PART 1
Glycodendrimers as a new tool for the
vaccination against pneumococcal diseases
Part 1. Preface
.1.1. Streptococcus pneumoniae: a worldwide pathogenic problem
Streptococcus pneumoniae, known as pneumococcus in medical microbiology, is a Gram-positive elongated coccus with a slightly pointed outer curvature. Usually, pneumococci are seen as pairs of cocci (diplococci), but they may also occur singly and in short
chains (Fig. 1.1). Streptococcus pneumoniae is a normal inhabitant of the human upper respiratory tract. The bacterium can cause pneumonia, usually of the lobar type, paranasal sinusitis and otitis media, or meningitis, which is usually secondary to one of the former infections. This bacterium has a capsule composed of polysaccharide that completely envelops the pneumococcal cells and that is an essential determinant of virulence during invasion, interfering with phagocytosis by leukocytes. 91 different capsule types of pneumococci have been identified and form the basis of
antigenic serotyping of the organism. Serotypes 6, 14, 18, 19, and 23 are the most prevalent, main causes for 60-80% of infections depending on the area of the world. Pneumococcal infection accounts for more deaths than any other vaccine-preventable bacterial disease. Those most commonly at risk for pneumococcal infection are children between 6 months and 4 years of age and adults over 60 years of age. Virtually every child will experience pneumococcal otitis media before the age of 5 years. It is estimated that 25% of all community-acquired pneumonia is due to pneumococcus (1,000 per 100,000 inhabitants). Until 2000, S. pneumoniae infections caused 100,000-135,000 hospitalizations for pneumonia, 6 million cases of otitis media, and 60,000 cases of invasive disease, including 3300 cases of meningitis every year and according to Centers for Disease Control and Prevention(CDC) caused more than 6,000 deaths annually. After 2000, with the use of PCV7 vaccine, severe pneumococcal disease dropped by nearly 80% among children under 5.
1.2. Vaccines against pneumococcal diseases
Given the 91 different capsular types of pneumococci, a comprehensive vaccine based on polysaccharide alone is not yet feasible. Thus, vaccines based on a subgroup of highly prevalent types have been formulated. At the moment there are two different types of vaccines against SP infections: the pneumococcal polysaccharide vaccine as commercial PPV23 and the pneumoccoccal conjugate vaccine as PCV13. The PPV23, commercialized as Pneumovax® from
Merck, is a 23-valent formulation: each 0.5 mL dose contains 25 µg of each of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. These serotypes represent 85-90% of those that cause invasive disease, and the vaccine efficacy is estimated at 60% . This formulation is currently recommended for use in all adults who are older than 65 years of age and for persons who are 2 years and older with high risk for diseases (HIV infection, or other immunocompromising conditions). It is also recommended for use in adults 19 through 64 years of age who smoke cigarettes or who have asthma. Unfortunately Pneumovax are not immunogenic in children under the age of 2 years where a significant amount of disease occurs. However, underutilization of this vaccine is so extensive and the pneumococcus remains the most common infectious agent leading to hospitalization in all age groups: in 2006, only about 57% of adults aged ≥65 years of age had received the vaccine.
PCV13, commercialized since February 2010 as Prevnar13® by Pfizer, is a 13-valent pneumococcal conjugate vaccine that replaces a previous conjugate vaccine (PCV7, Prevnar®), which protected against 7 pneumococcal types and has been in use since 2000 as a pediatric vaccine routinely given to infants. PCV13 contains polysaccharides of the capsular antigens of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually conjugated to the carrier protein CRM197 (CRM, cross-reactive material), a nontoxic mutant of diphtheria toxin. A 0.5 mL PCV13 dose contains approximately 2 µg of polysaccharide from each of 12 serotypes and approximately 4 µg of polysaccharide from serotype 6B; the total concentration of CRM197 is approximately 34 µg. PCV13 is approved for use among children aged 6 weeks-71 months. Since March 2009 also GlaxoSmithKline produces a decavalent conjugate vaccine, commercialized as Synflorix®, that contains 10 serotypes of pneumococcus (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F), eight of which are conjugated to a nonlipidated cell-surface lipoprotein (protein D) of non-typeable Haemophilus influenzae (NTHi) and two of which are conjugated to either tetanus or diphtheria toxoid.
Those vaccines induce antibodies to the specific types of pneumococcal capsule and have been shown to be effective against invasive disease, especially septicemia and meningitis. However, children exposed to a serotype not contained in the vaccine are not afforded any protection. This limitation, and the ability of capsular-polysaccharide conjugate vaccines to promote the spread of non-covered serotypes, has led to research into that type of vaccines: additional pneumococcal conjugate vaccine formulations are expected to be licensed soon.1
1.3. Biological action of polysaccharide and conjugate vaccines
The development of glycoconjugate vaccines that can produce saccharide specific antibodies in infants has its roots in the discovery that low molecular weight, non-immunogenic compounds
termed haptens can produce hapten specific antibodies in rabbits if covalently attached to horse gamma globulin.2 Application of this principle to carbohydrates led to the development of glycoconjugate vaccines licensed for immunization against H. influenzae type b, N. meningitidis types A, C, Y, and W135, and seven serotypes of pneumococci.3 While the exact mechanism by which PSs and their protein conjugates elicit humoral immune response is not fully understood, the prevailing concept is that high molecular weight PSs that are T-cell independent antigens, simultaneously interact with immunoglobulin receptors on the surface of B-cells (Fig. 1.2). This crosslinking initiates a cascade of events leading to the direct B-cell secretion of PS-specific IgG and IgM antibodies with no T-cell activation. An experimental support for this view comes from the fact that "small" polysaccharide as the O-polysaccharide region of LPSs are not immunogenic. Reexposure to PSs does not produce a booster (memory) response.
Figure 1.2
In contrast to pure PSs, PS-protein conjugates are recognized and internalized by the B-cells, where the protein is fragmented into peptides of 15- 20 amino acids. The peptide fragments, bearing the saccharide haptens are reexposed on the antigen presenting cells together with major histocompatibility complex (MHC) class II molecules to CD4+ helper T-lymphocytes. Interaction of the antigen-MHC complex with its specific receptors on the T-cells leads to differentiation of B-cells into antibody-secreting B-cells and antigen-specific memory B-cells. An indirect support to this concept is provided by the observation that small peptides corresponding to B-cell epitopes, can convert nonimmunogenic saccharides to immunogenic ones. Repeated exposure to the PS-protein or peptide conjugates results in a booster response of mainly IgG isotype. The saccharide-specific antibodies bind to extracellular antigens such as the PSs on bacterial surface can result in killing the bacteria.
1.4. Aim of this research
In the next two sections of Part 1 the complete synthesis of two different oligosaccharides that represent the repeating unit of SP19F (A) and SP14 (B) capsular polisaccharides (Fig. 1.3) will be presented. In each section problems and results of the synthetic strategy that had permitted the preparation of the desired oligosaccharides starting from commercial and easy available product as lactose and glucosamine will be described.
Figure 1.3
With the two target saccharides in hands, the final objective of this project was the preparation of a new type of glycoconjugates to be evaluated for its immunogenic properties toward Streptococcus Pneumoniae (Pn) 19F and 14 strains. In particular the targets were: (a) the preparation of the carbohydrate antigens as glycodendrimeric clusters of the CPS repeating units rather than as oligomers, as usually reported in the literature (b, Fig. 1.4); (b) their conjugation with the same protein carrier (CRM197), in order to obtain for the first time a chemically defined neo-glycoprotein potentially able to produce antibodies toward pneumococcal serogroup 19F and 14 (c, Fig. 1.4) and (d) evaluate a multivalence effect on biological targets arising from the presentation of the sugar “epitopes” on the surface of a multiantennary structure as a PAMAM dendron (see next Paragraphs). The chosen immunogenic carrier protein is the CRM197, a non-toxic mutant of diphtheria toxin, safely used in other human vaccines as PCV13.4
As an introduction to this aspect of the research, generalities about glycodendrimers chemistry and multivalence will complete this preface.
1.5. Dendrimers: an introduction
Dendrimers (also known as arborols or cascade molecules) and dendrons are repeatedly branched, roughly spherical large molecules. A dendrimer is typically symmetric around the core, and often adopts a spherical three-dimensional morphology. A dendron usually contains a single chemically addressable group called the focal point (Fig. 1.5).
Figure 1.5
The properties of dendrimers are dominated by the functional groups on the molecular surface, however, there are examples of dendrimers with internal functionality. Dendritic encapsulation of functional molecules allows for the isolation of the active site, a structure that mimics active sites in biomaterials. Also, it is possible to make dendrimers water soluble, unlike most polymers, by functionalizing their outer shell with charged species or other hydrophilic groups. Other controllable properties of dendrimers include toxicity, crystallinity, tecto-dendrimer formation, and chirality.
From 1978, when Vögtle et al.5 described a series of synthetic “cascade molecules”, starting from diverse primary monoamines and diamines, as the first tangible representatives of compounds exhibiting potentially perpetual branching, the chemistry of dendrimers has grown with new types of dendrimers and new synthetic methods discovered every year.
In 1985 Tomalia developed branched poly(amidoamines) (PAMAM), which he also designated as “starburst dendrimers” (Fig. 1.6) and generally propagated the name “dendrimer” (from the Greek dendron = tree and meros = part).6 Like the first cascade synthesis, the synthetic route again involved Michael addition (of methyl acrylate to ammonia).
Figure 1.6
The resulting ester was converted into the primary triamine by reaction with an excess of ethylenediamine. Repetition of the reaction sequence (iteration) by analogy with the cascade synthesis led to dendrimers of up to the tenth generation – with decreasing purity and perfection. Tomalia referred to the individual ester stages as half generations (0.5, 1.5, 2.5).
Tomalia’s exhaustive review paper with coloured illustrations did much to popularise the highly branched compounds and to ensure broad general acceptance of the family name “dendrimers”.7In general, dendrimer synthesis can be performed according to two major schemes: divergent and convergent growth.8 Divergent growth is based on the stepwise addition of low molecular mass building blocks starting from a multifunctional core molecule and results in a radial growth of the dendrimers. Convergent dendrimer synthesis, on the other hand, involves the coupling of preformed dendrons onto a central core molecule.9
1.6. Glycodendrimers
In molecules called “glycodendrimers”, saccharide portions are conjugated according to the principles of dendritic growth or they are ligated to dendrimers, respectively. The idea of supplementing carbohydrate chemistry by the concepts, which have made dendrimer chemistry the
intriguing field it is, has essentially been triggered by questions addressed in glycobiology. During recent years, it has become clear that in carbohydrate-protein interactions, which are essential molecular recognition events in cell biology, multivalency plays an important role.10 Therefore, chemists and biochemists have sought carbohydrates and glycoconjugates which can be used as molecular tools for the investigation and possibly manipulation of carbohydrate-protein interactions, especially with regard to the multivalency effect which has been observed.11 Four different classes of glycodendrimers can be distinguished according to the principal design of their molecular architectures (Fig. 1.7).
Figure 1.7
(i) The first example of glycodendrimer were molecules in which a non-carbohydrate dendritic core, as polyamidoamine (PAMAM) dendrimer, was built up first and was then functionalized with a coat of carbohydrate moieties in the periphery (Fig. 1.7 A).
(ii) Alternatively the convergent approach is followed, in which glyco-coated dendrons are synthesized first and eventually assembled on an oligofunctional core molecule (Fig. 1.7 B). (iii) The complexity of natural glycoconjugates is most closely resembled by glycodendrimers which are built from carbohydrate-derived building blocks (Fig. 1.7 C).
(iv) Finally, by reversing the architecture of glyco-coated dendrimers, carbohydrates can be used as the dendrimer core and this has been realized by the synthesis of carbohydrate-centered glycodendrimers (Fig. 1.7 D).
1.7. The use of glycodendrimers in biology: the multivalence effect
Protein-carbohydrate interactions are essential to many biological processes, individual interactions usually exhibit11 weak binding affinities (Kd values in the mM to µM range) as well as apparently relatively low selectivities between similar carbohydrate ligands. These characteristics and properties are at odds with the observed biological activities which demand interactions that are
both extremely selective and of high affinity. Nature’s answer to this problem is to use multivalency.10b Thus, multiple copies of the carbohydrate ligands are arranged on glycoprotein scaffolds or in patches of glycolipids on the surface of one cell, and multiple copies of lectins - or lectins each with multiple binding sites - are displayed at the surface of another cell. When these two surfaces come together, the individual interactions reinforce one another to give overall a high avidity. The glycoside cluster effect was defined initially by Lee and Lee12 as the ‘binding affinity enhancement exhibited by a multivalent carbohydrate ligand over and beyond that expected from the concentration increase resulting from its multivalency’. This enhancement in binding affinity can be the consequence of two different mechanisms13 at the molecular level: (i) a statistical effect in which the multivalent compound gives rise (Fig. 1.8a) to a highly localized concentration of the ligand at the receptor binding site; and (ii) a chelate effect in which the multivalent ligand cross-links binding sites either in adjacent receptors (Fig. 1.8b) or in a single multivalent receptor (Fig. 1.8c,d).
Figure 1.8
Although the observation that multivalency is important in protein-carbohydrate interactions formed the original rationale for developing a whole range of synthetically engineered glycoconjugate systems as neoglycoconjugates. An important aspect of multivalency that has been observed, in addition to high affinity, is the enhancement of the selectivity of a particular interaction. Small differences in the intrinsic binding affinity as monovalent binding affinity can be ‘amplified’ greatly on displaying the ligands in a multivalent fashion. Horan et al.14 have reported the increased binding selectivity of Bauhinia purpurea lectin to galactosides tethered to a gold surface in increasing surface densities. However, in their experiments, they also observe a change in the preferred ligand for the lectin on going from the monovalent solution binding through increasing surface density of multivalent ligands.
1.8. Synthesis of sugar-coated PAMAM dendrimers
The idea of the use of glycodendrimers is to substitute the complex carbohydrate interior of an oligoantennary glycoconjugate by a branched non-carbohydrate molecule which would basically only serve as a scaffold for the multiple presentation of sugar moieties, which are known as the principal carbohydrate epitopes in particular glycobiological systems. Analyzing different approaches to glycodendrimers, a divergent modification of pre-existing dendrimers is a convenient way to make polyvalent glycoconjugates in a minimal number of steps, assuming that the starting dendrimer is commercially or otherwise readily available. It is not surprising, therefore, that there have been many investigations along these lines and that many researchers have largely focused on two classes of dendrimers, as seen PAMAM dendrimers (Par. 1.5) and poly(propylene imine) as AstramolTM.15 Both types of dendrimers have tertiary amine- based skeletons, displaying primary amines around their peripheries to which other functional groups or molecules can be attached by means of urea, thiourea and amide bonds, for example. Talking about sugar moieties to be attached on dendrimers, numerous different spacer units have been used to link carbohydrate moieties to scaffolds in order to produce multivalent glycoconjugates. In classical dendrimers, the linker is often short and treated more or less as a connecting unit only, although the nature of the linker will have an impact on the accessibility of the sugar epitope both for a reagent during synthesis and for a tested receptor. Often, the choice of the linker is governed by the chemistry used and not by the desirable biological properties of the targeted product. It has been recognized that the spacer arms are not passive elements and influence the binding ability according to their chemical nature.16 However, it has been difficult to choose systematically a linker in order to guarantee proper ligand presentation according to exact geometrical criteria.
Synthesis of glycodendrimers based on PAMAM has been studied by several research groups. For example, a number of РАМАМ-based clusters with two to eight branches bearing α-D-mannose, β-D-galactose, β-cellobiose, and β-lactose residues have been prepared.17 These compounds were
characterized by 1Н and 13С- NMR data. The conjugation with the РАМАМ amino groups was carried out by the isothiocyanate method. The reaction of isothiocyanato-functionalized carbohydrate derivatives (Fig. 1.9a) with branched oligoamines such as PAMAM dendrimers proved to be a successful ligation technique for the synthesis of glycodendrimers.17 An advantage of this method is the possibility of using synthetic amino-spacered oligosaccharides for the attachment to amino-terminated dendrimers, while the use of toxic thiophosgene during the isothiocyanate synthesis is an obvious drawback. The activity of the resulting conjugates was not indicated. Synthesis of larger glycodendrimers based on РАМАМ bearing each 12, 24, and 48 glucose or galactose residues has been described. 18
Figure 1.9
As an alternative to thiourea bridge formation, there is also the use of peptide chemistry concerning the formation of an amide bond between PAMAM amine moieties and a carboxylic group on the sugar.19 Frequently carboxylic groups have been prepared and isolated as active esters (Fig. 1.9b) then coupled with dendrimers: N-hydroxysuccinimidyl and p-nitrophenol esters are the most common intermediates, but are also used 1-hydroxibenzotriazole (HOBt) or carbodiimide activated COOH groups. An example of this approach has been applied by Nifant’ev et al20 to prepare PAMAM neoglycoconjugates containing 4, 8, 32 and 64 terminal residues of BDI (Gal-α -(13)-Gal) and Neu5Ac (N-acetylneuraminic acid): sugar residues have been loaded on PAMAM
as aceyl protected p-nitrophenol esters then deprotected in good yields (from 50 to nearly 90%). Another attractive approach to PAMAM glycodendrimers is represented by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione (squarate)21 as the linker between two amino groups. The first stage of the method involves coupling a terminal amino group-containing saccharide with a squaric acid diester. At pH 7, the reaction was reported21 to stop at the monoester-monoamide level. Subsequent exposure of the monoester and the protein to pH 9 activates the second alkoxy group and anchors the saccharide-squaric acid construct to the amino group on PAMAM surface. Although squarate chemistry is well established in the conjugation of sugar to proteins, only a few examples using this method for the preparation of glycodendrimers have been reported (Scheme 1.1).22
Tetravalent hexylamino-terminated mannosylated wedges were treated with diethyl squarate in methanol, purified by GPC and clustered with tris(2-aminoethyl) amine in methanol containing DIPEA. After 10 days 62% of the dodecavalent glycocluster was isolated (Scheme 1.1). A recent finding is that pH and buffer concentration are of crucial importance to obtain reliable and high yielding conjugations23 which might encourage further use of this chemistry for the synthesis of multivalent glycoconjugates.
Scheme 1.1
Reagents and conditions. i: diethyl squarate, MeOH; ii: N(CH2CH2NH2)3, DIPEA, MeOH (62%).
Establishing the purity of such large molecules is not a trivial task. MALDI-TOF mass spectroscopy was possible for dendrimers smaller than 30000 Da, but larger molecules gave unreliable results. Whereas 1H-NMR spectra exhibited severely broadened lines for all of these compounds, 13C NMR spectroscopy revealed spectra with relatively simple and analogous sets of signals for each series of dendrimers.
