# Guest Covalent Capture by a Host: A Biomimetic Strategy for the Selective Functionalization of a Cavity

Nicolas Menard, Olivia Reinaud, Benoit Colasson

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Nicolas Menard, Olivia Reinaud, Benoit Colasson. Guest Covalent Capture by a Host: A Biomimetic Strategy for the Selective Functionalization of a Cavity. Chemistry - A European Journal, 2013, 19 (2), pp.642-653. 10.1002/chem.201202391. hal-05423202

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DOI: 10.1002/chem.201202391

# Guest Covalent Capture by a Host: A Biomimetic Strategy for the Selective Functionalization of a Cavity

Nicolas Menard, Olivia Reinaud, and Benoit Colasson*[a]

**Abstract:** A biomimetic strategy for the monofunctionalization of a calix[6]-arene core is described. It is based on host–guest chemistry (mimicking the Michaelis–Menten adduct in enzymes) and allows the finely tuned pre-organization of the substrate (an alkyne) with respect to the reactant (three azido groups introduced at the calixarene large rim). It is shown that the thermal Huisgen reaction implemented in this work proceeds under very mild conditions with total regioselectivity of the

cycloaddition process. The scope of the reaction was explored and the results suggest that such a supramolecular strategy is quite versatile and could be applied to the selective functionalization of other cavitands bearing different recognition patterns. A detailed structural, thermodynamic, and kinetic

study is also reported, highlighting interesting biomimetic features: The importance of the host–guest adduct strength, the high sensitivity of the reaction to the pre-organization of the reactive partners (alkyne vs. azide), and a significant impact of the embedment on the transition state. The self-coordination of the monofunctionalized products was also studied and an “*endo/exo*” switch of the internal side-chain could be triggered by adding competitive ligands.

**Keywords:** biomimetics • calixarenes • host–guest systems • regioselectivity • zinc

## Introduction

Selective desymmetrization is a classic challenge in organic synthesis. The selective transformation of one functional group among other identical groups is a difficult task and often mixtures are generated.[1] Consequently, the desired desymmetrized molecule is obtained in low-to-moderate yield. The synthesis and purification of larger desymmetrized molecules may be even more tedious. Nevertheless, the need for such structures is often required when one wants to introduce different reactive functionalities, add co-operativity, or explore multivalency in host–guest systems.[2] Highly symmetrical parent macrocycles such as resorcinarenes,[3] cucurbiturils,[4] cyclodextrins,[5] and calix[n]arenes[6] are often encountered in supramolecular chemistry as molecular scaffolds for hosts, sensors, or catalysts. Reliable and useful synthetic procedures allowing for the selective transformation of one and only one reactive site of these macromolecules are scarce. The monofunctionalization of cyclodextrins is often achieved in low-to-moderate yield because of the need for chromatographic separation.[7] Very recently, one procedure based on the covalent capture strategy

proved to be more selective.[8] Only a few procedures for the monofunctionalization of cucurbiturils are available.[9] As far as resorcinarene receptors are concerned, the modification of one wall out of four has been developed and extensively used by Rebek and co-workers.[10]

Synthetic procedures for the monofunctionalization of calix[6]arene macrocycles are rare. We previously reported a unique method for the selective monofunctionalization of a tris-azidocalix[6]arene[11] through a combination of the thermal Huisgen cycloaddition of azido compounds to acetylenes[12] and the host–guest properties of ZnII–calix[6]arene-based funnel complexes.[13] 4-Pentyn-1-amine ((C₃)NH₂) can coordinate to the ZnII ion, placing the acetylene in close vicinity to the three azido groups, thus pre-organizing the reactants. The cycloaddition occurs upon heating the complex to give the monofunctionalized complex [ZnM(C₃)NH₂]²⁺ (Scheme 1).[14] Subsequent cycloaddition reactions are prevented for two reasons: 1) Under such conditions, the intermolecular cycloaddition reaction is much slower as it requires much more drastic conditions (higher temperature and concentrations) and 2) the cavity of the calixarene is now occupied, preventing a second molecule of 4-pentyn-1-amine from displacing the now covalently linked amino chain and reacting intramolecularly.[15] This reaction is 100% selective towards monofunctionalization and creates exclusively the 1,5-triazole regioisomer.[11]

In this example, the “monoclick” reaction occurs because of the geometric fit between the acetylene and the azido groups. We intended to explore the scope of this methodology. To do so, we studied a variety of substrates that can be used to monofunctionalize the calixarene precursor. The acetylene guest can be constructed from three modifiable

<figure><img src="image_2.png" /></figure>

<footnote>[a] Dr. N. Menard, Prof. Dr. O. Reinaud, Dr. B. Colasson
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601
Université Paris Descartes, PRES Sorbonne Paris Cité
45 rue des Saints Pères, 75006 Paris (France)
Fax: (+33) 142-868-387
E-mail: benoit.colasson@parisdescartes.fr</footnote>

<footnote>Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201202391.</footnote>
<figure><img src="image_3.png" /><figcaption>Scheme 1. Reaction pathway for the monoclick reaction with 4-pentyn-1-amine (the structures of the complexes have been simplified for clarity).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Scheme 2. Schematic representation of the monoclick reaction with a reactive guest bearing a coordinating function X, a spacer, the reactive acetylenic group, and a substituent R. The coordinating chain of the monofunctionalized complex can be either *endo* or *exo* with respect to the cavity, depending on the nature of X, R, the spacer, and the nature of the competitive ligand L.</figcaption></figure>

parts: The coordinating group X (primary amine, alcohol), a spacer that can vary in length and in shape between X and the reactive acetylene function, and finally a terminal functionality R that can vary in size or in electronic properties (Scheme 2). We then investigated the relationship between the thermodynamically controlled complexation step and the kinetics of the cycloaddition step.

The monoclick reaction of such a guest provides a novel calix[6]arene-based ligand $M_{CnX}$ (Scheme 2), which differs from classical “funnel complexes” as an additional coordinating group X is covalently linked to the large rim of the calixarene. Such a system is prone to undergo self-coordination and offers an interesting example of an “ouroboros-like” molecule as defined by Durola and Rebek.[16] Hence, in addition to methodological studies yielding these monofunctionalized calixarene complexes, we wish to describe and discuss their specific host–guest properties. In this respect, we have studied the structural and conformational changes accompanying self-coordination and the factors controlling the intramolecular coordination to the zincation, that is, the “*endo/exo*” equilibrium of the internal coordinating arm.

## Results and Discussion

First, we evaluated the scope of the reaction by using primary amine ligands, which are the best guests for Zn–calixarene funnel complexes. The strong association to the zinc metal center provides unambiguous characterization of the intermediates and final compounds and facilitates discussion about the factors controlling reaction efficiency and structur-

al modifications. In the second part, this synthetic methodology has been applied to alcohols. The differences and similarities between the reactions with the amines and alcohols will be emphasized. In particular, the thermodynamics of the intramolecular coordination of the hydroxy arm will be discussed.

### Monoclick reactions with primary amines:

*Effect of spacer length:* The spatial proximity of the acetylene and the three azido groups appears to be one of the keys to monofunctionalization. The rate of reaction should be enhanced if the acetylene of the coordinated guest faces precisely the azido groups present on the large rim. Thus, we anticipated the existence of an optimal length for the spacer. To

optimize our system, we studied three different alkylamines that differ only in the number *n* of methylene groups in the spacer: $HC≡C(CH_2)_nNH_2$ (*n* = 3, 4, and 5): 4-Pentyn-1-amine (($C_3$)NH$_2$), 5-hexyn-1-amine (($C_4$)NH$_2$), and 6-heptyn-1-amine (($C_5$)NH$_2$).

*Synthesis and characterization of the three monoclick complexes:* The three pre-click host–guest adducts $HG_n$ were synthesized in situ from $[ZnX_6N_3](ClO_4)_2$ and the corresponding amines (see Figure 1) and characterized by ¹H NMR spectroscopy (see Figure 2A, C, and E) in CD₃CN. All the ¹H NMR spectra exhibit the same pattern and chemical shifts except for the resonances in the high-field region, which attests to the coordination of the amine to the metal center and its encapsulation in the cavity of the complex. The monoclick reactions were conducted in toluene at reflux with two equivalents of amine to provide the corresponding monofunctionalized $Zn^{II}$ complexes in high yields (95, 93, and 92% for $[ZnM_{(C_3)NH_2}](ClO_4)_2$, $[ZnM_{(C_4)NH_2}](ClO_4)_2$, and $[ZnM_{(C_5)NH_2}](ClO_4)_2$, respectively). The reactions were completed within 2 hours for *n* = 3 and 4, and 5 hours for *n* = 5. The products were isolated by precipitation with Et₂O and centrifugation or filtration, depending on the scale of the reaction.

The products were all characterized by positive electrospray mass spectroscopy (EIMS) after demetalation of the samples by addition of trifluoroacetic acid (TFA). In all cases, a single peak was observed corresponding to the protonated monofunctionalized ligands $[M_{C_nNH_2}+H]^+$. Neither the bis- nor tris-adduct was ever observed, which proves that the monofunctionalization reaction is extremely selective in all three cases. The products were characterized by
<figure><img src="image_5.png" /><figcaption>Figure 1. Top: Reaction pathway for the monoclick reactions of 4-pentyn-1-amine (n=3), 5-hexyn-1-amine (n=4), and 6-heptyn-1-amine (n=5). The corresponding complexation constant and the rate constant of the intramolecular cycloaddition are denoted as K<sub>n</sub> and k<sub>n</sub>, respectively. Bottom: Representation of the planar projection of the monofunctionalized complex [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> emphasizing the vertical symmetry plane σ<sub>v</sub>. For clarity, the bond between the amino group and the Zn<sup>II</sup> ion is not represented and the alkyl chain is represented out of the symmetry plane.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Figure 2. ¹H NMR spectra in CD₃CN of inclusion complexes HG<sub>n</sub> and the corresponding monofunctionalized complexes [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (n=3, 4, 5): A) HG<sub>3</sub> (500 MHz, 300 K), B) [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (250 MHz, 300 K), C) HG<sub>4</sub> (500 MHz, 300 K), D) [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (500 MHz, 300 K), E) HG<sub>5</sub> (500 MHz, 300 K), F) [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (500 MHz, 300 K), and G) [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (500 MHz, 353 K) (f: free amino-acetylene; s: residual solvent; the methylene groups of the alkyl chain are denoted as α, β, γ, δ, and ε, starting from the NH<sub>2</sub> group).</figcaption></figure>

the aromatic region attested to the formation of the triazole moiety. New upfield resonances appeared; the integration of these resonances indicates a guest/calixarene ratio of 1:1, which is consistent with a monofunctionalized calixarene in which the amino chain is coordinated to the Zn<sup>II</sup> ion through the cavity. The resonances of the monofunctionalized complexes [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> and [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> are sharp at 300 K, whereas the resonances of the monofunctionalized complex [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>, especially the resonances of the imidazole arms ImCH<sub>2</sub>, are broad at 300 K. However, at 353 K, the resonances sharpen. This indicates that [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> has a restricted conformational mobility at 300 K that is slow on the NMR timescale. Because this phenomenon was not observed for [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> or [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>, we can conclude that the longer alkyl chain imposes some distortion on the calixarene that hinders its conformational mobility (see below). In comparison with the pre-click inclusion complexes, the ¹H NMR spectra display a higher number of signals, consistent with a symmetry decrease from C<sub>3v</sub> to C<sub>s</sub> (Figure 1). Although the ImCH<sub>2</sub> protons resonate as a singlet in the inclusion complexes, after reaction they split into three signals: One singlet and two doublets experiencing a ²J coupling. This is consistent with the existence of a σ<sub>v</sub> symmetry plane, which differentiates the ImCH<sub>2</sub> that belongs to the σ<sub>v</sub> plane (one singlet integrating for 2H) and two pairs of protons (two doublets integrating for 4H). Likewise, the initial HAr<sub>N3</sub> signal is a singlet. After desymmetrization, the six protons are differentiated into

¹H NMR spectroscopy (Figure 2B, D, and G), evidencing further the high selectivity of the reaction. A new singlet in

three singlets integrating for two protons each: One singlet for HAr<sub>tria</sub> and two singlets for the remaining HAr<sub>N3</sub>. The
initial tBu singlet is also split into two singlets integrating for 9H (for the tBu that belongs to the symmetry plane) and 18H (for the two tBu on either side of the symmetry plane). In the same manner, the methoxy groups (OCH₃) are split from one singlet (9H) into two singlets integrating for 3H and 6H, respectively. This split is a general trend for all the other groups of protons (NCH₃, ImH) for which one set of protons belongs to the symmetry plane and the other two sets are on each side of the plane.

**Optimization of the chain length:** All three monofunctionalized complexes were obtained in high yields after reaction for 2 hours for (C₃)NH₂ and (C₄)NH₂, and 5 hours for (C₅)NH₂. We wondered what was the impact of the length of the spacer on the kinetics of the reaction. To answer this question, two competitive monoclick reactions were carried out: (C₃)NH₂ versus (C₄)NH₂ and (C₄)NH₂ versus (C₅)NH₂. The monoclick reaction is a two-step process: Inclusion of the amino guest followed by intramolecular Huisgen cycloaddition. To fully understand the origin of the anticipated difference in the reaction rates, the thermodynamic and kinetic parameters of these two steps were analyzed (see Figure 1 for the definition of the parameters Kₙ and kₙ).

The competitive complexation studies were carried out in CD₃CN at 300 K. A known mixture of 4-pentyn-1-amine (n=3) and 5-hexyn-1-amine (n=4) was added to a solution of precursor complex [ZnX₆N₃]²⁺ in CD₃CN to yield quantitatively a mixture of inclusion complexes HG₃ and HG₄ (Figure 3C), identified by comparison with the pure inclusion complexes (Figure 3A and B).

Relative integration of the upfield resonances of the coordinated guests provided the relative ratio of the inclusion complexes, which, corrected by the ratio of the free amines, provided the ratio of the binding constants: K₄/K₃=(1.81±0.20).[17] A similar competition experiment was conducted with (C₄)NH₂ and (C₅)NH₂, which provided the ratio K₄/K₅=(1.87±0.20) (for more details, see the Supporting Information). Therefore (C₄)NH₂ is the best amino-acetylene ligand of all three for inclusion. The formation of the host–guest complexes is enthalpically driven by the formation of a bond between NH₂ and the metal center.[18] The moderate selectivity of the host complex for (C₄)NH₂ is probably controlled by secondary CH–π interactions between the methylene groups and the calixarene cavity.

We then studied how the difference in the pre-organization of the host–guest inclusion complexes HGₙ influences the rate of the cycloaddition reaction by means of competitive monoclick reactions. A known mixture of host–guest inclusion complexes HG₃ and HG₄ and a large excess of the corresponding amines in toluene were heated at 110°C for 2 hours and, after isolation, the resulting mixture of monofunctionalized calixarenes [ZnM(C₃)NH₂]²⁺ and [ZnM(C₄)NH₂]²⁺ was analyzed by ¹H NMR spectroscopy in CD₃CN (Figure 3D–F). The kinetic law for each intramolecular reaction is given by Equation (1).

$$v_n = k_n [HG_n] = k_n K_n [C_n NH_2] [ZnX_6 N_3] / [H_2O] \quad (1)$$

During the reaction, the ratio [(C₃)NH₂]/[(C₄)NH₂] varies little and thus can be considered as a constant. Hence, we can write Equation (2).

$$v_4/v_3 = [ZnM_{(C_4)NH_2}]_\infty/[ZnM_{(C_3)NH_2}]_\infty = k_4 [HG_4]_0/k_3 [HG_3]_0 \quad (2)$$

From this we calculate Equation (3) (for more details, see the Supporting Information).

$$k_4/k_3 = ([ZnM_{(C_4)NH_2}]_\infty/[ZnM_{(C_3)NH_2}]_\infty)([HG_3]_0/[HG_4]_0) \quad (3) \\ = (1.25 \pm 0.10)$$

A similar experiment was conducted with amines (C₄)NH₂ and (C₅)NH₂, which provided k₄/k₅=(2.28±0.20).

Therefore, the highest rate constant for the intramolecular cycloaddition was reached with 5-hexyn-1-amine, which indicates that the geometry of the corresponding host–guest complex HG₄ is the closest to the transition state. In this case, the acetylene faces best the three azido groups. The effect is more significant when (C₄)NH₂ is compared with the longest chain (C₅)NH₂.

The trends in the values of the rate constants and the complexation constants follow the same pattern. Hence, the best ligand leads coincidentally to the highest reaction rate. 5-Hexyn-1-amine is unambiguously the best substrate for this monoclick reaction due to the synergy between the two phenomena. The synergy between thermodynamics and kinetics is often at work in enzymatic systems. It can be quantified in the Michaelis–Menten model by the ratio kcat/KM, kcat being the turnover number and KM the Michaelis constant. When the assumption of a rapid equilibrium is fulfilled, KM is equal to the dissociation constant of the enzyme–substrate complex.[19] In the system presented herein, the ratio kcat/KM is equivalent to kₙKₙ. Calculation gives k₄K₄/k₃K₃=2.2 and k₄K₄/k₅K₅=4.3. The highest value for k₄K₄ indicates a better specificity of the transformation of the trisazido–calixarene complex with the (C₄)NH₂ substrate. Ac-

<figure><img src="image_7.png" /><figcaption>Figure 3. ¹H NMR (CD₃CN, 500 MHz, 300 K) spectra in the high-field region. From bottom to top: A) HG₃, B) HG₄, C) HG₄+HG₃ with 10 equiv of a 1:1 mixture of (C₃)NH₂/(C₄)NH₂, D) [ZnM(C₃)NH₂]²⁺ + [ZnM(C₄)NH₂]²⁺ after heating of mixture C in toluene at 110°C, E) [ZnM(C₃)NH₂]²⁺, and F) [ZnM(C₄)NH₂]²⁺. The resonances of the β- and γ-methylene groups of the coordinated alkyl chain are indicated.</figcaption></figure>
<table><caption>Table 1. Chemical shifts (CD₃CN, 500 MHz, 300 K[¹]) of species investigated in this study.[b]</caption><thead><tr><th></th><th>α</th><th>β</th><th>γ</th><th>δ</th><th>ε</th><th>tBu</th><th>δ [ppm]<br>OCH<sub>3</sub></th><th>NCH<sub>3</sub></th><th>ImCH<sub>2</sub></th><th>HArN</th><th>ImH</th></tr></thead><tbody><tr><td>[ZnX<sub>6</sub>N<sub>3</sub>]<sup>2+</sup></td><td></td><td></td><td></td><td></td><td></td><td>1.38</td><td>3.65</td><td>3.65</td><td>5.04</td><td>5.91</td><td>6.88</td></tr><tr><td>(C<sub>3</sub>)NH<sub>2</sub></td><td>2.79</td><td>1.64</td><td>2.24</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HG<sub>3</sub></td><td>0.74</td><td>-0.82</td><td>-0.13</td><td></td><td></td><td>1.40</td><td>3.58</td><td>3.79</td><td>5.31</td><td>6.17</td><td>6.93</td></tr><tr><td>CIS<sub>3</sub></td><td>-2.05</td><td>-2.46</td><td>-2.37</td><td></td><td></td><td>0.02</td><td>-0.07</td><td>0.14</td><td>0.27</td><td>0.26</td><td>0.05</td></tr><tr><td>[ZnM<sub>(C<sub>3</sub>)NH<sub>2</sub></sub>]<sup>2+</sup></td><td>0.64</td><td>-0.66</td><td>1.15</td><td></td><td></td><td>1.43 (9H)<br>1.34 (18H)</td><td>3.80 (3H)<br>3.58 (6H)</td><td>3.80 (3H)<br>3.76 (6H)</td><td>5.34 (s)<br>5.02 (d1)<br>5.23 (d2)</td><td>6.00 (N<sub>3</sub>)<br>6.22 (N<sub>3</sub>)<br>6.03 (tria)</td><td>6.84 (1H)<br>6.85 (2H)</td></tr><tr><td>Δδ<sub>3</sub></td><td>-0.10</td><td>0.16</td><td>1.28</td><td></td><td></td><td>0.03 (9H)<br>-0.06 (18H)</td><td>0.22 (3H)<br>0.00 (6H)</td><td>0.01 (3H)<br>-0.03 (6H)</td><td>0.03 (s)<br>-0.29 (d1)<br>-0.08 (d2)</td><td>-0.17 (N<sub>3</sub>)<br>0.05 (N<sub>3</sub>)<br>-0.14 (tria)</td><td>-0.09 (1H)<br>-0.08 (2H)</td></tr><tr><td>(C<sub>4</sub>)NH<sub>2</sub></td><td>2.7</td><td>1.55</td><td>1.55</td><td>2.2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HG<sub>4</sub></td><td>0.75</td><td>-0.97</td><td>-0.73</td><td>1.22</td><td></td><td>1.39</td><td>3.57</td><td>3.79</td><td>5.34</td><td>6.21</td><td>6.93</td></tr><tr><td>CIS<sub>4</sub></td><td>-1.95</td><td>-2.52</td><td>-2.28</td><td>-0.98</td><td></td><td>0.01</td><td>-0.08</td><td>0.14</td><td>0.30</td><td>0.30</td><td>0.05</td></tr><tr><td>[ZnM<sub>(C<sub>4</sub>)NH<sub>2</sub></sub>]<sup>2+</sup></td><td>0.78</td><td>-1.09</td><td>-0.25</td><td>1.57</td><td></td><td>1.41 (9H)<br>1.34 (18H)</td><td>3.76 (3H)<br>3.57 (6H)</td><td>3.81 (3H)<br>3.77 (6H)</td><td>5.37 (s)<br>5.18 (d1)<br>5.28 (d2)</td><td>6.10 (N<sub>3</sub>)<br>6.24 (N<sub>3</sub>)<br>6.28 (tria)</td><td>6.87 (1H)<br>6.90 (2H)</td></tr><tr><td>Δδ<sub>4</sub></td><td>0.03</td><td>-0.12</td><td>0.48</td><td>0.35</td><td></td><td>0.02 (9H)<br>-0.05 (18H)</td><td>0.19 (3H)<br>0.00 (6H)</td><td>0.02 (3H)<br>-0.02 (6H)</td><td>0.03 (s)<br>-0.16 (d1)<br>-0.06 (d2)</td><td>-0.11 (N<sub>3</sub>)<br>0.03 (N<sub>3</sub>)<br>0.07 (tria)</td><td>-0.06 (1H)<br>-0.03 (2H)</td></tr><tr><td>(C<sub>5</sub>)NH<sub>2</sub></td><td>2.71</td><td>1.55</td><td>1.55</td><td>1.55</td><td>2.17</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>HG<sub>5</sub></td><td>0.75</td><td>-1.08</td><td>-0.92</td><td>0.52</td><td>1.80</td><td>1.40</td><td>3.56</td><td>3.80</td><td>5.34</td><td>6.22</td><td>6.92</td></tr><tr><td>CIS<sub>5</sub></td><td>-1.96</td><td>-2.63</td><td>-2.47</td><td>-1.03</td><td>-0.37</td><td>0.02</td><td>-0.09</td><td>0.15</td><td>0.30</td><td>0.31</td><td>0.04</td></tr><tr><td>[ZnM<sub>(C<sub>5</sub>)NH<sub>2</sub></sub>]<sup>2+</sup></td><td>0.70</td><td>-1.11</td><td>-0.66</td><td>0.70</td><td>2.09</td><td>1.42 (9H)<br>1.38 (18H)</td><td>3.71 (3H)<br>3.62 (6H)</td><td>3.82 (3H)<br>3.83 (6H)</td><td>5.37<sup>[a]</sup> (s)<br>5.35<sup>[a]</sup> (d1)<br>5.42<sup>[a]</sup> (d2)</td><td>6.16<sup>[a]</sup> (N<sub>3</sub>)<br>6.18<sup>[a]</sup> (N<sub>3</sub>)<br>6.70<sup>[a]</sup> (tria)</td><td>6.92 (1H)<br>7.02 (2H)</td></tr><tr><td>Δδ<sub>5</sub></td><td>-0.05</td><td>-0.03</td><td>0.26</td><td>0.18</td><td>0.29</td><td>0.02 (9H)<br>-0.02 (18H)</td><td>0.15 (3H)<br>0.06 (6H)</td><td>0.02 (3H)<br>0.03 (6H)</td><td></td><td></td><td>0.00 (1H)<br>0.10 (2H)</td></tr></tbody></table>

[a] With the exception of the peaks corresponding to CH₂Im, HAr<sub>N3</sub>, and HAr<sub>tria</sub> of [ZnM<sub>(C<sub>3</sub>)NH<sub>2</sub></sub>]<sup>2+</sup>, which are broad at 300 K and were recorded at 353 K. [b] First row is precursor complex [ZnX<sub>6</sub>N<sub>3</sub>]<sup>2+</sup>. Each of the other three rows (n=3, 4, 5) is divided into five sub-rows: A) Free amine (n=3, 4, 5); B) HG<sub>n</sub> (n=3, 4, 5); C) CIS (difference between δ<sub>FreeAmine</sub> in and out, or for the calixarene: difference between HG<sub>n=3,4,5</sub> and the initial complex; D) Monofunctionalized complex [ZnM<sub>CnNH<sub>2</sub></sub>]<sup>2+</sup> (n=3, 4, 5); E) Δδ<sub>n</sub> (difference between the chemical shifts of the monofunctionalized complexes [ZnM<sub>CnNH<sub>2</sub></sub>]<sup>2+</sup> and the corresponding inclusion complex HG<sub>n</sub>. The methylene groups of the alkyl chain are denoted as α, β, γ, δ, and ε, starting from the NH<sub>2</sub> group).

cordingly, n=4 corresponds to the optimized length for the spacer.

*Origin of the observed specificity based on a conformational analysis:* As pre-organization is key for this reaction, we studied the host–guest symmetry changes and the adaptation process that both the calixarene host and the guest alkyl chain must undergo to yield the monofunctionalized complex. To this end we analyzed the conformations of both the inclusion complexes and the monofunctionalized complexes in CD₃CN (see Figure 2 and Table 1).

1) Conformational comparison of the inclusion complexes HG<sub>n</sub>: In all cases, the aromatic walls of the calixarene cavity shield the amino guest upon complexation and lead to an upfield shift of the resonances of the alkyl chain.[13a,20] We defined the complex-induced shift (CIS) value as the difference between the chemical shifts of a group of protons in the inclusion complex HG<sub>n</sub> and in the free ligand. The CIS values are reported in Table 1. In all cases, H<sub>β</sub> has the greatest CIS value, which means that it is the most shielded. Nevertheless, from one guest

to another, the difference in CIS is 0.05 ppm at most, which indicates that the shielding effects are very similar. Therefore all three guests are positioned at the same height inside the calixarene cavity. Upon inclusion of the guests, some resonances of the calixarene unit remain almost unchanged. For instance, there is little effect of the guest complexation on the tBu (ca. 0.01 ppm) or ImH CIS (ca. 0.04 ppm). However, the resonances of ImCH₂ are different to those of the precursor complex, but are almost identical for the three amino guests (CIS ≈0.28 ppm). The same is true for methoxy groups (CIS≈-0.08 ppm) and HAr<sub>N3</sub> (CIS≈0.28 ppm). These data highlight a slight change in the conformation of the calixarene upon complexation of the guests. However, for all three, the conformational change is almost identical, which suggests that the difference in the reactivities of the three amino-alkynes does not come from the distortion of the calixarene at the complexation step.

2) Conformational analysis of the monoclick products [ZnM<sub>CnNH<sub>2</sub></sub>]<sup>2+</sup>: Unlike the inclusion complexes, there are clear differences between the spectra of the monoclick products, which indicates that the three products have
significantly different conformations (Figure 2). These
differences can be observed by measuring the differences
between the chemical shifts of the monoclick product
and the corresponding inclusion complex (Δδ<sub>n</sub>). To pic-
ture completely the influence of the chain length on the
structural properties, two conformational features need
to be discussed: Distortion of the calixarene cone and re-
stricted conformational mobility.

*Distortion of the calixarene cone:* After the click reaction, the ArBu units are differentiated but in all three cases the Δδ values are very close to zero for all complexes. Therefore the ArBu units see little conformational change following the cycloaddition step, which is consistent with the fact that these aromatic units are projected away from the calixarene cavity and are little affected by modifications to the inside of the cavity (Figure 4). The aromatic unit that sees the

<figure><img src="image_8.png" /><figcaption>Figure 4. Schematic representations of A) the pivot of the Ar<sub>tria</sub> unit by an α<sub>p</sub> angle (side-view, the tBu and N<sub>3</sub> groups have been omitted for clarity) and B) the equilibrium of the helix inversion around the metal center and the distortion of the calixarene structure (top view, the N<sub>3</sub> groups have been omitted for clarity).</figcaption></figure>

greatest environmental change is the Ar<sub>tria</sub> unit because it
becomes covalently linked to the coordinated amino chain.
In all three cases, the Δδ<sub>n</sub> value for its methoxy group
(Table 1, column OCH<sub>3</sub> (3H)) is positive. This indicates that
the methoxy groups are pushed away from the cavity forcing
the Ar<sub>tria</sub> unit to pivot towards the inside of the shielding
cavity (Figure 4A). Interestingly, Δδ<sub>n</sub> for the methoxy group
increases as the alkyl chain becomes shorter (0.15, 0.19, and
0.22 ppm for n=5, 4, and 3, respectively), which indicates
that with a shorter chain, the aromatic unit pivots more to-
wards the inside of the cavity. Although the signs of the Δδ<sub>n</sub>
values for HAr<sub>tria</sub> can hardly be interpreted in terms of con-
formational change because the chemical transformation of
azido to triazole has a great influence on the chemical shifts,
their relative values can be compared. The Δδ<sub>n</sub> values for
HAr<sub>tria</sub> (0.48, 0.07, and −0.14 ppm for n=5, 4, and 3, respec-

tively) and the H<sub>tria</sub> chemical shifts (7.33, 7.24, and 7.15 ppm for *n*=5, 4, and 3, respectively) decrease significantly when the spacer is shorter, which also indicates that the Ar<sub>tria</sub> aromatic unit rotates more towards the inside of the cavity when the chain is shorter.

The rotation of the two remaining Ar<sub>N3</sub> units can be meas-
ured by the average chemical shift of their aromatic protons
HAr<sub>N3</sub>. In all cases, the Ar<sub>N3</sub> units are in very similar posi-
tions (the average chemical shift for the HAr<sub>N3</sub> protons is
6.11 ppm for [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> and 6.17 ppm for both
[ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> and [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>). Therefore there is no sig-
nificant impact of the length of the spacer on the pivot of
the remaining Ar<sub>N3</sub> units.

The symmetry decrease from C₃ᵥ to Cₛ gives rise to newly
differentiated protons that no longer resonate at the same
frequency because they perceive different environments.
The difference in the chemical shifts of protons that are no
longer equivalent is an indication of the difference in the en-
vironment of these protons. Therefore a greater splitting in
the ¹H NMR spectrum is indicative of a greater distortion of
the calixarene cone. The splitting between the two sets of
methoxy groups is greater when the chain is shorter (0.09,
0.19, and 0.22 ppm for *n*=5, 4, and 3, respectively). This
phenomenon can also be observed for the two HArN₃ signals
(0.02, 0.14, and 0.22 ppm for *n*=5, 4, and 3, respectively),
the two ImCH₂ doublets (0.07, 0.10, and 0.21 ppm for *n*=5,
4, and 3, respectively), and, to a lesser extent, the two tBu
signals (0.04, 0.07, and 0.09 ppm for *n*=5, 4, and 3, respec-
tively). Therefore shorter chains accentuate the distortion of
the cone of the calixarene.

*Resonance broadening and restricted conformational mobility:* With *n*=5 only, a new phenomenon occurs: At 300 K, the resonances of ImCH<sub>2</sub>, HAr<sub>tria</sub>, and HAr<sub>N3</sub> of [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> are broad (Figure 2F) whereas they are sharp for [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> and [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> at the same temperature. In addition, the resonances of the ArCH<sub>ax</sub> protons, which are broad for [ZnM<sub>(C<sub>3</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> and [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>, become even broader and can barely be seen in the spectrum (Figure 2F), which is indicative of a different conformational mobility of [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>. Indeed, at 353 K, all these resonances are sharper (Figure 2G). This reflects the fact that the longer chain imposes a constrained conformation on the calixarene structure and more precisely on the coordination arms (through the coordination of the amino group to the Zn<sup>II</sup> ion) and the Ar<sub>tria</sub> unit (through the triazole group). Thus, [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> has a restricted conformational mobility that impacts on the helix inversion (see below).

*Dynamics of the helix inversion at the metal center:* We focused then on the influence of self-coordination on helix inversion, a conformational change experienced at the Zn¹¹ cation coordination sphere (Figure 4B).[21] To study the dynamics of such a process, we conducted ¹H NMR experiments at various temperatures in CD₃CN and in CDCl₃, a noncoordinating solvent (see the Supporting Information).
<figure><img src="image_9.png" /><figcaption>Figure 5. ¹H NMR spectra of monofunctionalized complex [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> (CD<sub>3</sub>CN, 500 MHz) at various temperatures (s: residual solvent).</figcaption></figure>

The ¹H NMR spectrum of [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> in CD<sub>3</sub>CN (Figure 2D) is that of a C<sub>5</sub>-symmetric species, which means that at 300 K the helicoidal inversion is fast on the NMR time-scale. The loss of C<sub>5</sub> symmetry in favor of the C<sub>1</sub>-symmetric species can be observed when the solution is cooled to 240 K (Figure 5). Indeed, there are six signals for the six [HAr<sub>N3</sub>+HAr<sub>tria</sub>] protons. There is also a clear splitting of the β- and γ-protons, which indicates that they have become diastereotopic. The ImCH<sub>2</sub> protons are divided into three AB systems. As previously observed for the tris-imidazole funnel complexes, the NMR data are consistent with the helicoidal structure of the coordination arms around the Zn<sup>II</sup> ion.[21a] This helicoidal chirality is transferred to the whole calixarene structure that twists around its axis. In CD<sub>3</sub>CN, the inversion barrier between the two helices is ΔG̸=̄=(50.7±0.4) kJmol<sup>-1</sup> and was calculated for the CH<sub>2</sub>(γ) protons at their coalescence temperature (T<sub>c</sub>= (260±2) K). The spectrum of [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> in CD<sub>3</sub>CN is broad at 300 K (Figure 2F) and greatly resembles the spectrum of [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> in CD<sub>3</sub>CN at 280 K. This indicates that the helicoidal inversion is slower for [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> than for [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>. Indeed, the inversion barrier between the two helices is ΔG̸=̄=(54.9±0.4) kJmol<sup>-1</sup> for [ZnM<sub>(C<sub>5</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> in CD<sub>3</sub>CN (calculated for the CH<sub>2</sub>(γ) protons at their coalescence temperature T<sub>c</sub>=(265±2) K), which is greater than that for [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>. This result is also consistent with the competition study. In this case the spacer is too long so the chain is packed in the cavity and impacts on the mobility of the whole structure. Thus, the competition experiments are plainly corroborated by the conformational analysis of the monofunctionalized products by reasoning on a late transition state. A C<sub>3</sub> spacer is too short and imposes a distortion on the calixarene cone whereas a C<sub>5</sub> spacer is too long and imposes constrained conformations on the global structure. Both spacers compel energetically unfavorable constraints on their corresponding transition states, which explains their high energies relative to the optimal C<sub>4</sub> spacer.

In conclusion to this extensive conformational analysis,
we have shown that the length of the spacer is a key ele-

ment for the monoclick reaction. The optimal four-atom
chain length ensures the best inclusion of the guest as well
the highest reactivity of the host–substrate complex. In addi-
tion, the nature of the spacer has a great impact on the con-
formation of the monofunctionalized complex and on its
conformational mobility.

**Monofunctionalization with other amino guests:** Having determined that the optimal length for the spacer is *n*=4, we decided to test other amino guests with four carbon atoms between the amino and the acetylene groups. The benzyl-amine-based guest (ArCH<sub>2</sub>NH<sub>2</sub>) presents a more rigid spacer. The other two substrates have the same aliphatic spacer as 5-hexyn-1-amine and the acetylene is substituted by *p*-methoxyphenyl (*p*MeOAr(C<sub>4</sub>NH<sub>2</sub>) or *p*-nitrophenyl (*p*NO<sub>2</sub>Ar(C<sub>4</sub>NH<sub>2</sub>); Figure 6).

<figure><img src="image_10.png" /><figcaption>Figure 6. Aminoalkynes with a C<sub>4</sub> spacer.</figcaption></figure>

**Monofunctionalization with ArCH<sub>2</sub>NH<sub>2</sub>:** The monoclick reaction proceeds in high yield with ArCH<sub>2</sub>NH<sub>2</sub> (95%). The reaction is complete within 2 hours, like the corresponding aliphatic 5-hexyn-1-amine. The inclusion complex was obtained quantitatively by adding 1.1 equivalents of 3-ethynylbenzylamine to the [ZnX<sub>6</sub>N<sub>3</sub>]<sup>2+</sup> complex. The resonances of the guest protons are all shifted upfield, which is consistent with the inclusion of the guest in the cavity. The strongest shielding effects can be observed on H<sub>a</sub> (CIS = -2.84 ppm) and H<sub>d</sub> (CIS = -2.50 ppm), which is stronger than with an alkyl spacer (see Figure 7 for assignment). Protons H<sub>b</sub> and H<sub>c</sub> are also shielded, but to a lesser extent, which indicates that their positions are lower in the calixarene cavity. However, the CIS values for the calixarene protons are all very close to the CIS values for HG<sub>4</sub> (see the Supporting Information, Table 4.1), which means that the rigidity of the guest ligand does not have an impact on the conformation of the host–guest complex.

The ¹H NMR (CD₃CN, 500 MHz, 300 K) spectrum of [ZnM<sub>ArCH<sub>2</sub>NH<sub>2</sub></sub>]<sup>2+</sup> is consistent with a C<sub>5</sub>-symmetrical complex. All the chemical shifts of the calixarene structure are similar to those of the [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup> complex. In this case, the signals from the ArCH<sub>ax</sub> protons are very sharp doublets, whereas they give broad resonances in [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>. This indicates that they waver in a more restricted space in [ZnM<sub>ArCH<sub>2</sub>NH<sub>2</sub></sub>]<sup>2+</sup>. Consequently, we can conclude that
<figure><img src="image_11.png" /><figcaption>Figure 7. Top: Reaction pathway for the monoclick reaction with 3-ethynylbenzylamine. Bottom: ¹H NMR (CD₃CN, 500 MHz, 300 K) spectra of the inclusion complex HG<sub>ArCH2NH2</sub> and the monofunctionalized complex [ZnM<sub>ArCH2NH2</sub>]<sup>2+</sup> (f: free ligand; s: residual solvent; w: water).</figcaption></figure>

[ZnM<sub>ArCH2NH2</sub>]<sup>2+</sup> is more rigid than [ZnM<sub>(C₄)NH₃</sub>]<sup>2+</sup>. The rigidity of the guest is then transferred to the monofunctionalized calixarene structure. ¹H NMR studies indicate that all six aromatic units are oriented very similarly to [ZnM<sub>(C₄)NH₃</sub>]<sup>2+</sup> but the calixarene structure is much more distorted (see the Supporting Information for more details).

**Monofunctionalization with substituted acetylenes:** The monoclick reaction also proceeds in high yields with pNO₂Ar(C₄)NH₂ (91%) and pMeOAr(C₄)NH₂ (92%) substituted acetylenes but the reaction requires 5 hours to go to completion. This indicates that the substitution of the acetylene has a strong effect on the kinetics of the reaction.[22] This difference originates from a less favorable coordination step ($K_4/K_{pNO_2Ar(C_4)NH_2}$ = (1.60 ± 0.20)), see the Supporting Information for competition experiments) because of the bulky phenyl group in this position, as well as a less favorable intramolecular reaction ($k_4/k_{pNO_2Ar(C_4)NH_2}$ = (2.60 ± 0.20)). The discrimination based on host–guest adduct formation can be even more drastic: A bulkier group (anthracene) prevents the coordination of the amino guest and no conversion for the monoclick reaction is observed. In both cases the spacer is the same (n=4) but the acetylene is differently functionalized. The chemical shifts of the monofunctionalized complexes are all very similar to the chemical shifts of [ZnM<sub>(C₄)NH₃</sub>]<sup>2+</sup>. This indicates that the three monofunctionalized complexes [ZnM<sub>pOCH₃(C₄)NH₃</sub>]<sup>2+</sup>, [ZnM<sub>pNO₂Ar(C₄)NH₃</sub>]<sup>2+</sup>, and [ZnM<sub>(C₄)NH₃</sub>]<sup>2+</sup> have very similar conformations. Therefore the nature of the spacer is what determines the conforma-

tion of the calixarene structure. The functionalization of the triazole at the 5-position has little impact on this structure.

**Conclusions on the monoclick reactions with amine substrates:**

The monofunctionalized reaction developed with amine substrates is an easy, clean, and efficient method for selectively monofunctionalizing calix[6]arenes in high yields. We have established that there is an optimal length of four carbon atoms for the spacer, which originates from the synergy between the thermodynamically controlled complexation step and the kinetically controlled intramolecular cycloaddition step. For the same optimal length, different structures for the spacer and for the alkyne substituent can be used for the monoclick reaction provided they are not too bulky. The monoclick reaction works with

different amines, despite distorting the calixarene structure. The distortion has a low energetic cost, which indicates that we are dealing with a very flexible cavity system. The conformation of the monofunctionalized calixarene is imposed by the nature of the spacer. The spacer has a steric limit because it should fit into the cavity and not differ too much from the optimum n=4. There is also a steric limit for the group functionalizing the acetylene. Too much hindrance prevents coordination of the substrates and inhibits the intramolecular reaction.

**Monoclick reactions with primary alcohols:** Amines proved to be suitable for structural analysis because the intramolecular coordination is fully observed both in coordinating and noncoordinating solvents, thus avoiding a mixture of *endo* and *exo* isomers. Primary alcohols are weaker ligands than primary amines but their coordination to the Zn¹¹ ion can still be observed in noncoordinating solvents.[14a] Starting with the conditions found for the amine substrates, the synthesis was optimized for the alcohols. This optimization is discussed below. The synthesis of a calixarene complex monofunctionalized by a hydroxy group enabled us to study the “*endo/exo*” dynamic equilibrium that operates under certain conditions.

**Synthesis and characterization of the monoclick product [ZnM<sub>(C₄)OH</sub>]<sup>2+</sup>:** 5-Hexyn-1-ol is the strict equivalent to 5-hexyn-1-amine as the only modified parameter is the coordinating group. Because water competes with the alcohol for
coordination at the metal center, the reaction was conducted
in dry toluene at reflux. The optimized conditions required
the use of ten equivalents of 5-hexyn-1-ol (vs. 2 equiv for 5-
hexyn-1-amine) and 4 h of reflux (vs. 2 h for 5-hexyn-1-
amine). Unlike with primary amines, the resulting mono-
functionalized complex [ZnM(C₄)OH]²⁺ was generally isolated
with some starting material [ZnX₆N₃]²⁺ and a difunctional-
ized complex, which shows that the monoclick reaction with
5-hexyn-1-ol is less selective than with 5-hexyn-1-amine.
After purification by column chromatography (during which
decoordination of the metal cation occurred), pure mono-
functionalized ligand M(C₄)OH was obtained. Upon recom-
plexation with Zn(ClO₄)₂·(H₂O)₆ in CH₂Cl₂/CH₃CN, pure
monofunctionalized calixarene [ZnM(C₄)OH]²⁺ was isolated in
a yield of 70–95 % (over two steps) depending on the run.

The monofunctionalized ligand M<sub>(C<sub>4</sub>OH</sub> was characterized
by EIMS spectrometry (a single peak was observed at m/z =
1350.7 for [M<sub>C<sub>4</sub>OH</sub>+H]<sup>+</sup>). The <sup>1</sup>H NMR spectrum of the
ligand is broad in the usual solvents (CDCl<sub>3</sub>, CD<sub>3</sub>CN,
[D<sub>6</sub>]DMSO) at 300 K but the resonances are sharper at
360 K in [D<sub>6</sub>]DMSO (Figure 8). The appearance of a singlet

placed by one molecule of CD₃CN (exo situation for the
chain). A second minor species can also be detected.
Indeed, shielded resonances were also observed at –0.25
and –1.03 ppm, attesting to the existence of a self-coordi-
nated complex (endo situation for the chain). At 300 K, the
exo to endo ratio, evaluated by integration of the signals of
the alkyl chain, is 93:7. This ratio was evaluated at different
temperatures between 270 and 340 K (see the Supporting
Information). Self-coordination of the hydroxy chain is fa-
vored at lower temperatures. The van't Hoff plot for the
equilibrium of the displacement of the coordinated hydroxy
chain by an acetonitrile molecule (endo/exo equilibrium)
gave the following thermodynamic parameters: Δ<sub>1</sub>H° =
11 kJmol<sup>–1</sup> and Δ<sub>2</sub>S° = 59 JK<sup>–1</sup>mol<sup>–1</sup>. The positive enthalpic
change reflects the better affinity of the zinc cation for an
alcohol compared with a nitrile.[13a] The favorable change in
entropy can be rationalized by the expulsion of the alkyl
chain outside of the cavity due to the decoordination and
gain of conformational flexibility of both the chain and the
calixarene core. In CD₃CN, the addition of one equivalent
of a primary amine like propylamine yields quantitatively

the inclusion complex
$[ZnM(C_4OH)(PrNH_2)]^{2+}$, which
means that both the self-coordi-
nated hydroxy chain and the
acetonitrile molecule are dis-
placed. This displacement is
enthalpically driven by the
strength of the Zn–N bond.
The behavior of complex
$[ZnM(C_4OH)]^{2+}$ is very similar in
CD_3OD because the two com-
plexes (endo and exo) coexist at
300 K, the exo complex being
largely favored (see the Sup-
porting Information). In the
noncoordinating solvent CDCl_3
at 325 K, the upfield resonances
of the β- and γ-methylenes at
–1.08 and –0.21 ppm, respec-
tively, as well as the absence of
resonances at 3.40 and
2.70 ppm for the α- and δ-meth-

ylenes, suggest the presence of only the self-coordinated
complex. The only competing ligand in the media is residual
water, which is not strong enough to displace the intramo-
lecularly coordinated hydroxy chain.

Thus, the host–guest properties of the [ZnM(C₄)OH]²⁺ com-
plex are very different to those of its amino analogue. The
hydroxy chain can be chased out of the cavity by an exoge-
nous alcohol, nitrile, or primary amine (Scheme 3). Never-
theless, compared with classic funnel complexes like
[ZnX₆tBu₆]²⁺, the affinity of [ZnM(C₄)OH]²⁺ for alcohols and
nitriles is greatly reduced due to the control of the accessi-
bility of the metal center by the self-coordination of the in-
ternal arm. In contrast, the amino-functionalized calixarene
remains self-coordinated in all tested conditions.

<figure><img src="image_12.png" /><figcaption>Figure 8. Bottom: ¹H NMR (500 MHz) spectrum of ligand M<sub>C4OH</sub> ([D<sub>6</sub>]DMSO, 360 K). Top: ¹H NMR (500 MHz) spectrum of complex [ZnM<sub>C4OH</sub>]²⁺ with zoom of the signals of the encapsulated β- and γ-methylene groups in the upfield region (CD<sub>3</sub>CN, 300 K) (s: residual solvent; w: water).</figcaption></figure>

at 7.64 ppm (integrating for 1H) for H<sub>tria</sub> on the newly
formed triazole group, as well as the resonances for the one
clicked alkyl chain attest to the monofunctionalization of
the calixarene. In addition, the splitting of the tBu, OCH<sub>3</sub>,
and ImCH<sub>2</sub> groups is consistent with a C<sub>s</sub>-symmetrical spe-
cies.

The monofunctionalized complex [ZnM<sub>(C<sub>4</sub>OH)</sub>]<sup>2+</sup> was char-
acterized by EIMS (m/z = 1512.6 for [ZnM<sub>(C<sub>4</sub>OH</sub>+ClO<sub>4</sub>]<sup>+</sup>).
The <sup>1</sup>H NMR spectrum in CD<sub>3</sub>CN at 300 K displays sharp
resonances and evidences one major product, consistent
with a monofunctionalized calixarene (Figure 8). In contrast
to [ZnM<sub>(C<sub>4</sub>NH<sub>2</sub>)</sub>]<sup>2+</sup>, the resonances of the alkyl chain in
[ZnM<sub>(C<sub>4</sub>OH)</sub>]<sup>2+</sup> are not shielded. This indicates that the hy-
droxy group is not coordinated to the zinc cation and is dis-
<figure><img src="image_13.png" /><figcaption>Scheme 3. Favorable displacement of the self-coordinated hydroxy chain of complex [ZnM<sub>C<sub>4</sub>OH</sub>](ClO<sub>4</sub>)<sub>2</sub> by coordinating solvents MeCN, MeOH, or PrNH<sub>2</sub> in stoichiometric proportions in any solvent.</figcaption></figure>

Variable-temperature (VT) NMR experiments were con-
ducted on [ZnM<sub>(C<sub>4</sub>OH)</sub>]<sup>2+</sup> in CDCl<sub>3</sub> (see the Supporting Infor-
mation). The results are very similar to those obtained with
[ZnM<sub>(C<sub>4</sub>NH<sub>3</sub>)</sub>]<sup>2+</sup>. Indeed, at 325 K, the spectrum of
[ZnM<sub>(C<sub>4</sub>OH)</sub>]<sup>2+</sup> is characteristic of a C<sub>8</sub>-symmetrical species in
which the helicoidal inversion of the imidazole arms is fast
on the NMR timescale. At 300 K, the signals broaden and at
266 K the spectrum is characteristic of a C<sub>1</sub>-symmetrical spe-
cies in which the helicoidal inversion has become very slow.
The inversion barrier was calculated for protons H<sub>γ</sub> at their
coalescence temperature (302 K): ΔG<sup>‡</sup> = (59.7 ±
0.5) kJmol<sup>-1</sup>. The inversion barrier is the same as for
[ZnM<sub>(C<sub>4</sub>NH<sub>3</sub>)</sub>]<sup>2+</sup> in the same solvent (ΔG<sup>‡</sup> = (59.3 ±
0.5) kJmol<sup>-1</sup>). Although we have observed that the inver-
sion barrier of the helicoidal equilibrium is sensitive to the
length of the spacer, there is little impact of the nature of
the coordinating group.

Monoclick reactions with other alcohol substrates: As in the case of the amines, the spacer can be slightly modified. Other alcohols such as 3-ethynylbenzyl alcohol (ArCH₂OH) and 4-pentyn-1-ol ((C₃)OH) allow a change in the spacer (Figure 9). Acetylene can also be substituted by a 4-nitrophenyl group, for example, pNO₂ArC₄OH. The monoclick reaction was conducted under the same conditions (10 equiv of 4-pentyn-1-ol or 3-ethynylbenzyl alcohol at reflux in dry toluene for 4 h). In both cases, the major product was the monofunctionalized product, but the precursor complex and the difunctionalized product were also usually observed. After purification by column chromatography and recom-

<figure><img src="image_14.png" /><figcaption>Figure 9. Hydroxy-acetylenes bearing different spacers and functionali-
ties.</figcaption></figure>

plexation with Zn(ClO₄)₂·(H₂O)₆, the corresponding mono-
functionalized complexes [ZnM(C₃)OH]²⁺ and [ZnM(ArCH₂OH)]²⁺
were obtained in good yields of 75–95%. Both complexes
were characterized by EIMS (m/z = 1498.6 for [ZnM(C₃)OH +
ClO₄]⁺ and m/z = 1546.6 for [ZnM(ArCH₂OH + ClO₄)]⁺). In con-
trast to [ZnM(C₄)OH]²⁺, the ¹H NMR spectra of [ZnM(C₃)OH]²⁺
and [ZnM(ArCH₂OH)]²⁺ in CD₃CN evidence only one C₈-sym-
metrical species with the pendant hydroxy chain out of the
cavity and an acetonitrile molecule coordinated to the Zn¹¹
ion. No self-coordinated complexes were observed.

The reaction conducted with pNO₂ArC₄OH was much
cleaner and much more selective. Indeed, the pure mono-
functionalized complex [ZnM<sub>pNO₂ArC₄OH</sub>]<sup>2+</sup> was easily isolat-
ed after precipitation. It was characterized by EIMS (m/z =
1633.6 for [ZnM<sub>pNO₂ArC₄OH</sub> + ClO₄]<sup>+</sup>). Its ¹H NMR spectrum
in CD₃CN at 300 K evidences a 75:25 mixture of the *exo*
and *endo* complexes. VT NMR experiments in CD₃CN in
the range 270–325 K gave the following thermodynamic pa-
rameters for the *endo/exo* equilibrium: Δ<sub>γ</sub>H° = 3.8 kJmol<sup>-1</sup>
and Δ<sub>γ</sub>S° = 22 JK<sup>-1</sup>mol<sup>-1</sup>. Compared with [ZnM<sub>(C<sub>4</sub>)OH</sub>]<sup>2+</sup>, the
self-coordination is enthalpically less favored, likely caused
by the steric hindrance exerted by the *p*-nitrophenyl group
at the large rim. The change in entropy is also much less
positive, which can hypothetically be explained by the re-
duced mobility of the alkyl chain in the vicinity of the *p*-ni-
trophenyl group. These results evidence a stronger self-coor-
dination effect with the *p*-nitrophenyl group compared with
other alcohols, which diminishes the possibility of the dis-
placement of the self-coordinated hydroxy chain in
[ZnM<sub>pNO₂ArC₄OH</sub>]<sup>2+</sup> by a second pNO₂ArC₄OH molecule
and thus rationalizes the better selectivity of the monoclick
reaction. In CD₃CN, both the coordinated acetonitrile mole-
cule and the self-coordinated hydroxy chain were classically
displaced by the addition of one equivalent of propylamine
to yield quantitatively the inclusion complex
[ZnM<sub>pNO₂ArC₄OH</sub>(PrNH₂)]<sup>2+</sup>.

**Conclusion**

Selective functionalization of a macrocyclic structure pre-
senting multiple and equally reactive sites is a real synthetic
challenge. It generally represents a limiting step for append-
ing valuable and spatially well-defined functions. In this
paper we have presented a strategy based on host–guest
chemistry that allows for the selective reaction of one out of
three equivalent units of a calix[6]arene core. The strategy
was inspired by natural systems, that is, enzymes that are
well known to perform highly selective reactions under very
mild conditions. The key points for enzymatic transforma-
tions are the formation of an enzyme–substrate complex
and preorganization of the reactive partners (reactant/sub-
strate) in the active-site pocket, which lead to low activation
energy and high selectivity. The reaction implemented here
is the thermal Huisgen cycloaddition, which, when noncata-
lyzed, requires high temperatures and leads to low regiose-
lectivities. To take advantage of the host properties of the
calix[6]arene-based ZnII funnel complexes, three azido groups were introduced at the large rim of the cavity (the reactant) and a coordinating moiety was appended to the alkyne substrates through a spacer (substrate). To explore the potentialities of this supramolecular strategy, four parameters were varied: 1) The nature of the coordinating moiety (primary amine vs. alcohol), 2) the length of the spacer from (CH₂)₃ to (CH₂)₅, 3) the shape of the spacer (linear alkane vs. phenyl nucleus), and 4) the substituent at the terminal site of the alkyne substrate.

Detailed structural and kinetic studies have evidenced in-
teresting biomimetic features. The better the guest, the
higher the reaction rate, which is related to thermodynamics
through the host–guest binding constant K. The less sterical-
ly constrained the product, the higher the rate constant,
which proves that embedment also has a significant impact
on the transition state.

The high sensitivity of the system is nicely illustrated by
the fact that a change of only one methylene group in the
spacer length has a significant impact. The optimal C₄ length
allows both a better preorganization and a lower-energy
transition state in comparison with C₃ and C₅. Nevertheless,
the flexibility of the host makes possible the efficient em-
bedment of either a linear alkyl chain or a cyclic moiety.
The efficiency of the confined reaction also permits alkynes
bearing a sterically encumbered aromatic substituent at
their terminal position to be grafted. All these results show
that such a pre-association mimicking the Michaelis–Menten
enzyme–substrate complex allows for the selective orienta-
tion of reactant versus substrate and a good pre-organiza-
tion leading to lower activation energies. This is illustrated
by the remarkably mild conditions under which this thermal
Huisgen reaction proceeds compared with classical bimolec-
ular reactions and also by the remarkable regioselectivity of
the cycloaddition.

Finally, it is important to note that another key point of
this synthetic strategy is the self-inhibition of the process.
After covalent capture of a single guest, the intramolecular
coordination link lowers the affinity of the metal center for
exogenous ligands. With very good donors such as amines,
the *endo* coordination is very strong and quasi-quantitative
yields of monofunctionalized product are obtained. With the
weaker donor alcohols, the transformation is less selective
because the formation of around 5–10% of dialkylated
products was observed. This is attributable to the more
labile alcohol–ZnII link that leads to more facile competition
for the *endo* binding of a second equivalent of guest alcohol.
Interestingly, the study related to such *endo–exo* competitive
binding revealed that the self-coordination process of the
appended alcohol function can be switched on and off by
the presence of a more or less competing ligand. The equi-
librium is shifted towards the *endo* adduct in pure CDCl₃
whereas the *exo* situation is obtained in the presence of one
equivalent of a primary amine. The “ouroboros-like” behav-
ior of this system actually offers the possibility of controlling
the access to the cavity by using different kinds of stimuli.
In this context, the use of an electrochemical input tuning

the redox state of a copper ion is under investigation in our
laboratory.

In conclusion, an in-depth analysis of the main factors governing the selectivity and efficiency of the supramolecular covalent capture process has shown that such a strategy is quite versatile and should be applicable to other cavitands associated with other recognition patterns. Also, this strategy could possibly be extended to other covalent capture reactions. Interestingly, such a covalent capture strategy has been recently applied to the synthesis of a carbonic anhydrase II mutant.[23]

**Experimental Section**

**General procedure for the monoclick reaction with amines:** Precursor complex [ZnX₆N₃](ClO₄)₂ (200 mg, 0.13 mmol, 1.0 equiv) was added to a solution of the appropriate amine (2 to 3 equiv) in dry toluene (10 mL). The resulting mixture was heated at reflux under argon for 2 h (for (C₃)NH₂ and (C₄)NH₂) and 5 h (for (C₅)NH₂). Toluene was evaporated under reduced pressure and the residue was redissolved in a minimum volume of acetonitrile (ca. 3 mL). The calixarene was then precipitated with diethyl ether (30 mL). The solid was filtered off, washed with diethyl ether (3 × 10 mL), and dried under vacuum.

**General procedure for the monoclick reaction with alcohols:** Precursor complex [ZnX₆N₃](ClO₄)₂ (200 mg, 0.13 mmol, 1.0 equiv) was added to a solution of the appropriate alcohol (10 equiv) in dry toluene (10 mL). The resulting mixture was heated at reflux under argon for 4 h. Toluene was evaporated under reduced pressure and the residue was redissolved in a minimum volume of acetonitrile (ca. 3 mL). The calixarene was then precipitated with diethyl ether (30 mL). The solid was filtered off, washed with diethyl ether (3 × 10 mL), and dried under vacuum. In the case of 6-(4-nitrophenyl)-5-hexyn-1-ol, the pure monofunctionalized complex was obtained without further purification. In the case of 5-hexyn-1-ol, 4-pentyn-1-ol, and 3-ethynylbenzyl alcohol, the crude product was purified by column chromatography on basic alumina (CH₂Cl₂/CH₃OH/NH₃ (27%), 99:1:0.01) to obtain the pure monofunctionalized calixarene ligand. The monofunctionalized ligand (20 mg, 1.0 equiv) was dissolved in a 1:1 mixture of CH₂Cl₂ and CH₃CN (1 mL). A solution of zinc perchlorate hexahydrate (1.0 equiv) predissolved in a minimum amount of THF was added to a solution of the ligand. After stirring for 15 min, diethyl ether was added (10 mL). The resulting precipitate was filtered off and dried under vacuum to provide the corresponding ZnII complex as a white solid in 94–98% yield.

Full characterization data for all the monoclick products are presented in the Supporting Information.

**Acknowledgements**

This project was supported by the CNRS (Institut de Chimie), the Minis-
tère de l'Enseignement Supérieur et de la Recherche, and the Agence
Nationale pour la Recherche (Cavity-zyme(Cu) Project ANR-2010-
BLAN-7141).

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[19] At room temperature, the exchange of two amine guests in the receptor takes place within less than a minute. At 110°C, the time-scale for the cycloaddition reaction is around 2 h.

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[21] a) O. Sénèque, Y. Rondelez, L. Le Clainche, C. Inisan, M.-N. Rager, M. Giorgi, O. Reinaud, Eur. J. Inorg. Chem. **2001**, 2597–2604; b) O. Sénèque, M. Giorgi, O. Reinaud, Supramol. Chem. **2003**, *15*, 573–580.

[22] Because the reactions with the two substrates pNO₂Ar(C₄)NH₂ and pOMeAr(C₄)NH₂ need 5 h to go to completion, we privilege steric rather than electronic factors for the discussion about the reaction rates.

[23] J. S. Wischeler, D. Sun, N. U. Sandner, U. Linne, A. Heine, U. Koert, G. Klebe, Chem. Eur. J. **2011**, *17*, 5842–5851.

Received: July 5, 2012

Revised: September 24, 2012

Published online: November 20, 2012