Peptide Dendrimers Aplication and Synthesis

Reviews in Molecular Biotechnology 90 2002.
195 229
Peptide dendrimers: applications and synthesis

Kristen Sadler, James P. TamU
Department of Microbiology and Immunology, Vanderbilt Uni ersity, A5119 MCN, Nash ille, TN 37232, USA
Abstract Peptide dendrimers are radial or wedge-like branched macromolecules consisting of a peptidyl branching core andror covalently attached surface functional units. The multimeric nature of these constructs, the unambiguous composition and ease of production make this type of dendrimer well suited to various biotechnological and biochemical applications. Applications include use as biomedical diagnostic reagents, protein mimetics, anticancer and antiviral agents, vaccines and drug and gene delivery vehicles. This review focuses on the different types of peptide dendrimers currently in use and the synthetic methods commonly employed to generate peptide dendrimers ranging from stepwise solid-phase synthesis to chemoselective and orthogonal ligation. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Review; Peptide dendrimer; Multiple antigenic peptide; MAP; Ligation chemistry
1. Introduction Dendrimers are polymers with three distinct structural features: a central core; surface functionalities; and branching units that link the two. Peptide dendrimers can be broadly dened as any dendrimer that contains peptide bonds. This definition would, in theory, include a dendrimer with an amino acid core, branching units, surface functional groups or any combination of the three as a peptide dendrimer. This denition could be furU
Corresponding author. Tel.: q1-615-343-1465; fax: q1615-343-1467. E-mail address: james.tam@mcmail.vanderbilt.edu J.P. Tam..
ther broadened if we consider all types of amino acids including naturally occurring -amino acids as well as unnatural amino acids that have been utilized in peptide dendrimer synthesis as both branching units and surface functional groups. For example, unnatural amino acids such as amino acids have also been used as branching units Esfand and Tomalia, 2001.. In practice, the majority of peptide dendrimers being referred to in the literature and currently in use are based on the multiple antigen peptide MAP. system that consists of only two of the three structural features: branching units and surface functional groups. Interestingly, these molecules contain both -peptide and -peptide Tam, 1988, 1996.. In addition, they are often synthesized without a
1389-0352r02r$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 5 2 0 1 . 0 0 0 6 1 - 7
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K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229 Table 1 Current and potential applications of polyvalent dendrimers Vehicles for delivery of Nucleic acids Encapsulated drugs Covalently linked drugs, e.g. anticancer agents Diagnostic reagents in Serodiagnosis Biosensor systems Magnetic resonance imaging Vaccines against Bacteria Virus Parasites Inhibitors of Pathogenic infections Inammatory response Autoimmune disease Cancer metastasis Modication of Cell cell interactions Gene expression, e.g. alteration of transcription factors binding to DNA
core and more appropriately termed dendrons rather than classical dendrimers. However, for convenience we will refer to all branched polypeptide constructs as dendrimers.
2. General characteristics of peptide dendrimers By virtue of their dendritic architecture, peptide dendrimers are frequently utilized as protein and liposomal mimetics as well as biomaterials in the life sciences and biomedical applications. Dendrimer design exploits two traits that are frequently observed in many naturally occurring systems: globular structure and polyvalency. Polyvalent interactions occur through the association of two or more ligands and receptors of the same type. Multivalent molecules are commonly found in nature ranging from small molecules of polysaccharides, nucleic acids and peptides to protein aggregates on virions, bacteria and other cells that aid in adhesion and cell cell interactions. The properties of multiple simultaneous interactions can differ markedly from those displayed by the constituents during monovalent interactions, suggesting that dendrimers with multiple functional sites can be used in the design of new drugs and research reagents. Such strategies to cluster ligands and functional groups on dendrimeric structures are attracting considerable attention in different areas from the production of biomaterials to vaccine technology. Dendrimeric materials are nding applications as antiviral agents Zanini and Roy, 1998., vehicles for delivery of nucleic acids Haensler and Szoka, 1993; Kukowska-Latallo et al., 1996; DeLong et al., 1997. and drugs Esfand and Tomalia, 2001., as biomedical tools such as magnetic resonance imaging contrast agents Kim and Zimmerman, 1998., protein mimetics, vaccines directed towards bacterial, viral and parasitic pathogens, as well as diagnostic reagents and molecular inhibitors. In the rst step of infection most pathogens such as bacteria and virus particles adhere to potential host cells via polyvalent interactions Mammen et al., 1998.. Therefore, there may be a role for multivalent dendrimers capable of mimicking receptors and ligands in preventing
infections. Similarly, cell cell interactions that are mediated by specic receptor ligand associations can be studied in ne detail through the use of dendrimers. Pathophysiological responses such as inammation and autoimmune reactions could be prevented by therapeutic use of dendrimers. Some of the current and potential applications of synthetic dendrimers in biomedicine and biotechnology are outlined in Table 1. Peptide dendrimers vary from low molecular weight species of 2 kDa to large protein-like constructs ) 100 kDa. The size and complexity of the individual dendrimers are determined by two factors, the number of layers of branching units often referred to as the generation number. and the surface supporting the terminal functional groups which can be large peptides or proteins of substantial size. Typically, peptide dendrimers have generation numbers between 2 and 32. Similar to other dendrimers, synthesis of peptide dendrimers is tightly controlled with products of consistent size, architecture and composition.
K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229
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Peptide dendrimers can be divided into three types. The rst are grafted peptide dendrimers. These are conventional dendrimers with either unnatural amino acids or organic groups as the branching core and peptide or proteins attached as surface functional groups. Of the three, the grafted peptide dendrimer is the largest in terms of size because they generally contain high generation numbers of branching cores. In contrast, peptide dendrimers of the second type are essentially branching polyamino acids. Consequently, they tend to be the smallest by size with the core consisting of natural amino acids and the terminal amino acids acting as surface functional groups. The third type consisting of mostly peptides has been traditionally known as peptide dendrimers. In this group, with MAPs as the most well known example, the core consists of amino acids and the surface functional groups are also peptidyl chains. This review provides some details on the rst two types of peptide dendrimers, however, we will largely focus on the third type because this type is used most frequently in biological and biochemical applications. 2.1. Grafted peptide dendrimers with unnatural amino acids and organic cores Peptide dendrimers based on the commercially available poly amidoamine., or PAMAM core may be considered classical dendrimers and one of the most widely used Esfand and Tomalia, 2001.. They are synthesized by a divergent method involving a two-step iterative reaction sequence that produces concentric shells of dendritic alanine units. Various functional groups, including peptides, have been covalently linked to the PAMAM dendrimer and used for different applications. Anticancer drugs may be encapsulated by PAMAM dendrimers with polyethylene glycol. grafts prior to delivery in order to increase efcacy Kojima et al., 2000.. Addition of sugars such as lactose and fructose to the PAMAM dendrimer produces glycodendrimers that are potential high-afnity ligands for sugar receptors Andre et al., 1999; Kawase et al., 2000.. Excellent reviews by Esfand and Tomalia 2001., Kim and Zimmerman 1998. outline the potential
biomedical applications of PAMAM dendrimers with carbohydrate and nucleic acid functional units. Peptides have also been attached to the PAMAM core. In one of the most promising studies an articial photosynthetic system has been constructed with amphipathic -helix peptides acting as surface functional groups attached to the core through a thioether linkage Sakamoto et al., 2001a,b.. The PAMAM dendrimer has also been used to generate a biochemical reagent Singh, 1998. by reacting a PAMAM core with calf intestine alkaline phosphatase and a Fab fragment of an anti-creatine kinase MB isoenzyme antibody. 2.2. Peptide dendrimers with polyamino acids as branching units and as surface functional sites Water-soluble amphipathic peptide dendrimers comprised only of three or four generations of lysine residues have been constructed to aid in the delivery of DNA to intact cells. Dendrimers consisting of a branched lysine core and 8 or 16 terminal surface amines may be mixed in solution with plasm id D NA and left to form dendrimer DNA complexes. The complexes are subsequently taken up into cells and the gene products expressed with high efciency compared to cells exposed to DNA alone Choi et al., 1999; Toth et al., 1999; Shah et al., 2000.. In a similar vein, lipidated peptide dendrimers consisting of polyamide groups 4, 8 or 16 terminal lysines. were linked through the rst generation lysine to three alkyl chains C 14 . and their interaction with charged and neutral liposomes was studied Purohit et al., 2001.. It was found that the interaction efciency of the dendrimers was greater than 88% and that the amount of dendrimer adsorbed decreased as the dendrimer size increased. The mechanism of interaction is unclear but adsorption is most likely due to hydrophobic interactions rather than electrostatic because the dendrimers interacted with equal efciency with negatively and positively charged liposomes. Lipidated peptide dendrimers have also been synthesized by the addition of 2-amino tetradecanoic acid to a polylysine support to create a dendrimer with 16 surface alkyl chains Florence et al., 2000..
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This peptide dendrimer was used to study the uptake of lipidated molecules by the mucosal surfaces of the digestive tract in mice. 2.3. Peptide dendrimers with amino acid branching units and surface peptidyl chains Peptide dendrimers comprised of amino acid cores and surface peptide chains Fig. 1. have been used most extensively in biological applications. The underlying design principle is that the polyvalency of the peptide dendrimers enhances the afnity of peptide-specic interactions with peptides, proteins and carbohydrates, thus enabling the study of receptor ligand interactions, the introduction of increased binding afnities and also development of new techniques for afnity purication and diagnostic tests. An example showing the ability of multimeric peptides to enhance binding afnity was described by Fassina et al. 1992a. where analytical HPLC and solid phase binding assays were used to evaluate the binding interactions between two complementary peptides in monomeric and octameric forms. Binding afnity was found to increase when one or both of the sequences were synthesized as octameric dendrimers. The interaction between the multimeric peptides was of similar afnity to that of polyclonal antibody and multimeric peptide illustrating that weak interactions may be magnied through the use of peptide dendrimers allowing more complete analysis. This may be potentially useful for characterizing the weak interactions between cell-surface receptors and ligands. Some of the biochemical applications of peptide dendrimers are listed in Table 2.
Fig. 1. Schematic illustration of peptide dendrimers that vary in size.
3. Peptide dendrimers in immunoassays and serodiagnosis Many immunoassays rely on the attachment of antigens to a solid surface such as plastic microtitre wells and their detection by addition of specic reagents. Short synthetic peptides readily available through chemical synthesis are generally considered to be ideal antigens because of the high specicity they confer. Unfortunately, most
short peptides are poor antigens due to their inability to bind to solid surfaces. The multimeric nature of peptide dendrimers has been found to aid in overcoming such deciencies by providing increased surface-binding character and sensitivity of detection Tam and Zavala, 1989; Marguerite et al., 1992; Briand et al., 1992; Marsden et al., 1992.. Adesida et al. 1999. exploited the increased afnity of protein interaction with peptide dendrimers compared to monomeric peptides in a study focusing on the interaction of immunoglobulins with a non-epitopic peptide in both direct and indirect peptide-antibody binding assays. In serodiagnosis where serum samples from naturally immunized or infected individuals are tested for the presence of specic antibody, the multimeric arrangement of dendrimers may also improve the detection of low afnity antibodies during early stages of infection, particularly those of the IgM isotype Habluetzel et al., 1991; Marsden et al., 1992.. In a dramatic example, a dendrimer shows a sensitivity increase of over 10 8-fold when compared to a peptide antigen in an enzyme-linked immunosorbent assay. Immunosorbent assays may be superceded in the near future by biosensor technology where dendrimers are bound directly to sensory chips and the strength of antibody interaction monitored with great accuracy Gomara et al., 2000a.. The use of dendrimers in biosensor technology requires further
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examination, particularly to optimize immobilization and ensure that factors such as steric hindrance Francis et al., 1991; Le et al., 1998. and interchain aggregation do not impede immobilization nor antibody recognition. The introduction
of spacer residues such as glycine between the lysine core and peptide sequence of interest Marsden et al., 1992. may reduce inhibitory interchain aggregation. The dendrimers must also be bound to the sensory chips in the correct
Table 2 Diagnostic and biochemical uses of peptide dendrimers Application Immunoassays and serodiagnosis Malaria Cirrhosis HIV-1 References
Tam and Zavala 1989., Habluetzel et al. 1991. Avila et al. 2001. Briand et al. 1992. Marsden et al. 1992., Estaquier et al. 1993. Vogel et al. 1994., Shin et al. 1997. Marguerite et al. 1992. Sabbatini et al. 1993a., Caponi et al. 1995. Marchini et al. 1994. Jackwood and Hilt 1995. Gomara et al. 2000a,b. Adesida et al. 1999. Tres and Kierszenbaum 1996.
Schistosoma mansoni Systemic lupus erythematosus Epstein-Barr virus Infectious bronchitis virus Hepatitis A virus Non-epitopic peptide Sperm antigen Epitope mapping and ligand binding Bluetongue virus Systemic lupus erythematosus Hepatitis C virus P. falciparum Pf72rHsp70-1 antigen NKTag, tumour antigen Inhibitors Macroautophagia and proteolysis Tumour growth and metastasis Enzyme inhibitors HIV-1 fusion and infection Interleukin 6 Sporozoite, malaria Fibronectin Antagonist of C5a Articial proteins Minicollagen Synthetic enzyme Biochemical studies Afnity purication of antibodies Presentation of T-cell epitopes Afnity purications
Yang et al. 1992. Sabbatini et al. 1993b. Simmonds et al. 1993. Dat et al. 2000. Jaso-Friedmann et al. 1996.
Miotto et al. 1994., Mortimore et al. 1994. Nomizu et al. 1993., Kim et al. 1994. Iwamoto et al. 1996., Manki et al. 1998. Fassina and Cassani 1993. Yahi et al. 1994a,b, 1995., Weeks et al. 1994. Wallace et al. 1994. Sinnis et al. 1994. Ingham et al. 1994. Kaneko et al. 1995.
Fields et al. 1993. Hahn et al. 1990.
Butz et al. 1994., Yang et al. 1999. Grillot et al. 1993. Fassina 1992., Fassina et al. 1992a,b. Yao et al. 1994. Sheldon et al. 1995.
Intracellular delivery
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orientation, for this ligation reactions may be exploited for example thiol exchange, hydrazide or sulfhydryl group coupling Schuck, 1997..
enhanced and a potentially useful therapeutic agent produced.
5. Peptide dendrimers as mimetics 4. Peptide dendrimers as inhibitors Another promising application of peptide dendrimers has been realized in the general design of inhibitors. Multimerization of binding elements such as charge in a dendrimer is controllable and provides an unambiguous structure that can be rened by analogs with relative ease. The inhibition of entry of malaria parasites into hepatocytes Sinnis et al., 1994. is a ne example of this application. Sinnis et al. 1994. have taken advantage of the fact that a cationic conserved sequence of malaria circumsporozoite CS. protein known as region II-plus requires aggregation for binding to liver hepatocyte proteoglycans. A tetramer containing region II-plus was synthesized and subsequently found to inhibit CS protein binding to hepatocytes in mice. A peptide dendrimer based on an eight-residue sequence from the third variable region V3 loop. of gp120, the HIV-1 surface envelope glycoprotein, has been shown to inhibit HIV-1 infection of both CD4q and CD4y cells Yahi et al., 1995; Carlier et al., 2000.. The increased binding afnity of dendrimers compared to monomeric peptides is becoming an important factor in the design of peptides aimed at inhibiting metastasis of various types of cancer cells. One of the most advanced studies has shown that a dendrimer containing 16 copies of a peptide from laminin a protein of the basement membrane extracellular matrix. signicantly inhibited tumor growth in vivo Nomizu et al., 1993.. This inhibitory activity increased as the peptide copy number increased, i.e. 16 ) 8 ) 4. The enhanced anti-tumor effects of the 16-mer were subsequently partly attributed to the ability of the multimer to compete more successfully with laminin for the laminin receptor on tumor cells thereby blocking the binding of malignant cells to blood vessel walls Iwamoto et al., 1996.. Therefore, by covalently linking multiple copies of an anti-tumor peptide the binding afnity has been Peptide dendrimers have proven to be useful substitutes for proteins and even DNA in a number of autoimmune disease model systems. Dendrimers based on peptides from various proteins have been used to generate autoimmune conditions in normal animals that are comparable to the condition systemic lupus erythematosus SLE. James and Harley, 1998; Mason et al., 1999; Farris et al., 1999.. Mason showed that inoculation of mice with a MAP consisting of eight copies of a peptide derived from the spliceosomal Sm protein resulted in epitope spreading, a response usually attributed to an immune response against self proteins and DNA. Anti-nuclear antibodies were detected in some animals illustrating that the MAP acted as a protein and DNA mimetic. Putterman and Diamond 1998. identied a peptide mimetic for double stranded DNA that elicited anti-DNA antibody production. Mice developed a lupus-like syndrome creating an experimental model for SLE. Using dendrimers Khalil et al. 2001. replicated the T cell-dependent nature of SLE using a peptide immunogen instead of a DNA immunogen and characterized the T cell response. Unlike other examples mentioned thus far, it was determined that the increased response to the dendrimer was dependent more on the amino acid sequence than on valency. A plausible explanation for the observed results is that the presence of the dendrimer backbone allowed more effective processing and presentation of the correct T helper epitopes. During a similar study where the specicity of antibodies was examined, it was found that dendrimers themselves could not only bind to antibodies but also directly interact with the 60-kDa Ro nuclear protein in various assays Scoeld et al., 1999.. Furthermore, it was concluded that Ro and La, components of a single ribonucleoprotein particle, directly interact. The protein-binding sequences within Ro were identied using den-
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drimers; the dendrimers were an important tool for exploring both intramolecular and intermolecular protein-protein interactions. Even though the afnity of interaction may not be absolutely determined because the use of dendrimers may increase avidity in immunoassays by several logs Tam, 1996., the relative afnities may be determined. In the context of SLE, the peptide dendrimer provides useful reagents for probing the extent of immune tolerance in the B and T cell compartments to nuclear antigens. The weak antimicrobial activity of monomeric peptides has been observed to increase after multimerization. An 11-residue peptide representing a sequence from human lactoferrin an iron-binding glycoprotein found in neutrophils and secretions. was incorporated into peptide dendrimers consisting of 1, 2, 4, 8 or 16 copies and the dendrimers were tested for antibacterial activity Azuma et al., 1999.. An increase in antimicrobial activity correlated with an increase in the peptide copy number. The dendrimer containing 16 copies of the peptide was as effective as the traditional antibiotic gentamycin against Gram-negative and Gram-positive bacteria. In this case, the authors suggest that the monomeric peptide is too short to span the membrane and create an ionic pore whereas the dendrimers are large enough to span the membrane and form a pore or mimic pore formation thus resulting in microbicidal activity. To advance the dendritic design a step further, Lu et al. 2001. designed an inactive tetrapeptide
with a basic and hydrophobic motif derived from -strand topology of tachyplesins and protegrins which exhibited signicant antimicrobial activity when synthesised as a dendrimer. The tetrameric dendrimer is similar in molecular weight and antimicrobial potency as protegrins or tachyplesins. More importantly, each peptide dendrimer assayed was found to have higher antimicrobial activity against fungi, Gram-negative and Grampositive bacteria than linear tandem repeating peptides.
6. Peptide dendrimers as immunogens Possibly the most widely known biological application of peptide dendrimers is the use of MAPs as immunogens. Immunogens are distinguished from antigens by their ability to induce an immune response in vivo. MAPs, typically of the form shown in Fig. 2, have enormous potential as immunogens and a signicant portion of this potential has already been realized. The advantages of using MAPs as immunogens include: Simplicity in design and synthesis; Versatility for investigating various immune responses; Reliability of generating site-specic antibodies; and Generation of site-specic antibodies in the laboratory.
Fig. 2. a. Schematic representation of a multiple antigen peptide MAP., incorporating eight peptide monomers. b. An increase in the number of Lys branching units increases the number of surface amine groups.
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Since the rst report of the synthesis and potential of MAPs by Tam 1988. MAPs have been employed routinely as immunogens for antibody production and cytotoxic immune responses.1 Before we review the application of MAPs as immunogens, several points regarding peptide sequence selection and design of MAPs will be presented. 6.1. Summary of immune responses This overview aims to briey summarize the role of peptide epitopes in the induction of immune responses and to alert the reader to the importance of epitope selection. In general, there are two types of immune responses, humoral and cell-mediated. Induction of a humoral response results in the production of antibodies and relies upon the activation of both B cells and T helper cells. Cytotoxic T cells and T helper cells, on the other hand, mediate cellular immune responses. For maximal antibody production a B cell must receive two signals. It must bind a B cell epitope to the surface immunoglobulin with high afnity. The B cell epitope is usually a peptide or protein segment approximately nine residues in length. Secondly, the B cell must interact with an activated T helper cell leading to B cell maturation and production of antibody with the same specicity as the surface immunoglobulin. The T helper cell is in turn activated by interaction with another type of cell, the antigen presenting cell APC.. The role of the APC is to engulf extracellular material, including foreign peptides and proteins, and degrade the proteins and peptides into short segments. These short segments are subsequently presented on the APC surface in association with MHC class II molecules. The T helper cell is activated if it encounters an APC presenting a peptide-MHC complex that binds with high afnity to the T cell receptor TCR.. The peptide associated with the MHC molecule is referred to as a T helper epitope. Without the presence of both the B cell epitope and T helper epitope
antibody production is generally short-lived and the highly desirable memory response is lacking. Activation of T helper cells is also required for the induction of a cell-mediated response. In this case the T helper cells interact with and provide cytokines for the stimulation of cytotoxic T cells. The main role of the cytotoxic T cells is to recognise and kill host cells which are expressing nonself peptides associated with MHC class I proteins such as peptides from intracellular bacteria and viruses. These peptides are referred to as cytotoxic T lymphocyte CTL. epitopes. The majority of host cells are capable of degrading intracellular proteins and presenting CTL epitopes on the surface when bound to MHC class I molecules. On the rst occasion that a cytotoxic T cell encounters a host cell expressing a non-self epitope it undergoes maturation aided by T helper cells. and is then capable of killing other host cells expressing the same CTL epitope. In summary, humoral responses require B and T helper epitopes and cell-mediated responses require CTL and T helper epitopes. An effective synthetic immunogen must contain peptide sequences that activate the appropriate type of immune response and the exibility of the MAP system allows incorporation of multiple epitopes in to the same construct. 6.2. Design of MAPs for induction of an antibody response B cell epitopes alone are poor immunogens and have traditionally been conjugated to large carrier proteins that provide strong T helper cell epitopes. The evolution of the MAP system now allows B cell and T helper epitopes to be assembled directly onto a small polylysine core. In contrast to conventional peptide-protein conjugates where peptide epitopes account for less than 20% of the total weight, MAPs consist of concentrated epitopes and possess an immunologically silent lysine core Posnett et al., 1988; Del Giudice et al., 1990.. In some cases there is signicant difference and a generally better quality of antisera in using MAPs vs. peptide protein conjugates McLean et al., 1991; Wang et al., 1991; Estaquier et al., 1993; Molnar et al., 1993;
A recent literature survey showed that Tams 1988 paper has been cited over 600 times.
K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229 Table 3 Utilization of peptide dendrimers for antibody and CTL production Target Bacteria Tandem repeat motifs, Leishmania SLT-1 protein, Escherichia coli P30 antigen, Toxoplasma gondii Glucosyltransferase, Streptococcus Toxin T, Bordetella pertussis Toxin B, Vibrio cholera Membrane protein 1, C. trachomatis Membrane protein, Neisseria meningitidis Cyclase, Saccharomyces cere isiae Exoproteases, Pseudomonas aeruginosa Gingipains, Porphyromonas gingi alis Porin protein, N. meningitidis Virus Surface antigen, Hepatitis B virus Reductase, Herpes simplex virus VP2 and VP7, bluetongue virus VP1 protein, foot-and-mouth disease virus gp120, HIV References
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Liew et al. 1990. Boyd et al. 1991. Darcy et al. 1992., Godard et al . 1994. Smith et al. 1993., Smith et al. 1994a. Felici et al. 1993. Halimi and Rivaille 1993. Zhong et al. 1993. Christodoulides and Heckels 1994. Shi et al. 1994. Coin et al. 1997. Genco et al. 1998. Christodoulides et al. 1999.
Nef, HIV-1 Polymerase, inuenza virus Hemagglutinin, inuenza virus NSm protein, bunyamwera virus B19, parvovirus Core proteins, vaccinia virus gp46, HTLV-1 V3 loop, HIV-1 MVF, measles virus gp51, bovine leukemia virus Fusion protein, respiratory syncytial virus VP3, Hepatitis A virus V3, feline immunodeciency virus VP1 protein, JC virus Parasite CS protein, Plasmodium berghei
Tam and Lu 1989., Manivel et al. 1993. Lankinen et al. 1989. Li and Yang 1990. Francis et al. 1991., Lugovskoi et al. 1992. Brown 1992. Wang et al. 1991., Defoort et al. 1992a,b. Nardelli et al. 1992a,b, 1994., Nardelli and Tam 1993. Levi et al. 1993., Kelker et al. 1994. De Santis et al. 1994., Huang et al. 1994. Fraisier et al. 1994., Vogel et al. 1994. Estaquier et al. 1992, 1993. Nieto et al. 1992. Toth et al. 1993., Naruse et al. 1994. Zeng et al. 2000. Nakitare and Elliott 1993. Saikawa et al. 1993., Anderson et al. 1995. Vanslyke and Hruby 1994. James et al. 1995. Cruz et al. 2000. Olszewska et al. 2000a,b. Kabeya et al. 1996. Hsu et al. 1999. Pinto et al. 1998. Rigby et al. 1996. Aoki et al. 1996.
CS protein, P. malariae CS protein, P. falciparum
Romero et al. 1988., Migliorini et al. 1993. Zavala and Chai 1990., Chai et al. 1992. Widmann et al . 1992., Valmori et al. 1994. Del Giudice et al. 1990. Munesinghe et al. 1991., Pessi et al. 1991. Valmori et al. 1992., Nardin and Nussenzweig 1993. Calvo-Calle et al. 1992, 1993. De Oliveira et al. 1994., Ahlborg 1995. Nardin et al. 1998, 2001., Moreno et al. 1999.
204 Table 3 Continued. Target CS protein, P. yoelii
References Wang et al. 1995., Marussig et al. 1997. Franke et al. 2000. Del Guercio et al. 1997. Auriault et al. 1991., Khan et al . 1994. Pancre et al. 1994. Reynolds et al. 1994. Ferru et al. 1997. Argiro et al. 2000.
CS protein , P. i ax P28 antigen, Schistosoma mansoni Triose-phosphate isomerase, S. mansoni Sm28GST and sTPI, S. mansoni Sm37-GAPDH and Sm10-DLC, S. mansoni Other Trp protein, Drosophila UL47, UL8, Vmw63 and UL42 Lutropin, human Ribosomal protein, plant Fatty acid binding protein, human Transforming growth factor , human Alkyltransferase, human Cytochrome P-4501A, rat Mast cell protease-5 , mouse ATPase, plant Calcium channel, rabbit Sperm myoglobin, whale Sperm protein, human Protein kinase p34cdc2 , eukaryote Membrane domain of band 3, human Angiotensin II type-1 receptor, rat Actin-fragment kinase, plasmodia Seed protein, plant IGF binding protein, bovine Glutamate receptor, rat Glucose transporter, rat RNA-binding protein, rodent Insulin receptor, human Myotrophin, human Iron regulatory factor, human Protein G, human Tyrosine kinase, sponge Chaperonin polypeptide, eukaryote Sperm protein , Xenopus pP344 retinal cell, chicken Myosin phosphatase, chicken Prion protein, mouse Ameloginin, mouse Histones, mouse GTPase, rat Neurexins, rat T-tubule, rabbit Calcium release channel, rabbit Anion transport protein band 3, human Guanylin, human Prolin, human
Wong et al. 1989. McLean et al. 1990., Parry et al. 1993. Sinclair et al. 1994., Marsden et al. 1994. Troalen et al. 1990. Szymkowski and Deering 1990. St John et al. 1991. Lu et al. 1991. Pegg et al. 1991. Edwards et al. 1991. McNeil et al. 1992. Suzuki et al. 1992. Malouf et al. 1992. McLean et al. 1992. Vanage et al. 1992, 1994. Kamo et al. 1992. Kang et al. 1992. Zelezna et al. 1992, 1994. Gettemans et al. 1993. Monsalve et al. 1993. Arnold et al. 1993. Molnar et al. 1993. Nagamatsu et al. 1993. Henderson et al. 1993. Itoh et al. 1993. Sil et al. 1993. Emery-Goodman et al. 1993., Gray et al. 1993. Raymond et al. 1993. Schacke et al. 1994. Lingappa et al. 1994. Bauer et al. 1994. Iio et al. 1994. Shimizu et al. 1994. ORourke et al. 1994. Simmer et al. 1994. Meziere et al. 1994. Wilson et al. 1994. Perin 1994. Stout et al. 1994. Callaway et al. 1994. Crandall and Sherman 1994. Kuhn et al. 1994., Cetin et al. 1994. Finkel et al. 1994.
K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229 Table 3 Continued. Target Type V collagen, human Phosphatase 2A, human DR molecules, human Elan, human Gangliosides, human 5 -reductase 2, human Heme oxtgenase-1, human Spliceosome protein, human Cyclin, human Phospholipase A2 , human Kainate receptor, goldsh Myelin basic protein, murine Serotonin transporter, rat Ras p21, human Poly ADP-ribose. polymerase, eukaryote Seed proteins, Vigna angularis Sm BrB protein, primate pRL1a, murine leukemia N-myc oncoprotein References Moradi-Ameli et al. 1994. Zolnierowicz et al. 1994. Demotz et al. 1994. Nara et al. 1994. Helling et al. 1994. Eicheler et al. 1994. Kutty et al. 1994., Smith et al. 1994b. James et al. 1995. Digweed et al. 1995. Sa et al. 1995. Wo and Oswald 1995. Zhou and Whitaker 1996. Zhou et al. 1996. Schott et al. 1996. Duriez et al. 1997. Kajiwara and Tomooka 1998. Arbuckle et al. 1998. Manki et al. 1998. Yang et al. 1999.
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Vogel et al., 1994.. A comparative study by McLean et al. 1991. identied what has now become a general trend in the properties of antisera elicited by different constructs, sera obtained from animals inoculated with MAPs is of a higher titer and produced more rapidly than those obtained with protein conjugates which are in turn higher than those obtained with resin-linked peptides. The MAP format allows investigators to overcome the immunodominance conferred by linker and carrier proteins and elicit antibody responses to weakly immunogenic sub-dominant peptide sequences. Examples of the use of MAPs as immunogens are listed in Table 3. There are many possible arrangements of linking B and T helper epitopes on MAPs, the most popular approach to date is to link the epitopes in a tandem formation. The main drawback with this method is that new immunodominant epitopes may be formed within the tandem peptide and the MAP is not processed and presented as the intended design. One empirical solution suggested by Lo-Man et al. 1994. is to insert a longer T epitope to preserve the diversity of T cell recognition. Alternatively convergent strategies may be employed where the dendrimer contains copies of each peptide sequence on sep-
arate arms thereby decreasing the possibility of neo-epitope formation and increasing the response to the desired epitopes. Tandem, or diepitope, MAP constructs have been extensively studied in the malaria synthetic vaccine model. Ten MAP constructs were synthesized, each containing a combination of described B and T cell epitopes, the constructs were designed to evaluate the relevance of the number of copies, stoichiometry, and orientation of T and B sequences in a diepitope model Tam et al., 1990.. In this case it was concluded that there was no advantage in using octameric instead of tetrameric MAPs, and that having the B cell epitope at the N-terminal produced a more efcient immunogen. The degree of protection against challenge of immunized mice with 2000 Plasmodium berghei sporozoites correlated with the antibody levels obtained by the immunization protocol. Tetra- and octameric MAPs have been utilized in most studies to date. The number of branches required tends to depend largely on the amino acid residues: with peptides ) 15 amino acids we have found that there is no real advantage in using more than four branches. A similar conclusion was reached by Francis et al. 1991. where guinea pigs were immunized with constructs con-
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taining one, two, four or eight copies of a footand-mouth disease virus peptide. It was determined that in this case presentation in a tetrameric structure was sufcient for an optimal antibody response. Diepitope MAPs were synthesized in the design of a synthetic vaccine model for hepatitis B using a different chemical approach Tam and Lu, 1989.. A B cell epitope and T helper epitope were used in mono- and diepitope congurations, in the latter the two peptides were connected in alternating forms on the lysine core instead of in tandem. Only when the B cell epitope was presented in the MAP form with the T helper epitope was it immunogenic in rabbits. Therefore, these results conrmed that a diepitope MAP format may overcome the poor immunogenicity of a linear peptide. Mono- and diepitope MAP congurations were further analyzed in a synthetic model for the human immunodeciency virus type 1 HIV-1. Nardelli et al., 1992a.. Monoepitope MAPs consisting of four copies of a major neutralizing epitope of HIV-1 were synthesized along with diepitope MAPs that also contained a known T helper epitope at the C-terminus of the B cell epitopes. The diepitopes were immunogenic in all species tested whereas the monoepitope MAPs elicited species-specic responses. The use of universal or promiscuous T helper epitopes which are recognized by three or more strains of mice are particularly useful for diepitope constructs and have been used to enhance the immunogenicity of B cell epitopes in a number of MAPs McLean et al., 1992; Munesinghe et al., 1991; Marguerite et al., 1992.. Finally, Levi et al. 1993. have demonstrated that boosting with B cells and T helper cell epitope MAP constructs induced a higher antibody titer for HIV-1 and improved cellular responses than boosting with recombinant gp160 protein. The use of chemoselective ligation through oxime bond formation is gaining notoriety as a feasible method for immunogen production. Selfadjuvanting tetrameric MAP constructs incorporating B cell and T helper epitopes from different sources induce strong antibody responses Nardin et al., 1998; Zeng et al., 2000..
6.3. Cell-mediated responses induced by lipidated MAPs Induction of a cell-mediated response relies upon the delivery of CTL epitopes to an intracellular endosomal pathway that results in the presentation of peptides in association with class I MHC molecules. Others have found the value of lipidated peptide in eliciting cellular responses because of the increased efciency of delivery of peptides to the intracellular compartment. Longchain lipidic amino acids have been developed by Gibbons et al. 1990. in combination with detergent-solubilized liposome as a delivery system. This approach was applied to the MAP format in successful vaccine models against HIV-1 Defoort et al., 1992a,b; Nardelli et al., 1992a, 1994. and Chlamydia trachomatis Zhong et al., 1993; Pancre et al., 1994.. A cytotoxic response was induced by a lipidated MAP containing a peptide sequence from gp120 envelope protein of HIV-1, III-B isolate which overlaps a B cell epitope, a T helper epitope and a CTL epitope. This response was detected after a single inoculation without extraneous adjuvant, was superior to the response induced by a full cycle of inoculations with the non-lipidated MAP in complete Freunds adjuvant and was still detectable 7 months later. The lipidated MAP was found to produce systemic antibody and cellular responses regardless of the route of inoculation employed Nardelli et al., 1992b, 1994.. The ability of MAPs containing the lipid moiety tripalmitoyl-S-glyceryl cysteine P3 C. to induce mucosal antibody response via oral administration adds a new dimension to applications of the MAP constructs, and it may be particularly useful in preventing transmission of pathogens, such as HIV, through mucosal surfaces. 6.4. Possible reasons for increased immunogenicity The multimeric MAP approach has been shown to increase the immunogenicity of weakly immunogenic monomeric peptides, there are several possible reasons for the observed enhanced activity. The conformation of the peptide epitopes within the MAP may contribute to the longevity
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and strength of the responses induced by MAPs. MAPs attain a somewhat unnatural polymeric structure when compared to a linear peptide which may aid in resistance to in vivo proteolytic degradation leading to a longer immunogen halflife. This persistence of immunogen in the systemic circulation possibly induces longer lasting immune responses. The increase in immunogenicity could also be due to new T helper epitopes being generated within the MAP format. Even though the lysine core itself is immunologically silent the contribution of one or two residues to the attached peptide sequences could result in the production of new epitopes. This would provide a plausible explanation for the fact that the antibody response to the repetitive NANP peptide from Plasmodium falciparum CS protein is restricted to a single mouse strain when administered as a linear peptide but administration of the peptide in a MAP format results in antibody production in a further ve strains that differ in MHC haplotype Pessi et al., 1991.. Another alternative is that the MAP construct allows the desired epitopes to be processed and presented by APCs in a more efcient manner. For instance all residues in a linear peptide may be accessible to degrading enzymes, whereas the MAP conguration may actually protect certain residues from enzymatic cleavage resulting in the presentation of different epitopes. In many cases the more immunogenic diepitope MAP constructs are synthesized with the desired T helper epitope in close proximity to the branching lysine core. The design of a MAP provides a scaffold for close packing of peptide sequences that may allow the stabilization of the secondary structure and the reverse turns of peptides. Furthermore, the distal end of the peptide away from the core of MAPs is more exposed and exible than the proximal end, for these reasons we may expect the immunogenicity of the B cell epitope to be greater if the epitope is located at the distal, N-terminal site. The multimeric nature of the MAP system may also contribute to the well-documented increases in immunogenicity. The higher copy number of closely packed epitopes could lead to greater B
cell surface cross-linking through surface immunoglobulins resulting in increased activation and antibody production. 6.5. Potential application: peptide dendrimers as synthetic accines The majority of vaccines currently approved for human use consist of either heat-killed or liveattenuated infectious agents. Despite the successes of such vaccines, development by conventional methods is limited by several factors, namely: Hazardous production; Cold chain required for storage; Presence of contaminating materials; Risk of reversion to infectious state; and Side effects of vaccination. Subunit vaccines consisting of either whole recombinant proteins or synthetic peptides are appealing alternatives because they are safe, selective and chemically dened. Peptides have further advantages in that short peptide sequences may be synthesized that only contain the epitope of interest and deleterious epitopes may be omitted. Large quantities of chemically puried peptide vaccines can be prepared with automated methods and peptide-based immunogens are more likely to be resistant to denaturation, and they can be easily stored and transported without refrigeration. A peptide dendrimer malaria vaccine recently entered a Phase I study in human volunteers Nardin et al., 2001.. The vaccine was assembled using chemoselective ligation via oxime bonds and consists of four peptide branches each containing B cell epitopes and a promiscuous T helper cell epitope from P. falciparum CS protein. Exogenous adjuvant was not required due to the inclusion of a P3 C moiety covalently linked to the polylysine core. The majority of the inoculated volunteers developed high titered antibody responses as well as T cells specic for the T helper epitope. The use of MAPs as vaccines may extend past the use of relatively small tetra- and octameric
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constructs that contain a minimal quantity of epitopes. MAP technology lends itself to the design of macromolecular structures that may mimic whole organisms or several different organisms. Ultimately several similar or different closely packed peptide immunogens prepared as MAPs could be anchored on lipid vesicles that mimic surface proteins. Immunogenicity could be broadened and enhanced by the inclusion of a non-covalent mixture of B cell and T helper and CTL epitopes from various proteins. At the same time the combination of adjuvant effects of liposome and the built-in lipid anchor may replace the need for additional adjuvant. Lipidated MAPs have been shown to become entrapped in liposomes in an efcient manner, nearly 80% of
lipidated MAPs were incorporated as compared to 2 5% without the lipid anchor Defoort et al., 1992a,b. in one example.
7. Synthesis overview Synthesis of peptide dendrimers embraces a broad range of chemistry from conventional solid-phase peptide synthesis schemes in organic solvents to the formation of regiospecic amide or non-amide bonds in aqueous solutions. Strategies for preparing peptide dendrimers can generally be divided into two categories, namely the divergent and convergent approaches Fig. 3.. The divergent strategy is a direct approach by which
Fig. 3. A schematic comparison of divergent and convergent synthesis methods.
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the dendrimer is built stepwise and diverges outward. Stepwise solid-phase synthesis has been used to prepare the dendrimer in a continuous operation on a solid support. However, the convergent strategy is an indirect, modular approach by which peptidyl surface functional groups and the branching unit are prepared separately. The
puried components are then linked together and peptide sequences converge to the branching unit as a dendrimer. Speed and efciency are advantages of the divergent stepwise strategy particularly by solidphase methods because intermediates are neither puried nor characterized. This strategy is suit-
Fig. 4. Examples of simple amino, carboxylic and hydroxylic cores.
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able for validation of concepts and for small peptide dendrimers that may be separated from their byproducts by powerful systems. In many cases stepwise synthesis is not practical, either because of the nature of the branching unit preventing integration into a stepwise strategy or because the resulting dendrimer is too large e.g. ) 15 kDa. to be separated from its byproducts with condence. An advantage of the convergent strategy is chemical unambiguity because the protected or unprotected peptide segments and the branching unit used in coupling reactions are puried prior to reaction, thereby limiting the range of byproducts and facilitating purication. A disadvantage lies in the operational complexity: the convergent strategy requires more steps than stepwise synthesis, including additional purication and characterization steps for intermediates. The convergent strategy could also pose signicant problems with regard to the solubility of peptide intermediates when protected peptide segments are being used. Thus, a recent trend has developed in which aqueous, soluble unprotected peptides are used that are convenient to handle, purify and characterize. Several laboratories have made extensive efforts to develop new methodologies of ligation chemistry that involve the fewest steps possible. These chemistries are based on chemoselectivity and orthogonality of unprotected intermediates for assembling peptide dendrimers. 7.1. Purication and characterization Methods for purication and characterization of peptide dendrimers are similar to those of peptides and proteins. Crude synthetic peptide dendrimers derived from the divergent strategy often require multiple methods that may include sequential steps of dialysis or gel-ltration chromatography, followed by RP-HPLC or high-performance ion-exchange chromatography. Even by these steps it may still not be possible to remove byproducts with a single modication or deletion of an amino acid from the desired product. Synthetic products assembled using a convergent strategy can be rened by most chromatographic
methods resulting in a homomeric product. Characterization methods for peptide dendrimers include the usual panel of techniques: amino acid analysis; SDS-PAGE; capillary zone electrophoresis; and enzymatic digestion. Mass spectrometric analysis has now become an indispensable tool for determining the molecular weights indicating product homogeneity of these complex molecules. 7.2. Types of dendrimer cores and branching units For the design of articial proteins the type of core selected is critical because it may confer rigidity and exert conformational inuence on the overall structure of the peptide dendrimer. Elaborate cores Unson et al., 1984; Sasaki and Kaiser, 1989; Ghadiri and Case, 1993; Chapman et al., 1994; Schneider and Kelly, 1995; Tsikaris et al., 1996; Wrighton et al., 1997; Fairlie et al., 1998; Frey, 1998; Goodman et al., 1998; Jefferson et al., 1998. are designed to induce or to assist folding into structural motifs such as -helices, -sheets, reverse-turns and loops which are often lacking in peptide monomers. However, most cores employed to date are simple small organic molecules - 1000 Da with the sole purpose of clustering or amplifying peptides to enhance the polyvalency effect. Selected examples are shown in Fig. 4. Such cores are often commercially available allowing many groups to investigate the use of peptide dendrimers in various applications. The most common cores are simple amino compounds, namely amino acids and dipeptides. The simplest amino cores are the trisethylene amine. ammonia types rst used in organic dendrimers that have recently been utilized in formation of peptide dendrimers. The use of heterocyclic compounds such as porphyrin and constrained and unusual amino acids has also been reported Sasaki and Kaiser, 1989.. Simple organic polycarboxylic acids have been used as branching units Roth and Heidemann, 1980; Kemp and Petrakis, 1981; Thakur et al., 1986; Fairlie et al., 1998; Goodman et al., 1998., however, many branching units contain a combination of functional groups. As a group, carbohydrates and their intermediates represent a diverse and attractive source of polyhydroxylic branching
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units because of their small sizes and the stereochemical variations of pendant hydroxyls Newkome et al., 1991; Whittaker et al., 1993; Murer and Seebach, 1995.. In addition, their hydrophilic nature favors convergent synthesis in aqueous solutions. The utility of hydroxylic and carboxylic branching units is currently limited by the need for a synthetic methodology to distinguish between these groups that also occur frequently in peptides. In the case of hydroxylic groups, they are further hindered by the less reactive nature when compared to amines and carboxylic acids. As a result, their use has not yet been widely explored in peptide dendrimers. 7.3. Polylysine branching units Polyamino acids consisting of several branches of a trifunctional acid represent the largest group of branching units being used today Tam, 1988; Rao and Tam, 1994.. By virtue of their trivalency, reactivity and synthetic expedience branching units derived from lysine and its homologs are the most popular. Lysine with N - and N -amino groups as reactive ends is a particularly suitable trifunctional amino acid to form a branching unit by a limited sequential propagation of lysines. These low molecular weight cores with 2 n reactive amino ends can then serve as attachment sites for peptides. Solid phase schemes are the method of choice for the synthesis of lysine
branching units Tam, 1988; Tam and Lu, 1989. where di-protected Lys, e.g. Boc LysBoc. or Fmoc LysFmoc., is used to produce a core of multiple levels of lysines. The diamino nature of lysine creates a situation where each additional level of Lys effectively doubles the number of sites to which peptide monomers may be attached. A typical branching lysine unit consists of three lysines to give a tetravalent peptide dendrimer Fig. 5.. Further divergence of the K 2 K unit to one or two additional levels will generate di-K 2 K and tetra-K 2 K dendrons with reactive ends of 8 and 16 amino groups, respectively, to which peptides can be attached. Peptides may be synthesised directly onto the lysine dendron using a divergent strategy or dendrons can be further functionalized with electrophiles and thiol nucleophiles for convergent ligation of peptides. The K 2 K-type branching unit is small in size when compared with the bulk of peptides layered around the surface. As an example, a dendrimer consisting of a branched unit and eight copies of peptide chains, each containing 14 amino acid residues, would have an average molecular weight of 13 14 kDa. Only a small fraction of the mass is due to the lysine unit; 93% of the mass is due to peptide sequences. Many variations of K 2 K cores have evolved. For example, a version containing a -alanine spacer Huang et al., 1994. at the -amine of Lys has produced a branching unit that has symmetri-
Fig. 5. Polylysine cores consisting of K 2 K and di K 2 K, where Xaa can be either OH, an amino acid, peptide or organic core see Fig. 4..
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Fig. 6. Different K 2 K, polyamino, linear and cyclic cores used for dendrimer assembly. Ahx s aminohexanoic acid.
cal branches. In another example, in order to limit the exibility of the branched lysyl template, ornithine, the lower homolog of Lys, as well as more constrained diamino acids have been used Roth and Heidemann, 1980; Hahn et al., 1990.. Non-branched linear polylysine cores Tsikaris et al., 1996. and cyclic peptides Goodman et al., 1998. have also been developed to impart different forms of dendrimeric architectures Fig. 6.. As eluded to earlier, the basic branching unit may be further modied to allow peptide attachment. As shown in Fig. 7, attachment of serine to the K 2 K dendron during solid-phase synthesis allows the production of a branching unit with N-terminal aldehyde moieties. This serine can be
readily converted to aldehyde at neutral pH in aqueous conditions and has been used by Spetzler and Tam 1995. as a convenient masked aldehyde precursor. Thiol-functionalized branching units are popular and convenient to prepare because of the relative ease with which reactive thiol moieties may be placed on peptide units in preparation for convergent syntheses Lu et al., 1991; Defoort et al., 1992b; Zhang and Tam, 1997b.. Drijfhout and Bloemhoff 1991. introduced a thiol-functionalized branching unit based on the K 2 K dendron by coupling N- S-acetylmercaptoacetyl.-glutamyl residues to the amino groups. Subsequently, Baleux and Dubois 1992. prepared a thiolate branching unit based on a similar design by coupling Cys to the amino groups Fig. 8.. 7.4. Di ergent stepwise strategy for peptide dendrimer synthesis Divergent methods used to prepare peptide dendrimers are operationally similar to the stepwise solid-phase syntheses of linear peptides rst developed by Merrield 1963.. Thus, protecting
Fig. 7. Solid phase synthesis of a K 2 K glyoxylyl core.
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Fig. 8. Synthesis of thiol-functionalized S-acetyl and CysNpys. di-K 2 K cores.
group schemes, resin supports, and cleavage methods found in peptide synthesis protocols are generally applicable to the divergent synthetic schemes of peptide dendrimers. Because this approach produces fully characterized dendrimers in a short time, often within a week, it is popular in biological research in which peptide dendrimers are used as tools and reagents for validating biochemical goals. Syntheses generally commence with the branching unit which is usually of the K 2 K type. In such cases two or three levels of diverging lysine are formed rst by coupling a suitable di-protected lysine, either a di-Boc or di-Fmoc derivative, to a small amino acid attached to a resin support to form a branched structure. The peptide sequence is then synthesized stepwise on this branched-lysine scaffolding. After peptide synthesis the peptide dendrimer is cleaved from the resin support, puried and characterized. Goodman Goodman et al., 1998; Jefferson et al., 1998. introduced a different divergent synthesis approach. In the synthesis of collagen models consisting three copies of homomeric peptide chains, solid-phase schemes began with monomers and then the Kemp tricarboxylic acid KTA. template Kemp and Petrakis, 1981. was used to link three monomers to form the peptide dendrimers Fig. 9.. Interchain aggregation may occur within the branched structure of dendrimers because the quantity of peptide monomers obviously increases with each additional level of lysine units. To
minimize this problem the divergent synthesis often starts with a low amino acid loading of approximately 0.1 mmolrg on a resin support. This is signicantly lower than the customary loading, typically 0.3 0.8 mmolrg resin, conventionally used in solid-phase peptide synthesis. Formation of interchain hydrogen bonds that occlude coupling reactions can also be minimized by using specic solvent combinations as well as coupling at elevated temperatures Tam and Lu, 1995.. Following synthesis and cleavage some peptide dendrimers have exhibited an unusual tendency to aggregate after cleavage from the resin due to the entrapment of aromatic scavengers. Dialysis under basic, denaturing conditions using 8 M urea is useful to remove such unwanted additives. After dialysis either RP-HPLC or high performance gel-ltration chromatography can be used to purify peptide dendrimers. A variation of the divergent synthesis scheme is the synthesis of two dissimilar peptide chains on different arms of a lysine branching unit. This is a particularly useful method for the production of immunogens containing different peptide epitopes. Methods to distinguish between - and -amines of lysine for such a purpose are based
Fig. 9. Synthesis of collagen models with three copies of homomeric peptide chains on the Kemp tricarboxylic acid core.
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on orthogonal protecting group schemes. Suitable protecting group combinations include: i. Boc Fmoc Merrield, 1963.; ii. Fmoc Aloc Kates et al., 1993.; iii. Fmoc Dde Bycroft et al., 1993.; and iv. Npys Fmoc Hahn et al., 1990; Ahlborg, 1995; Rajagopalan et al., 1995.. In general, the combination of Boc Fmoc chemistry is convenient because the protected amino acids are readily available. This chemistry utilizes both acidand base-driven deprotection methods Tam and Lu, 1989. in repetitive steps that elongate the two dissimilar peptide chains. In the K 2 K core, for example, when half of the reactive lysine amino chains e.g. -amino groups. are chemically blocked during synthesis it is possible to construct a peptide dendrimer containing two different peptide sequences Fig. 10.. The rst peptide chain can be assembled on the N -termini of the lysine matrix using Boc chemistry while the Fmoc groups of N -termini are not affected during the Boc synthesis. When the rst peptide is complete the second peptide chain can be synthesized on the N -termini using Fmoc chemistry. The heteromeric peptide dendrimer is then cleaved from the resin by HF that will also remove both the benzyl- Boc chemistry. and tertbutyl- Fmoc chemistry. protecting groups. In recent years the use of the Boc protecting group has become less popular because special apparatus must be used for deprotection by HF. Alternative methods have been developed and these include Boc substitution with i. Aloc that is removable by Pd o in the presence of a nucleophile such as morpholine, ii. Dde which is removable by 2% hydrazine in DMF, and iii. Npys 3-nitropyridino-2-sulfhydryl. which is removable under neutral conditions by a trivalency phosphine or a thiol. An example of the utilization of orthogonal protection was reported by Hahn et al. 1990. with the divergent synthesis of a chymotrypsin-like articial enzyme using three types of protecting groups on a Lys 2 Orn branching unit to form a peptide dendrimer consisting of four dissimilar chains. The rst two chains were assembled by alternating Boc and Npys chemistries on the differentially protected dipeptide Npys LysBoc. OrnFmoc.. By terminating each chain with an
Fig. 10. Schematic illustration of the synthesis of heteromeric peptide dendrimers on a di-K 2 K core.
acetylated amino acid and repeating the same chemistry, the next two chains were assembled on Boc LysNpys. coupled to the Orn side chain after the removal of Fmoc group. The strategic simplicity and exibility of divergent orthogonal synthesis also permits the incorporation of unusual moieties such as lipid chains and lipidic P3 C into the dendrimeric design Defoort et al., 1992b..
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7.5. Con ergent synthesis strategies for peptide dendrimers Convergent strategies differ from the divergent methods in that the branching unit and peptides are synthesized separately, puried individually and then coupled to form the dendrimer. Two different convergent strategies are typically used; one strategy exploits a protection scheme while the other does not. The choice of a convergent strategy is often arbitrary. Consideration should, however, be given to the nature of the peptide monomer, the complexity of the peptide dendrimer, and the expertise of a given laboratory. An additional factor is the orientation of the peptide that is to be attached and this is governed by the functional consequences. For example, peptides derived at or near C-termini can be arranged as in the native protein by linkage through the N-terminus to the branching unit. A variety of functional groups have been used in the development of numerous ligation protocols in order to provide site-specic and chemically unambiguous dendrimers. A detailed review of different ligation strategies and methodologies was published recently by Tam 2000.. 7.6. Con ergent synthesis with a protecting group scheme The convergent synthesis using protecting groups Blake, 1981; Yajima and Fujii, 1981; Yamashiro and Li, 1988; Hojo and Aimoto, 1992; Sakakibara, 1995; Albericio et al., 1997. generally follows conventional segment coupling schemes with enthalphic activation by a coupling reagent. The major difference between forming a branched dendrimer and forming a linear peptide by segment condensation is that two or more protected peptide segments are simultaneously coupled to a core to afford the former. Purication by gel permeation chromatography and RP-HPLC is facilitated by the substantial differences in the molecular weights of the desired product compared to incompletely coupled byproducts. Sasaki and Kaiser 1989. reported formation of such a peptide dendrimer using convergent synthesis where an articial hemeprotein containing four
Fig. 11. Convergent synthesis of an articial hemeprotein.
identical peptides tethered to a coproporphyrin core was assembled. The 15aa fully protected peptide segment was synthesized through a twosegment condensation 8aa q 7aa. and then coupled to the hydroxysuccinimide ester of the branching unit through its N-terminal Fig. 11.. A common approach is to use a less elaborate chemistry as illustrated by McLean et al. 1992. who described a rapid convergent scheme for the formation of MAPs. To obtain a 17aa protected peptide segment they used an Fmoc-tertbutyl protecting group scheme with a highly acid-labile resin. The cleaved protected peptide containing a free carboxylic acid was then coupled to a tetrameric K 2 K branching unit. However, the poor solubility of this protected peptide resulted in varying degrees of coupling in six MAPs, ranging from complete to no observed coupling. Although this difculty does not impede the biological goals it certainly indicates the unpredictable nature of using protected peptide segments in convergent schemes. 8. Chemoselective ligation strategies: unprotected peptide synthesis A recent advance in convergent synthesis has been the use of unprotected peptide segments
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instead of bulky protected peptides. One such strategy that links unprotected peptide monomers to a branching unit to form a dendrimer via non-amide bonds is regiospecic chemoselective ligation. This type of ligation exploits a pair of mutually reactive functional groups, usually a nucleophile and an electrophile, on the peptide and branching unit that only react specically with each other. Another strategy that links unprotected peptides via amide bond is orthogonal ligation Tam et al., 2000.. Orthogonal ligation is N-terminal specic, thus, a peptide containing an N-terminal Lys can be ligated in the presence of other N-terminal amino acid peptides. An advantage of these types of ligation is their specicity because peptide segments and branching units are used in their unprotected state they are more likely to be water soluble and amenable to purication by RP-HPLC. A plethora of chemoselective and orthogonal ligation methods and many variations have been developed Tam, 2000.. Furthermore, the nucleophile and electrophile can be placed on either the peptide segment or the branching unit creating numerous potential combinations. In addition, there are many readily available bifunctional groups that are used to modify K 2 K dendrons in preparation for ligation including: succinimide esters; maleimides; sulfenyl thiols; and chloroacetyl groups Keller and Rudinger, 1975; Carlsson et al., 1978; King et al., 1978, 1986; Yoshitake et al., 1979; Wunsch et al., 1985; Ottl et al., 1999.. Examples of the types of bonds that may be formed by regiospecic chemoselective ligation include hydrazone, oxime, thioether and thioester bonds. Each chemoselective, or orthogonal, strategy consists of an initial capture step to form a covalent intermediate between two peptide segments. Then, an amide bond is formed via an intramolecular acyl transfer resulting in the coupling of the two different segments. The strategies are categorized by two major chemistries: thioester and imine. 8.1. Thioester ligation In the thioester ligation strategy a thioester and an N-terminal cysteine are required. The
initial capture product is a thioester and an S, Nacyl shift results in formation of a cysteine residue at the ligation site Muir et al., 1994; Tam et al., 1995.. Other variations of cysteine ligation such as perthioester ligation have also been developed to make thioester a popular orthogonal ligation strategy Tam et al., 2001.. 8.2. Imine and imide ligation The imine chemoselective ligation strategy requires the presence of an acyl-aldehyde and an N-terminal weak amine. Commonly used imine ligation strategies include oximation, hydrazone formation and imide ligation. Oximation has been used in protein conjugation reactions Vilaseca et al., 1993; Rose, 1994. and in the synthesis of peptide dendrimers Shao and Tam, 1995; Pallin and Tam, 1996.. Hydrazone formation has also been employed in ligation reactions through the reaction of aldehyde and C-terminal hydrazide Gaertner et al., 1992.. For orthogonal imide Nterminal. ligation a C-terminal glycoaldehyde ester is required for reaction with either an Nterminal NT. cysteine pseudoproline. or NTserine or NT-threonine oxaproline.. The mechanism of imide ligation is similar to orthogonal thioester ligation with an imine capture, a ring closure to a thiazolidine NT-Cys. or oxazolidine NT-SerrThr. and then an S, N- or O, N-acyl shift to form an imide Liu and Tam, 1994.. Pseudoproline ligation has been successfully employed in the production of synthetic HIV-1 protease analogs Liu et al., 1996.. Each of these imine or imide ligations relies on the presence of aldehydes and since aldehydes are not found in amino acids several methods have been used to introduce aldehydes to amino acids. Two convenient methods are: 1. conversion of N-terminal Ser and Thr residues to aldehyde through sodium metaperiodate oxidation; and 2. conversion of masked acetals attached to amino groups. to aldehyde by TFA or concentrated HCl. The conversion of N-terminal Ser and Thr residues to aldehydes has been used extensively in protein conjugation and designing new proteins as well as for immunological applications Zhang and Tam, 1996; Zhang et al., 1998..
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Even though the two reactive species may be placed on either the branching unit or monomeric peptide, for synthetic convenience weak bases are often added to the N-terminal of a peptide and in such cases the peptide sequence is oriented with the N-terminal residues proximal to the branching unit. The reverse orientation may be achieved with a C-terminal hydrazide derivative Rao and Tam, 1994; Shao and Tam, 1995; Pallin and Tam, 1996; Zhang and Tam, 1996; Liu and Tam, 1997.. Combinations of two, and potentially more, reaction conditions can be exploited for ligating dissimilar peptide chains to a branching unit to form heteromeric peptide dendrimers Liu et al., 1999. in a process termed tandem ligation. 8.3. Tandem cyclization strategies for peptide dendrimers Constrained peptide dendrimers have been developed in an effort to generate molecules that more closely mimic native protein conformations and to improve biological activity. One such design contains multiple closed-chain branches which are cyclic peptides constrained by end-toend, end-to-side, or side-to-side linkages Liu and Tam, 1997; Spetzler and Tam, 1996; Zhang and Tam, 1997a,b.. A general approach to achieve such constrained peptide dendrimers is to attach multiple copies of a cyclic peptide to a branching unit. Conventionally, this scheme of convergent synthesis requires two types of manipulations: a cyclization step on a linear precursor and a coupling step for its attachment to a branching unit. This two-step process usually requires a multitiered protecting group scheme. To minimize the protection and deprotection procedures of con-
ventional convergent methods and also difculties due to the insolubility of intermediates and products, simplied tandem chemoselective methods have been developed based on unprotected peptide precursors in aqueous conditions. 8.4. Di ergent synthesis and chemoselecti e cyclization Spetzler and Tam 1996. developed a method for preparing peptide dendrimers with end-to-side chain cyclic peptide monomers that combines on-resin synthesis and off-resin cyclization. First, a divergent strategy of stepwise solid-phase synthesis was used to assemble the peptides linked to a K 2 K branching unit. This branched precursor was then cleaved from the resin and its protecting groups removed to permit an off-resin cyclization that formed the constrained dendrimer through a thiazolidine cyclization in an aqueous solution Fig. 12.. The use of this method may, however, be restricted by the competing intramolecular cyclizations among the peptide branches. Ring-chain tautomerization may be used to overcome this limitation where mutually reactive nucleophile electrophile pairs simultaneously self-assemble to form peptide dendrimers with multiple copies of cyclic peptides. 8.5. Chemoselecti e cyclization and con ergent ligation Using a slightly different approach, Pallin and Tam 1995. synthesized a constrained peptide dendrimer by rst cyclizing the monomeric peptide and then attaching the monomers to a branching unit as shown in Fig. 13. The linear
Fig. 12. Use of divergent synthesis and thiazolidine intramolecular cyclization to generate a peptide dendrimer.
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sequence KAKGIGPGRAFGK- Ala derived from the neutralizing determinant of the V3 loop of gp120 was selected and prepared by Fmoc chemistry resulting in the precursor KCys. AKO NH 2 .GIGPGRAFGKSer.- Ala. The Ser moiety attached to the rst Lys served as a masked aldehyde and was oxidized to an aldehyde. A cyclic peptide was formed by utilizing the intramolecular oxime formation from the reaction between the Lys-side chain tethered O-alkylhydroxylamine and the aldehyde. The thiol residue of Cys on the side chain of the third Lys moiety was then liberated and used to ligate to
the aldehyde moieties of a glyoxyyl di-K 2 K branching unit form a peptide dendrimer containing four copies of side-to-side chain cyclic peptides. This method for formation of side-to-side chain cyclic peptides differs from the previous example Fig. 12. in the form of cyclic peptide constraint and method of synthesis. 8.6. Orthogonal cyclization and con ergent thiol ligation Tandem ligation is a strategy consisting of two or more chemoselective and orthogonal ligations
Fig. 13. Synthesis of a cyclic peptide dendrimer through side-chain to side-chain oxime cyclization and thiazolidine ligation.
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that do not require a deprotection step in the synthetic scheme. Thus, tandem ligation represents a convergent scheme with the fewest synthetic steps and the most rapid approach for synthesis of high molecular weight peptides. Three tandem ligation methods for preparing constrained peptide dendrimers have been developed based on orthogonal cyclization and convergent thiol ligation Liu and Tam, 1997; Zhang and Tam, 1997b.. These are: cysteine-thioester cyclization and thioalkylation; perthioester cyclization and thiol addition; and imine cyclization and thioalkylation. A complete discussion of these methods is found in Tam 2000.. Even though orthogonal cyclization and convergent ligation methods also use a linear unprotected peptide precursor and a functionalized branching unit as building blocks for peptide dendrimers there are two major differences between these and the previously mentioned chemoselective ligation strategies. Firstly, orthogonal cyclization methods produce lactams and secondly, only one weak base nucleophile, fullls the function of two nucleophiles in chemoselective ligation. The rst reaction common to the three reaction schemes is the orthogonal lactamization of the N-terminal nucleophile and the C-terminal electrophile of the linear precursor to generate an end-to-end cyclic peptide Zhang and Tam, 1997b; Tam and Lu, 1998; Tam and Yu, 1998; Tam et al., 1999.. The second reaction ligates the cyclic peptide monomer to a functionalized branching unit through its thiol side chain resulting in a peptide dendrimer Liu and Tam, 1997; Zhang and Tam, 1997b.. These two reactions can be performed in tandem without purication when homogeneous intermediates are used. An advantage of these methods is their high efciency because no protection or deprotection steps are involved. Racemization is minimized because there is no enthalpic activation. Furthermore, the cyclization can occur at high peptide concentrations due to the high effective molarity of an intra- vs. inter-molecular reaction. The variation in the strategies now available for the preparation of peptide dendrimers allows investigators to select a method most suitable for their individual needs. The high specicity and
homogeneity conferred by convergent chemoselective methods may be benecial in some situations whereas the rapid production of divergent peptide dendrimers could be advantageous in others. The use of peptide dendrimers as biochemical reagents and immunogens is entering a new phase. The reliability and versatility of the dendrimer system are now well-documented and investigators are looking towards the utilization of peptide dendrimers as gold-standard reagents and as a new generation of totally synthetic vaccines. References
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Peptide Dendrimers Aplication and Synthesis

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Reviews in Molecular Biotechnology 90 2002.

Peptide dendrimers: applications and synthesis

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 1. Schematic illustration of peptide dendrimers that vary in size.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fields et al. 1993. Hahn et al. 1990.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

enhanced and a potentially useful therapeutic agent produced.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

CS protein, P. malariae CS protein, P. falciparum

204 Table 3 Continued. Target CS protein, P. yoelii

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 3. A schematic comparison of divergent and convergent synthesis methods.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 4. Examples of simple amino, carboxylic and hydroxylic cores.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 7. Solid phase synthesis of a K 2 K glyoxylyl core.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 8. Synthesis of thiol-functionalized S-acetyl and CysNpys. di-K 2 K cores.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

Fig. 11. Convergent synthesis of an articial hemeprotein.

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

K. Sadler, J.P. Tam r Re iews in Molecular Biotechnology 90 (2002) 195 229

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