amino acids, peptides and proteins in organic chemistry (volume 4: protection reactions, medicinal...

15Amino Acid-Based DendrimersZhengshuang Shi, Chunhui Zhou, Zhigang Liu, Filbert Totsingan,and Neville R. Kallenbach

15.1Introduction

Dendrimers are highly branched molecules consisting of a central core that isgenerationally grafted with layers of building blocks (Figure 15.1) [1, 2]. Thedendrimer generation is defined as the number of branching points from the coreto the surface of the dendrimer. Here, amino acid-based dendrimers, or peptidedendrimers, are loosely defined as molecules formed from amino acids emanatingfrom a branched template or scaffold, in which branched templates can be branchedamino acids such as lysine [3–5], glutamic and aspartic acid [6, 7], cis-4-amino-L-proline [8], or other building blocks, including cyclic peptides, poly(amido amines)(PAMAM) [9], polypropylene imines (PPI) [1], poly(aryl ethers) [10, 11], and a varietyof other organic branched motifs such as carbohydrates [12, 13]. In this chapter wefocus mainly on peptide dendrimers that are branched with amino acids (natural orunnatural) and exclusively composed of amide bonds. As a result, dendrimers thatare obtained by conjugating peptides/amino acids onto other scaffolds are discussedonly briefly.

15.2Peptide Dendrimer Synthesis: Divergent and Convergent Approaches

Dendrimers afford a precise and concentrated display of functionality on a scaffold.There are two general conceptually different synthetic routes for the construction ofdendrimers: divergent and convergent approaches (Figure 15.2) [15]. In the divergentmethod, the dendrimer is built up from its core outward – the most obvious scheme(Figure 15.2A); in the convergent method the dendrimer is assembled throughcoupling of preformed branching segments onto a central core unit (Figure 15.2B).The convergent synthetic strategy was devised by Frechet et al. [10, 11] to overcomea serious problemwith the divergent approach. Due to the progressive accumulation

Amino Acids, Peptides and Proteins in Organic Chemistry.Vol.4 – Protection Reactions, Medicinal Chemistry, Combinatorial Synthesis. Edited by Andrew B. HughesCopyright � 2011 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32103-2

j491

of defects during the process of performing the exponentially growing number ofreactions as the dendrimer expands from one generation to the next, it becomespractically impossible to synthesize defect-free dendrimers beyond the fifth or sixthgenerations [16]. For example, given a yield of 99.5% per reaction site, only0.995exp504¼ 8.0% of defect-free products would be obtained in the case ofsynthesis of a sixth generation PPI dendrimer constructed using the divergent

Figure 15.1 Dendrimer structure. (Adapted with permission from Fig. 1 of [14], copyright 2009,American Chemical Society.)

Figure 15.2 (A) Divergent approach. (B) Convergent approach. (Reproduced with permissionfrom Fig. 1 of [17], copyright 2001, P. Pomovski and Polish Biochemical Society.)

492j 15 Amino Acid-Based Dendrimers

strategy [16]. In practice, a two-stage strategy that combines the divergent andconvergent approaches has been developed [8].

15.2.1Synthesis of the First Peptide Dendrimers: Polylysine Dendrimers

The first peptide dendrimers appear to have been synthesized by Denkewalter et al.and Aharoni et al. at the Allied Corporation in the early 1980s [3–5]. They reportedthe synthesis of polylysine dendrimers from Na,Ne-di-(tert-butoxycarbonyl)-pro-tected lysine in a stepwise fashion (Scheme 15.1). This original synthesis wasaccomplished by a repetitive sequence of Na,Ne-di-(tert-butoxycarbonyl)-L-lysine-p-nitrophenyl ester couplings and trifluoroacetic acid-mediated deprotection reac-tions. The basic idea of this design is tomake use of the bifunctionality of the aminoacid lysine to form a core as well as to serve as branching units, which is possible asboth Na- and Ne-amino groups are available reactive ends. Subsequent coupling oflysine residues propagates additional generations of the dendrimer. Differentpeptide sequences can be assembled on this scaffold to create a large number ofdesired peptide dendrimer products.

Scheme 15.1 Synthesis of polylysine dendrimers. (Reproduced with permission from Scheme 1of [18], copyright 2009, Elsevier B.V.)

15.2 Peptide Dendrimer Synthesis: Divergent and Convergent Approaches j493

15.2.2Glutamic/Aspartic Acid, Proline, and Arginine Dendrimers

Mitchell et al. reported the synthesis of L-glutamic acid-based dendrimers in 1994(Scheme 15.2) [6]. L-Glutamic acid was protected and activated as the N-(benzyloxy-carbonyl)-L-glutamic acid bis(N-hydroxysuccinimide) ester, whichwas reacted with L-glutamic acid diethyl ester in dimethoxyethane. Selective deprotection of the benzy-loxycarbonyl protective group in the product with iodotrimethylsilane resulted in thefirst-generation dendrimers. The first-generation dendrimers were subsequentlyconverted to the second-generation dendrimers, duringwhich the benzyloxycarbonylprotective group was removed by hydrogenation as iodotrimethylsilane was noteffective. The final dendrimers were obtained by applying repetitive reactions to thesecond-generation dendrimers. The same group also prepared L-aspartic acid-baseddendrimers in which L-aspartic acid served as the branching unit and 1,3,5-benzenetricarbonyl core as the core unit [7].

A number of other peptide dendrimers composed of glutamic/asparticacid [19–23], proline [8, 24–26], or arginine [27–29] have been reported. Ranganathanet al. synthesized several series of polyglutamic/aspartic acid dendrimers includingthose with adamantane, an oxalyl group, or a benzenetricarbonyl unit as dendrimer

Scheme 15.2 Synthesis of glutamic acid dendrimers. (Reproduced with permission from Scheme3 of [18], copyright 2009, Elsevier B.V.)


core (Scheme 15.3) [19–21]. Kraatz et al. synthesized two series of glutamic aciddendrimers using a ferrocene core (Figure 15.3) [22, 23]. Arginine-rich peptidedendrimers in which arginines are grafted onto polylysine dendrimeric cores havealso been designed and synthesized (Scheme 15.4) [27–29]. Polyproline dendrimersbased on cis-4-amino-L-proline as branching units and either spermidine or a cyclicLys–Lys as the core have been constructed by Crespo et al. [8]. Interested readersmay

Aa

O

Aa

O CO2Me

CO2Me

CO2Me

CO2Me Aa

O

Aa

O Aa

Aa

Aa

AaCO2Me

CO2MeCO2Me

CO2MeCO2MeCO2MeCO2Me

CO2Me

AaO

Aa

O

Aa

Aa

Aa

Aa

AaAa

Aa

AaAa

Aa

AaAa

CO2MeCO2Me

CO2MeCO2MeCO2MeCO2MeCO2MeCO2Me

CO2Me

CO2MeCO2Me

CO2Me

CO2MeCO2Me

CO2MeCO2Me

N

O

N

O

N

OO

O

O

HN

OO

OMe

OMe OMe

MeO

N

ONHO

NH

O

HN

O

O

O

O

N

MeO

O

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OMe

OMe

O

N

OO

O

OMe

OMe

NHO

OO

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OHN

HN

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NH

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MeO

MeO

O

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HH

HH

HN

O

OO

N

O

NH O

O

O OOMe

OMe

OMe

OMe

OMe

OMe

O

H

H

H

N

NO

O NH

O

O

O

N

O

O

O

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O

O

O

OMeOMe

OMe

OMe

OMe

OMe

N

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H

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N

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NHO

NH

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O

HN

O

O

O

O

OMe

MeOMeO

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OMe

OMe

OMe

OMeOMe

OMe

OMe

OMe

OMeMeO

OMeO

H

H

H

H

H

Aa = Asp; Glu

Scheme15.3 Glutamic/aspartic acid peptidedendrimers prepared byRanganathan et al. (Adaptedwith permission from [19], copyright 1997, Elsevier B.V., [20] copyright 1998, Ovid, [21], copyright2000, Jhon Wiley and sons, Inc.)


Figure 15.3 (a) Structures of disubstitutedFc–peptide dendrimers G1, G2 and G3.(b) Molecular models of disubstitutedFc–glutamic acid dendrimers G1–G6 (Fc:yellow; carbon: green; hydrogen: white;

oxygen: red; nitrogen: blue). (c) Schematicrepresentation of disubstituted Fc-glutamic aciddendrimers G1–G6. (Adapted withpermission from [22, 23], copyright 2005/06,American Chemical Society.)


refer to the corresponding literature for the synthetic details of these interestingproducts.

15.2.3Synthesis of MAPs

In the late 1980s, Tam extended applications of polylysine peptide dendrimers in anattempt to develop potentialmultiple antigenic peptides (MAPs) [30, 31]. As indicatedby the name, the goal was to provide a rational system for polyvalent linkage ofdifferent types of peptide antigens and attachment of immunomodulatingmoleculesfor use as vaccines. There are two general approaches to the synthesis of MAPdendrimers: the direct divergent approach and an alternative indirect approach(Scheme 15.5) [32]. The indirect synthesis route combines the divergent approachwith various chemoselective ligation methods to couple different presynthesizedpeptides onto the polylysine dendritic matrix. The most commonly used ligationmethods are based on thiol chemistry and carboxyl chemistry [32]. Some chemo-selective ligation reactions tailored for polylysine or other modified polylysinescaffolds are summarized in Scheme 15.6, taken from Tam [32].

Scheme 15.4 Structure of the arginine dendrimer. (Reproduced with permission from Fig. 8 of [8],copyright 2005, American Chemical Society.)


The principle of multivalency has been widely discussed as a general strategy toenhance the avidity of weak binding ligands for a given target [34]. Increasing thenumber of copies of a ligand on a covalent scaffold can elicit dramatic enhancementsin the affinity of a single weak-binding ligand. The effect is similar to that involved inchelation, in which divalent metal ions are bound extremely tightly relative to anymonovalent ion.

The effect depends on the topology and affinity of each ligand site, as outlined inFigure 15.4. In the simplest case, corresponding to a dendrimer, sites are distributedroughly uniformly over the approximate surface of a sphere, rather than a circle as

Scheme 15.5 Schematic diagram for the preparation ofMAPs: (a) direct approach and (b) indirectapproach. (Reproduced with permission from Fig. 1 of [33], copyright 1999, John Wiley and Sons,Inc.)


1. Thiol chemistry

a. thioalkylation R1-SH + X-CH2-CO-R2

b. thiol addition R1-SH +

c. thiol-disulfide exchange R1-SH + Ar-S-S-R2

2. Carboxyl chemistry

a. hydrazone

b. oxime

c. thiazolidine

d. oxazolidine

R1-S-CH2-CO-R2

N-R2

O

O

N-R2

O

OR1S

R1-S-S-R2

R1

HC

O+ NH2-NH-R2 R1-CH=N-NH-R2

R1

HC

O+ NH2-O-R2 R1-CH=N-O-R2

R1

HC

O+

H2N R2

HS

R1

SNH

R2

R1

HC

O H2N R2

HO+ R1

ONH

R2

Scheme 15.6 Chemoselectivemethods for ligating unprotected peptides to formMAPs. (Adaptedwith permission from Table 3 of [32], copyright 1996, Elsevier B.V.)

Figure 15.4 Thermodynamicmodel of themultivalency effect. (Adaptedwithpermission from [34],copyright 2003, American Chemical Society.)


shown. However, the amplification factor can easily lead to nanomolar dissociationconstants for the assembly as a whole when single affinities are in the millimolar tomicromolar range.

The MAP strategy affords construction of multivalent assemblies with an excep-tional degree of flexibility as illustrated in Figure 15.5 [32, 33, 35, 36]. MAPscontaining monoepitopes, chimeric epitopes, and diepitopes can be generated atwill. Lipidated MAPs and MAPs containing cyclic peptide epitopes, which mimicprotein surface loops better (Scheme 15.7) [37], can be synthesized as well. Throughchemoselective ligations, the design and synthesis of MAPs that display heteroge-neous epitopes are also possible.

15.2.4Synthesis of Peptide Dendrimers Grafted on PAMAM and other Peptide Dendrimers

While its core matrix is not directly synthesized from amino acids, Tomalia�sPAMAM dendrimers represent an important class of macromolecular architecture

Figure 15.5 Schematic of different MAPdesigns comprising different peptidesrepresenting T and B cell epitopes, respectively,showing different ways to fuse these elementsinto MAP structures. Also shown is thepossibility of including fatty acids into such

structures either as single, straight chainalkyls or as the specific tripalmitatecysteinylstructure. (Reproduced with permissionfrom Fig. 21 of [36], copyright 2004, RoyalSociety of Chemistry.)

Solid-phase Synthesis

ClCH2CO

ClCH2CO

ClCH2CO

Lys

Lys-Ala-OHpH 8.5

SH

SH

cyclic peptide

Lys

ClCH2CO

SCH2CO

SCH2CO

SCH2CO

SCH2CO

Lys-Ala-OHLys

Lys

Scheme 15.7 Synthesis of cyclic peptide dendrimers through thioether linkage. (Reproduced withpermission from Fig. 8 of [37], copyright 1997, American Chemical Society.)


with a high degree of molecular uniformity, specific size and shape characteristics,and ahighly functionalized terminal surface constructed fromstable amidebonds [2].PAMAM dendrimers can be synthesized by the divergent method, which involvesa two-step iterative reaction sequence that yields successive generations of dendriteb-alanine units around a central core [38] (Scheme 15.8). While the PAMAMcore–shell increases with each generation, the surface groups amplify exponentiallyas in the previous cases discussed.

Efforts have beenmade to attach surface peptide groups to the PAMAMdendrimercore, forming peptide dendrimer conjugates [39]. An example of such a conjugationis the coupling of cysteine-containing peptides with a maleimide-derivatizedPAMAMdendrimer using solution-phase reaction [40, 41]. Alternatively, solid-phasesynthesis of peptide–dendrimer conjugates may be achieved starting from the initialcore [42, 43].

Other scaffolds have been designed with different core compositions. One suchdesign involves assembling peptide dendrimers on a poly(ethylene glycol) resin fromsimple amino acid-based monomers using a cyanoethylation/hydrogenationstrategy [44]. Peptide dendrimers constructed by coupling peptide segments ontopolyphenylene scaffolds have been synthesized, affording shape-persistent, MAPcandidates [45]. Examples of the use of carbohydrates as potential templates formultivalent peptide assembly have also been reported [12, 13]. A novel amino acid-based dendrimer was prepared from 3,5-bis(2-tert-butyloxycarbonyl aminoethoxy)benzoic acid methyl ester [46, 47]. Sasaki and Kaiser�s design of an artificialhemeprotein via attachment of four identical peptides onto a coproporphyrin core

H2NNH2

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O

H2NNH2

a)

b)N

N

HN

HN

NH

NH

O

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O

O

NH2

NH2

NH2

NH2

Core Generation 0

Generation 0

OMe

O

H2NNH2

a)

b)

Generation 1, 2, 3, ...

Scheme 15.8 Synthesis of PAMAM dendrimers by exhaustive Michael addition of amino groupswith methyl acrylate, followed by amidation of the resulting esters with ethylenediamine [38].(Copyright 2001, Elsevier B.V.)


can be regarded as another example of peptide dendrimers [48]. Additional examplesof the application of amino-, hydroxylic-, and carboxylic-based organic templates asdendrimer scaffolds using either the divergent or convergent approach are sum-marized in two previous reviews [49, 50].

15.3Applications of Peptide Dendrimers

15.3.1Initial Efforts on MAPs

In efforts to apply polylysine dendrimers as potential MAPs, increasing the func-tionality of small peptideswas hoped to enhance their immunogenicity and thereforetheir applicability as vaccines [51]. As compared to conventional peptide–proteinconjugates in which antigens account for only a small percentage of the total mass,MAPs potentially affordmore concentrated epitopes attached to an immunologicallysilent lysine core. Despite many articles reporting initially encouraging results usingMAPs, no vaccine based on these appears to be in use today as far as we can discover.

15.3.2Peptide Dendrimers as Antimicrobial Agents

Naturally occurring antimicrobial peptides are found in all orders of life [52]. Whileover 1000 antimicrobial peptides have been identified, the problem is that they tend tobe highly cytotoxic, hemolyzing red blood cells. Efforts to dissociate the hemolyticactivity from the antimicrobial activity of these peptides are an important part of thesearch for new antibiotics capable of overcoming multidrug-resistant bacterialstrains that are prevalent in hospitals and elsewhere.

Tam et al. have designed and synthesized several sets of dendrimeric peptides withtwo to eight copies of tetrapeptides or octapeptides derived from a putative microbialsurface recognition motif that occurs naturally in protegrins and tachyplesins [53].The resulting dendrimeric peptides are broadly active against four Gram-negativebacteria, three Gram-positive bacteria and three fungi with minimal inhibitionconcentration in the low- or submicromolar range. Hemolytic assays showed thatthey are not toxic to human erythrocytes with an observable hemolytic index (HI) inthe range of above 2000. These results suggest that dendrimer peptides are prom-ising antibacterial drug candidates, maintaining the antimicrobial potency whilereducing cytotoxicity.

Recently, Kallenbach et al. have observed that a series of very short peptides withsimple repeat sequences (RW)n (n¼ 3–5) are potent antimicrobial agents with IC50

values in themicromolar range [54]. Based on this observation, a dendrimeric peptide(RW)4D (Figure 15.6) was designed and synthesized [55]. Compared to the antimi-crobial dendrimeric peptides designed by Tam [53], (RW)4D is simpler and moreeconomical to produce, since it contains four copies of a dipeptide RW only. The


activity of (RW)4D againstEscherichia coli (D31) orStaphylococcus aureus is in the samerange as that of the linear chains from (RW)n (n¼ 3–5). Significantly, however, ascompared to (RW)n (n¼ 3–5), (RW)4D shows much lower hemolytic activity whenevaluated by the HI. Surprisingly, (RW)4D is more effective against Gram-negativethan Gram-positive bacteria (Figure 15.6), reversing the trend seen in naturalantimicrobial peptides. Furthermore, (RW)4D is active against E. coli biofilms [56].

Biofilms are a major threat to human health, mediating infection via in-dwellingcatheters or implanted devices. Recently, Reymond et al. [57, 58] identified twofucosyl-peptide dendrimers, FD2 (C-Fuc-LysProLeu)4(LysPheLysIle)2LysHisIleNH2

and PA8 (O-Fuc-LysAlaAsp)4(LysSerGlyAla)2LysHisIleNH2, through affinity screen-ing toward fucose-specific lectin, LecB. As a human pathogen, the bacteriumPseudomonas aeruginosa can synthesize a fucose-specific lectin, LecB, that is sus-pected of playing a role on the formation of biofilms. They examined the effect ofdendrimers on inhibiting P. aeruginosa biofilm formation and found that FD2 cancompletely inhibit P. aeruginosa biofilm formation at 50 mM (IC50� 10mM) and it isalso effective at dispersing preformed biofilms. Interestingly, FD2 is not toxic to thebacteriumother than biofilm inhibition and dispersion. Another recent study byZhuet al. [59] has tested the inhibitory effects of a series of branchedHis–Lys dendrimerson a number of Candida species and some other fungi. They found that the four-branched dendrimer H2K4b is the most effective among all tested on fungal growthinhibition. It is important to note that these dendrimers are both active as antifungalagents andnucleic acid delivery agents. The results are consistentwith the hypothesisthat both activities result from some common properties of the histidine-richdendrimer peptide – their endosomal-disrupting properties. A number of otherdendrimer peptides have been reported to be effective against a variety of bacterialstrains including multidrug-resistant strains [60–64]. It is generally observed thatbacterial resistance to peptides or the dendrimers develops much less than to

HN

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(RW)3 (RW)4 (RW)5 (RW)4D

13.1 10.4 12.3

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1

10

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1000

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Figure 15.6 HI values. The HI, defined as theratio of HD50 to IC50, indicates the selectivity.Note the HI is in log scale and the values arelabeled at the top of each bar. E. coli (D31),having a second outer membrane and

containing negatively chargedlipopolysaccharide, are Gram-negative; S.aureus have a thicker outer cell wall made ofpeptidoglycan and are Gram-positive.

15.3 Applications of Peptide Dendrimers j503

standard antibiotics such as vancomycin. This is especially true for bacterial cells inbiofilms, which enhance antibiotic resistance very significantly.

15.3.3Peptide Dendrimers as Protein/Enzyme Mimics

The intrinsically multivalent nature of dendrimeric architecture is reflected in thepresence of multiple copies of a functional group attached to a compact surface.Given multiple copies of a catalytic group positioned at such a surface, peptidedendrimers might serve as potentially effective enzyme mimics [65, 66]. Reymondet al. have recently identified a series of peptide dendrimers built from His–Serrepeats using diaminopropionic acid (Dap) as branching units (Figure 15.7) [67].These dendrimers display enzyme-like Michaelis–Menten kinetics upon catalyzingthe hydrolysis of pyrene trissulfonate esters. Furthermore, the catalytic activity ofthese molecules shows a strong positive dendritic effect, with the catalytic efficiencyof these molecules increasing with the generation number (Figure 15.7).Upon increasing the generation from G1 (Ac-His-Ser)2Dap-His-Ser-NH2 to G4(Ac-His-Ser)16(Dap-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2, kcat/KM increases quadratically with contributions from both kcat and KM. Mutational

Figure 15.7 Catalysis of ester hydrolysis of peptide dendrimers G1–G4 and the dendritic effect onthe hydrolysis reaction. (Reproduced with permission from Scheme 1 and Fig. 2 of [67], copyright2004, American Chemical Society.)


studies on the third-generation dendrimer (Ac-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2 revealed that the catalytic proficiency is related to the locationof histidines within the dendrimers as well as to the identities of other amino acids.The most effective catalytic dendrimer found [68] is the threonine mutant (Ac-His-Thr)8(Dap-His-Thr)4(Dap-His-Thr)2Dap-His-Thr-NH2 with a kcat/kuncat¼ 90 000,corresponding to a 5-fold improvement over the original third-generation dendrimer(Figure 15.8).

Investigating the potential of peptide dendrimers to serve as protein mimics [69],the same group has recently identified a variety of vitaminB12 dendrimer ligands thatcould potentially serve as a vitamin B12 transporter [70]. Reymond et al. prepareda split-and-mix combinatorial peptide dendrimer library and screened their affinityfor cobalamin. The dendritic ligands found through screening have metal-bindingresidues at the core and a polyanionic shell of glutamates in the outer layers.

15.3.4Peptide Dendrimers as Ion Sensors and MRI Contrast Agents

Functionalized peptide dendrimers can target specific analytes and therefore findapplications as sensors [71]. Vogtle et al. modified the surface of a polylysinedendrimer with 24 chromophoric dansyl units and employed it as ion sensors(Figure 15.9) [72, 73]. Dansyl units show an intense fluorescence band in the visibleregion, and 21 amide groups contained inside the interior of each dendrimer caninteractwithCo2þ , Ni2þ , Zn2þ , and lanthanide ions, includingNd3þ , Eu3þ , Gd3þ ,Tb3þ , Er3þ , and Yb3þ . Upon titration of each ion into a dendrimer solution, thefluorescence of the dansyl units is quenched to various degrees (Figure 15.9).

Figure 15.8 Threonine mutant dendrimer and its improved catalytic activity on ester hydrolysisreactions. (Reproduced with permission from [68], copyright 2006, American Chemical Society.)


As lanthanide ions are paramagnetic, their presence can enhance the relaxationrate of water protons and therefore improve the signal-to-noise ratio of the imageobtained [74]. Lanthanide ions in general, in particular Gd3þ , are excellent magneticresonance imaging(MRI) contrast agents. Gadomer 17 was designed as a dendriticMRI contrast agent (Figure 15.10) [75]. It has a trimesoyl triamide core to which 18lysine residues are attached. On the surface of the dendrimer, 24 Gd3þ -DOTA(tetraazacyclododecanetetraacetic acid) can bind.

In another example, a symmetric polylysine-based dendrimer was prepared bynative chemical ligation (Figure 15.11) [76]. This dendrimer has two polylysinedendritic wedges. On one wedge, diethylenetriaminepentaacetic acid (DTPA), wasattached to the polylysine scaffold and could be used as an efficient chelating agent forlanthanide ions, in particular Gd3þ ; on the other wedge, an oligopeptide RGDS thatbinds to avb3 integrins was attached to allow specific targeting. The system reportedhas potential to be developed as a MRI contrast agent specific for cardiovasculardisease.

Figure 15.9 (Top) Structure of the dendrimerand the schematic representation of thedendrimer as ion sensor. (Bottom) Titrationresults of the dendrimers with different ions as

indicated by fluorescence quench. (Adaptedwith permission from [72] copyright 2000, RoyalSociety of Chemistry, [73] copyright 2002,American Chemical Society.)


15.3.5Peptide Dendrimers as DNA/RNA Delivery Vectors

Treatment of inherited disorders through gene therapy relies on the development ofsafe and efficient gene delivery vectors [14, 77–81]. Many investigations of viral andnonviral gene delivery systemshave been carried out in recent years. Compared to theinherent danger and limitations of viral delivery systems, nonviral systems are saferand have gained a lot of interest recently despite the fact that nonviral systems areconsidered to be less effective. In the search for synthetic nonviral vectors, efforts

Figure 15.10 Structure and the computer generated model of Gadomer 17. (Reproduced withpermission from [75], copyright 2002, Wiley-VCH.)

Figure 15.11 Nonsymmetric dendrimers prepared from native chemical ligation. (Reproducedwith permission from Scheme 1 of [76], copyright 2006, Royal Society of Chemistry.)


have been devoted to several different types of molecular systems. These includecationic polymers, cationic dendrimers (including peptide and nonpeptide dendri-mers), cationic liposomes, and cell-penetrating peptides, including those equippedwith nuclear localization sequences. Here, we discuss the development of syntheticvectors based on peptide dendrimers. For the purpose of vector development, vectorsneed to bind and carry DNA/RNA, and protect them from enzymatic degradation;they should interact with target cell surfaces and transport DNA/RNA acrossmembranes; they should allow their cargo DNA/RNA to escape from the endosomefollowing internalization; and they should release therapeutic DNA/RNA at theappropriate location in the target cell and hopefully produce the desired perturbationof gene expression.

Peptide dendrimers in principle can satisfy many of the above requirements.Cationic peptide dendrimers bind negatively charged DNA/RNA with high affinityand show strong protection of DNA/RNA against enzymatic digestion. Differentmodifications of peptide dendrimers such as the incorporation of amino acids withlong lipid chains (Figure 15.12) [82] and attachment of different penetrating peptidesonto dendrimer surfaces [83–85] have been explored in an effort to take advantage ofdifferent endocytotic pathways and overcome themammalian cell membrane barrier.There are four major internalization pathways used by mammalian cells [14, 36, 80]:macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, aswell as clathrin- and caveolin-independent endocytotic pathways (Figure 15.13).

Tung et al.designed a series of peptides derived fromapolylysine dendritic scaffold(Figure 15.14) [83]. Their ideawas based on the observation that a peptide fragment ofthe HIV tat protein, RKKRRQRRR, is able to ferry various molecules across cellularand nuclear membranes. They reasoned that the peptide might help to improveplasmid DNA delivery efficiency if multiple copies of the peptides can be incorpo-rated onto a branched scaffold. The results showed that all designed peptides interact

Figure 15.12 Structure of a lipid dendrimer, and computer-generated models of the dendrimer,a segment of DNA with 32 base pairs, and the dendrimer/DNA complexes (two different views).(Adapted with permission from [82, 86], copyright 2003, 2005, Elsevier B.V.)


Figure 15.13 Internalization pathway of gene/dendrimer cargo. (Reproduced with permissionfrom Fig. 2 of [14], copyright 2009, Maik Naukc/Interperiodica.)

Figure 15.14 Sequences and structures ofsynthesized peptides (bA is b-alanine and theundulating line represents the TAT peptide) andthe transfection of COS-1 cell with differentpeptides at various charge ratios(L: lipofectamine; D: naked DNA control)

as well as gel-shift assays indicating the bindingof synthesized peptides with luciferase DNAat various charge ratios. (Adapted withpermission from Figs 1–3 of [83], copyright2002, Elsevier B.V.)


with plasmid DNA as revealed by gel-shift assays (Figure 15.14). The dendrimerpeptide with eight tat peptide chains attached showed the optimal transfectionactivity.

Bayele et al. [87] have recently designed two polylysine dendrimers that containlipidated amino acids, S-72 andS-73 (Figure 15.15a). The uniqueness of this design isthat they incorporated two functional moieties into the dendrimers: (i) a positivelycharged lysine module for nucleic acid interactions and (ii) a neutral lipid moiety formembrane transit. The results showed that the dendrimers can indeed serve asnucleic acid carriers and deliver the desired oligonucleotides into the nucleus(Figure 15.15).

Toth andMinchin�s group have recently constructed a small combinatory library ofsynthetic DNA vectors (Figure 15.16) [84, 85]. The vectors consist of variouscombinations of the cell penetrating peptide TAT, the SV40 large T protein nuclearlocalization signal (NLS), and a cationic dendrimer of seven lysine residues (DEN).The vectors were assessed for their capability to form complexes with plasmid DNA

Figure 15.15 (a) Structures of lipidic peptide dendrimers and nuclear delivery of oligonucleotideby dendrimers. (b) Transfection assays on different cell types. (Adapted with permission from Figs1, 4, and 5 of [87], copyright 2005, Wiley Liss, Inc. and The American Pharmacist Association.)


Figure 15.16 Sequences and structures of the dendrimers and gel-shift assays of the dendrimer/DNAcomplexes aswell as theDNAuptakeof different dendrimer/DNAcomplexes at various chargeratios. (Adapted with permission from [84, 85], copyright 2007, 2009, Elsevier B.V.)


using several biophysical assays all of which indicate positive results (Figure 15.16).A test of eachmember�s capability to deliver exogenous DNA into humanHeLa dellsrevealed that the best system among them is the one containing all three function-alities (i.e., TAT, NLS, and DEN). Fluorescence microscopy indicated that with thehelp of the DEN–NLS–TAT dendrimer the exogenous DNA was delivered butconfined to intracellular compartments. Incubation of the dendrimer with a fuso-genic agent such as chloroquine enhanced transgene expression by more than 500-fold. The results point to the potential importance of multifactorial assembly indesign of nonviral gene delivery carriers.

Mixson et al. have recently investigated a series of branched peptides composedmainly of histidine and lysine residues asDNA/RNAvectors [88–90]. They found thatH2K4bTwith a histidine-rich tail is an optimal DNA vector [89] as the dendrimer byitself can effectively transport plasmids into a variety of cells without any covector.They interpret the successful effect of adding a histidine-rich tail to the dendrimer asdue to the histidine-rich sequence�s capability to buffer the acidic endosome andthereby induce its lysis. This helps exogenous DNA escape the endosomal pathwayand thus DNA degradation by lysosomes is avoided. The optimal plasmid DNAcarrier H2K4bT has four branches and a tail. In contrast, the optimal smallinterfering RNA carriers H3K8b, H3K8b(þRGD), and similar structural analogsconsist of eight terminal branches [90]. They have further tested both types of vectorsasDNA/RNAcarriers in several in vivo studies. In tumor-bearingmousemodels, theyobserved successful delivery of corresponding genes or small interfering RNAs anddemonstrated inhibitory effects on tumor xenografts [91–94]. Taken together, thesedata suggest that development of nonviral DNA/RNA vectors through amino acidsequence modification and peptide branching with lysine residues may lead to aneffective strategy for cancer treatments.

15.3.6Other Application of Peptide Dendrimers

Fassina has developed a procedure for the direct immobilization on preactivatedaffinity solid supports of a peptide dendrimer with a multimeric tripeptide ligand(Met–Tyr–Phe) [95]. The peptide ligand was assembled on an eight-branchedheptalysine dendritic scaffold and the resulting dendrimers were coupled to pre-activated resin supports. The modified solid supports were then packed onto a glasscolumn and applied to the affinity purification of bovine neurophysin from a crudemixture. Another important application of peptide dendrimers was reported byGrinstaff et al. [96–98]. They used cross-linkable precursor dendritic molecules toform a hydrogel and applied the cross-linked biomolecules to seal ocular woundscreated in surgeries, such as corneal incision during a typical cataract procedure(Figure 15.17). The results suggest potential applications of peptide dendrimers assoft materials for specific biomedical purposes. Thus, since the initial design ofpeptide dendrimers as MAPs [30], the potential of these molecules for vaccinedevelopment has proven limited in practice. However, several fruitful biochemicaland medical applications of various designed peptide dendrimers have been pur-


sued [18, 99–103]. Peptide dendrimers have been reported to serve a variety ofpurposes in applications ranging from protein/enzymemimics [48, 65, 66], to metalion sensors [72, 73], MRI contrast agents [75], drug transport vehicles, DNA/RNAdelivery vectors [91–94], and antimicrobial agents [53, 55, 62, 63].

15.4Conclusions

Peptide dendrimers represent a unique class of molecules constructed from aminoacids with branched structures. Their properties differ from linear polypeptides dueto their intrinsically multivalent construction. Display of functional groups orstructures on dendrimeric scaffolds has opened up new vistas for synthesis, study,and application.While numerous peptide dendrimers have been developed in recentdecades, there is still additional potential to explore the structural diversity andwealthof applications for this unique class of structures [17].

Figure 15.17 Synthesis of cross-linkableprecursor dendritic molecules and theirreaction with poly(ethylene glycol dialdehyde)to form an idealized cross-linked hydrogel.The left photograph shows thesynthesized hydrogel and the right the

result of a surgery wound repaired by thehydrogel sealant (incision is between the twopurple dots and the gel is within the blueborder). (Reproduced with permission fromFigs 1 and 2 and Scheme 1 of [96], copyright2004, American Chemical Society.)

15.4 Conclusions j513

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