, 2009, multivalent cooperative catalysts · catalysis than their monovalent counterpart has been...

Current Organic Chemistry, 2009, 13, 000-000 1

1385-2728/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Multivalent Cooperative Catalysts

Leonard J. Prins, Fabrizio Mancin and Paolo Scrimin*

University of Padova, Department of Chemical Sciences and ITM-CNR, Padova Section, 35131 Padova, Italy

Abstract: Multivalent systems are well known for their enhanced ability to bind multivalent counterparts. This contribu-

tion addresses the question whether they can also behave as cooperative catalysts. Analyzing examples mostly (but not

only) from our own laboratory we show what conditions are required for obtaining cooperativity in catalysis. Systems

considered range from simple, discrete catalysts to dendrimers and monolayer-protected gold nanoparticles. Reactions

taken into considerations for our analysis are the hydrolyses of carboxylate- and phosphate esters.

1. INTRODUCTION

It is a widely accepted paradigm that multivalent systems [1], i.e. systems topologically presenting a collection of iden-tical (or similar) functional groups on their periphery, show enhanced binding ability against multivalent counterparts. This is the case of systems governing cell-cell, protein-protein, protein-cell interactions, for instance [2]. A large amount of data show how affinity constants between these systems may increase by several order of magnitude with respect to those of the monovalent ones. This is the result of cooperativity between the several units present in the recog-

nition site.

The ability of multivalent systems to perform better in catalysis than their monovalent counterpart has been less studied, notwithstanding the fact that most, if not all, of the biological catalysts exert their catalytic activity as the result of the cooperative action of several functional groups present in their catalytic site. Thus, catalysis takes advantage of a concerted action of a collection of functional groups as mo-lecular recognition does. However, catalysis is much more complex to achieve than molecular recognition and, as a re-sult, the design and synthesis of catalysts in which collection of functional groups work cooperatively still remains a major challenge for scientists. The aim of this review is to analyze catalytic, multivalent systems, mostly studied by our group during the last several years, to pinpoint properties required for such systems to become better catalysts than their mono-

valent counterparts.

One of the earliest multivalent systems studied was con-stituted by aggregates of lipids or surfactants forming vesi-cles or micelles. In studying micellar catalysis Fred Menger [3], reviewing the field more than fifteen years ago, wrote: “…groups of molecules, properly assembled, can obviously accomplish much more than an equal number of molecules functioning separately”. A careful dissection of the contribu-tion to the catalytic activity of these systems led, however, the groups of Bunton [4], Romsted [5] and others [6] to reach the conclusion that these systems were not performing much better than the monomeric ones. In fact, although

*Address correspondence to this author at the University of Padova, De-partment of Chemical Sciences and ITM-CNR, Padova Section, 35131

Padova, Italy; Tel: -----------; Fax: -------------; E-mail:

experimental data show very impressive rate accelerations, the analysis of their kinetic behavior indicated that the rate accelerations observed were mostly due to local concentra-tion effects (including the ‘apparent’ reduction in reaction volume, but also the change of pH at the reaction loci). Thus cooperativity in these multivalent systems remains a contro-versial issue whose analysis is complicated by the fact that catalytic mechanisms with these systems proceed via pre-binding of the substrate (like in enzyme catalysis). The cor-rect analysis of the cooperativity in catalysis thus requires that molecular recognition (binding) and catalysis be ana-lyzed separately. As we will see the separation of the two processes is not always an easy task to perform. Further, by working under saturation conditions a multivalent catalyst may bind several substrate units. For proper comparison with monovalent systems this fact must be considered to avoid misinterpretation of the kinetic data.

For the ease of comprehension we will analyze multiva-lent systems starting with the simplest ones and then increase complexity ending with dendrimers, polymers, and function-

alized metal nanoparticles.

2. ASSESSING COOPERATIVITY IN CATALYSIS WITH MULTIVALENT SYSTEMS

Quite often false claims of cooperativity in catalysis with multivalent systems are due to a non-correct interpretation of the kinetic data concerning the activity of these systems. We will consider a multivalent catalyst as a cooperative catalyst whenever: a) its activity is larger than the summation of the contribution of the individual components (this is often re-ferred to as positive cooperativity), or b) the individual com-ponents play different roles not attainable with a monomeric catalyst resulting in rate acceleration. The following exam-

ples should clarify the concept.

Let’s first consider the kinetic behavior obtained by in-creasing the concentration of a multivalent catalyst and a monomeric one. In Fig. (1) the rate profile obtained with a micellar system, compared with a monomeric one bearing the same functional group, is reported [7]. The reactivity of the monomeric catalyst increases linearly with concentration while that of the micellar one (but for the inflection at low concentration due to the formation of the aggregates) follows a Michaelis-Menten-type profile. The micellar system is ob-viously much more active than the monomeric one in the

2 Current Organic Chemistry, 2009, Vol. 13, No. 11 Prins et al.

concentration interval explored. The enzyme-like reactivity profile indicates binding of the substrate to the catalyst but doesn’t provide any indication whether the catalyst is a co-operative catalyst or not. The rate acceleration could be sim-ply due to a higher local concentration of catalytic units rather than to their enhanced activity (in other words, cata-lysts and substrates are confined to a smaller volume which results in an increased rate). To assess cooperativity we need to determine the rate acceleration by changing the concentra-tion of catalytically active units within the multivalent cata-lyst. This can be done by using a multivalent catalyst, in which active and non-active components are mixed together, and the mole fraction of the active component is increased. Under these conditions a non-cooperative catalyst will give a linear increase of the rate constant with concentration (Fig. 2, trace a) while a cooperative catalyst will deviate from linear-

ity (Fig. 2, traces b and c).

Alternatively one should demonstrate that different spe-cies within the multivalent catalyst play different roles in a concerted manner. A very simple example is that provided by a hydrolysis catalyst in which the same functional unit may act as a base or nucleophile in its non-protonated form and as an acid when protonated. Fig. (3) shows an example of such a catalyst (trace b). In this Figure the rate constant is plotted as a function of pH. Trace a is that typically observed with a monomeric catalysts for which the deprotonated spe-cies is catalytically more active than the protonated one. Ac-cordingly, the curve is just the kinetic version of a titration. Curve b goes through a maximum at a pH corresponding to the pKa of the kinetically relevant species. But maximum activity is observed when both protonated and deprotonated species are present and they must work cooperatively.

3. SIMPLE, MINIMAL, MULTIVALENT CATALYSTS

The simplest example of a multivalent catalyst is that provided by a system presenting at least two catalytic units. Although within the scope of this review such a catalyst may appear trivial, it is quite useful to start our overview from

very simple systems because even such catalysts may pro-vide important hints in understanding multivalency in cataly-

sis.

The catalysts we will consider have all in common the ability to catalyze the cleavage of a carboxylate or phosphate ester. Reactions mechanisms for these systems are reasona-bly well understood and hence, their analysis is facilitated.

Fig. (1). Kinetic behavior of monomeric (a) and multivalent micel-

lar (b) catalysts plotted as a function of the total catalyst concentra-

tion.

Fig. (2). Kinetic profiles observed with multivalent catalysts upon

changing the mole fraction of active component. Profile a is ob-

served with a non-cooperative catalysts while profiles b and c are

typical of cooperative catalysts. The difference between profile b

and c is that, with the latter, maximum efficiency was obtained at a

mole fraction of active component lower than one.

Fig. (3). A kinetic profile of a cooperative kinetic process involving

an acid and its conjugate base (b); line a reports the profile ob-

served for a non-cooperative process involving the conjugate base of a protic catalyst. Inset: the log version of both curves.

b

a

b

a

c

b

a

pH

logk ab

Multivalent Cooperative Catalysts Current Organic Chemistry, 2009, Vol. 13, No. 11 3

While carboxylate ester constitute a simple proving ground for such catalysts, phosphates are quite more challenging, being rather stable compounds. Their hydrolysis at physio-logical pH and room temperature proceeds with a half life of 10

10 years (dimethyl phosphate) [8]. Excellent catalysts both

natural (enzymes) and artificial for phosphate esters hydroly-sis, typically require at least two metal ions for activity [9,10]. These two metal ions work cooperatively playing

distinct roles in the cleavage process.

3.1. Peptide-Based Catalysts

The group of Baltzer, exploiting the conformational pref-erence of designed 42 amino acid sequences assuming a he-lix-loop-helix conformation (Fig. 4), has systematically modified specific residues in key positions of the oligopep-tide in order to catalyze the hydrolysis and transesterification

reactions of p-nitrophenyl esters [11,12].

Imidazole-functionalized peptides obtained by introduc-ing several histidines in the sequence were able to provide substrate recognition and accelerations exceeding three or-ders of magnitude compared to N-methylimidazole. Se-

quences like that depicted in Fig. (4) hydrolyze 2,4-dinitrophenyl acetate with a second order rate constant of 0.18 M

-1 s

-1 at pH 3.1 compared with 9.9 10

-5 M

-1 s

-1 for N-

methylimidazole. In this case it has been demonstrated that the reaction mechanism takes advantage of the cooperativity of two adjacent histidines, one acting as the nucleophile and the other one as a general base as shown in Fig. (5). This

mechanism corresponds to that reported in Fig. (3) above.

Our contribution to these simple catalysts has been the synthesis of the artificial amino acid ATANP [13] and its incorporation in oligopeptides [14]. ATANP is characterized by the presence of a lateral 1,4,7-triazacyclononane unit, which forms 1:1 complexes with metal ions such as Cu(II) and Zn(II) with strong affinities. As said above these metals play an essential role in accelerating the hydrolysis of DNA and RNA. Thus, in collaboration with Baltzer’s group a 42-mer peptide analogous to the one reported in Fig. (4) was synthesized, with the difference that these new sequences incorporated up to 4 copies of ATANP (Fig. 6). Also these new peptides form helix-loop-helix motifs and bind Zn(II) ions with the triazacyclononane subunits present in the lat-

eral arms of ATANP.

It was observed that metal complexation causes a de-crease in the helical content of the peptide. However, even upon a partial unfolding of the structure, an acceleration of the cleavage of HPNPP (2-hydroxypropyl-p-nitrophenyl phosphate, an RNA model substrate) was observed with sig-

Fig. (4). The helix-loop-helix-forming 42-mer peptide motif used

by Baltzer for the design of catalysts for the cleavage of carboxy-late esters (the dimeric form is shown here).

Fig. (6). Hairpin-folded 42-mer peptide incorporating four ATANP

amino acids active in the cleavage of HPNPP as the Zn(II) com-plex.

Fig. (5). Cooperative mechanism involving two His in the cleavage of a carboxylate ester reported by Baltzer.

HN

HN

R

O

O

NO2

N

NH

His-30

His-34

HN

HNR

O-

O

NO2

N

N

His-30

His-34

N

HN

His-30

N

N

His-34 O

R

HN

HN

R

O

O-

N

NH

His-30

His-34


nificant rate acceleration with respect to the mononuclear catalyst. In this case too cooperativity is rather evident and is elicited by the convergence of several metal centers where the binding of the substrate occurs, i.e. to one of the two

faces of the double-helical hairpin.

A very appealing method for the construction of very short, but highly organized peptides, is the use of C -tetrasubstituted amino acids. Research by the group of Toni-olo [15,16] has demonstrated that most of the C -tetrasubstituted amino acids, of which -aminoisobutyric acid (Aib) is the simplest example, are very strong promoters of the 310/ -helical conformations. The main factors that determine the type of helix formed are the main-chain length, fraction of Aib, and amino acid sequence. Although in low polarity solvents the formation of a stable 310-helix is quantitative, in polar solvents, like water, folding generally results mostly in -helices [17]. The heptapeptide of Fig. (7), containing five Aib residues, prevailingly adopts a 310-

helical conformation also in aqueous solution [15].

The sequence contains also two ATANPs (see above) for metal ions complexation. The dinuclear Zn(II) complex turned out to be a good catalyst for the cleavage of both the RNA model substrate HPNPP [18] and plasmid DNA [19]. In the latter case, cooperative action between the metal cen-ters was demonstrated by comparison of the activity of the mononuclear versus the dinuclear complex. Tentatively, a mechanism was proposed which requires the formation of a supramolecular DNA-peptide complex as shown in Fig. (7). The k for the cleavage process (~1 10

-5 s

-1) allows the esti-

mation of a rate acceleration of about ten million fold over the uncatalyzed cleavage process. In this case the pH vs. rate profile goes through a maximum suggesting complementary

roles for the two metal ions involved in catalysis. This result

also supports cooperativity.

3.2. Tripodal Catalysts

Tripodal platforms also constitute simple examples of

multivalent catalysts. Analogously to the multiple-imidazole-

functionalized peptide of Baltzer reported above mixtures of

catalysts having up to 3 imidazole units connected to a 1,3,5-

triethylbenzene scaffold were prepared simply by connecting

histamine and N,N-dimethylethylendiamine in varying

amounts to the scaffold (Fig. 8).

The ratio between the four resulting catalysts was deter-

mined by the ratio of the reagents added [20]. Direct screen-

ing for catalytic activity and subsequent deconvolution then

gave direct access to the individual contribution of each cata-

lyst. The mixtures were tested for activity in the cleavage of

p-nitrophenylacetate (PNPA) and the resulting pseudo-first

order rate constants plotted against the relative amount of

imidazole subunits present in the mixture. The straight lines

obtained at both pH = 7 and 8 clearly indicate that, in this

particular system, cooperativity between imidazoles is com-

pletely absent. The simple functionalization of the platform

with the three arms is, accordingly, not enough to elicit co-

operativity between them.

A further elaboration of the system was the use of simple

dipeptides containing an histidine and a carboxylate either

from a terminal amino acid or from aspartic acid. In the cata-

lytic site of many esterases these functional groups operate in

a concerted fashion as general acid and general base (or nu-

cleophile) in the catalytic process.

Fig. (7). The octapeptide functionalized with five Aib and two copies of the triazacyclonane-bearing amino acid ATANP is active in the

cleavage of plasmid DNA. On the right is reported the bell-shaped rate profile as a function of pH.


These were the functional groups of the catalyst shown in Fig. (9), incorporating three AspHis-dipeptides on the 1,3,5-triethylbenzene scaffold [21]. For the synthesis we used a new synthetic methodology which allows us to functionalize any molecular scaffold with peptides on solid support. As part of a combinatorial search for small peptide-catalysts we studied the catalytic activity in the hydrolysis of p-nitrophenylbutyrate (PNPB) at pH 6.5 ([cat]: 0.1 mM; [PNPB]: 20 μM). We observed a significant 53-fold rate enhancement compared to the uncatalyzed background reac-tion, and a 20-fold rate increase with respect to the catalytic activity of the monomeric unit. This could be evidence for cooperativity in this small molecular system although the kinetic analysis carried out so far has not provided clear-cut

evidence on this regard.

However cooperativity between metal centers was ob-served in a tripodal system obtained by connecting several ligand amino acids (analogous to ATANP) to a tris-aminoethylamine (TREN) platform and inducing proximity between the metal complexes by using an allosteric metal center (Fig. 10) [22,23].

At variance with the previous system close proximity be-

tween the arms was ensured by a fourth metal ion. Its indis-

pensable role was confirmed by acylation of the amines of

the platform thus blocking their ability to bind this metal ion:

the resulting catalyst was not behaving in a cooperative fash-

ion. Allosteric control of conformation worked also nicely

for the obtainment of a three helix bundle comprising three

peptide sequences each containing an ATANP unit [24].

Cooperative catalysis was clearly apparent with the RNA

model substrate HPNPP but not in the case of an oligonu-

cleotide catalyst. One may speculate that in this latter case

several phosphate groups bind to the three metal complexes

thus spoiling the system of any possible cooperativity in ca-

talysis.

This observation brings about an important point: a mul-tivalent catalyst must be able to converge its catalytic units on the functional group to be transformed. Thus this section message is that structurally well-defined structures (as con-formationally stable peptides) behave cooperatively in ca-talysis. More loose systems like the tripodal ones on the con-trary do not, unless they are put to order, like, for instance,

by an allosteric regulator.

4. DENDRIMERIC CATALYSTS

In the final part of this review we will discuss multivalent structures which cooperative phenomena have been fre-quently observed: dendrimers, polymers, and nanoparticles. We will focus the attention on how cooperativity is ex-pressed by these systems and what its origin is. Dendrimers are monodispersed, hyperbranched polymers which expose a high surface density of functional groups that depends on the generation. The first catalytic peptide dendrimers were re-ported by Reymond and co-workers for the hydrolytic cleav-age of esters [25,26,27]. These dendrimers were prepared by disulfide dimerization of second-generation dendritic pep-tides containing all possible combinations of the catalytic triad of the serine proteases (aspartic acid, histidine, and ser-ine) resulting in 21 dimeric dendrimers (Fig. 11). Screening with a fluorogenic substrate showed that peptide dendrimers with triad amino acids having eight histidine groups at the surface were catalytically active and displayed enzyme-like Michaelis-Menten kinetics with substrate binding (KM ~ 0.1 mM) and rate acceleration (kcat/kuncat ~ 10

3). From a system-

atic alanine-scan it was concluded that catalysis most likely originated from cooperative action of two imidazoles, whereas the serine residues did not appear to play a signifi-

cant role [26].

Therefore, in a subsequent study a series of dendrimers of different generation were prepared in which histidine-units were repeated in each generation (Fig. 12) [28]. The

Fig. (8). Catalysts containing up to 3 imidazole moieties connected to a 1,3,5-triethylbenzene scaffold. On the right, the rate dependence as a

function of the composition.

pH 8

20 40 60 80 1000

NH

HN

NH

N

N

N

NH

HN

NH

N

N

NH

N

NH

HN

NH

N

NHN

NH

N

NH

HN

NH

NHN

NHN

NH

N


catalytic activity was studied using a pyrene trisulfonate es-ter as a substrate. A systematic study of the dendritic effect in peptide dendrimer catalysis revealed that the catalytic rate constant kcat and substrate binding constant 1/KM both in-creased with increasing generation number. The dendrimers showed rate accelerations up to kcat/kuncat = 20000 and KM values around 0.1 mM. The reactivity of histidine side chains

within the dendrimer is increased up to 4500-fold when compared to 4-methylimidazole. A bell-shaped pH-rate pro-file with a maximum around pH 5.5 in the dendrimer-catalyzed reactions suggests that catalytic activity results from cooperative action of two histidines (adding both a nu-cleophilic and a general base component to catalysis, analo-

gous to what reported in Fig. 3).

As a first entry into dendrimer synthesis, our group has functionalized 3

rd generation commercial DAB

(poly(propylene imine)) dendrimers [29] with a triazacy-clononane (TACN)-bearing acetate via amide bond forma-tion (see Fig. 13). In order to specifically address the issue of cooperativity between different metal ions present on the periphery of the dendrimer we have prepared derivatives

with different degrees of functionalization [30]. In this way the relative concentration of the complexes on the surface of each dendrimer could be changed thus allowing us to test how this affects the activity of the different catalysts. Usu-ally, the activity of increasing generation of functional den-drimers is compared but this is not a reliable way to assess cooperativity, since both the size and the number of func-

Fig. (9). Structure of tripodal catalyst bearing AspHis dipeptides.

Fig. (10). Tripodal metallocatalysts subject to allosteric control by a remote metal ion. On the right, the kinetic profile as a function of the

equivalents of Zn(II) added. The filled symbols refer to the tripodal catalyst while the open ones refer to the mixture of its components.

Fig. (11). Dendrimeric-peptide catalyzed ester hydrolysis.

NH

HN

HN

O

HN

O

NHAc

HNN

O

NH

O

NHAc

N

NH

O

HN

HOOC

COOH

HOOC

O

NHAc

HN

N

ZnII

N

HN

NH

NH

O

HN

O

O

O

NH

NH

NHN

NH

O

NH

NH

NH

N

HN

O NH

NH

NH

N

ZnII

ZnII

ZnII

Allosteric

site

Catalytic site

ZnII

N

HN

NH

NH

O

HN

O

O

O

NH

NH

NHN

NH

O

NH

NH

NH

N

HN

O NH

NH

NH

N

ZnII

ZnII

ZnII

Allostericsite

Catalytic site

6

1 2 3

Zn equivalentsII

4 5

8

10

12

k / 1

0 s

ob

s

6-1

dendrimer

pH=6.0, rtNO

O

N

O

HOOH

dendrimer:

Cys

SS

Cys

A1

A1'

A2 A2

A3

A3A3

A3

Cap

Cap

Cap

Cap

A2' A2' A3

A3A3

A3

Cap

CapCap

Cap

A1, A2, A3 = Asp, His, Ser


tional groups present on the periphery are increased. This makes it difficult to correlate the activity with the concentra-tion of the active functions at the reaction loci (i.e. the den-drimer interfacial region). By reporting the reaction rates determined with dendrimers with different catalyst (TACN)-loadings on the surface but at a constant catalyst (TACN) concentration we obtained the upward curved profile re-ported in Fig. (13), right. The fitting of this curve gave us the

critical information that two metal ions are involved in the catalytic process. Likely this is also the catalytically active complex in the dendrimers. The data clearly indicate that in spite of the fact that more than two metal ions are potentially accessible to the substrate the most efficient catalytic process is that that takes advantage of the cooperative action of two

metal centers.

Fig. (12). A strong positive effect in peptide dendrimer catalyzed ester hydrolysis. Higher generation dendrimers show a stronger substrate

binding and stronger catalysis, resulting in a large enhancement of the specificity constant kcat/KM (graph at lower right). Figure taken from

ref. [27].

Fig. (13). Triazacyclononane-functionalized dendrimers bearing increased amounts of ligand units. On the right, the kinetic profile obtained

as a function of the mole fraction of metallocatalyst present.

N

NN

N

NH

NH C

O NH

NH

N

n

C

O

H3C

16-n

xCl-

n=0 (0% funct.)n=3 (20.5% funct.)n=5 (32.5% funct.)n=8 (53.0% funct.)n=12 (75.0% funct.)n=16 (100% funct.)


In subsequent work, we studied the origin of this positive dendritic effect in more detail [31]. A series of peptide den-drons and dendrimers of increasing generation functionalized at the periphery with TACN were prepared. Kinetic studies showed that these metallodendrimers very efficiently cata-lyze the cleavage of the RNA model compound HPNPP, with dendrimer D32, containing 32 endgroups, exhibiting a rate acceleration of around 80 000 (kcat/kuncat) operating at a concentration of 600 nM. Although these are impressive numbers, a careful analysis revealed their true value. A theo-retical model was developed to explain the positive dendritic effect displayed by multivalent catalysts in general. In this model the classical Michaelis-Menten model (describing interactions between an enzyme and a single substrate) was extended to cover a multivalent catalyst involving multiple simultaneous binding and catalytic events. Simulation of the catalytic behavior of a series of multivalent models and analysis of the saturation profile and the Michaelis-Menten parameters kcat and KM revealed that the positive dendritic effect is an intrinsic property of multivalent catalysts. The positive dendritic effect can be explained without the need to recall reasons such as changes in the catalytic site, increased substrate binding constant, or changes in the microenviron-ment. Rather it appears that the efficient catalytic behavior of multivalent catalysts is mainly determined by two factors: the number of catalytic sites occupied by substrate molecules under saturation conditions, and the efficiency of the multi-valent system to generate catalytic sites in which multiple catalytic units act cooperatively on the substrate. In other words, clustering of multiple catalytic units in close proxim-ity enhances the apparent concentration of dinuclear cata-lytic sites, which makes substrate binding appear stronger (Fig. 14). Obviously, under saturation conditions, this effect

is lost and in fact, often the catalytic constant per catalytic unit is identical for dendrimers of different generation. None-theless, catalytic sites that cannot be saturated (for example because of poor accessibility) negatively affect the global catalytic constant of the system. Analysis of this type will be of increasing importance for the design and evaluation of new multivalent systems.

5. POLYMERIC CATALYSTS

An approach pursued in the design of multivalent catalyst is that of functionalizing a polymer. Polymers, at variance with dendrimers, are not so well defined in terms of molecu-lar weight and conformation. The difficulty in conformation control reflects itself in the uncertainty in knowing the loca-tion in the space of the functional groups, their proximity and, ultimately, their ability to cooperate one with the other. Nevertheless functional polymers have been widely used as cooperative catalysts and as enzyme mimetic systems behav-ing sufficiently well to warrant them the name synzymes [32]. After the excitation generated at the beginning research on this field somehow faded to be revitalized in the late ‘90s by Suh and his group [33]. The target they have selected is the hydrolysis of the peptide and phosphate bonds that very well fits within the scope of this review. The first systems reported was designed as a mimic of a protease and was ob-tained by constructing an artificial active site comprising three convergent salicylate residues on the backbone of a highly branched, polyethylenimine polymer [34]. To over-come the problem of the correct folding of the polymer and, hence, of the appropriate location of functional groups, they assembled three molecules of 4-bromoacetylsalicylate around an Fe(III) ion and then crosslinked with PEI. Once an amino group of PEI was linked to one of the three salicylates

Fig. (14). The effect of the clustering of catalytic units on the creation of catalytic sites in multivalent models E2 and E4. Each catalytic unit is

depicted by a black circle ( ), each catalytic site by a line connecting two catalytic units (-).

# cat.units = 12

# cat.sites = 6

# cat.units = 12

# cat.sites = 18

# S bound = 6

S

S

S

S

S

S

# S bound = 6

S

S

SS

S

S

saturation

S S

saturation

E2

E4

# cat.units = 12

# cat.sites = 6

# cat.units = 12

# cat.sites = 18

# S bound = 6

S

S

S

S

S

S

# S bound = 6

S

S

SS

SS

S

S

saturation

S S

saturation

E2

E4


through a covalent bond, other amino groups of PEI were located in the right positions to attack the remaining two salicylates, thus completing the cross-linkage. After removal of Fe(III) ion by treatment with acid, a water-soluble poly-mer with catalytic sites comprising three proximal salicylates was obtained. The polymer was incubated with -globulin and it catalyzed its degradation with a half-life of 1 h at pH 7 and 50 °C. When salicylates were attached to PEI randomly, little catalytic activity was observed, indicating that the pro-teolytic activity arose from collaboration among proximal salicylates. This example illustrates very well how stringent is the requirement of inducing proximity between catalytic units in a polymer in order to obtain an efficient catalyst. Polymerization is also a viable way to fix the conformation of a polymer. And in fact Suh et al. used to prepare a similar catalyst, poly(chloromethylstyrene-co-divinylbenzene) (PCD) and poly(aminomethylstyrene-co-divinylbenzene) (PAD), which are cross-linked polystyrenes with styryl resi-dues containing chloromethyl and aminomethyl groups [35]. Using the same catalytic units they succeeded in the cleavage of albumin with optimum activity at pH ca. 3. The concept was also extended to imidazole-functionalized PCD poly-mers by attaching imidazole to PCD through the carbon (C-2) atom [36]. The imidazole-containing PCD hydrolyzed albumin with a half-life of 1200 sec at pH 7 and 25 °C. The optimum activity was manifested at pH ca. 8. When the con-tent of the imidazole was reduced by 4.4 times, the prote-olytic activity was reduced by 24 times. Hence there was not a linear dependence of reactivity on imidazole functionaliza-tion of the polymer implicating collaboration of two or more imidazoles in the catalytic process. The mechanism proposed (see Fig. 15) is very similar to the one reported in Fig. (3) and involves cooperation between two imidazoles: one as a nucleophile and the other as a general acid.

A clever approach for the design of a catalytic site on a polymer was reported, among others, by the group of Wulff with the use ‘molecularly imprinted polymers’ [37,38]. For this, a crosslinked polymer is formed around a molecule that acts as a template. The monomer mixture contains functional monomers that can interact with the template through cova-

lent or noncovalent interaction. After removal of the tem-plate, an imprint containing functional groups in a certain orientation remains in the highly cross-linked polymer. The shape of the imprint and the arrangement of the functional groups are complementary to the structure of the template. This corresponds to an implementation of the concepts re-ported above, as not only multiple functions are put in the catalytic site, but also diverse ones. The final goal is obvi-ously to arrive to an array of functions acting in a concerted manner, i.e. cooperatively. If the template is a model of the transition state of the reaction the polymer is supposed to catalyze, it may stabilize it via complexation, thus increasing the rate of the process. The concept of transition state stabili-zation in (enzyme) catalysis, first introduced by Pauling [39], was very successfully applied to catalytic antibodies. The most recent advances in the field of imprinted polymers con-cern the formation of a polymer with carboxypeptidase A activity [40]. By using a phosphate diester as a mimic of the transition state of a carbonate ester hydrolysis Wulff and Liu have imprinted a polymer with amidinium and a metal ion binding site. By using diarylcarbonates as substrates and Cu(II) as the metal ion, a rate acceleration over the uncata-lyzed reaction of up to 217,000 was observed. Most impor-tant, the imprinting factor (or, in other words, the cooperativ-ity factor) ranged from 50 to 80 to illustrate the great advan-tage of the use of an imprinted polymer over a non imprinted one. The mechanism suggested, involving the cooperativity

by the amidinium and the metal ion is illustrated in Fig. (16).

Switching to the cleavage of phosphate esters, recently Hollfelder et al. [41] reported the use of PEI derivatized with alkyl, benzyl and guanidinium groups to obtain catalysts with enzyme-like properties and rate accelerations (kcat/kuncat) of up to 10

4 for the standard RNA-model substrate HPNPP

in the absence of any added metal. The authors have shown that that the efficacy of the catalysts is determined by the PEI derivatization pattern that exert a synergistic effect. This means that the catalysts act cooperatively because the com-bination of all these parts in the polymer increases catalysis by more than the sum of each modification taken independ-ently. Highest rate was observed at pH 7.8 corresponding to

Fig. (15). Mechanism suggested by Wulff for the cleavage of a carbonate

ester in the catalytic site of an imprinetd polymer (the best results were obtained by using Cu(II)).

Fig. (16). Poly(chloromethylstyrene-co-divinylbenzene) (PCD)

polymer functionalized with imidazoles catalyzing the cleavage of

peptide bonds with a cooperative mechanism reported by Suh.

PCD PCDPCD PCD

O

O

O

NH

NH N

NH2

NH2

(II)Zn

+

OH-


the combination of two effects: the decrease of the binding at higher pH due to the deprotonation of the amines and the decrease of the [OH-] at low pH. Catalysis is thus due to substrate binding by positively charged amine groups and the presence of hydroxide ions in active sites in a relatively hy-drophobic environment provided by the presence of the long hydrocarbon chains.

The very same PEI polymer was functionalized again by Suh et al. by random attachment of lauryl groups and Ni(II) complexes of terpyridine to obtain a metallocatalysts for HPNPP cleavage [42]. In water, the lauryl and the terpyridyl residues are expected to form hydrophobic clusters on the backbone of PEI. An artificial active site comprising two or more metal ions can form in the cluster, whose structure de-pends on the contents of lauryl and terpyridyl residues. In this case too, the cooperativity between two (or more) metal ions was suggested as the possible mechanism for the accel-

erated rate of cleavage of the substrate (Fig. 17).

6. NANOPARTICLE-BASED CATALYSTS

Clusters of gold atoms covered by a monolayer of or-

ganic molecules constitute an interesting example of self-

assembled multivalent systems [43]. Multivalency in these

systems is ensured by the presence, on the monolayer cover-

ing the gold core, of several identical functions that, in prin-

ciple, could cooperate in a catalytic process [44]. Typically,

individual molecules are anchored on the gold surface via a

thiol that ensures a strong (ca. 20 kcal/mole) interaction with

the metal. Depending on the size of the nanoparticles these

thiols are more (large and less curved clusters) or less (small

and more curved clusters) tightly packed. Furthermore the

use of mixtures of thiols with different functional groups

may allow the modulation of their interaction and reactivity.

Our first attempt to obtain a cooperative catalysts for the

cleavage of a carboxylate ester was performed by incorpora-

tion of imidazole subunits in the self assembled monolayer

(SAM) [45]. The system (Fig. 18) consisted of a mixed

monolayer of dodecanethiol and the N-methylimidazole-

terminated thiol. Proton NMR studies revealed a 1:1 ratio of

the thiols in the monolayer. The catalytic activity of the Au-

nanoparticles was tested on the substrate 2,4-

dinitrophenylacetate (DNPA) in methanol-water (6:4) solu-

tions in the pH-range 4.5-7.2. The resulting dependence of

the second-order rate constants is shown in Fig. (18) together

with those obtained for the monomeric catalyst. Packing of

the imidazole units on the Au-nanoparticle surface induces a

modest 30-fold rate acceleration with respect to the mono-

mer. However, the bell-shaped dependence of k2 with a

maximum in the proximity of the pKa (similar to curve b in

Fig. 1), supports cooperativity between two methylimida-

zoles in the DNPA cleavage by the nanocluster (general

acid/base or nucleophilic catalysis). The kinetic behavior is

very similar to that reported by Baltzer et al. who studied

four helix bundle-forming peptides bearing multiple imida-

zole subunits and discussed in Section 3.1. The simple con-

clusion from these studies is that by confining the catalytic

Fig. (17). PEI-based polymer functionalized with Ni(II) complexes

and made lipophilic by derivatization with lauryl groups reported as a cooperative catalysts in the cleavage of HPNPP by Suh.

Fig. (18). Imidazole-functionalized gold nanoparticles (Au-MPCs) showing cooperative behavior in the cleavage of the activate ester DNPA (see right, bell-shaped curve) as compared to a non-cooperative one shown by a monomeric catalyst (N-methylacetylhistamine).

NiNi

P

OO

O

O

O

-H

Ni

OH

PEI

S

S

O

NH

NNm

n

S

S

O

NH

NN

S

Au

Au-MPC

N-methylacetylhistamine

5 6 7 84

1.0

1.2

1.4

1.6

1.8 0.2

0.1


units in an environment like that provided by the monolayer

covering the gold nanocluster we managed to elicit coopera-

tivity between them. This is probably due to their reduced

mobility.

Slightly more complex systems were those constituted by dipeptides designed to induce cooperativity between car-boxylates and imidazoles by using histidine and a carboxy-late either from a terminal amino acid or from aspartic acid. The concept is very similar to the one reported in Section

3.2.

Nanoparticles functionalized with these peptides gave positive proof of cooperativity [46]. The complementary role

of a carboxylate and an imidazolium ion was demonstrated by studying the hydrolysis at low pH. The thiol used for the passivation contains a HisPhe-OH terminal sequence and was used in conjunction with a tris-ethyleneglycol methyl ether (TEG)-containing thiol, which makes the system water-soluble. As a reference catalyst, Ac-HisPhe-OH was used, which contains the same functional groups but is unable to

aggregate.

Fig. (19) reports the activity against pH of these func-tional nanoparticles and the monomeric catalyst in the hy-drolysis of 2,4-dinitrophenylbutanoate (DNPB). At all pH values the Au-nanoparticle catalyst outcompetes the mono-meric catalyst, but, extremely interestingly, the two curves

Fig. (19). Dipeptide-functionalized gold nanoparticles effective in the cleavage of DNPB (see rate plot on the right, open circles) and show-

ing at a low pH regime a mechanism involving both imidazole and carboxylate and not present in the monovalent catalysts based on the ace-

tylated dipeptide.

Fig. (20). Dodecapeptide-functionalized gold nanoparticles showing cooperative behavior in the cleavage of DNPB (circles) and their

monomeric counterparts (diamonds).

S

NH

S

O

NH

m

n

O

O

O

O

HN

N

HN

O

COOH

S

NH

OO

O

O

S

O

NH

HN

N

HN

OCOOH

Au

pH2 4 6 8 10 12

Lo

gk2

ap

p

-3

-2

-1

0

HN

H

NO

C NH

O

HN

O

OH

NHO

NH2

HN

O

NH

O

N

NH

HN

O

N

H

O

HN

O

NH

O

HN

H2 NNH

H

N

OOH

O

NH

NH2

HN

S

O

O

HN

S

O

O

O

O

HN

S

O

O

O

O

H

N

SO

OO

NHNH

O

C

HN

O

NHO OH

NH

O

NH2NHO

HN

O

NHN

NHO

HN

O

NHO

HN

ONH

H2 N

NH

NHO

OH

O

HN

H2N

NH

S

O

O

HN

S

O

O

O

O

HN

S

O

O

O

Au


show strikingly different profiles. The monomeric catalyst behaves as a system in which a catalytically relevant nucleo-phile is generated with pKa 6.6, which is consistent with the basicity of the imidazole. This is the logarithmic version of curve a of Fig. (1). On the contrary, the nanoparticle shows a more complex profile: a first nucleophilic species is gener-ated with pKa 4.2, then the curve flattens up to pH 7 where a second nucleophile is generated with pKa 8.1. These pKa values can be assigned to the carboxylic acid and the imida-zolium, respectively. The reason of the higher value of the pKa of the imidazolium in the nanoparticle is due to the ani-onic nature of the nanoparticle that disfavors the deprotona-tion of the imidazolium cation. What is particularly remark-able is the high activity, at acidic pH, of the nanoparticles-based catalyst showing over 300-fold rate acceleration with respect to the acetylated dipeptide. Mechanistically, this has been interpreted by involving a carboxylate anion in the cleavage that acts as a general base deprotonating a water molecule and a protonated imidazole acting as a general acid. The absence of this mechanism in the monomeric sys-tem clearly indicates that this behavior results from the con-finement of the dipeptide on the monolayer covering the nanoparticle. Any influence of the TEG units appears to be highly unlikely.

Moving on towards real ‘nanozymes’, i.e. nanoparticle-based models of enzymes, a dodecapeptide was grafted on Au-nanoparticles in a collaborative study with the group of Baltzer (see Fig. 20) [47]. A combination of a His, two Arg, and a Lys residue was expected to enable nucleophilic, gen-eral-acid, and/or general base catalysis, but also stabilization of the negatively charged transition state that arises along the pathway of ester hydrolysis. Au-nanoparticles were func-tionalized in a similar way as before and the catalytic activity of the resulting nanosystem in the hydrolysis of DNPB was studied as a function of pH. The results are reported in Fig. (20) (top, right) together with those related to the activity of the monomeric S-acetylated peptide. At low pH values the nanoparticle-based catalyst behaves very similarly to the previous dipeptide-based system, although the dodecapep-tide-nanoparticle has an additional 10-fold gain in activity. This amounts to a 3000-fold rate acceleration over that ex-

erted by the simple dipeptide. The larger catalytic efficiency is ascribed to a stronger acidity of the protonated imidazole group, which in the dodecapeptide has a pKa value 0.9 units lower than in the dipeptide. This is due to the fact that now the nanoparticle has no longer a net negative charge as this is compensated by the presence of the cationic guanidinium

groups.

At higher pH values, the activity of the peptide-nanoparticle increases significantly with respect to the dipep-tide-NP reaching an additional 40-fold rate acceleration. This can be ascribed to the presence of an additional nucleophile

(the phenoxide of tyrosine) with an apparent pKa of 9.9.

When addressing phosphate cleavage with these systems we prepared an ATANP-functionalized thiol and grafted it on the gold nanoparticle in a 1:1 mixture with dodecanethiol [48].

By studying the cleavage of the usual RNA-model sub-strate HPNPP, we obtained, by working at a fixed concentra-tion of nanoparticle and progressively adding Zn(II) ions, the sigmoidal profile shown in Fig. (21). Once again this curve provides strong support in favor of a cooperative mechanism between several metal ions. Indeed this catalysts was also

active with dinucleotides, UpU in particular.

A step further in the improvement of the catalytic unit was that to functionalize the monolayer with a more elabo-rate ligand in which the action of the metal ion could be as-sisted by that of ancillary groups (able to H-bond with the substrate, for instance). Accordingly, we prepared a bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) functionalized thiol (Fig. 22) as the BAPA-Zn(II) complex is known to be able to elicit the cooperation between metal Lewis acid acti-vation and hydrogen-bonding to achieve increased hydrolytic activity toward phosphate diesters [49,50].

The nanoparticles

were prepared following a recent protocol [51] reported by us. The resulting Zn(II) nanoparticle proved to be one of the most effective catalysts reported so far for the cleavage of the DNA model phosphate, bis-p-nitrophenyl phosphate (BNPP) [52]. The second-order rate constant observed was more than 60,000 higher than that of the base-catalyzed reac-tion and more than 100-fold better than that of the mono-

Fig. (21). Triazacyclononane-functionalized nanoparticles behaving cooperatively in the cleavage of HPNPP as evidenced by the sigmoidal

reactivity profile shown on the right reporting the reactivity as a function of the equivalents of Zn(II) added.

S

S

NH

m

S

Au

O

O

HN

N

NH

HN

n

S

N

H

OO

HN

N

NH

HN

S

NHO

O

NH

N

N

H HN4.0

3.0

2.0

1.0

0.5 1.0

Zn equivalentsII

10

k /

s

4-1


meric complex. The dependence of the rate constants on the equivalents of Zn(II) added, showed clearly also in this case, the occurrence of two catalytically relevant situations: the first one, at low Zn(II) loading, involving a mononuclear complex and a second one at Zn(II) saturation, involving a dinuclear complex. Both condition represent a significant

increase in rate with respect to the monomeric complex.

However the most striking result was that provided by the cleavage of plasmid DNA. The hydrolytic cleavage of this substrate is easily detectable as the compact supercoiled conformation of the polymer (form I) converts into the re-laxed circular one (form II) following a single cut in one of the two strands. However, if a second cut occurs in the other strand in close proximity to the previous one a further change occurs with the linearization of the double strand (form III). Statistically this second cleavage occurs in the proper position only in 1% of the cases. Strikingly, in our case linear DNA started forming after only 10% of the origi-nal supercoiled DNA was cleaved and already with 16% of it cleaved it was the most abundant form present (Fig. 23). This clearly indicates that the multivalent nanoparticle was

able to perform multiple cuts on the double strand, the obvi-ous consequence of its multivalent nature. One may specu-late that the nanoparticle by sticking to the polymer is able to hydrolyze at the same time several phosphate bonds belong-ing to both strands of the polymer thus linearizing it very efficiently, a behavior never observed with other catalysts. This is a very important result because for the first time a multivalent catalyst takes advantage of its multivalency on two counts. First, it cleaves efficiently a specific functional group by converging several cooperating catalytic units on it. Second, using the same mechanism it cleaves simultaneously several functional groups on a multivalent target, the polym-

eric DNA back bone.

CONCLUSIONS

We have tried to show in this overview, how to turn mul-tivalent catalysts into cooperative ones. The clear message is that maximum cooperativity is the result of a decrease of the conformational freedom of the multivalent catalysts and of the enhanced proximity of the putative catalytic units con-verging in the catalytic site. With small, discrete multivalent catalysts, cooperativity is more difficult to obtain. Coopera-tivity not only provides very large rate accelerations, but also induces quite new reaction mechanisms, which are not ac-cessible with monomeric, analogous systems. The best per-forming multivalent catalysts are dendrimers, imprinted polymers, and functional gold nanoparticles. The latter pre-sent the enormous advantage of being self-assembled sys-tems, thus reducing to the minimum the synthetic efforts for

their preparation.

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Fig. (22). BAPA-functionalized nanoparticle, very effective in the cleavage of BPNPP, a DNA-model substrate. On the right, the rate profile observed using increasing equivalents of Zn(II) ions showing also in this case a sigmoidal-like profile.

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AuS

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ONPNPO H

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k·1

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+ f

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form III

form II

[Au-MPC-Zn(II)], μM

0

4

8

12

16

0 1 6 10 15

form

II

+ f

orm

III

, %

form III

form II

[Au-MPC-Zn(II)], μM


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