built up from two Greek words “dendros” meaning “tree” or “branch” and “meros” meaning “part” in Greek. Later refinement and development of synthetic tools enabled the scientists also to synthesise macromolecular structures relying on the original “Vögtle cascade motif”.

Parallel to polymer chemists taking this new class of compounds into use, dendritic structures also started to emerge in the “biosphere”, where J. P. Tam in 1988 developed intriguing dendritic structures based on branched natural amino acid monomers thereby creating macromolecular dendritic peptide structures commonly referred to as “Multiple Antigen Peptide”. The Multiple Antigen Peptide is, as we shall see later, a special type of dendrimer.

Dendrimers are also sometimes denoted as “arboroles”, “arborescent polymers” or more broadly “hyperbranched polymers”, although dendrimers having a well-defined finite molecular structure, should be considered a sub-group of hyperbranched polymers. After the initial reports the papers published on the synthesis, design and uses of dendrimers in chemistry as well as in biological field has had an exponential increase in numbers.

1.2 Terms and Nomenclature in Dendrimer Chemistry

Dendrimer chemistry, as other specialised research fields, has its own terms and abbreviations. Furthermore, a more brief structural nomenclature is applied to describe the different chemical events taking place at the dendrimer surface. In the following section a number of terms and abbreviations common in dendrimer chemistry will be explained, and a brief structural nomenclature will be introduced.

Hyperbranched polymers is a term describing a major class of polymers mostly achieved by incoherent polymerisation of ABn (n ≥ 2) monomers, often utilising one-pot reactions. Dendrimers having a well-defined finite structure belongs to a special case of hyperbranched polymers (see Figure 1.3). To enhance the availability of dendritic structures, hyperbranched polymers are for some purposes used as dendrimer “mimics”, because of their more facile synthesis. However, being polydisperse, these types of polymers are not suitable to study chemical phenomena, which generally require a well-defined chemical motif enabling the scientist to analyse the chemical events taking place. The physicochemical properties of the undefined hyperbranched polymers are intermediate between dendrimers and linear polymers.

Dendrigrafts are class of dendritic polymers like dendrimers that can be constructed with a well-defined molecular structure, i.e. being monodisperse. However, in contrast to dendrimers, dendrigrafts are centred around a linear polymer chain, to which branches consisting of copolymer chains are attached. These copolymer chains are further modified with other copolymer chains and so on, giving a hyper-branched motif built up by a finite number of combined polymers. Whereas the dendrimer resembles a tree in structure, the core part of a dendrigraft to some extent resembles the structure of a palm-tree.

Dendrons is the term used about a dendritic wedge without a core, the dendrimer can be prepared from assembling two or more dendrons. As we shall see later, dendrons are very useful tools in the synthesis of dendrimers by the segment coupling strategy (convergent synthesis). A class of dendrons, which is commercially available and has been applied with great success in the covalent and non-covalent assembly of dendrimers, are the “Fréchet-type dendrons”. These are dendritic wedges built up by hyperbranched polybenzylether structure, like the Fréchet-type dendrimers. These dendrons have been used in the creation of numerous of dendrimers having different structures and functions.

Generation is common for all dendrimer designs and the hyperbranching when going from the centre of the dendrimer towards the periphery, resulting in homostructural layers between the focal points (branching points). The number of focal points when going from the core towards the dendrimer surface is the generation number (Figure 1.4). That is a dendrimer having five focal points when going from the centre to the periphery is denoted as the 5th generation dendrimer. Here, we abbreviate this term to simply a G5-dendrimer, e.g. a 5th generation polypropylene imine and a polyamidoamine dendrimer is abbreviated to a “G5-PPI-” and “G5- PAMAM” dendrimer, respectively. The core part of the dendrimer is sometimes denoted generation “zero”, or in the terminology presented here “G0”. The core structure thus presents no focal points, as hydrogen substituents are not considered focal points. Thus, in PPI dendrimers, 1,4-diaminobutane represents the G0 core-structure and in PAMAM Starburst dendrimers ammonia represents the G0 core-structure. Intermediates during the dendrimer synthesis are sometimes denoted half-generations, a well-known example is the carboxylic acid-terminated PAMAM dendrimers which, as we shall see later, sometimes have properties preferable to the amino-terminated dendrimers when applied to biological systems.

Shell: The dendrimer shell is the homo-structural spatial segment between the focal points, the “generation space”. The “outer shell” is the space between the last outer branching point and the surface. The “inner shells” are generally referred to as the dendrimer interior.

Pincer: In dendrimers, the outer shell consists of a varying number of pincers created by the last focal point before reaching the dendrimer surface. In PPI and PAMAM dendrimers the number of pincers is half the number of surface groups (because in these dendrimers the chain divides into two chains in each focal point).

End-group is also generally referred to as the “terminal group” or the “surface group” of the dendrimer. The word surface group is slightly more inaccurate, in the sense that the dendrimer branches can sometimes fold into the interior of the dendrimer. Dendrimers having amine end-groups are termed “amino-terminated dendrimers”.

MAP-dendrimers stand for “Multiple Antigen Peptide”, and is a dendron-like molecular construct based upon a polylysine skeleton. Lysine with its alkylamino side-chain serves as a good monomer for the introduction of numerous of branching points. This type of dendrimer was introduced by J. P. Tam in 1988, has predominantly found its use in biological applications, e.g. vaccine and diagnostic research. MAP was in its original design a “tree shaped” dendron without a core. However, whole dendrimers have been synthesised based upon this motif either by segmental coupling in solution using dendrons or stepwise by solid-phase synthesis.

PPI-dendrimers stand for “Poly (Propylene Imine)” describing the propyl amine spacer moieties in the oldest known dendrimer type developed initially by Vögtle. These dendrimers are generally poly-alkylamines having primary amines as endgroups, the dendrimer interior consists of numerous of tertiary tris-propylene amines. PPI dendrimers are commercially available up to G5, and has found widespread applications in material science as well as in biology. As an alternative name to PPI, POPAM is sometimes used to describe this class of dendrimers. POPAM stands for POly (Propylene AMine) which closely resembles the PPI abbreviation. In addition, these dendrimers are also sometimes denoted “DAB-dendrimers” where DAB refers to the core structure which is usually based on DiAminoButane.

PEI-dendrimers is a less common sub-class of PPI dendrimers based on Poly (Ethylene Imine) dendritic branches. The core structure in these dendrimers are diamino ethane or diamino propane.

PAMAM-dendrimers stand for PolyAMido-AMine, and refers to one of the original dendrimer types built up by polyamide branches with tertiary amines as focal points. After the initial report by Tomalia and co-workers in the mid-1980s PAMAM dendrimers have, as the PPI dendrimers, found wide use in science. PAMAM dendrimers are commercially available, usually as methanol solutions. The PAMAM dendrimers can be obtained having terminal or surface amino groups (full generations) or carboxylic acid groups (half-generations). PAMAM dendrimers are commercially available up to generation 10.

Starburst dendrimers is applied as a trademark name for a sub-class of PAMAM dendrimers based on a tris-aminoethylene-imine core. The name refers to the star- like pattern observed when looking at the structure of the high-generation dendrimers of this type in two-dimensions. These dendrimers are usually known under the abbreviation PAMAM (Starburst) or just Starburst.

Fréchet-type dendrimers is a more recent type of dendrimer developed by Hawker and Fréchet based on a poly-benzylether hyperbranched skeleton. This type of dendrimer can be symmetric or built up asymmetrically consisting of 2 or 3 parts of segmental elements (dendrons) with, e.g. different generation or surface motif. These dendrimers usually have carboxylic acid groups as surface groups, serving as a good anchoring point for further surface functionalisation, and as polar surface groups to increase the solubility of this hydrophobic dendrimer type in polar solvents or aqueous media.

“Black ball” nomenclature: Because of the large molecular structure of a dendrimer, the full picture of, e.g. reactions taking place on the dendrimer surface or in the outer shell can be difficult to depict. A way to facilitate the depiction of these macromolecules is by showing the inner (and unmodified) part of the dendrimer as a “black ball”. Depending on whether the reaction takes place at the surface groups or in the outer shell, the appropriate part of the molecular motif, e.g. the outer pincers, may be fully drawn out to give a concise picture of a reaction involving the outer shell (see Figure 1.5). In this way the picture of reactions taking place at the dendrimer surface or in the outer shell is greatly simplified.

1.3 Dendrimer Design

After the initial reports and development of these unique well-defined structures, chemists have begun to develop an excessive number of different designs of dendrimers for a wide variety of applications. Newkome and co-workers developed the unimolecular micelle consisting of an almost pure hydrocarbon scaffold, Majoral and Caminade introduced the multivalent phosphorus to create intriguing new dendrimeric designs and dendrimers having new properties. Other third period elements like silicon and sulfur have been implemented in the dendritic structures resulting in dendrimers having properties quite different from the classical PAMAM and PPI designs. The monomers applied in the build-up of a dendrimer are generally based on pure synthetic monomers having alkyl or aromatic moieties, but biological relevant molecules like carbohydrates, amino acids and nucleotides have been applied as monomers as well (Figure 1.6).

Using biological relevant monomers as building blocks presents an intriguing opportunity to incorporate biological recognition properties into the dendrimer. As we shall see, also metal ions serve as good focal points and have found extensive use in various functional dendrimer designs as well as in the synthesis of dendrimers by self-assembly.

1.4 Dendrimer Synthesis

Divergent dendrimer synthesis: In the early years of dendrimers, the synthetic approach to synthesise the two major dendrimer designs, the PPI and PAMAM, relied on a stepwise “divergent” strategy. In the divergent approach, the construction of the dendrimer takes place in a stepwise manner starting from the core and building up the molecule towards the periphery using two basic operations (1) coupling of the monomer and (2) deprotection or transformation of the monomer end-group to create a new reactive surface functionality and then coupling of a new monomer etc., in a manner, somewhat similar to that known from solid-phase synthesis of peptides or oligonucleotides.

For the poly (propyleneimine) dendrimers, which are based on a skeleton of poly alkylamines, where each nitrogen atom serves as a branching point, the synthetic basic operations consist of repeated double alkylation of the amines with acrylonitrile by “Michael addition” results in a branched alkyl chain structure. Subsequent reduction yields a new set of primary amines, which may then be double alkylated to provide further branching etc. (Figure 1.7)

PAMAM dendrimers being based on a dendritic mixed structure of tertiary alkylamines as branching points and secondary amides as chain extension points was synthesised by Michael alkylation of the amine with acrylic acid methyl ester to yield a tertiary amine as the branching point followed by aminolysis of the resulting methyl ester by ethylene diamine.

The divergent synthesis was initially applied extensively in the synthesis of PPI and PAMAM dendrimers, but has also found wide use in the synthesis of dendrimers having other structural designs, e.g. dendrimers containing third period heteroatoms such as silicium and phosphorous. Divergent synthesis of dendrimers consisting of nucleotide building blocks has been reported by Hudson and coworkers. The divergent stepwise approach in the synthesis of nucleotide dendrimers and dendrons is interesting from a biochemical perspective as it may mimic the synthesis of naturally occuring lariat and forked introns in microbiology.

(Continues…)Excerpted from Dendrimers in Medicine and Biotechnology by U. Boas, J.B. Christensen, P.M.H. Heegaard. Copyright © 2006 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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