NMR study of the binding between host Ox3 and protonated symmetrical diamines. The binding properties of Ox3 towards the monocationic form of linear symmetrical diamines H2N-(CH2)n-NH2 (with n ranging from 3 to 8) were first evaluated in CDCl3/CD3CN (9:1). The addition of aliquots of diamine and picric acid (PicH) (1:1 ratio) to Ox3 led, in all cases, to the intra-cavity complexation of the monoammonium ion, as evidenced by the presence of highfield signals (i.e. below 1 ppm) in the 1 H NMR spectra (see Figure -b for cadaverine CadNH2, n = 5). With all guests, host-guest exchange was slow on the NMR chemical shift timescale and the signals of the host-guest complexes were assigned through COSY experiments. The shifts of the host ArH and ArCH2 signals following complexation are characteristic of the more open conformation adopted by Ox3 when an ammonium ion is bound in the cavity (see the structure displayed in Figure ). For the shorter diamines (n ≤ 6), integration of the 1 H signals showed the exclusive formation of 1:1 complexes over the course of the titration. The complexes were obtained quantitatively after the addition of a slight excess of the ammonium salt, confirming a high binding affinity (log Ka = 4.2 ± 0.2, 4.1 ± 0.2, 4.7 ± 0.2 and > 5 for diamines with n = 3, 4, 5, and 6 respectively). With the longer diamines (n = 7 and 8), NMR signals corresponding to the 2:1 host-guest complex (<10%) were also observed. Similar results, albeit with slightly smaller affinity constants, were obtained when the binding studies were conducted in CDCl3/CD3OD (4:1): log Ka = 3.7 ± 0.2, 3.7 ± 0.2, 4.0 ± 0.2 and 4.3 ± 0.3 for diamines with n = 3, 4, 5, and 6 respectively. Addition of an excess of PicH to the host-guest complexes led to the protonation of the free amino group dangling outside of the host cavity. For n = 3 and 4, the resulting diammonium ions were not soluble in CDCl3/CD3CN (9:1). For n = 5 and 6, the binding properties of Ox3 were only weakly affected by the protonation of the second amino group and similar binding affinities to those obtained with monoammonium ions were determined (log Ka = 4.8 ± 0.2 and >5 with n = 5 and 6 respectively). With n = 7 and 8, 2:1 host-guest complexes were also detected over the course of the titration (identified through COSY experiments and integration of appropriate signals). 20 In silico studies by molecular mechanics conformational analysis were performed for the 1:1 complex Ox3⸧CadNH 3 + (Figure ) and the 2:1 complex (Ox3) 2 ⸧ + H 3 N-(CH 2 ) 8 -NH 3 + (Figure ). In both cases, the host displays an open-cone conformation with the oxygen of the ether bridges oriented toward the center of the cavity. The bound ammonium groups are deeply inserted in the cavity of the receptor, establishing three H-bonds with the ether bridges. These results are consistent with the data obtained by NMR spectroscopy. Moreover, the minimized structure of Ox3⸧CadNH 3 + shows that a five methylene spacer (n = 5) is long enough for the free amine group of the guest to protrude from the cavity and thus be accessible to reactants in the solution. Host-guest exchange dynamics. Interestingly, for the diamines with n ≤ 6, the number of 1 H signals for the complexed monoammonium guest was less than expected in the spectra recorded at 298 K. In the case of cadaverine (n = 5), only three high-fielded signals instead of five were observed for the methylene groups (Figure ). The five expected signals were however clearly observed in the spectrum recorded at 223 K (Figure ) and their chemical shifts confirm a fast exchange process at 298 K between the CH2 and CH2 protons and between the CH2 and CH2 protons (see inset Figure for the assignment of the methylene protons). At 298 K, the exchange process was slow on the NMR timescale for the longer guests (n = 7 and 8), as distinct high field signals were observed for all the guest methylene groups. Two possible degenerate processes can be envisaged to explain the reduced number of signals for the shorter guests at 298 K: an associative process (Figure , path a) consisting in the folding of the guest carbon chain in the cavity followed by an internal proton transfer from one amino group to the other (guest tumbling) or a dissociative process with reversal of the guest upon re-complexation (Fig 5, path b). The dissociative mechanism can be ruled out as slow in-out host-guest exchange is observed in all cases. It is noteworthy that the tumbling process was not observed with a rigid diamine (i.e. 1,4-phenylenediamine = 1,4-Ph(NH2)2) unable to fold in the cavity, even at 323 K, or when the diamines were fully protonated. In the latter case, the associative process is probably prevented because of the electrostatic repulsion that would result from the folding of the dicationic guest in the cavity. (9:1) with three different reagents (i.e. 3,5-bis(trifluoromethyl)phenyl isothiocyanate, di-tertbutyl dicarbonate and acetic anhydride). Control experiments in the absence of Ox3 were first performed with 1 equiv. of 3,5-bis(trifluoromethyl)phenyl isothiocyanate added to cadaverine either in absence or in presence of 1 equiv. of PicH. In both cases, a statistical ca. 1:2:1 mixture of unreacted starting material, monothiourea and dithiourea was obtained, showing that both amine groups react independently. The functionalization of the various diamines was then investigated in the presence of molecular container Ox3 (Scheme 1) and, as a representative example, Figure displays the 1 H spectra recorded during the process for cadaverine with the isothiocyanate. The host-guest complex with a reactive amine group protruding from the cavity (Step 1, Scheme 1 and Figure ) was first obtained through the addition of the diamine (0.9 equiv.) to host Ox3 in the presence of PicH (0.9 equiv.). A slight excess of the host was used to ensure that no free monoammonium ion remains in the bulk solution. The subsequent addition of the reagent (0.9 equiv.) led to the formation of the complexed monofunctionalized product (Step 2, Scheme 1 and Figure ). Finally, addition of the base 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) (1 to 2 equiv.) 24 triggered the release of the monofunctionalized product through deprotonation of its ammonium group (Step 3, Scheme 1 and Figure ). The quantitative regeneration of free molecular container Ox3 was observed in all cases, opening the way to its reuse for an additional cyclic three-step monofunctionalization of the diamine (vide infra). Scheme 1. Iteroselective monofunctionnalization of diamines through a three-step sequence involving their supramolecular protection by Ox3. Figure . H NMR spectra (298 K, 400 MHz, CDCl 3 /CD 3 CN 9:1) of Ox3 (3 mM) (a); after the successive addition of 0.9 equiv. of cadaverine and 0.9 equiv. of PicH (b); 0.9 equiv. of 3,5-bis(trifluoromethyl)phenyl isothiocyanate (after 3 hours) (c) and 1.3 equiv. of DBU (d). S: residual solvents; w: residual water; g: grease; *: DBU/DBU.H + .