peptide dendrimers: applications and synthesis · peptide dendrimers: applications and synthesis...

Ž .Reviews in Molecular Biotechnology 90 2002 195�229

Peptide dendrimers: applications and synthesis

Kristen Sadler, James P. Tam�

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 coreand�or covalently attached surface functional units. The multimeric nature of these constructs, the unambiguouscomposition and ease of production make this type of dendrimer well suited to various biotechnological andbiochemical applications. Applications include use as biomedical diagnostic reagents, protein mimetics, anticancerand antiviral agents, vaccines and drug and gene delivery vehicles. This review focuses on the different types ofpeptide dendrimers currently in use and the synthetic methods commonly employed to generate peptide dendrimersranging 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 distinctstructural features: a central core; surface func-tionalities; and branching units that link the two.Peptide dendrimers can be broadly defined as anydendrimer that contains peptide bonds. This def-inition would, in theory, include a dendrimer withan amino acid core, branching units, surface func-tional groups or any combination of the three as apeptide dendrimer. This definition could be fur-

� Corresponding author. Tel.: �1-615-343-1465; fax: �1-615-343-1467.

ŽE-mail address: [email protected] J.P..Tam .

ther broadened if we consider all types of aminoacids including naturally occurring �-amino acidsas well as unnatural amino acids that have beenutilized in peptide dendrimer synthesis as bothbranching units and surface functional groups.For example, unnatural amino acids such as �-amino acids have also been used as branching

Ž .units Esfand and Tomalia, 2001 . In practice, themajority of peptide dendrimers being referred toin the literature and currently in use are based on

Ž .the multiple antigen peptide MAP system thatconsists of only two of the three structural fea-tures: branching units and surface functionalgroups. Interestingly, these molecules contain

Ž .both �-peptide and �-peptide Tam, 1988, 1996 .In addition, they are often synthesized without a

1389-0352�02�$ - see front matter � 2002 Elsevier Science B.V. All rights reserved.Ž .PII: S 1 3 8 9 - 0 3 5 2 0 1 0 0 0 6 1 - 7

( )K. Sadler, J.P. Tam � Re�iews in Molecular Biotechnology 90 2002 195�229196

core and more appropriately termed ‘dendrons’rather than classical dendrimers. However, forconvenience we will refer to all branched poly-peptide constructs as dendrimers.

2. General characteristics of peptide dendrimers

By virtue of their dendritic architecture, pep-tide dendrimers are frequently utilized as proteinand liposomal mimetics as well as biomaterials inthe life sciences and biomedical applications.Dendrimer design exploits two traits that are fre-quently observed in many naturally occurring sys-tems: globular structure and polyvalency.

Polyvalent interactions occur through the asso-ciation of two or more ligands and receptors ofthe same type. Multivalent molecules are com-monly found in nature ranging from smallmolecules of polysaccharides, nucleic acids andpeptides to protein aggregates on virions, bacteriaand other cells that aid in adhesion and cell�cellinteractions. The properties of multiple simulta-neous interactions can differ markedly from thosedisplayed by the constituents during monovalentinteractions, suggesting that dendrimers with mul-tiple functional sites can be used in the design ofnew drugs and research reagents. Such strategiesto cluster ligands and functional groups on den-drimeric structures are attracting considerable at-tention in different areas from the production ofbiomaterials to vaccine technology. Dendrimericmaterials are finding applications as antiviral

Ž .agents Zanini and Roy, 1998 , vehicles for deliv-Žery of nucleic acids Haensler and Szoka, 1993;

Kukowska-Latallo et al., 1996; DeLong et al.,. Ž .1997 and drugs Esfand and Tomalia, 2001 , as

biomedical tools such as magnetic resonanceŽimaging contrast agents Kim and Zimmerman,

.1998 , protein mimetics, vaccines directed to-wards bacterial, viral and parasitic pathogens, aswell as diagnostic reagents and molecular inhibi-tors. In the first step of infection most pathogenssuch as bacteria and virus particles adhere topotential host cells via polyvalent interactionsŽ .Mammen et al., 1998 . Therefore, there may be arole for multivalent dendrimers capable ofmimicking receptors and ligands in preventing

Table 1Current and potential applications of polyvalent dendrimers

Vehicles for delivery of

Nucleic acidsEncapsulated drugsCovalently linked drugs, e.g. anticancer agents

Diagnostic reagents inSerodiagnosisBiosensor systemsMagnetic resonance imaging

Vaccines againstBacteriaVirusParasites

Inhibitors ofPathogenic infectionsInflammatory responseAutoimmune diseaseCancer metastasis

Modification ofCell�cell interactionsGene expression, e.g. alteration of transcription factors

binding to DNA

infections. Similarly, cell�cell interactions that aremediated by specific receptor�ligand associationscan be studied in fine detail through the use ofdendrimers. Pathophysiological responses such asinflammation and autoimmune reactions could beprevented by therapeutic use of dendrimers. Someof the current and potential applications of syn-thetic dendrimers in biomedicine and biotech-nology are outlined in Table 1.

Peptide dendrimers vary from low molecularweight species of 2 kDa to large protein-likeconstructs �100 kDa. The size and complexity ofthe individual dendrimers are determined by twofactors, the number of layers of branching unitsŽ .often referred to as the generation number andthe surface supporting the terminal functionalgroups which can be large peptides or proteins ofsubstantial size. Typically, peptide dendrimershave generation numbers between 2 and 32. Simi-lar to other dendrimers, synthesis of peptide den-drimers is tightly controlled with products of con-sistent size, architecture and composition.

( )K. Sadler, J.P. Tam � Re�iews in Molecular Biotechnology 90 2002 195�229 197

Peptide dendrimers can be divided into threetypes. The first are grafted peptide dendrimers.These are conventional dendrimers with eitherunnatural amino acids or organic groups as thebranching core and peptide or proteins attachedas surface functional groups. Of the three, thegrafted peptide dendrimer is the largest in termsof size because they generally contain high gener-ation numbers of branching cores. In contrast,peptide dendrimers of the second type are essen-tially branching polyamino acids. Consequently,they tend to be the smallest by size with the coreconsisting of natural amino acids and the termi-nal amino acids acting as surface functionalgroups. The third type consisting of mostly pep-tides has been traditionally known as peptidedendrimers. In this group, with MAPs as the mostwell known example, the core consists of aminoacids and the surface functional groups are alsopeptidyl chains. This review provides some detailson the first two types of peptide dendrimers,however, we will largely focus on the third typebecause this type is used most frequently in bio-logical and biochemical applications.

2.1. Grafted peptide dendrimers with unnaturalamino acids and organic cores

Peptide dendrimers based on the commerciallyŽ .available poly amidoamine , or PAMAM core

may be considered classical dendrimers and oneŽof the most widely used Esfand and Tomalia,

.2001 . They are synthesized by a divergent methodinvolving a two-step iterative reaction sequencethat produces concentric shells of dendritic �-alanine units. Various functional groups, includ-ing peptides, have been covalently linked to thePAMAM dendrimer and used for different appli-cations. Anticancer drugs may be encapsulated by

Ž .PAMAM dendrimers with poly ethylene glycolgrafts prior to delivery in order to increase effi-

Ž .cacy Kojima et al., 2000 . Addition of sugars suchas lactose and fructose to the PAMAM den-drimer produces glycodendrimers that are poten-tial high-affinity ligands for sugar receptorsŽ .Andre et al., 1999; Kawase et al., 2000 . Excel-

Ž .lent reviews by Esfand and Tomalia 2001 , KimŽ .and Zimmerman 1998 outline the potential

biomedical applications of PAMAM dendrimerswith carbohydrate and nucleic acid functionalunits.

Peptides have also been attached to the PA-MAM core. In one of the most promising studiesan artificial photosynthetic system has been con-structed with amphipathic �-helix peptides actingas surface functional groups attached to the core

Žthrough a thioether linkage Sakamoto et al.,.2001a,b . The PAMAM dendrimer has also been

Žused to generate a biochemical reagent Singh,.1998 by reacting a PAMAM core with calf intes-

tine alkaline phosphatase and a Fab fragment ofan anti-creatine kinase MB isoenzyme antibody.

2.2. Peptide dendrimers with polyamino acids asbranching units and as surface functional sites

Water-soluble amphipathic peptide dendrimerscomprised only of three or four generations oflysine residues have been constructed to aid inthe delivery of DNA to intact cells. Dendrimersconsisting of a branched lysine core and 8 or 16terminal surface amines may be mixed in solutionwith plasmid DNA and left to formdendrimer�DNA complexes. The complexes aresubsequently taken up into cells and the geneproducts expressed with high efficiency compared

Žto 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 ofŽ .polyamide groups 4, 8 or 16 terminal lysines

were linked through the first generation lysine toŽ .three alkyl chains C and their interaction with14

charged and neutral liposomes was studiedŽ .Purohit et al., 2001 . It was found that the inter-action efficiency of the dendrimers was greaterthan 88% and that the amount of dendrimeradsorbed decreased as the dendrimer size in-creased. The mechanism of interaction is unclearbut adsorption is most likely due to hydrophobicinteractions rather than electrostatic because thedendrimers interacted with equal efficiency withnegatively and positively charged liposomes. Lipi-dated peptide dendrimers have also been synthe-sized by the addition of 2-amino tetradecanoicacid to a polylysine support to create a dendrimer

Ž .with 16 surface alkyl chains Florence et al., 2000 .


This peptide dendrimer was used to study theuptake of lipidated molecules by the mucosalsurfaces of the digestive tract in mice.

2.3. Peptide dendrimers with amino acid branchingunits and surface peptidyl chains

Peptide dendrimers comprised of amino acidŽ .cores and surface peptide chains Fig. 1 have

been used most extensively in biological applica-tions. The underlying design principle is that thepolyvalency of the peptide dendrimers enhancesthe affinity of peptide-specific interactions withpeptides, proteins and carbohydrates, thus en-abling the study of receptor�ligand interactions,the introduction of increased binding affinitiesand also development of new techniques for af-finity purification and diagnostic tests. An exam-ple showing the ability of multimeric peptides toenhance binding affinity was described by Fassina

Ž .et al. 1992a where analytical HPLC and solidphase binding assays were used to evaluate thebinding interactions between two complementarypeptides in monomeric and octameric forms.Binding affinity was found to increase when oneor both of the sequences were synthesized asoctameric dendrimers. The interaction betweenthe multimeric peptides was of similar affinity tothat of polyclonal antibody and multimeric pep-tide illustrating that weak interactions may bemagnified through the use of peptide dendrimersallowing more complete analysis. This may bepotentially useful for characterizing the weak in-teractions between cell-surface receptors and lig-ands. Some of the biochemical applications ofpeptide dendrimers are listed in Table 2.

3. Peptide dendrimers in immunoassays andserodiagnosis

Many immunoassays rely on the attachment ofantigens to a solid surface such as plastic mi-crotitre wells and their detection by addition ofspecific reagents. Short synthetic peptides readilyavailable through chemical synthesis are generallyconsidered to be ideal antigens because of thehigh specificity they confer. Unfortunately, most

Fig. 1. Schematic illustration of peptide dendrimers that varyin size.

short peptides are poor antigens due to theirinability to bind to solid surfaces. The multimericnature of peptide dendrimers has been found toaid in overcoming such deficiencies by providingincreased surface-binding character and sensitiv-

Žity of detection Tam and Zavala, 1989; Mar-guerite et al., 1992; Briand et al., 1992; Marsden

. Ž .et al., 1992 . Adesida et al. 1999 exploited theincreased affinity of protein interaction with pep-tide dendrimers compared to monomeric peptidesin a study focusing on the interaction of immuno-globulins with a non-epitopic peptide in both di-rect and indirect peptide-antibody binding assays.

In serodiagnosis where serum samples fromnaturally immunized or infected individuals aretested for the presence of specific antibody, themultimeric arrangement of dendrimers may alsoimprove the detection of low affinity antibodiesduring early stages of infection, particularly those

Žof the IgM isotype Habluetzel et al., 1991; Mars-.den et al., 1992 . In a dramatic example, a den-

drimer shows a sensitivity increase of over 108-foldwhen compared to a peptide antigen in an en-zyme-linked immunosorbent assay. Immunosor-bent assays may be superceded in the near futureby biosensor technology where dendrimers arebound directly to sensory chips and the strengthof antibody interaction monitored with great ac-

Ž .curacy Gomara et al., 2000a . The use of den-drimers in biosensor technology requires further


examination, particularly to optimize immobiliza-tion and ensure that factors such as steric hin-

Ž .drance Francis et al., 1991; Le et al., 1998 andinterchain aggregation do not impede immobiliza-tion nor antibody recognition. The introduction

of spacer residues such as glycine between thelysine core and peptide sequence of interestŽ .Marsden et al., 1992 may reduce inhibitory in-terchain aggregation. The dendrimers must alsobe bound to the sensory chips in the correct

Table 2Diagnostic and biochemical uses of peptide dendrimers

Application References

Immunoassays and serodiagnosisŽ . Ž .Malaria Tam and Zavala 1989 , Habluetzel et al. 1991

Ž .Avila et al. 2001Ž .Cirrhosis Briand et al. 1992Ž . Ž .HIV-1 Marsden et al. 1992 , Estaquier et al. 1993

Ž . Ž .Vogel et al. 1994 , Shin et al. 1997

Ž .Schistosoma mansoni Marguerite et al. 1992Ž . Ž .Systemic lupus erythematosus Sabbatini et al. 1993a , Caponi et al. 1995Ž .Epstein-Barr virus Marchini et al. 1994

Ž .Infectious bronchitis virus Jackwood and Hilt 1995Ž .Hepatitis A virus Gomara et al. 2000a,bŽ .Non-epitopic peptide Adesida et al. 1999

Ž .Sperm antigen Tres and Kierszenbaum 1996

Epitope mapping and ligand bindingŽ .Bluetongue virus Yang et al. 1992

Ž .Systemic lupus erythematosus Sabbatini et al. 1993bŽ .Hepatitis C virus Simmonds et al. 1993

Ž .P. falciparum Pf72�Hsp70-1 antigen Dat et al. 2000Ž .NKTag, tumour antigen Jaso-Friedmann et al. 1996

InhibitorsŽ . Ž .Macroautophagia and proteolysis Miotto et al. 1994 , Mortimore et al. 1994Ž . Ž .Tumour growth and metastasis Nomizu et al. 1993 , Kim et al. 1994Ž . Ž .Iwamoto et al. 1996 , Manki et al. 1998

Ž .Enzyme inhibitors Fassina and Cassani 1993Ž . Ž .HIV-1 fusion and infection Yahi et al. 1994a,b, 1995 , Weeks et al. 1994

Ž .Interleukin 6 Wallace et al. 1994Ž .Sporozoite, malaria Sinnis et al. 1994Ž .Fibronectin Ingham et al. 1994Ž .Antagonist of C5a Kaneko et al. 1995

Artificial proteinsŽ .Minicollagen Fields et al. 1993Ž .Synthetic enzyme Hahn et al. 1990

Biochemical studiesŽ . Ž .Affinity purification of antibodies Butz et al. 1994 , Yang et al. 1999Ž .Presentation of T-cell epitopes Grillot et al. 1993

Ž . Ž .Affinity purifications Fassina 1992 , Fassina et al. 1992a,bŽ .Yao et al. 1994

Ž .Intracellular delivery Sheldon et al. 1995


orientation, for this ligation reactions may beexploited for example thiol exchange, hydrazide

Ž .or sulfhydryl group coupling Schuck, 1997 .

4. Peptide dendrimers as inhibitors

Another promising application of peptide den-drimers has been realized in the general design ofinhibitors. Multimerization of binding elementssuch as charge in a dendrimer is controllable andprovides an unambiguous structure that can berefined by analogs with relative ease. The inhibi-tion of entry of malaria parasites into hepatocytesŽ .Sinnis et al., 1994 is a fine example of this

Ž .application. Sinnis et al. 1994 have taken advan-tage of the fact that a cationic conserved se-

Ž .quence of malaria circumsporozoite CS proteinknown as region II-plus requires aggregation forbinding to liver hepatocyte proteoglycans. Atetramer containing region II-plus was synthe-sized and subsequently found to inhibit CS pro-tein binding to hepatocytes in mice. A peptidedendrimer based on an eight-residue sequence

Ž .from the third variable region V3 loop of gp120,the HIV-1 surface envelope glycoprotein, hasbeen shown to inhibit HIV-1 infection of both

� � ŽCD4 and CD4 cells Yahi et al., 1995; Carlier.et al., 2000 .

The increased binding affinity of dendrimerscompared to monomeric peptides is becoming animportant factor in the design of peptides aimedat inhibiting metastasis of various types of cancercells. One of the most advanced studies has shownthat a dendrimer containing 16 copies of a pep-

Žtide from laminin a protein of the basement.membrane extracellular matrix significantly in-

Ž .hibited tumor growth in vivo Nomizu et al., 1993 .This inhibitory activity increased as the peptidecopy number increased, i.e. 16�8�4. The en-hanced anti-tumor effects of the 16-mer weresubsequently partly attributed to the ability of themultimer to compete more successfully withlaminin for the laminin receptor on tumor cellsthereby blocking the binding of malignant cells to

Ž .blood vessel walls Iwamoto et al., 1996 . There-fore, by covalently linking multiple copies of ananti-tumor peptide the binding affinity has been

enhanced and a potentially useful therapeuticagent produced.

5. Peptide dendrimers as mimetics

Peptide dendrimers have proven to be usefulsubstitutes for proteins and even DNA in a num-ber of autoimmune disease model systems. Den-drimers based on peptides from various proteinshave been used to generate autoimmune condi-tions in normal animals that are comparable to

Ž .the condition systemic lupus erythematosus SLEŽJames and Harley, 1998; Mason et al., 1999;

.Farris et al., 1999 . Mason showed that inocula-tion of mice with a MAP consisting of eightcopies of a peptide derived from the spliceosomalSm protein resulted in epitope spreading, a re-sponse usually attributed to an immune responseagainst self proteins and DNA. Anti-nuclear anti-bodies were detected in some animals illustratingthat the MAP acted as a protein and DNAmimetic.

Ž .Putterman and Diamond 1998 identified apeptide mimetic for double stranded DNA thatelicited anti-DNA antibody production. Mice de-veloped a lupus-like syndrome creating an experi-mental model for SLE. Using dendrimers Khalil

Ž .et al. 2001 replicated the T cell-dependent na-ture of SLE using a peptide immunogen insteadof a DNA immunogen and characterized the Tcell response. Unlike other examples mentionedthus far, it was determined that the increasedresponse to the dendrimer was dependent moreon the amino acid sequence than on valency. Aplausible explanation for the observed results isthat the presence of the dendrimer backboneallowed more effective processing and presenta-tion of the correct T helper epitopes.

During a similar study where the specificity ofantibodies was examined, it was found that den-drimers themselves could not only bind to anti-bodies but also directly interact with the 60-kDa

ŽRo nuclear protein in various assays Scofield et.al., 1999 . Furthermore, it was concluded that Ro

and La, components of a single ribonucleoproteinparticle, directly interact. The protein-binding se-quences within Ro were identified using den-


drimers; the dendrimers were an important toolfor exploring both intramolecular and intermolec-ular protein-protein interactions. Even though theaffinity of interaction may not be absolutely de-termined because the use of dendrimers mayincrease avidity in immunoassays by several logsŽ .Tam, 1996 , the relative affinities may be de-termined. In the context of SLE, the peptidedendrimer provides useful reagents for probingthe extent of immune tolerance in the B and Tcell compartments to nuclear antigens.

The weak antimicrobial activity of monomericpeptides has been observed to increase after mul-timerization. An 11-residue peptide representing

Ža sequence from human lactoferrin an iron-bind-ing glycoprotein found in neutrophils and secre-

.tions was incorporated into peptide dendrimersconsisting of 1, 2, 4, 8 or 16 copies and thedendrimers were tested for antibacterial activityŽ .Azuma et al., 1999 . An increase in antimicrobialactivity correlated with an increase in the peptidecopy number. The dendrimer containing 16 copiesof the peptide was as effective as the traditionalantibiotic gentamycin against Gram-negative andGram-positive bacteria. In this case, the authorssuggest that the monomeric peptide is too shortto span the membrane and create an ionic porewhereas the dendrimers are large enough to spanthe membrane and form a pore or mimic poreformation thus resulting in microbicidal activity.To advance the dendritic design a step further,

Ž .Lu et al. 2001 designed an inactive tetrapeptide

with a basic and hydrophobic motif derived from�-strand topology of tachyplesins and protegrinswhich exhibited significant antimicrobial activitywhen synthesised as a dendrimer. The tetramericdendrimer is similar in molecular weight and anti-microbial potency as protegrins or tachyplesins.More importantly, each peptide dendrimer as-sayed was found to have higher antimicrobialactivity against fungi, Gram-negative and Gram-positive bacteria than linear tandem repeatingpeptides.

6. Peptide dendrimers as immunogens

Possibly the most widely known biological ap-plication of peptide dendrimers is the use ofMAPs as immunogens. Immunogens are distin-guished from antigens by their ability to inducean immune response in vivo. MAPs, typically ofthe form shown in Fig. 2, have enormous poten-tial as immunogens and a significant portion ofthis potential has already been realized. The ad-vantages of using MAPs as immunogens include:

� Simplicity in design and synthesis;� Versatility for investigating various immune

responses;� Reliability of generating site-specific anti-

bodies; and� Generation of site-specific antibodies in the

laboratory.

Ž . Ž . Ž .Fig. 2. a Schematic representation of a multiple antigen peptide MAP , incorporating eight peptide monomers. b An increase inthe number of Lys branching units increases the number of surface amine groups.


Since the first report of the synthesis and po-Ž .tential of MAPs by Tam 1988 MAPs have been

employed routinely as immunogens for antibodyproduction and cytotoxic immune responses.1 Be-fore we review the application of MAPs as im-munogens, several points regarding peptide se-quence selection and design of MAPs will bepresented.

6.1. Summary of immune responses

This overview aims to briefly summarize therole of peptide epitopes in the induction of im-mune responses and to alert the reader to theimportance of epitope selection. In general, thereare two types of immune responses, humoral andcell-mediated. Induction of a humoral responseresults in the production of antibodies and reliesupon the activation of both B cells and T helpercells. Cytotoxic T cells and T helper cells, on theother hand, mediate cellular immune responses.

For maximal antibody production a B cell mustreceive two signals. It must bind a B cell epitopeto the surface immunoglobulin with high affinity.The B cell epitope is usually a peptide or proteinsegment approximately nine residues in length.Secondly, the B cell must interact with an acti-vated T helper cell leading to B cell maturationand production of antibody with the same speci-ficity as the surface immunoglobulin. The T helpercell is in turn activated by interaction with an-other type of cell, the antigen presenting cellŽ .APC . The role of the APC is to engulf extracel-lular material, including foreign peptides and pro-teins, and degrade the proteins and peptides intoshort segments. These short segments are subse-quently presented on the APC surface in associa-tion with MHC class II molecules. The T helpercell is activated if it encounters an APC present-ing a peptide-MHC complex that binds with high

Ž .affinity to the T cell receptor TCR . The peptideassociated with the MHC molecule is referred toas a T helper epitope. Without the presence ofboth the B cell epitope and T helper epitope

1 A recent literature survey showed that Tam’s 1988 paperhas been cited over 600 times.

antibody production is generally short-lived andthe highly desirable memory response is lacking.

Activation of T helper cells is also required forthe induction of a cell-mediated response. In thiscase the T helper cells interact with and providecytokines for the stimulation of cytotoxic T cells.The main role of the cytotoxic T cells is to recog-nise and kill host cells which are expressing non-self peptides associated with MHC class I pro-teins such as peptides from intracellular bacteriaand viruses. These peptides are referred to as

Ž .cytotoxic T lymphocyte CTL epitopes. The ma-jority of host cells are capable of degrading intra-cellular proteins and presenting CTL epitopes onthe surface when bound to MHC class I molecules.On the first occasion that a cytotoxic T cell en-counters a host cell expressing a non-self epitope

Ž .it undergoes maturation aided by T helper cellsand is then capable of killing other host cellsexpressing the same CTL epitope.

In summary, humoral responses require B andT helper epitopes and cell-mediated responsesrequire CTL and T helper epitopes. An effectivesynthetic immunogen must contain peptide se-quences that activate the appropriate type of im-mune response and the flexibility of the MAPsystem allows incorporation of multiple epitopesin to the same construct.

6.2. Design of MAPs for induction of an antibodyresponse

B cell epitopes alone are poor immunogens andhave traditionally been conjugated to large car-rier proteins that provide strong T helper cellepitopes. The evolution of the MAP system nowallows B cell and T helper epitopes to be assem-bled directly onto a small polylysine core. Incontrast to conventional peptide-protein conju-gates where peptide epitopes account for lessthan 20% of the total weight, MAPs consist ofconcentrated epitopes and possess an immunolog-

Žically silent lysine core Posnett et al., 1988; Del.Giudice et al., 1990 . In some cases there is

significant difference and a generally better qual-ity of antisera in using MAPs vs. peptide�protein

Žconjugates McLean et al., 1991; Wang et al.,1991; Estaquier et al., 1993; Molnar et al., 1993;


Table 3Utilization of peptide dendrimers for antibody and CTL production

Target References

BacteriaŽ .Tandem repeat motifs, Leishmania Liew et al. 1990Ž .SLT-1 protein, Escherichia coli Boyd et al. 1991Ž . Ž .P30 antigen, Toxoplasma gondii Darcy et al. 1992 , Godard et al . 1994Ž . Ž .Glucosyltransferase, Streptococcus Smith et al. 1993 , Smith et al. 1994aŽ .Toxin T, Bordetella pertussis Felici et al. 1993

Ž .Toxin B, Vibrio cholera Halimi and Rivaille 1993Ž .Membrane protein 1, C. trachomatis Zhong et al. 1993

Ž .Membrane protein, Neisseria meningitidis Christodoulides and Heckels 1994Ž .Cyclase, Saccharomyces cere�isiae Shi et al. 1994Ž .Exoproteases, Pseudomonas aeruginosa Coin et al. 1997Ž .Gingipains, Porphyromonas gingi�alis Genco et al. 1998

Ž .Porin protein, N. meningitidis Christodoulides et al. 1999

VirusŽ . Ž .Surface antigen, Hepatitis B virus Tam and Lu 1989 , Manivel et al. 1993

Ž .Reductase, Herpes simplex virus Lankinen et al. 1989Ž .VP2 and VP7, bluetongue virus Li and Yang 1990Ž . Ž .VP1 protein, foot-and-mouth disease virus Francis et al. 1991 , Lugovskoi et al. 1992

Ž .Brown 1992Ž . Ž .gp120, HIV Wang et al. 1991 , Defoort et al. 1992a,bŽ . Ž .Nardelli et al. 1992a,b, 1994 , Nardelli and Tam 1993

Ž . Ž .Levi et al. 1993 , Kelker et al. 1994Ž . Ž .De Santis et al. 1994 , Huang et al. 1994

Ž . Ž .Fraisier et al. 1994 , Vogel et al. 1994Ž .Nef, HIV-1 Estaquier et al. 1992, 1993

Ž .Polymerase, influenza virus Nieto et al. 1992Ž . Ž .Hemagglutinin, influenza virus Toth et al. 1993 , Naruse et al. 1994Ž .Zeng et al. 2000

Ž .NSm protein, bunyamwera virus Nakitare and Elliott 1993Ž . Ž .B19, parvovirus Saikawa et al. 1993 , Anderson et al. 1995

Ž .Core proteins, vaccinia virus Vanslyke and Hruby 1994Ž .gp46, HTLV-1 James et al. 1995

Ž .V3 loop, HIV-1 Cruz et al. 2000Ž .MVF, measles virus Olszewska et al. 2000a,b

Ž .gp51, bovine leukemia virus Kabeya et al. 1996Ž .Fusion protein, respiratory syncytial virus Hsu et al. 1999Ž .VP3, Hepatitis A virus Pinto et al. 1998Ž .V3, feline immunodeficiency virus Rigby et al. 1996Ž .VP1 protein, JC virus Aoki et al. 1996

ParasiteŽ . Ž .CS protein, Plasmodium berghei Romero et al. 1988 , Migliorini et al. 1993

Ž . Ž .Zavala and Chai 1990 , Chai et al. 1992Ž . Ž .Widmann et al . 1992 , Valmori et al. 1994Ž .CS protein, P. malariae Del Giudice et al. 1990Ž . Ž .CS protein, P. falciparum Munesinghe et al. 1991 , Pessi et al. 1991

Ž . Ž .Valmori et al. 1992 , Nardin and Nussenzweig 1993Ž .Calvo-Calle et al. 1992, 1993Ž . Ž .De Oliveira et al. 1994 , Ahlborg 1995

Ž . Ž .Nardin et al. 1998, 2001 , Moreno et al. 1999


Ž .Table 3 Continued

Target References

Ž . Ž .CS protein, P. yoelii Wang et al. 1995 , Marussig et al. 1997Ž .Franke et al. 2000

Ž .CS protein , P. �i�ax Del Guercio et al. 1997Ž . Ž .P28 antigen, Schistosoma mansoni Auriault et al. 1991 , Khan et al . 1994

Ž .Pancre et al. 1994Ž .Triose-phosphate isomerase, S. mansoni Reynolds et al. 1994

Ž .Sm28GST and sTPI, S. mansoni Ferru et al. 1997Ž .Sm37-GAPDH and Sm10-DLC, S. mansoni Argiro et al. 2000

OtherŽ .Trp protein, Drosophila Wong et al. 1989

Ž . Ž .UL47, UL8, Vmw63 and UL42 McLean et al. 1990 , Parry et al. 1993Ž . Ž .Sinclair et al. 1994 , Marsden et al. 1994Ž .Lutropin, human Troalen et al. 1990

Ž .Ribosomal protein, plant Szymkowski and Deering 1990Ž .Fatty acid binding protein, human St John et al. 1991

Ž .Transforming growth factor � , human Lu et al. 1991Ž .Alkyltransferase, human Pegg et al. 1991

Ž .Cytochrome P-4501A, rat Edwards et al. 1991Ž .Mast cell protease-5�, mouse McNeil et al. 1992Ž .ATPase, plant Suzuki et al. 1992Ž .Calcium channel, rabbit Malouf et al. 1992Ž .Sperm myoglobin, whale McLean et al. 1992Ž .Sperm protein, human Vanage et al. 1992, 1994

cdc2 Ž .Protein kinase p34 , eukaryote Kamo et al. 1992Ž .Membrane domain of band 3, human Kang et al. 1992

Ž .Angiotensin II type-1 receptor, rat Zelezna et al. 1992, 1994Ž .Actin-fragment kinase, plasmodia Gettemans et al. 1993

Ž .Seed protein, plant Monsalve et al. 1993Ž .IGF binding protein, bovine Arnold et al. 1993Ž .Glutamate receptor, rat Molnar et al. 1993

Ž .Glucose transporter, rat Nagamatsu et al. 1993Ž .RNA-binding protein, rodent Henderson et al. 1993

Ž .Insulin receptor, human Itoh et al. 1993Ž .Myotrophin, human Sil et al. 1993

Ž . Ž .Iron regulatory factor, human Emery-Goodman et al. 1993 , Gray et al. 1993Ž .Protein G, human Raymond et al. 1993

Ž .Tyrosine kinase, sponge Schacke et al. 1994Ž .Chaperonin polypeptide, eukaryote Lingappa et al. 1994

Ž .Sperm protein , Xenopus Bauer et al. 1994Ž .pP344 retinal cell, chicken Iio et al. 1994

Ž .Myosin phosphatase, chicken Shimizu et al. 1994Ž .Prion protein, mouse O’Rourke et al. 1994

Ž .Ameloginin, mouse Simmer et al. 1994Ž .Histones, mouse Meziere et al. 1994

Ž .GTPase, rat Wilson et al. 1994Ž .Neurexins, rat Perin 1994

Ž .T-tubule, rabbit Stout et al. 1994Ž .Calcium release channel, rabbit Callaway et al. 1994

Ž .Anion transport protein band 3, human Crandall and Sherman 1994Ž . Ž .Guanylin, human Kuhn et al. 1994 , Cetin et al. 1994Ž .Profilin, human Finkel et al. 1994


Ž .Table 3 Continued

Target References

Ž .Type V collagen, human Moradi-Ameli et al. 1994Ž .Phosphatase 2A, human Zolnierowicz et al. 1994

Ž .DR molecules, human Demotz et al. 1994Ž .Elafin, human Nara et al. 1994

Ž .Gangliosides, human Helling et al. 1994Ž .5�-reductase 2, human Eicheler et al. 1994

Ž . Ž .Heme oxtgenase-1, human Kutty et al. 1994 , Smith et al. 1994bŽ .Spliceosome protein, human James et al. 1995

Ž .Cyclin, human Digweed et al. 1995Ž .Phospholipase A , human Sa et al. 19952

Ž .Kainate receptor, goldfish Wo and Oswald 1995Ž .Myelin basic protein, murine Zhou and Whitaker 1996

Ž .Serotonin transporter, rat Zhou et al. 1996Ž .Ras p21, human Schott et al. 1996

Ž . Ž .Poly ADP-ribose polymerase, eukaryote Duriez et al. 1997Ž .Seed proteins, Vigna angularis Kajiwara and Tomooka 1998

Ž .Sm B�B� protein, primate Arbuckle et al. 1998Ž .pRL1a, murine leukemia Manki et al. 1998Ž .N-myc oncoprotein Yang et al. 1999

.Vogel et al., 1994 . A comparative study byŽ .McLean et al. 1991 identified what has now

become a general trend in the properties of anti-sera elicited by different constructs, sera obtainedfrom animals inoculated with MAPs is of a highertiter and produced more rapidly than those ob-tained with protein conjugates which are in turnhigher than those obtained with resin-linked pep-tides. The MAP format allows investigators toovercome the immunodominance conferred bylinker and carrier proteins and elicit antibodyresponses to weakly immunogenic sub-dominantpeptide sequences. Examples of the use of MAPsas immunogens are listed in Table 3.

There are many possible arrangements oflinking B and T helper epitopes on MAPs, themost popular approach to date is to link theepitopes in a tandem formation. The main draw-back with this method is that new immunodomi-nant epitopes may be formed within the tandempeptide and the MAP is not processed and pre-sented as the intended design. One empirical

Ž .solution suggested by Lo-Man et al. 1994 is toinsert a longer T epitope to preserve the diversityof T cell recognition. Alternatively convergentstrategies may be employed where the dendrimercontains copies of each peptide sequence on sep-

arate ‘arms’ thereby decreasing the possibility ofneo-epitope formation and increasing the re-sponse to the desired epitopes.

Tandem, or diepitope, MAP constructs havebeen extensively studied in the malaria syntheticvaccine model. Ten MAP constructs were synthe-sized, each containing a combination of describedB and T cell epitopes, the constructs were de-signed to evaluate the relevance of the number ofcopies, stoichiometry, and orientation of T and B

Ž .sequences in a diepitope model Tam et al., 1990 .In this case it was concluded that there was noadvantage in using octameric instead of tetra-meric MAPs, and that having the B cell epitopeat the N-terminal produced a more efficient im-munogen. The degree of protection against chal-lenge of immunized mice with 2000 Plasmodiumberghei sporozoites correlated with the antibodylevels obtained by the immunization protocol. Te-tra- and octameric MAPs have been utilized inmost studies to date. The number of branchesrequired tends to depend largely on the aminoacid residues: with peptides �15 amino acids wehave found that there is no real advantage inusing more than four branches. A similar conclu-

Ž .sion was reached by Francis et al. 1991 whereguinea pigs were immunized with constructs con-


taining one, two, four or eight copies of a foot-and-mouth disease virus peptide. It was de-termined that in this case presentation in a te-trameric structure was sufficient for an optimalantibody response.

Diepitope MAPs were synthesized in the designof a synthetic vaccine model for hepatitis B using

Ž .a different chemical approach Tam and Lu, 1989 .A B cell epitope and T helper epitope were usedin mono- and diepitope configurations, in thelatter the two peptides were connected in alter-nating forms on the lysine core instead of intandem. Only when the B cell epitope was pre-sented in the MAP form with the T helper epi-tope was it immunogenic in rabbits. Therefore,these results confirmed that a diepitope MAPformat may overcome the poor immunogenicityof a linear peptide.

Mono- and diepitope MAP configurations werefurther analyzed in a synthetic model for the

Ž .human immunodeficiency virus type 1 HIV-1Ž .Nardelli et al., 1992a . Monoepitope MAPs con-sisting of four copies of a major neutralizingepitope of HIV-1 were synthesized along withdiepitope MAPs that also contained a known Thelper epitope at the C-terminus of the B cellepitopes. The diepitopes were immunogenic in allspecies tested whereas the monoepitope MAPselicited species-specific responses. The use of uni-versal or promiscuous T helper epitopes whichare recognized by three or more strains of miceare particularly useful for diepitope constructsand have been used to enhance the immunogenic-ity of B cell epitopes in a number of MAPsŽMcLean et al., 1992; Munesinghe et al., 1991;

. Ž .Marguerite et al., 1992 . Finally, Levi et al. 1993have demonstrated that boosting with B cells andT helper cell epitope MAP constructs induced ahigher antibody titer for HIV-1 and improvedcellular responses than boosting with recombi-nant gp160 protein.

The use of chemoselective ligation through ox-ime bond formation is gaining notoriety as afeasible method for immunogen production. Self-adjuvanting tetrameric MAP constructs incor-porating B cell and T helper epitopes from dif-ferent sources induce strong antibody responsesŽ .Nardin et al., 1998; Zeng et al., 2000 .

6.3. Cell-mediated responses induced by lipidatedMAPs

Induction of a cell-mediated response reliesupon the delivery of CTL epitopes to an intracel-lular endosomal pathway that results in the pre-sentation of peptides in association with class IMHC molecules. Others have found the value oflipidated peptide in eliciting cellular responsesbecause of the increased efficiency of delivery ofpeptides to the intracellular compartment. Long-chain lipidic amino acids have been developed by

Ž .Gibbons et al. 1990 in combination with deter-gent-solubilized liposome as a delivery system.This approach was applied to the MAP format in

Žsuccessful vaccine models against HIV-1 Defoort.et al., 1992a,b; Nardelli et al., 1992a, 1994 and

ŽChlamydia trachomatis Zhong et al., 1993; Pancre.et al., 1994 .

A cytotoxic response was induced by a lipidatedMAP containing a peptide sequence from gp120envelope protein of HIV-1, III-B isolate whichoverlaps a B cell epitope, a T helper epitope anda CTL epitope. This response was detected aftera single inoculation without extraneous adjuvant,was superior to the response induced by a fullcycle of inoculations with the non-lipidated MAPin complete Freund’s adjuvant and was still de-tectable 7 months later. The lipidated MAP wasfound to produce systemic antibody and cellularresponses regardless of the route of inoculation

Ž .employed Nardelli et al., 1992b, 1994 . The abil-ity of MAPs containing the lipid moiety tripalmi-

Ž .toyl-S-glyceryl cysteine P C to induce mucosal3antibody response via oral administration adds anew dimension to applications of the MAP con-structs, and it may be particularly useful in pre-venting transmission of pathogens, such as HIV,through mucosal surfaces.

6.4. Possible reasons for increased immunogenicity

The multimeric MAP approach has been shownto increase the immunogenicity of weakly im-munogenic monomeric peptides, there are severalpossible reasons for the observed enhanced activ-ity. The conformation of the peptide epitopeswithin the MAP may contribute to the longevity


and strength of the responses induced by MAPs.MAPs attain a somewhat unnatural polymericstructure when compared to a linear peptidewhich may aid in resistance to in vivo proteolyticdegradation leading to a longer immunogen half-life. This persistence of immunogen in the sys-temic circulation possibly induces longer lastingimmune responses.

The increase in immunogenicity could also bedue to new T helper epitopes being generatedwithin the MAP format. Even though the lysinecore itself is immunologically silent the contribu-tion of one or two residues to the attached pep-tide sequences could result in the production ofnew epitopes. This would provide a plausible ex-planation for the fact that the antibody responseto the repetitive NANP peptide from Plasmodiumfalciparum CS protein is restricted to a singlemouse strain when administered as a linear pep-tide but administration of the peptide in a MAPformat results in antibody production in a further

Žfive strains that differ in MHC haplotype Pessi et.al., 1991 . Another alternative is that the MAP

construct allows the desired epitopes to beprocessed and presented by APCs in a moreefficient manner. For instance all residues in alinear peptide may be accessible to degradingenzymes, whereas the MAP configuration mayactually protect certain residues from enzymaticcleavage resulting in the presentation of differentepitopes. In many cases the more immunogenicdiepitope MAP constructs are synthesized withthe desired T helper epitope in close proximity tothe branching lysine core.

The design of a MAP provides a scaffold forclose packing of peptide sequences that may al-low the stabilization of the secondary structureand the reverse turns of peptides. Furthermore,the distal end of the peptide away from the coreof MAPs is more exposed and flexible than theproximal end, for these reasons we may expectthe immunogenicity of the B cell epitope to begreater if the epitope is located at the distal,N-terminal site.

The multimeric nature of the MAP system mayalso contribute to the well-documented increasesin immunogenicity. The higher copy number ofclosely packed epitopes could lead to greater B

cell surface cross-linking through surface im-munoglobulins resulting in increased activationand antibody production.

6.5. Potential application: peptide dendrimers assynthetic �accines

The majority of vaccines currently approved forhuman use consist of either heat-killed or live-attenuated infectious agents. Despite the suc-cesses of such vaccines, development by conven-tional 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 re-combinant proteins or synthetic peptides are ap-pealing alternatives because they are safe, selec-tive and chemically defined. Peptides have furtheradvantages in that short peptide sequences maybe synthesized that only contain the epitope ofinterest and deleterious epitopes may be omitted.Large quantities of chemically purified peptidevaccines can be prepared with automated meth-ods and peptide-based immunogens are morelikely to be resistant to denaturation, and theycan be easily stored and transported without re-frigeration.

A peptide dendrimer malaria vaccine recentlyentered a Phase I study in human volunteersŽ .Nardin et al., 2001 . The vaccine was assembledusing chemoselective ligation via oxime bondsand consists of four peptide branches each con-taining B cell epitopes and a promiscuous T helpercell epitope from P. falciparum CS protein. Ex-ogenous adjuvant was not required due to theinclusion of a P C moiety covalently linked to the3polylysine core. The majority of the inoculatedvolunteers developed high titered antibody re-sponses as well as T cells specific for the T helperepitope.

The use of MAPs as vaccines may extend pastthe use of relatively small tetra- and octameric


constructs that contain a minimal quantity ofepitopes. MAP technology lends itself to the de-sign of macromolecular structures that may mimicwhole organisms or several different organisms.Ultimately several similar or different closelypacked peptide immunogens prepared as MAPscould be anchored on lipid vesicles that mimicsurface proteins. Immunogenicity could bebroadened and enhanced by the inclusion of anon-covalent mixture of B cell and T helper andCTL epitopes from various proteins. At the sametime the combination of adjuvant effects of lipo-some and the built-in lipid anchor may replacethe need for additional adjuvant. Lipidated MAPshave been shown to become entrapped in lipo-somes in an efficient manner, nearly 80% of

lipidated MAPs were incorporated as comparedŽto 2�5% without the lipid anchor Defoort et al.,

.1992a,b in one example.

7. Synthesis overview

Synthesis of peptide dendrimers embraces abroad range of chemistry from conventionalsolid-phase peptide synthesis schemes in organicsolvents to the formation of regiospecific amideor non-amide bonds in aqueous solutions. Strate-gies for preparing peptide dendrimers can gener-ally be divided into two categories, namely the

Ž .divergent and convergent approaches Fig. 3 . Thedivergent strategy is a direct approach by which

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


the dendrimer is built stepwise and diverges out-ward. Stepwise solid-phase synthesis has beenused to prepare the dendrimer in a continuousoperation on a solid support. However, the con-vergent strategy is an indirect, modular approachby which peptidyl surface functional groups andthe branching unit are prepared separately. The

purified components are then linked together andpeptide sequences converge to the branching unitas a dendrimer.

Speed and efficiency are advantages of the di-vergent stepwise strategy particularly by solid-phase methods because intermediates are neitherpurified nor characterized. This strategy is suit-

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


able for validation of concepts and for smallpeptide dendrimers that may be separated fromtheir byproducts by powerful systems. In manycases stepwise synthesis is not practical, eitherbecause of the nature of the branching unit pre-venting integration into a stepwise strategy or

Žbecause the resulting dendrimer is too large e.g..�15 kDa to be separated from its byproducts

with confidence.An advantage of the convergent strategy is

chemical unambiguity because the protected orunprotected peptide segments and the branchingunit used in coupling reactions are purified priorto reaction, thereby limiting the range ofbyproducts and facilitating purification. A disad-vantage lies in the operational complexity: theconvergent strategy requires more steps thanstepwise synthesis, including additional purifica-tion and characterization steps for intermediates.The convergent strategy could also pose signifi-cant problems with regard to the solubility ofpeptide intermediates when protected peptidesegments are being used. Thus, a recent trend hasdeveloped in which aqueous, soluble unprotectedpeptides are used that are convenient to handle,purify and characterize. Several laboratories havemade extensive efforts to develop new method-ologies of ligation chemistry that involve thefewest steps possible. These chemistries are basedon chemoselectivity and orthogonality of unpro-tected intermediates for assembling peptide den-drimers.

7.1. Purification and characterization

Methods for purification and characterizationof peptide dendrimers are similar to those ofpeptides and proteins. Crude synthetic peptidedendrimers derived from the divergent strategyoften require multiple methods that may includesequential steps of dialysis or gel-filtration chro-matography, followed by RP-HPLC or high-per-formance ion-exchange chromatography. Even bythese steps it may still not be possible to removebyproducts with a single modification or deletionof an amino acid from the desired product. Syn-thetic products assembled using a convergentstrategy can be refined by most chromatographic

methods resulting in a homomeric product. Char-acterization methods for peptide dendrimers in-clude the usual panel of techniques: amino acidanalysis; SDS-PAGE; capillary zone electrophore-sis; and enzymatic digestion. Mass spectrometricanalysis has now become an indispensable toolfor determining the molecular weights indicatingproduct homogeneity of these complex molecules.

7.2. Types of dendrimer cores and branching units

For the design of artificial proteins the type ofcore selected is critical because it may conferrigidity and exert conformational influence on theoverall structure of the peptide dendrimer.

ŽElaborate cores Unson et al., 1984; Sasaki andKaiser, 1989; Ghadiri and Case, 1993; Chapmanet al., 1994; Schneider and Kelly, 1995; Tsikaris etal., 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 assistfolding into structural motifs such as �-helices,�-sheets, reverse-turns and loops which are oftenlacking in peptide monomers. However, mostcores employed to date are simple small organicmolecules �1000 Da with the sole purpose ofclustering or amplifying peptides to enhance thepolyvalency effect. Selected examples are shownin Fig. 4. Such cores are often commercially avail-able allowing many groups to investigate the useof peptide dendrimers in various applications.

The most common cores are simple aminocompounds, namely amino acids and dipeptides.

ŽThe simplest amino cores are the tris ethylene.amine ammonia types first used in organic den-

drimers that have recently been utilized in forma-tion of peptide dendrimers. The use of hetero-cyclic compounds such as porphyrin and con-strained and unusual amino acids has also been

Ž .reported Sasaki and Kaiser, 1989 .Simple organic polycarboxylic acids have been

Žused 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 combi-nation of functional groups. As a group, carbohy-drates and their intermediates represent a diverseand attractive source of polyhydroxylic branching


units because of their small sizes and the stere-Žochemical variations of pendant hydroxyls New-

kome et al., 1991; Whittaker et al., 1993; Murer.and Seebach, 1995 . In addition, their hydrophilic

nature favors convergent synthesis in aqueoussolutions. The utility of hydroxylic and carboxylicbranching units is currently limited by the needfor a synthetic methodology to distinguishbetween these groups that also occur frequentlyin peptides. In the case of hydroxylic groups, theyare further hindered by the less reactive naturewhen compared to amines and carboxylic acids.As a result, their use has not yet been widelyexplored in peptide dendrimers.

7.3. Polylysine branching units

Polyamino acids consisting of several branchesof a trifunctional acid represent the largest group

Žof branching units being used today Tam, 1988;.Rao and Tam, 1994 . By virtue of their trivalency,

reactivity and synthetic expedience branchingunits derived from lysine and its homologs are themost popular. Lysine with N�- and N�-aminogroups as reactive ends is a particularly suitabletrifunctional amino acid to form a branching unitby a limited sequential propagation of lysines.These low molecular weight cores with 2n reac-tive amino ends can then serve as attachmentsites for peptides. Solid phase schemes are themethod of choice for the synthesis of lysine

Ž .branching units Tam, 1988; Tam and Lu, 1989Ž .where di-protected Lys, e.g. Boc�Lys Boc or

Ž .Fmoc�Lys Fmoc , is used to produce a core ofmultiple levels of lysines. The diamino nature oflysine creates a situation where each additionallevel of Lys effectively doubles the number ofsites to which peptide monomers may be at-tached. A typical branching lysine unit consists ofthree lysines to give a tetravalent peptide den-

Ž .drimer Fig. 5 . Further divergence of the K K2unit to one or two additional levels will generatedi-K K and tetra-K K dendrons with reactive2 2ends of 8 and 16 amino groups, respectively, towhich peptides can be attached. Peptides may besynthesised directly onto the lysine dendron usinga divergent strategy or dendrons can be furtherfunctionalized with electrophiles and thiol nucle-ophiles for convergent ligation of peptides.

The K K-type branching unit is small in size2when compared with the bulk of peptides layeredaround the surface. As an example, a dendrimerconsisting of a branched unit and eight copies ofpeptide chains, each containing 14 amino acidresidues, would have an average molecular weightof 13�14 kDa. Only a small fraction of the mass isdue to the lysine unit; 93% of the mass is due topeptide sequences.

Many variations of K K cores have evolved.2For example, a version containing a �-alanine

Ž .spacer Huang et al., 1994 at the �-amine of Lyshas produced a branching unit that has symmetri-

ŽFig. 5. Polylysine cores consisting of K K and di K K, where Xaa can be either OH, an amino acid, peptide or organic core see2 2.Fig. 4 .


Fig. 6. Different K K, polyamino, linear and cyclic cores used2for dendrimer assembly. Ahx�aminohexanoic acid.

cal branches. In another example, in order tolimit the flexibility of the branched lysyl template,ornithine, the lower homolog of Lys, as well asmore constrained diamino acids have been usedŽ .Roth and Heidemann, 1980; Hahn et al., 1990 .

ŽNon-branched linear polylysine cores Tsikaris et. Žal., 1996 and cyclic peptides Goodman et al.,

.1998 have also been developed to impart differ-Ž .ent forms of dendrimeric architectures Fig. 6 .

As eluded to earlier, the basic branching unitmay be further modified to allow peptide attach-ment. As shown in Fig. 7, attachment of serine tothe K K dendron during solid-phase synthesis2allows the production of a branching unit withN-terminal aldehyde moieties. This serine can be

readily converted to aldehyde at neutral pH inaqueous conditions and has been used by Spetzler

Ž .and Tam 1995 as a convenient masked aldehydeprecursor.

Thiol-functionalized branching units are popu-lar and convenient to prepare because of therelative ease with which reactive thiol moietiesmay be placed on peptide units in preparation for

Žconvergent syntheses Lu et al., 1991; Defoort et.al., 1992b; Zhang and Tam, 1997b . Drijfhout and

Ž .Bloemhoff 1991 introduced a thiol-functional-ized branching unit based on the K K dendron by2

Ž .coupling N- S-acetylmercaptoacetyl -glutamylresidues to the amino groups. Subsequently,

Ž .Baleux and Dubois 1992 prepared a thiolatebranching unit based on a similar design by cou-

Ž .pling Cys to the amino groups Fig. 8 .

7.4. Di�ergent stepwise strategy for peptide dendrimersynthesis

Divergent methods used to prepare peptidedendrimers are operationally similar to the step-wise solid-phase syntheses of linear peptides first

Ž .developed by Merrifield 1963 . Thus, protecting

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


Ž .Fig. 8. Synthesis of thiol-functionalized S-acetyl and Cys Npysdi-K K cores.2

group schemes, resin supports, and cleavagemethods found in peptide synthesis protocols aregenerally applicable to the divergent syntheticschemes of peptide dendrimers. Because this ap-proach produces fully characterized dendrimersin a short time, often within a week, it is popularin biological research in which peptide den-drimers are used as tools and reagents for validat-ing biochemical goals.

Syntheses generally commence with thebranching unit which is usually of the K K type.2In such cases two or three levels of diverginglysine are formed first by coupling a suitabledi-protected lysine, either a di-Boc or di-Fmocderivative, to a small amino acid attached to aresin support to form a branched structure. Thepeptide sequence is then synthesized stepwise onthis branched-lysine scaffolding. After peptidesynthesis the peptide dendrimer is cleaved fromthe resin support, purified and characterized.

ŽGoodman Goodman et al., 1998; Jefferson et.al., 1998 introduced a different divergent synthe-

sis approach. In the synthesis of collagen modelsconsisting three copies of homomeric peptidechains, solid-phase schemes began with monomers

Ž .and then the Kemp tricarboxylic acid KTA tem-Ž .plate Kemp and Petrakis, 1981 was used to link

three monomers to form the peptide dendrimersŽ .Fig. 9 .

Interchain aggregation may occur within thebranched structure of dendrimers because thequantity of peptide monomers obviously increaseswith each additional level of lysine units. To

minimize this problem the divergent synthesis of-ten starts with a low amino acid loading of ap-proximately 0.1 mmol�g on a resin support. Thisis significantly lower than the customary loading,typically 0.3�0.8 mmol�g resin, conventionallyused in solid-phase peptide synthesis. Formationof interchain hydrogen bonds that occlude cou-pling reactions can also be minimized by usingspecific solvent combinations as well as coupling

Ž .at elevated temperatures Tam and Lu, 1995 .Following synthesis and cleavage some peptidedendrimers have exhibited an unusual tendencyto aggregate after cleavage from the resin due tothe entrapment of aromatic scavengers. Dialysisunder basic, denaturing conditions using 8 Murea is useful to remove such unwanted additives.After dialysis either RP-HPLC or high perfor-mance gel-filtration chromatography can be usedto purify peptide dendrimers.

A variation of the divergent synthesis scheme isthe synthesis of two dissimilar peptide chains ondifferent arms of a lysine branching unit. This is aparticularly useful method for the production ofimmunogens containing different peptide epi-topes. Methods to distinguish between �- and�-amines of lysine for such a purpose are based

Fig. 9. Synthesis of collagen models with three copies ofhomomeric peptide chains on the Kemp tricarboxylic acidcore.


on orthogonal protecting group schemes. SuitableŽ .protecting group combinations include: i

Ž . Ž .Boc�Fmoc Merrifield, 1963 ; ii Fmoc�AlocŽ . Ž . ŽKates et al., 1993 ; iii Fmoc�Dde Bycroft et

. Ž . Žal., 1993 ; and iv Npys�Fmoc Hahn et al., 1990;.Ahlborg, 1995; Rajagopalan et al., 1995 . In gen-

eral, the combination of Boc�Fmoc chemistry isconvenient because the protected amino acids arereadily available. This chemistry utilizes both acid-

Žand base-driven deprotection methods Tam and.Lu, 1989 in repetitive steps that elongate the two

dissimilar peptide chains. In the K K core, for2example, when half of the reactive lysine amino

Ž .chains e.g. �-amino groups are chemicallyblocked during synthesis it is possible to constructa peptide dendrimer containing two different

Ž .peptide sequences Fig. 10 . The first peptidechain can be assembled on the N�-termini of thelysine matrix using Boc chemistry while the Fmocgroups of N�-termini are not affected during theBoc synthesis. When the first peptide is completethe second peptide chain can be synthesized onthe N�-termini using Fmoc chemistry. The het-eromeric peptide dendrimer is then cleaved fromthe resin by HF that will also remove both the

Ž . Žbenzyl- Boc chemistry and tertbutyl- Fmoc.chemistry protecting groups. In recent years the

use of the Boc protecting group has become lesspopular because special apparatus must be usedfor deprotection by HF. Alternative methods havebeen developed and these include Boc substitu-

Ž . otion with i Aloc that is removable by Pd in theŽ .presence of a nucleophile such as morpholine, ii

Dde which is removable by 2% hydrazine in DMF,Ž . Ž .and iii Npys 3-nitropyridino-2-sulfhydryl which

is removable under neutral conditions by a triva-lency phosphine or a thiol.

An example of the utilization of orthogonalŽ .protection was reported by Hahn et al. 1990

with the divergent synthesis of a chymotrypsin-likeartificial enzyme using three types of protectinggroups on a Lys Orn branching unit to form a2peptide dendrimer consisting of four dissimilarchains. The first two chains were assembled byalternating Boc and Npys chemistries on the dif-

Ž .ferentially protected dipeptide Npys�Lys Boc �Ž .Orn Fmoc . By terminating each chain with an

Fig. 10. Schematic illustration of the synthesis of heteromericpeptide dendrimers on a di-K K core.2

acetylated amino acid and repeating the samechemistry, the next two chains were assembled on

Ž .Boc�Lys Npys coupled to the Orn side chainafter the removal of Fmoc group. The strategicsimplicity and flexibility of divergent orthogonalsynthesis also permits the incorporation of un-usual moieties such as lipid chains and lipidic P C3

Žinto the dendrimeric design Defoort et al.,.1992b .


7.5. Con�ergent synthesis strategies for peptidedendrimers

Convergent strategies differ from the divergentmethods in that the branching unit and peptidesare synthesized separately, purified individuallyand then coupled to form the dendrimer. Twodifferent convergent strategies are typically used;one strategy exploits a protection scheme whilethe other does not. The choice of a convergentstrategy is often arbitrary. Consideration should,however, be given to the nature of the peptidemonomer, the complexity of the peptide den-drimer, and the expertise of a given laboratory.An additional factor is the orientation of thepeptide that is to be attached and this is governedby the functional consequences. For example,peptides derived at or near C-termini can bearranged as in the native protein by linkagethrough the N-terminus to the branching unit. Avariety of functional groups have been used in thedevelopment of numerous ligation protocols inorder to provide site-specific and chemically un-ambiguous dendrimers. A detailed review of dif-ferent ligation strategies and methodologies was

Ž .published recently by Tam 2000 .

7.6. Con�ergent synthesis with a protecting groupscheme

The convergent synthesis using protectingŽgroups Blake, 1981; Yajima and Fujii, 1981; Ya-

mashiro and Li, 1988; Hojo and Aimoto, 1992;.Sakakibara, 1995; Albericio et al., 1997 generally

follows conventional segment coupling schemeswith enthalphic activation by a coupling reagent.The major difference between forming a brancheddendrimer and forming a linear peptide by seg-ment condensation is that two or more protectedpeptide segments are simultaneously coupled to acore to afford the former. Purification by gelpermeation chromatography and RP-HPLC isfacilitated by the substantial differences in themolecular weights of the desired product com-pared to incompletely coupled byproducts. Sasaki

Ž .and Kaiser 1989 reported formation of such apeptide dendrimer using convergent synthesiswhere an artificial hemeprotein containing four

Fig. 11. Convergent synthesis of an artificial hemeprotein.

identical peptides tethered to a coproporphyrincore was assembled. The 15aa fully protectedpeptide segment was synthesized through a two-

Ž .segment condensation 8aa�7aa and then cou-pled to the hydroxysuccinimide ester of the

Ž .branching unit through its N-terminal Fig. 11 .A common approach is to use a less elaborate

Ž .chemistry as illustrated by McLean et al. 1992who described a rapid convergent scheme for theformation of MAPs. To obtain a 17aa protectedpeptide segment they used an Fmoc-tertbutyl pro-tecting group scheme with a highly acid-labileresin. The cleaved protected peptide containing afree carboxylic acid was then coupled to a te-trameric K K branching unit. However, the poor2solubility of this protected peptide resulted invarying degrees of coupling in six MAPs, rangingfrom complete to no observed coupling. Althoughthis difficulty does not impede the biological goalsit certainly indicates the unpredictable nature ofusing protected peptide segments in convergentschemes.

8. Chemoselective ligation strategies: unprotectedpeptide synthesis

A recent advance in convergent synthesis hasbeen the use of unprotected peptide segments


instead of bulky protected peptides. One suchstrategy that links unprotected peptide monomersto a branching unit to form a dendrimer vianon-amide bonds is regiospecific chemoselectiveligation. This type of ligation exploits a pair ofmutually reactive functional groups, usually a nu-cleophile and an electrophile, on the peptide andbranching unit that only react specifically witheach other. Another strategy that links unpro-tected peptides via amide bond is orthogonal liga-

Ž .tion Tam et al., 2000 . Orthogonal ligation isN-terminal specific, thus, a peptide containing anN-terminal Lys can be ligated in the presence ofother N-terminal amino acid peptides. An advan-tage of these types of ligation is their specificitybecause peptide segments and branching unitsare used in their unprotected state they are morelikely to be water soluble and amenable to puri-fication by RP-HPLC. A plethora of chemoselec-tive and orthogonal ligation methods and many

Ž .variations have been developed Tam, 2000 . Fur-thermore, the nucleophile and electrophile can beplaced on either the peptide segment or thebranching unit creating numerous potential com-binations.

In addition, there are many readily availablebifunctional groups that are used to modify K K2dendrons in preparation for ligation including:succinimide esters; maleimides; sulfenyl thiols;

Žand 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 thatmay be formed by regiospecific chemoselectiveligation include hydrazone, oxime, thioether andthioester bonds. Each chemoselective, or orthogo-nal, strategy consists of an initial capture step toform a covalent intermediate between two pep-tide segments. Then, an amide bond is formed viaan intramolecular acyl transfer resulting in thecoupling of the two different segments. Thestrategies are categorized by two majorchemistries: thioester and imine.

8.1. Thioester ligation

In the thioester ligation strategy a thioesterand an N-terminal cysteine are required. The

initial capture product is a thioester and an S,N-acyl shift results in formation of a cysteine residue

Žat the ligation site Muir et al., 1994; Tam et al.,.1995 . Other variations of cysteine ligation such

as perthioester ligation have also been developedto make thioester a popular orthogonal ligation

Ž .strategy Tam et al., 2001 .

8.2. Imine and imide ligation

The imine chemoselective ligation strategy re-quires the presence of an acyl-aldehyde and anN-terminal weak amine. Commonly used imineligation strategies include oximation, hydrazoneformation and imide ligation. Oximation has been

Žused in protein conjugation reactions Vilaseca et.al., 1993; Rose, 1994 and in the synthesis of

Žpeptide dendrimers Shao and Tam, 1995; Pallin.and Tam, 1996 . Hydrazone formation has also

been employed in ligation reactions through thereaction of aldehyde and C-terminal hydrazideŽ . ŽGaertner et al., 1992 . For orthogonal imide N-

.terminal ligation a C-terminal glycoaldehyde es-ter is required for reaction with either an N-

Ž . Ž .terminal NT cysteine pseudoproline or NT-Ž .serine or NT-threonine oxaproline . The mechan-

ism of imide ligation is similar to orthogonalthioester ligation with an imine capture, a ring

Ž .closure to a thiazolidine NT-Cys or oxazolidineŽ .NT-Ser�Thr and then an S,N- or O,N-acyl shift

Ž .to form an imide Liu and Tam, 1994 . Pseu-doproline ligation has been successfully employedin the production of synthetic HIV-1 protease

Ž .analogs Liu et al., 1996 . Each of these imine orimide ligations relies on the presence of aldehy-des and since aldehydes are not found in aminoacids several methods have been used to intro-duce aldehydes to amino acids. Two convenient

Ž .methods are: 1 conversion of N-terminal Serand Thr residues to aldehyde through sodium

Ž .metaperiodate oxidation; and 2 conversion ofŽ .masked acetals attached to amino groups to

aldehyde by TFA or concentrated HCl. The con-version of N-terminal Ser and Thr residues toaldehydes has been used extensively in proteinconjugation and designing new proteins as well as

Žfor immunological applications Zhang and Tam,.1996; Zhang et al., 1998 .


Even though the two reactive species may beplaced on either the branching unit or monomericpeptide, for synthetic convenience weak bases areoften added to the N-terminal of a peptide and insuch cases the peptide sequence is oriented withthe N-terminal residues proximal to the branch-ing unit. The reverse orientation may be achieved

Žwith a C-terminal hydrazide derivative Rao andTam, 1994; Shao and Tam, 1995; Pallin and Tam,

.1996; Zhang and Tam, 1996; Liu and Tam, 1997 .Combinations of two, and potentially more, reac-tion conditions can be exploited for ligating dis-similar peptide chains to a branching unit to form

Ž .heteromeric peptide dendrimers Liu et al., 1999in a process termed tandem ligation.

8.3. Tandem cyclization strategies for peptidedendrimers

Constrained peptide dendrimers have been de-veloped in an effort to generate molecules thatmore closely mimic native protein conformationsand to improve biological activity. One such de-sign contains multiple closed-chain brancheswhich are cyclic peptides constrained by end-to-

Žend, end-to-side, or side-to-side linkages Liu andTam, 1997; Spetzler and Tam, 1996; Zhang and

.Tam, 1997a,b . A general approach to achievesuch constrained peptide dendrimers is to attachmultiple copies of a cyclic peptide to a branchingunit. Conventionally, this scheme of convergentsynthesis requires two types of manipulations: acyclization step on a linear precursor and a cou-pling step for its attachment to a branching unit.This two-step process usually requires a multi-tiered protecting group scheme. To minimize theprotection and deprotection procedures of con-

ventional convergent methods and also difficultiesdue to the insolubility of intermediates andproducts, simplified tandem chemoselective meth-ods have been developed based on unprotectedpeptide precursors in aqueous conditions.

8.4. Di�ergent synthesis and chemoselecti�ecyclization

Ž .Spetzler and Tam 1996 developed a methodfor preparing peptide dendrimers with end-to-sidechain cyclic peptide monomers that combineson-resin synthesis and off-resin cyclization. First,a divergent strategy of stepwise solid-phase syn-thesis was used to assemble the peptides linked toa K K branching unit. This branched precursor2was then cleaved from the resin and its protectinggroups removed to permit an off-resin cyclizationthat formed the constrained dendrimer through athiazolidine cyclization in an aqueous solutionŽ .Fig. 12 . The use of this method may, however,be restricted by the competing intramolecular cy-clizations among the peptide branches. Ring-chaintautomerization may be used to overcome thislimitation where mutually reactive nucleophile�electrophile pairs simultaneously self-assemble toform peptide dendrimers with multiple copies ofcyclic peptides.

8.5. Chemoselecti�e cyclization and con�ergentligation

Using a slightly different approach, Pallin andŽ .Tam 1995 synthesized a constrained peptide

dendrimer by first cyclizing the monomeric pep-tide and then attaching the monomers to abranching unit as shown in Fig. 13. The linear

Fig. 12. Use of divergent synthesis and thiazolidine intramolecular cyclization to generate a peptide dendrimer.


sequence KAKGIGPGRAFGK-�Ala derivedfrom the neutralizing determinant of the V3 loopof gp120 was selected and prepared by Fmoc

Ž .chemistry resulting in the precursor K CysŽ . Ž .AK O�NH GIGPGRAFGK Ser -�Ala. The Ser2

moiety attached to the first Lys served as a maskedaldehyde and was oxidized to an aldehyde. Acyclic peptide was formed by utilizing the intra-molecular oxime formation from the reactionbetween the Lys-side chain tethered O-al-kylhydroxylamine and the aldehyde. The thiolresidue of Cys on the side chain of the third Lysmoiety was then liberated and used to ligate to

the aldehyde moieties of a glyoxyyl di-K K2branching unit form a peptide dendrimer contain-ing four copies of side-to-side chain cyclic pep-tides. This method for formation of side-to-sidechain cyclic peptides differs from the previous

Ž .example Fig. 12 in the form of cyclic peptideconstraint and method of synthesis.

8.6. Orthogonal cyclization and con�ergent thiolligation

Tandem ligation is a strategy consisting of twoor more chemoselective and orthogonal ligations

Fig. 13. Synthesis of a cyclic peptide dendrimer through side-chain to side-chain oxime cyclization and thiazolidine ligation.


that do not require a deprotection step in thesynthetic scheme. Thus, tandem ligation repre-sents a convergent scheme with the fewest syn-thetic steps and the most rapid approach forsynthesis of high molecular weight peptides. Threetandem ligation methods for preparing con-strained peptide dendrimers have been developedbased on orthogonal cyclization and convergent

Žthiol ligation Liu and Tam, 1997; Zhang and.Tam, 1997b . These are: cysteine-thioester cy-

clization and thioalkylation; perthioester cycliza-tion and thiol addition; and imine cyclization andthioalkylation. A complete discussion of these

Ž .methods is found in Tam 2000 . Even thoughorthogonal cyclization and convergent ligationmethods also use a linear unprotected peptideprecursor and a functionalized branching unit asbuilding blocks for peptide dendrimers there aretwo major differences between these and the pre-viously mentioned chemoselective ligation strate-gies. Firstly, orthogonal cyclization methods pro-duce lactams and secondly, only one weak basenucleophile, fulfills the function of two nucle-ophiles in chemoselective ligation.

The first reaction common to the three reac-tion schemes is the orthogonal lactamization ofthe N-terminal nucleophile and the C-terminalelectrophile of the linear precursor to generate

Žan 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 thecyclic peptide monomer to a functionalizedbranching unit through its thiol side chain result-

Žing in a peptide dendrimer Liu and Tam, 1997;.Zhang and Tam, 1997b . These two reactions can

be performed in tandem without purification whenhomogeneous intermediates are used. An advan-tage of these methods is their high efficiencybecause no protection or deprotection steps areinvolved. Racemization is minimized becausethere is no enthalpic activation. Furthermore, thecyclization can occur at high peptide concentra-tions due to the high effective molarity of anintra- vs. inter-molecular reaction.

The variation in the strategies now availablefor the preparation of peptide dendrimers allowsinvestigators to select a method most suitable fortheir individual needs. The high specificity and

homogeneity conferred by convergent chemo-selective methods may be beneficial in some situ-ations whereas the rapid production of divergentpeptide dendrimers could be advantageous in oth-ers.

The use of peptide dendrimers as biochemicalreagents and immunogens is entering a new phase.The reliability and versatility of the dendrimersystem are now well-documented and investiga-tors are looking towards the utilization of peptidedendrimers as ‘gold-standard’ reagents and as anew generation of totally synthetic vaccines.

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