Dendrimers as articial enzymes

Jacob Kofoed and Jean-Louis Reymond

Dendrimers are regular tree-like macromolecules accessible by chemical synthesis from a variety of building blocks. Their topology enforces a globular shape that offers a unique opportunity to design articial enzymes. Catalytic groups such as metal complexes and cofactors can be placed at the dendrimer core to exploit microenvironment and selectivity effects of the dendritic shell. In a second approach, attaching catalytic groups in multiple copies at the end of the dendritic branches may lead to cooperativity effects. Finally, exploration of dendritic structural space by screening combinatorial libraries of peptide dendrimers for catalytic activity can lead to discovery of functional dendrimers with enzyme-like properties, in a process mimicking natural selection.
Addresses Department of Chemistry & Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland Corresponding author: Reymond, Jean-Louis ( jean-louis.reymond@ioc.unibe.ch)

as a single copy at the core of a dendrimer or in multiple copies at the dendrimer surface [11]. Herein, we review selected examples of these approaches. We also discuss recent studies aimed at discovering articial enzymes by exploring the structural diversity of synthetic peptide dendrimers. The proximity and microenvironment effects occurring within dendrimers is harnessed for catalysis by functional selection from combinatorial peptide dendrimer libraries, an approach that mimics the genetic mutation and natural selection processes leading to enzymes.

Dendrimers with a catalytic site at the core

Enzymes consist of a catalytic pocket equipped with precisely positioned functional groups for catalysis, surrounded by a protein shell. Several studies of dendrimers as enzyme-like catalysts have focused on the combination of catalytic groups at the dendritic core with dendritic branches to modulate catalyst selectivity [12]. Brunner and co-workers prepared a so-called dendrizyme by growing chiral dendritic branches around an achiral metal chelate to catalyze the cyclopropanation of styrene 1 with ethyl diazoacetate 2 to form the cyclopropane carboxylates 3ad (Figure 1) [8,9]. Ligand 4, with two chiral branches, gave products with 50% enantiomeric excess (ee) (3a,b) and 44% ee (3c,d) without stereoselectivity, but the more dendritic complex 5 gave no more than 11% ee. Studies by other authors with different metal complexes and reactions gave similar results, with approximately 23 fold modulation of either reaction rate or selectivity upon attachment of dendritic branches to a catalytic core. For example, Suslick and co-workers have reported dendrimer metalloporphyrins as oxidation catalysts [13]. A manganese containing porphyrin was surrounded by four dendritic branches and used for the epoxidations of alkenes in dichloromethane solution using iodosylbenzene as oxidant. The turnover numbers of the dendritic catalyst were similar to porphyrin alone (24 s1), yet the dendrimer displayed a 23-fold increased selectivity towards terminal alkenes over cyclic or disubstituted alkenes when compared with the porphyrin alone. Metalligand-based dendritic catalysts for other reaction types such as DielsAlder reactions [14], aldol reactions [15] and organometallic couplings have also been described [16,17]. Catalysis by organic cofactors at the dendrimer core has also been studied. Seebach and co-workers investigated the reaction of phenyl methyl ketene with methanol to give methyl 2-methylphenylacetate, under catalysis by a chiral diamine (1 mol% in toluene) at the core of a chiral Frechet-type dendrimer. This resulted in higher enantioselectivity (50% ee) compared with that of the nonwww.sciencedirect.com

Current Opinion in Chemical Biology 2005, 9:656664 This review comes from a themed issue on Model systems Edited by Paolo Scrimin and Lars Baltzer

1367-5931/$ see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2005.10.013

Introduction
Dendrimers are regular tree-like molecules. Their topology enforces a globular or disk-shaped structure and enables macromolecular properties such as a controlled microenvironment around the dendritic core and cooperativity effects between surface groups. Since the initial synthesis of dendrimers by Vogtle [1], Tomalia [2] and Newkome [3], the dendrimer concept has proven exceptionally successful at delivering functional macromolecules in preparatively useful amounts from a variety of building blocks and coupling chemistries. Applications range from biology and medicine to industrial catalysis and polymers [4]. Dendrimer-based catalysis has been the object of numerous studies [57] since initial reports by Brunner et al. in 1994 to create a so-called dendrizyme [8,9], and polycarbosilane dendrimers reported by van Koten and co-workers catalyzing the Karasch reaction [10]. These systems focused on modulating the efciency and selectivity of catalytic groups by placing them either
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Figure 1

Brunners dendrizyme ligands reported in 1994, active for the cyclopropanation of styrene.

dendritic amine catalyst (15% ee) [18]. Diederich and coworkers prepared thiazolio-cyclophanes decorated by dendritic branches as mimics for pyruvate decarboxylase [19]. This construct promoted an aldehyde to ester oxidation by a avin cofactor, but the reaction was two orders of magnitude slower than with the thiazolio-cyclophane system without dendritic branches. Smith and co-workers and Lopez and co-worker placed a tertiary amine at the core of a poly-L-lysine [20] or Frechet-type [21] dendrimer to catalyze the Henry reaction (aldol-type addition of nitroethane to benzaldehyde). However, the dendritic catalysts were, approximately 10-fold less efcient than the tertiary amine alone and decreased the syn/anti stereoselectivity ratio. Breslow and co-workers investigated a pyridoxamine reagent attached covalently at the core of poly(amidoamine) (PAMAM) [2] dendrimers of increasing size, which displayed up to 1000-fold higher reactivity for transamination with pyruvate and phenylpyruvate in aqueous medium [22]. The higher reactivity of the dendritic pyridoxamine reagent compared with that of pyridoxamine alone might reect acidbase catalysis by the dendrimer amine backbone and hydrophobic substrate binding. Dendritic shells can be used to create a microenvironment favourable for catalysis or provide shielding for functional groups at the dendritic core [23]. Frechet and co-workers demonstrated this principle in the catalysis of the elimination of hydrogen iodide from an iodoalkane to form an alkene, using a dendrimer posseswww.sciencedirect.com

sing a polar core [24]. The elimination reaction takes place within the polar dendrimer interior, but not in the surrounding hydrocarbon solvent. In a similar study, a polar interior dendrimer with three nucleophilic 4-(pyrrolidino)pyridine groups at the core catalyzed the esterication of the tertiary alcohol linalool with pivalic anhydride and triethylamine in cyclohexane solvent, under which conditions the non-dendritic catalysts 4(pyrrolidino)pyridine was much less active [25]. Dendronized polymer versions of the system were as active as the dendrimer catalyst, suggesting a microenvironment effect. In a converse example, the hydrophobic core of dendrimer 9, equipped with a benzophenone photosensitizer, could catalyze the [4+2] cycloaddition of singlet oxygen to cyclopentadiene 6, with methanol as a polar solvent (Figure 2) [26]. The hydrophobic dendritic microenvironment allowed binding of the non-polar cyclopentadiene substrate 6 but not the more polar unstable cycloaddition product 7. The diol 8 formed in situ by reduction of 7 with thiourea. Up to 40% conversion of cyclopentadiene 6 was observed in the presence of 0.l mol% of the dendritic benzophenone 9, whereas less than 10% product was formed with the same amount of a nondendritic benzophenone activator. Shielding effects from dendritic shells have also been used in non-catalytic applications. Examples include dendritic shielding of a palladium-porphyrin for oxygen sensing in vivo [27], of a metal-free porphyrin chromophore to effect two-photon activation for singlet oxygen
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Figure 2

Frechets light-driven dendritic catalysis. Singlet oxygen is generated in the benzophone core of dendrimer 9 and reacts subsequently with 6 in a [4+2] cycloaddition reaction to form 7, which is reduced in situ to form diol 8.

generation [28], and of ironsulfur clusters to modulate their redox properties [29].

Dendrimers with multiple catalytic groups

A characteristic feature of dendrimers is the multivalent display of functional groups at the end of dendritic branches. Poly(propyleneimine) [30] (PPI) and PAMAM [2] are commercially available with 4, 8, 16, 32 and 64 amino groups. Such multivalence is also realized in dendrimers based on lysine [31], 3,5-dihydroxy-benzylalcohol [32], [tris(2-ethoxycarbonyl ethoxymethyl) aminomethane] [33], 2,2-bis(hydroxymethyl)-propionic acid (e.g. 9, Figure 2) [34], P=N-P=S linkages [35] and glycerol [36,37]. The dendritic multivalent display of catalytic groups, in particular catalytic metal complexes, has been studied to create macromolecular catalysts that are easily separable from products by nanoltration or precipitation [38]. This technical advantage is very useful independently of any rate or selectivity effect of the dendrimer structure on the catalyst. Nevertheless, a few reports indicate that dendritic multivalent display of catalysts sometimes results in higher catalytic performance. Detty and co-workers reported a strong positive dendritic effect in the oxidation of bromide
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by H2O2 using dendritic phenylselenides, with a dendrimer with 12 selenide groups showing a 75-fold higher rate constant per selenide group compared with the monoselenide catalyst [39]. Jacobsen and co-workers reported the PAMAM dendrimer 12 with four Co-Salen complexes (Figure 3) [40]. This dendrimer catalyzed the hydrolytic kinetic resolution of (rac)-1,2-epoxycyclohexane 10 with 24-fold higher reactivity per Co-Salen unit and higher enantioselectivity (99.2% ee of diol product 11 at 43% conv.) compared with the metal complex alone (98.2% ee at 29% conv.), indicating a positive cooperative effect. Unfortunately, the dendritic multivalent display of catalytic groups also often reduces reactivity. Recent examples include PPI-dendrimers functionalized with imidazoles for the hydrolysis 2,4-dinitrophenyl acetate in water [41], and with phosphazene bases for base-promoted carboncarbon-bond forming reactions such as Michael and Nitroaldol additions [42]. Quaternary ammonium PPI dendrimers at millimolar concentrations were reported by Ford and co-workers to accelerate the decarboxylation of 6-nitrobenzisoxazole-3-carboxylate at pH 11.4 by 650-fold over the same reaction in the absence of dendrimer [43]. The rate of
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Figure 3

The dendritic version 12 of Jacobsens epoxide hydrolysis catalyst shows cooperativity (25-fold higher reactivity per group) and comparable enantioselectivity for the hydrolyis of racemic epoxide 10 (99.2% ee of diol product 11 at 43% conv.).

decarboxylation is sensitive to medium and thought to be accelerated within the non-polar interior of the quaternary ammonium dendrimer. The dendrimers also accelerated the hydrolysis of p-nitrophenyl hexanoate at pH 9.4 by 14-fold. Mazugaki and co-workers recently reported PPI-dendrimers with 16 decanoylamides at the surface and quaternized with excess methyl iodide. These quaternary ammonium iodide catalysts accelerate the MukaiyamaAldol reaction of trimethylsilyl methyl ketene acetal with various aldehydes at 60 8C in toluene [44]. Catalysis was attributed to an enhanced reactivity of the iodide anion as a Lewis base when encapsulated within the polar environment of the quaternary ammonium dendrimer.

pitation, as is often observed with linear synthetic peptides. Peptide dendrimers have been used previously for the multivalent display of ligands, and have been used as diagnostic reagents, protein mimetics, anticancer and antiviral agents, vaccines and drug and gene delivery vehicles [46,47]. Polylysine dendrimers decorated with various endgroups are also being evaluated as drugs, for example multivalent aryl sulfonates can be used for the prevention of HIV infections [48]. The rst catalytic peptide dendrimers were reported by Reymond and co-workers for an ester hydrolysis reaction (Figure 4) [49]. The peptide dendrimers were prepared by disulde dimerization of second-generation dendritic peptides containing all possible combinations of the catalytic triad amino acids aspartate, histidine and serine, resulting in six half-dendrimers and 21 dimeric dendrimers. Dendrimer families were prepared with different branching diamino acids, (1,3-diamino-2-propyl)oxyacetic acid, 2-aminomethyl-b-alanine and (S)-2,3-diamino propionic acid [50], or 3,5-diaminobenzoic acid [51]. Screening with uorogenic ester substrate 13 showed that peptide dendrimers with catalytic triad amino acids and displaying eight histidine groups at the surface (positions 0 A3 and A3 ) were catalytically active and displayed enzyme-like MichaelisMenten kinetics with substrate binding (KM $0.1 mM) and rate acceleration (kcat/kuncat $103).
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Catalytic peptide dendrimers

This section reviews recent efforts to create articial enzymes using peptide dendrimers, capitalizing on the simplied synthesis and enforced globular shape of dendritic peptides [45]. This strategy circumvents the difcult problem of protein folding, and should render peptide dendrimers relatively more stable towards denaturing conditions (high temperature or non-aqueous conditions) and biodegradation relative to natural proteins. The enforced globular shape is also optimally suited to favor intramolecular interactions between amino acids that lead to macromolecular function, rather than intermolecular interactions leading to aggregation and preciwww.sciencedirect.com

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Figure 4

Peptide dendrimer catalyzed ester hydrolysis. Box: the 21-peptide dendrimers with the catalytic surface histidines and the core disulfide bridge marked in red. Dendrimer DD, containing 3,5-diaminobenzoic acid B, is a branching diamino acid with an N-acetylated N-terminal aspartate residue. DD catalyzed the enantioselective ester hydrolysis. Vnet/Vuncat is the apparent rate acceleration observed with 200 mM substrate and 5 mM (2.5 mol%) peptide dendrimer. In the mutagenesis study, peptides Ala-1 to Ala-4 denote sequential replacement of histidine residues by alanine. Dendrimer DG had one core serine mutated to alanine, and GG has both residues mutated to alanine.

When 3,5-diaminobenzoic acid was used as branching unit, the dendrimer with four histidine residues in the intermediate positions within the dendrimer branches showed catalytic activity and enantiomeric discrimination for the chiral 2-phenylpropionic ester derivative 14 (Figure 4). An alanine-scan study showed that replacement of only one of the four catalytic histidine residues lowered activity by 75%, whereas replacement of all four histidine residues completely abolished activity. By contrast, replacing the two core serine residues by alanine did not affect catalysis, suggesting that cooperative catalysis occurs between histidine residues without participation of serine. Catalysis probably involves nucleophilic catalysis by histidine; however, dendrimers containing combinations of histidine with aromatic or hydrophobic amino acids were not catalytically active. Similar results were obtained with peptide dendrimers with a hydrophobic
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core and catalytic residues at the surface, which were prepared using a chiral, optically pure (1,3-diamino-2propyl)oxyacetic acid with orthogonally protected amino groups as branching unit [52]. A systematic exploration of peptide dendrimers as enzyme models should take the mechanism of natural evolution by mutation and selection into account. Indeed natural enzymes and their exquisite specicities and rate accelerations are the products of the genetically driven exploration of the large combinatorial space of amino acid sequences over eons of time, a process which can be partly reproduced in the laboratory to modify existing enzymes [53]. The fundamental evolutionary steps of diversity generation and functional selection have been recently demonstrated for synthetic peptide dendrimers on the basis of combinatorial chemistry [54].
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Figure 5

Combinatorial library of peptide dendrimers. Synthesis by split-and-mix protocol on solid support (tentagel) produced 65 536 different dendrimer sequences. Screening using fluorescent beads (picture at center) identified hydrolysis of ester 16a. Sequencing and resynthesis led to catalytic peptide dendrimers. The branching diamino acid is (S)-2,3-diamino propionic acid. All dendrimers are acetylated at the N-terminus.

A split-and-mix combinatorial library of peptide dendrimers was prepared with eight variable amino acid positions occurring as pairs in four successive branches of a third-generation peptide dendrimer (Figure 5). Sixteen different amino acids were used, placing four different amino acids at each position, to generate a library of 48 = 65 536 different dendrimers. Direct on-bead screening for ester hydrolysis of the uorogenic butyrate ester 16a gave a few uorescent beads, which were selected by visual inspection, isolated, and sequenced. The peptide dendrimers were resynthesized and puried, and the resulting peptide dendrimers showed the expected esterase-like activity with the pyrene trisulfonate ester 16a and its acetate and nonanoate analogs 16b and 16c, conrming the combinatorial strategy. A similar screening for binding to vitamin B12 gave dendrimers binding to vitamin B12. Most of the esterolytic peptide dendrimers above display multiple histidine residues at their surface. A systematic study of a dendritic effect in peptide dendrimer catalysis showed that the catalytic rate constant kcat and substrate binding constant 1/KM increased with increasing generation number (Figure 6) [55]. Isothermal calorimetry measurements showed that binding of product 17 was not affected by dendrimer size, suggesting hydrophobic binding of the substrates at the acyl chain due to a hydrophobic core microenvironment in the higher genwww.sciencedirect.com

eration dendrimers. The dendrimers showed rate acceleration up to kcat/kuncat = 20 000 and KM value around 0.1 mM. The reactivity of histidine side chains within the dendrimer is increased up to 4500-fold when compared with 4-methyl imidazole, which also weakly promotes the reaction. A bell-shape pH-rate prole around pH 5.5 in the dendrimer-catalyzed reactions suggests that cooperativity between the histidine residues involves both pKamodulation by proximity effects and electrostatic substrate binding.

Conclusion
Although still very far from rivalling natural or modied enzymes, dendritic catalysts are capable of rate accelerations and selectivities comparable with those observed with enzyme models based on antibodies [56], non-catalytic proteins, [57,58] and small peptides [59]. However, by radically departing from the linear topology of natural proteins, dendrimers might circumvent inherent limitations of protein-based designs, in particular thermal instability and solvent incompatibility due to unfolding and aggregation. Dendrimers might also show increased stability towards biodegradation. A combinatorial exploration of dendritic structural space on the basis of peptide dendrimers seems promising for the development of dendrimers as articial enzymes. By contrast to the situation with natural enzymes, there is currently an almost complete lack of structural characterization [60] or
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Figure 6

A strong positive effect in a peptide dendrimer catalyzed ester hydrolysis reaction. Higher generation dendrimers bind substrates 16a tighter (higher 1/KM) and show stronger catalysis (higher kcat), resulting in a large enhancement of the specificity constant kcat/KM (graph at lower right).

molecular modelling [61] studies with dendrimers. These will be required in the future to lay structurefunction relationships for dendrimers on a rm basis and allow further progress.

2.

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Acknowledgements
This work was supported by the University of Berne, the Swiss National Science Foundation, the COST action D25, and the Marie Curie Training Network IBAAC.

4.

5. 6. 7. 8.

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Current Opinion in Chemical Biology 2005, 9:656664

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