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2 Mimics of the Michaelis–Menten Complexes of Zinc(II) Enzymes with Polyimidazolyl Calixarene-Based Ligands

2 Mimics of the Michaelis–Menten Complexes of Zinc(II) Enzymes with Polyimidazolyl Calixarene-Based Ligands

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Figure 12.1 Top left: Schematic representation of the 3D structure of a mononuclear metalloenzyme with its active site in inset (adamalysine, a Zn-matrix metallopeptidase from

snake venom, PDB codes: 1IAG). Schematized supramolecular models of metalloenzyme

active sites based on calix[6]arenes and relevant bioinspired ditopic and self-assembled

receptors with allosteric properties.

complex one Zn(II) metal ion to yield 4-coordinate mononuclear complexes where

all three imidazoles wrap the metal dication in a helical way (Figure 12.2). The

apical binding site is oriented toward the center of the cavity and occupied by a

guest ligand. The calixarene adopts a cone conformation that is locked since the

aromatic units cannot undergo flipping around the methylene bridges of the calix

small rim due to the metal binding to the three nitrogen arms. Nevertheless, the

aromatic units still stand alternatively in in and out positions relative to the cavity.

This flattened conformation is the opposite of the one observed for the free ligand,

as the anisole X substituents now adopt an in position relative to the three others.

Altogether, they form a door controlling the cavity entrance.

12.2.1 A Bis-aqua Zn(II) Complex Modeling the Active Site

of Carbonic Anhydrase

Several chemical systems have been developed by various groups to reproduce

the [Zn(His)3 (OH2 )][2+] coordination core encountered in many hydrolytic Zn

enzymes.4 Surprisingly, dicationic zinc aqua model complexes have proved

extremely difficult to stabilize and most classical models only succeeded in

stabilizing Zn-hydroxo species because of the high Lewis acidity of the Zn(II)

center bound to only four neutral ligands.19 In strong contrast, the reaction of the

calix[6]tris(imidazole) ligand with Zn(H2 O)6 (ClO4 )2 in THF readily yields a very

stable dicationic zinc–aqua complex.20 The unusual stability of the aqua complex



Figure 12.2 Shaping the calix[6]arene core into a Zn(II) funnel complex.





O8 3.01














(Wat 338)

Figure 12.3 XRD structures of (from right to left) the active site of carbonic anhydrase II21 and the calix-based model compound (bottom and side views, respectively).20

(Reproduced with permission. Copyright the Royal Society of Chemistry: Ref. 25.)

is actually due to second coordination sphere and cavity effects: the water ligand

is hydrogen-bonded to the oxygen atoms of the calix small rim that defines an

electron-rich environment; it is further stabilized by a strong hydrogen bond to a

second water guest suspended in the heart of the cavity, itself stabilized by OH/π

interactions with the aromatic walls of the cavity. This complex shows remarkable

similarities with the active site of carbonic anhydrase (Figure 12.3), which makes

it the first structural model for the Zn–aqua species found in enzymes, nicely

illustrating the importance of the microenvironment for the stabilization of reactive


In this calix[6]arene-based system, the cone constrains the metal ion in a tetrahedral geometry, precluding a second guest as small as a water molecule to coordinate

the metal center in endo position. The corollary of this feature is that such a model

will not allow mimicking the 5-coordinate intermediate formed during enzymatic

catalysis. Indeed, no hydrolytic activity has been observed. On the other hand, this

model has allowed studying, for the first time, the binding properties of such a

constrained and highly Lewis acidic Zn(II) center toward a variety of exogenous

ligands (vide infra).




Structural Key Features of the Zn(II) Funnel Complexes

The exceptional stability of this calixarene-based Zn–aqua complex is best illustrated by its reluctance to deprotonation in the presence of an amine. Instead,

both water molecules are displaced by a primary amine yielding the 4-coordinate

adduct depicted in Figure 12.4. Similar ternary complexes are readily formed by a

wide variety of small organic coordinating molecules (L).20, 22 In each case, X-ray

diffraction analysis shows a Zn(II) center in the regular tetrahedral environment

provided by the tris(imidazole) core and the guest ligand L. With protic guests

such as amines, alcohols, or primary amides, hydrogen bonds always connect their

acidic protons to one or two calixarene phenoxyl units as illustrated in Figure 12.4

by the XRD structure of the heptylamine complex. The guest conformation often

undergoes gauche interactions for an optimized filling of the calixarene cavity

with stabilizing CH/π interactions between the guest alkyl chain and the aromatic

walls of the host. The ethanol23 and acetaldehyde ternary complexes, which have

been characterized by XRD as well as in solution, provide interesting models for

substrate binding in liver alcohol dehydrogenase (LADH),24 a zinc enzyme catalyzing the reversible dehydrogenation of alcohols to aldehydes via hydride transfer

to NAD+ .

Due to their helical shape, these funnel complexes are chiral. The helicity, which

originates from the metal binding of the three imidazolyl arms, is transmitted to the

calixarene core, hence providing a chiral environment that ultimately is experienced

by the guest. In solution, both enantiomers are in equilibrium. Interestingly, it

has been shown that a chiral guest can control the equilibrium between the two

helical forms of the complexes, thereby transmitting its own chirality to the whole

calixarene-based system.26




(2 sphere)


Δδ (ppm)



to Zn


(1 sphere)

CH-π, van der walls




L = HeptNH2





4 2









–1.0 –1.2










Positon of the protons



Figure 12.4 Left: XRD structure of the heptylamine dicationic Zn(II) funnel complex.22

Bottom: Representative high-field shifted 1 H signature of an included guest [L =

CH3 (CH2 )6 NH2 , CDCl3 , 300 K, 500 MHz].22 Right: Mapping of the δ shifts corresponding to heptylamine complexes as a function of the large rim substitution pattern of

the calix[6]arene cavity.25



12.2.3 Hosting Properties of the Zn(II) Funnel Complexes:

Highly Selective Receptors for Neutral Molecules


H NMR spectroscopy has proved to be a powerful tool to monitor the presence

of a coordinating molecule (L) inside the cavity as the in/out exchange is generally slow on the NMR analysis time scale.15 The up-field shifts ( δ) measured

for the guest protons, due to the shielding effect of the π electron ring current,

are dependent on their spatial position in the aromatic cavity of the calixarene

(Figure 12.4). The relative capacity of guest ligands to bind to the Zn(II) center

was thus evaluated by 1 H NMR spectroscopy through competition experiments in

a noncoordinating solvent (Figure 12.5). The equilibrium constants KL /EtOH show

that the selectivity of the inner-cavity binding is based on (1) the σ -donor property

of the guest ligand, (2) the possible establishment of hydrogen bonds between the

guest and the small rim, (3) the relative host/guest geometries, and (4) the charge of

the ligand.

• With primary amines, coordination to the metal center is stoichiometric and

quantitative at millimolar (mM) concentrations. Amides and alcohols are also

excellent guests, better than nitriles. Coordination of aldehydes and carboxylic

acids is much weaker, although detected. Neither ether nor ketone yields

detectable coordinated species.

• Steric hindrance at the level of the coordinating atom and at its α position

is a major factor of selectivity: whereas primary amines are the best ligands,

secondary amines do not coordinate the metal center at all! Coordination of

1-propanol is 30 times stronger than 2-propanol.

Figure 12.5 Ligand exchange at the Zn(II) center of the funnel complexes based on the

hexa-tBu-calix[6]tris(imidazole) ligand. The equilibrium constants KL /EtOH (exchange of

L = EtOH for L at 298 K in CDCl3 ) are given in parentheses. *KEtOH/2H2O = 0.4mol ·

L−1 . When no endo coordination can be detected, KL/2H2O < 10−5 mol· L-1 .20, 22



• Neither a methyl substituent in 2-position nor a long alkyl chain precludes

coordination at the metal center. However, benzo- and benzyl-nitrile are too

sterically demanding to yield a stable adduct with the hexa-tBu ligand.

• The calix cavity is surprisingly reluctant to host anions in spite of the presence

of the dicationic metal center. Adding halides or hydroxides27 in excess either

leads to decoordination or induces a rearrangement of the calixarene macrocycle to give dimers and trimers with the coordinated bridging anions outside

the cavity. The reason actually stems from the second coordination sphere

defined by the calix oxygen-rich small rim28 : in the conformation adopted by

the tris(imidazole)-based complexes (Figure 12.2), all six oxygen atoms of

the macrocycle point their lone pairs toward the cone axis, at the level of the

coordinating atom. If an anion were to occupy that position, a strong electrostatic repulsion would result and lead to the destabilization of the host–guest


As shown schematically in Figure 12.2, the para-substituents of the anisole

units are oriented in an in position relative to the cavity, thus constituting a door

that closes the entrance of the host. Removing these three bulky tBu groups29 or

replacing them by smaller ones allows the cavity to host larger guests. For example,

the tris(anilino) derivative30 (corresponding to X = NH2 in Figure 12.2) accepts

much larger guests, for example, dimethyldopamine, tryptamine, and benzylamine,

than the parent compound (X = tBu), for which endo coordination of these bulky

amines has never been detected (vide infra).


Induced Fit: Recognition Processes Benefit from Flexibility

Highly specific enzymes generally display a relatively rigid pocket for the selective recognition of a unique substrate. In contrast, enzymes that contribute to the

metabolism of drugs and xenobiotics, such as cytochromes P450, must face the efficient binding of a wide variety of substrates within the same active pocket. It has

recently been recognized that this class of P450 enzymes has indeed a very flexible

proteic backbone that allows the active pocket to shrink or expand depending on

the substrate size.31 The importance of such a behavior has been well recognized

for other enzyme–substrate complexes as well as for drug–receptor complexes.32

Interestingly, the calix[6]arene-based Zn(II) receptors are also capable of inducedfit behaviors for guest binding. This is well illustrated by the comparison of the

XRD structures shown Figure 12.6.

Replacement of three tBu groups by three small NH2 substituents at the large rim

(X in Figure 12.2) not only opens wider the entrance of the cavity but also allows

spectacular induced-fit behaviors. The XRD structures of the ternary complexes

obtained with dimethyldopamine and benzylamine evidence aniline walls standing

almost parallel to each other to allow the large aromatic cores of the guest to fit

in. Surprisingly, in the related aqua complex, the second water guest present in the

parent hexa-tBu system is absent in the aniline host. The stabilization of the acidic

water ligand is now ensured by a direct OH–π interaction with the bent aniline unit



7.53 Å

3.12 Å

6.10 Å

6.57 Å

Figure 12.6 Induced-fit process undergone by the tris(aniline) derivative that allows

larger and smaller guests to bind compared to the parent tBu compounds, as illustrated by

XRD structures. From left to right: The dimethyldopamine, benzylamine, and mono-aqua

Zn(II) complexes and the bis-aqua derivative based on the hexa-tBu ligand for comparison

purposes.30 (Reproduced with permission. Copyright the Royal Society of Chemistry:

Ref. 25.)

that shrinks the cavity size, thus adapting it to the smallness of the guest ligand. In

contrast, when stacked together, the bulky tBu groups define a larger cavity space,

thus requiring the hosting of a second water molecule to stabilize the structure (see

Figure 12.6, right). In other words, the increased flexibility allows the optimization

of noncovalent attractive interactions within the cavity, that is, hydrogen bonds

and OH–π and CH–π interactions with the aromatic walls of the more or less

flattened cone core with small as well as with large guests. Such a behavior stands

in strong contrast to cyclodextrine33 or resorcinarene-based34 receptors, which, due

to their rigidity, can display strong binding to organic guests, at the very condition,

however, that there is a good fit between the guest size and the cavity size (described

as the 55% rule by Rebek).35 Hence, a rigid receptor presents a disadvantage as

only a restricted number of guests will display a strong affinity for the receptor:

only those whose size fits with the 55% rule. In the calix[6]-based system, the high,

but controlled, flexibility of the host turns out to be an advantage, with a cavity

that adapts to the size and nature of the guest for an optimized host–guest binding



Multipoint Recognition

Para-substituents can also be involved in molecular recognition. The Zn(II) funnel complex based on the same tris(aniline) ligand, for example, displays a high

propensity to interact at the level of the large rim aniline door with a variety of

cations, such as a second metal ion,36 a single proton, or an ammonium,37 thus

giving rise to stable tricationic structures. Thanks to the establishment of multiple hydrogen bonds at the level of the tris(aniline) door, this receptor is able to

discriminate between mono- and polyamines; for example, it binds much better

1,3-propyldiamine than butylamine, provided, however, the former is monoprotonated. The remarkable selectivity of this multipoint recognition system is best

illustrated by the regioselective binding of an unsymmetrical triamine, as illustrated

in Figure 12.7.



Figure 12.7 Left: Side view of the XRD refined structure13 of tricationic complex B.

Right: Regioselective binding of N-(2-aminoethyl)propane-1,3-diamine to the Zn(II) funnel complex based on the tris(aniline) ligand (X = NH2 in Figure 12.2) and acid–base

control of its directionality. (1) and (8) are arbitrary numbers used to differentiate the

two primary N atoms of the triamino guest. Supramolecular protection allowing the

regioselective carbamoylation of N-(2-aminoethyl)propane-1,3-diamine (Boc2 O stands for


Here, the funnel wraps and orients with a high selectivity an unsymmetrical

triamine guest as a function of its protonation state. This host–guest adduct thus

behaves as a bistable system: the guest reversibly changes from an “up” position to

a “down” position depending on the acidity of the medium, depicting an acid–basecontrolled directional switch. This remarkably discriminative recognition process

has been used for orienting an electrophilic reagent (Boc2 O) at a single site (N1)

with an unprecedented chemo- and regioselectivity, very much like in the SSAT

enzyme,38 an N -acetyl transferase that monoacetylates the triamine spermidine in

vivo on N1. Indeed, in the funnel system like in the enzyme, the microenvironment

not only guarantees specific binding but also positions the substrate molecule relative to the reactive species. As a result, a substrate presenting several sites susceptible to react is regio- and chemoselectively transformed. This case study also shows

that a funnel-like receptor can be used as a supramolecular protecting tool allowing

a transformation that is hardly feasible with conventional covalent chemistry.


Implementation of an Acid–Base Switch for Guest Binding

The second generation of ligands (Figure 12.1) was developed in order to introduce

an additional functionality to the metal complexes.39 The ligands present a fourth



Figure 12.8 Acid–base switch for guest binding by the calix[6]N3 ArOH based Zn(II)

complexes. Bottom, from left to right: Proposed mechanism for the peptidase activity of

astacin and serralysin Zn-enzyme families, XRD structures of the monocationic phenoxide

complex with a noncoordinated MeOH guest, and the neutral chlorophenoxide complex

presenting a self-included imidazolyl arm.41

donor group covalently linked to one nitrogenous arm, which provides an additional

cap to the system. This fourth donor can play the role of a redox-active function,

such as a phenoxide, and participate in the oxidation of a substrate, hence providing a good functional model of the radical copper enzyme, galactose oxidase.40

It can also act as a hemilabile arm and control the inner binding.41 Indeed, with

ligand calix[6]N3 ArOH (schematically illustrated in Figure 12.8), three different

protonation states for the corresponding Zn(II) complexes have been characterized: [Zn(II)N3 ArOH]2+ , [Zn(II)N3 ArO]+ , and [Zn(II)(OH)N3 ArO]. Whereas the

dicationic 5-coordinate species is very sensitive to guest binding, the monocationic

complex binds a guest ligand with a lower affinity due to a decrease of the Zn(II)

Lewis acidity.

The neutral species can be obtained upon reaction with a base to yield a hydroxo

complex or with an anion such as a chloride that coordinates the metal center from

the outside of the calixarene cavity. The simultaneous binding of two anionic donors

induces an impressive conformational reorganization of the system. One imidazole

arm is released by the metal center. The other one undergoes self-inclusion into

the π-basic calixarene cavity, thus precluding any guest inclusion. As a result,

the calix[6]N3 ArOH-based Zn(II) complexes act as an acid–base switch for guest

binding. Several aspects of this system appear reminiscent of Zn-peptidases of



the astacin and serralysin families.4 For these enzymes, in addition to the three

histidine residues, a side chain tyrosine coordinates the metal ion and its role

has been questioned. This model system suggests that one role is to accurately

control the activity of the enzymes as the pH varies, acting as an off switch upon

a pH rise.





Tren-Based Calix[6]arene Receptors

An alternative approach for the elaboration of biomimetic receptors consists of capping the hydrophobic cavity of the calix[6]arene skeleton by a tripodal aza subunit.

Tris(2-aminoethyl)amine (tren) provides an attractive platform for the elaboration

of such an aza cap. In this context, three different tren-based calix[6]arenes (i.e.,

calix[6]tren,42 calix[6]trenamide,43 and calix[6]trenurea44 ) have been synthesized

(Figure 12.9). These three molecular receptors display a calix[6]arene framework

constrained in a cone conformation by either a covalent trisamino, trisamido, or

trisureido tren-based cap. In all cases, the grid-like nitrogenous cap closes the

conic cavity at the narrow rim, leaving a single entrance controlled by the flexible

tBu door.

5 steps

3 steps


20 % overall yield

51 % overall yield

3 steps

49 % overall yield




Figure 12.9 Structures of the three different tren-based calix[6]arenes. X6 H3 Me3 stands

for 1,3,5-trimethoxy-calix[6]arene.42 – 44




Versatility of a Polyamine Site45 Highly Resistant Funnel Complexes With Zn2+ , Cu2+ , and

Cd2+ , the calix[6]tren ligand leads to funnel complexes with the metal ion

coordinated by the tren cap and an exchangeable neutral molecule bound to the

metal in the calixarene pocket, as depicted in Figure 12.10.46 The XRD structure

of a Zn2+ complex displays a 5-coordinate metal ion bound to a guest EtOH ligand

and to the tren cap in an asymmetrical environment (right inset, Figure 12.10).

Due to both a strong chelate effect and a full cavity-controlled access to the metal

center, the Zn complexes appeared remarkably resistant and better hosts than

their parent tris(imidazolyl) calix[6]-based complexes. They are resistant to bases,

acids, and even electrophiles (e.g., NBu4 OH, AcOH, and MeI). Most interestingly,

they proved not only capable of hosting a variety of small guest ligands such as

alcohols, nitriles, amides, or primary amines, but also allowed the binding of large

guests such as imidazole, benzylamine, and dodecyldiamine under experimental

conditions for which the parent systems (Figure 12.5) underwent decoordination

of Zn. Complexation of Ammonium Ions In the absence of metal ion,

this calix-cryptand is insensitive to neutral species such as alcohols, amides, and

amines in chloroform solutions at millimolar (mM) concentrations. However,

Figure 12.10 Versatility of the calix[6]tren receptor as a tetracation, a monocation, or a

Zn(II) complex.45

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2 Mimics of the Michaelis–Menten Complexes of Zinc(II) Enzymes with Polyimidazolyl Calixarene-Based Ligands

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