Dynamic covalent self-assembly and self-sorting processes in the formation of imine-based macrocycles and macrobicyclic cages

Investigating the self-assembly and self-sorting behaviour of dynamic covalent organic architectures makes possible the parallel generation of multiple discrete products in a single one pot procedure. We here report the self-assembly of covalent organic macrocycles and macrobicyclic cages from dialdehyde and polyamine components via multiple [2 + 2] and [3 + 2] polyimine condensations. Furthermore, component self-sorting processes have been monitored within the dynamic covalent libraries formed by these macrocycles and macrobicyclic cages. The progressive assembly of the final structures involves intermediates which undergo component selection and self-correction to generate the final thermodynamic constituents. The homo-self-sorting observed seems to involve entropic factors, as the homoleptic species present a higher symmetry than the competing heteroleptic ones. This study not only emphasizes the importance of an adequate design of the components of complex self-sorting systems, but also verifies the conjecture that systems of higher complexity may generate simpler outputs through the operation of competitive self-sorting.


Instrumentation and Measurement
NMR spectra were recorded on Bruker Avance 400 (400 MHz for 1 H and 100 MHz for 13 C), Bruker Avance III plus 400 (400 MHz for 1 H) and Bruker Ascend Spectroscope Avance Neo-500 MHz (500 MHz for 1 H and 125 MHz for 13 C). MestReNova 10 software was used for the treatment of the NMR spectra. Chemical shifts are given in ppm. Residual solvent peaks were taken as reference (CDCl3:7.26 ppm for 1 H and 77.16 ppm for 13 C). The quantitative 1 H NMR was measured by using hexamethyldisilane as internal standard. The error in 1 H-NMR integration amounts to about 5%. Peaks are described as singlet (s), doublet (d). Unless otherwise noted, spectra were recorded at 23 °C.
HRMS-ESI (High-Resolution Mass Spectrometry-Electro-Spray Ionisation) mass spectra were recorded by direct injection into a ThermoFisher Exactive Plus EMR Orbitrap mass spectrometer.
X-Ray diffraction data was obtained on a PHOTON-III CPAD (Bruker) equipped with a CCD detector. The structures were solved with the SHELXT 2014/5 structure solution program and refined with the SHELXL-2014/7 refinement package. 1 Artwork representations were processed using MERCURY software. 2 X-Ray diffraction data collection was carried out on a Bruker PHOTON-III DUO Kappa CPAD diffractometer.
Commercially available chemicals were generally purchased from Sigma-Aldrich, Alfa Aesar, Fluorochem, TCI and were used without further purification. Solvents and reagent of pharmaceutical grade quality were purchased from Carlo Erba, and solvents of spectroscopic grade were purchased from Sigma-Aldrich and Fisher Chemical. CDCl3 was purchased from Euriso-TOP and filtered through basic alumina to remove traces of acidity before use. S3 2. List of the selected dialdehydes, polyamines, macrocycles and macrobicyclic cages Scheme S1. All of aldehydes, amines and macrocycles, macrobicyclic cages that have been used in the text.

Experimental methods for time-dependent 1 H NMR monitoring studies a) Preparation of stock solutions
CDCl3 was filtered through basic alumina oxide to remove traces of acidity before use. A stock solution of internal standard was made by adding 1.5 µL hexamethyldisilane into 2 mL CDCl3. The stock solutions of amines (NON, T) and dialdehydes were freshly prepared in CDCl3 and quantified referencing to the internal standard (hexamethyldisilane). For all the cases, dialdehyde and polyamine were mixed in a proper stoichiometric ratio in CDCl3 containing hexamethyldisilane as internal standard. The operation details were described as following:

b) formation of a single macrocycle
In an NMR tube, 30 mM solution of NON 72 µL (2 equiv.) was added into 528 µL CDCl3 solution containing 2 equiv. dialdehyde and 40 µL internal standard solution. The final concentration of dialdehyde and NON was S4 3.6 mM. The reaction solution was monitored over time at 23-25 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

c) formation of a single macrobicyclic cage
In an NMR tube, 30 mM solution of T 48 µL (2 equiv.) was added into 552 µL CDCl3 solution containing 3 equiv. dialdehyde (for instance pPh, Py, BiPh) and 40 µL internal standard solution. The final concentration of dialdehyde and T was 3.6 mM and 2.4 mM, respectively. The reaction solution was monitored over time at 23-25 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

d) self-sorting of 2pPh+2BiPh+4NON
In an NMR tube, 30 mM solution of NON 156 µL (4 equiv.) was added into 494 µL CDCl3 solution containing 2 equiv. dialdehyde pPh, 2 equiv. dialdehyde BiPh and 40 µL internal standard solution. The final concentration of each dialdehydes and NON were 3.6 mM and 7.2 mM, respectively. The reaction solution was monitored over time at 23-25 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

e) self-sorting of 3pPh+3BiPh+4T
In an NMR tube, 30 mM solution of T 96 µL (4 equiv.) was added into 504 µL CDCl3 solution containing 3 equiv. dialdehyde pPh, 3 equiv. dialdehyde BiPh and 40 µL internal standard solution. The final concentration of each dialdehydes and T were 3.6 mM and 4.8 mM, respectively. The reaction solution was monitored over time at 23-25 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

f) self-sorting of 2pPh+2BiPh+2TriPh+6NON
In an NMR tube, 30 mM solution of NON 65 µL (6 equiv.) was added into 585 µL CDCl3 solution containing dialdehyde pPh, BiPh, TriPh (2 equiv. of each) and 40 µL internal standard solution. The final concentration of each dialdehydes were 1.0 mM and NON was 3.0 mM. The reaction solution was monitored over time at 40 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

h) self-sorting of 3pPh+3BiPh+3TriPh+6T
In an NMR tube, 30 mM solution of T 43 µL (6 equiv.) was added into 607 µL CDCl3 solution containing dialdehyde pPh, BiPh, TriPh (3 equiv. of each) and 40 µL internal standard solution. The final concentration of each dialdehydes and T were 1.0 mM and 2.0 mM, respectively. The reaction solution was monitored over time at 40 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

i) self-sorting of 3pPh+3BiPh+3TriPh+2T+6NON
In an NMR tube, 20 mM solution of T 22 µL (2 equiv.) and 20 mM solution of NON 43 µL (6 equiv.) were added into 585 µL CDCl3 solution containing dialdehyde pPh, BiPh, TriPh (3 equiv. of each) and 40 µL internal standard solution. The final concentration of each dialdehydes were 1.0 mM. The reaction solution was monitored over time at 40 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

j) self-sorting of 3Py+3pPh+3BiPh+3TriPh+2T+9NON
In an NMR tube, 20 mM solution of T 44 µL (2 equiv.) and 100 mM solution of NON 40 µL (9 equiv.) were added into 570 µL CDCl3 solution containing dialdehyde Py, pPh, BiPh, TriPh (3 equiv. of each) and 40 µL internal standard solution. The final concentration of each dialdehydes were 2.0 mM. The reaction solution was monitored over time at 40 °C, until the equilibrium was reached. The composition of species was calculated according to the internal standard and presented on basis of imine integration data.

Experimental methods for time-dependent HRMS monitoring studies
The 30 mM stock solutions of amines (NON, T) and dialdehydes (pPh, BiPh) were freshly prepared in 50%-50% CHCl3/MeOH. For all the cases, HRMS kinetic experiments were carried out under dialdehyde concentration of 2 mM and were monitored as a function of time.

NOTE
The time dependent course of the reactions was sensitive to the experimental conditions. The data are only qualitative as significant variations in rates have been obtained, possibly due to sensitivity to solvent acidity despite the filtration of the solvent over an alumina column. The reaction times t1/2 were markedly affected by the aluminium oxide used to treat the CDCl3 (acidity and water) used as the solvent. To verify how could different qualities of alumina can affect the reaction courses, three different kinds of experiments were carried out: 1) experiments with fresh opened aluminum treated CDCl3; 2) experiments with a dated aluminum treated CDCl3. We verified that with different qualities of alumina giving these different time dependence curves, the sequence of reaction processes remained the same. For example, in the case of pPh/NON system, the halfconsumption t1/2 C of pPh could vary from 270 min (for dated alumina treated CDCl3) to 720 min (for fresh alumina treated CDCl3) and the half formation time t1/2 F of the pPh2(NON)2 macrocycle from 660 min to 2200 min in the same conditions. We verified that with different qualities of alumina giving these different time dependence curves, the sequence of reaction processes remained the same, with the sequence of three intermediates, and finally, yielded nearly 99% of pPh2(NON)2 macrocycle as illustrated in Figs. S6 and S8 for more detailed information. Unless otherwise noted, the CDCl3 is treated with fresh opened aluminum before using for 1 H NMR kinetic experiments.

Synthesis of Macrobicyclic Cage TriPh3T2
[1,1':4':1'']Terphenyl-4,4''-dicarbaldehyde (TriPh) (33.15 mg; 0.12 mmol) was dissolved in 8 mL CHCl3. Then, the solution was further diluted with 3 mL MeOH. Thereafter, a MeOH (2 mL) solution of Tris(2aminoethyl)amine (T) (11.07 mg; 0.076 mmol) was added in five portions during 20 minutes. The reaction mixture was stirred at room temperature for 3 days. After removal of the solvent by centrifugation, the crude product was washed with 1 mL CHCl3 and MeOH, then dried in vacuum to afford macrobicyclic cage TriPh3T2 as yellow solids (30.21 mg, 76 %).                  u.) the formation of several intermediates and of the final macrobicyclic cage pPh3T2                 Fig. S45. HRMS-ESI kinetic evolution of the species generated during the during the self-sorting process of 2pPh + 2BiPh + 4NON (50%-50% CHCl3/MeOH, r.t) as a function of time over 1440 min. NB: The relative combined ion percentage is obtained by the ratio of combined ion count of each species to the sum of combined ion counts for all species at each time point. These data do not provide quantitative information about the relative amounts of each species identified by its mass, but, taken separately, they display the evolution of a given identified species during the course of the reaction. The curves are added to guide the eye.    a. Unable to read the mass-to-charge ratio (m/z) abundance as it possesses extremely close m/z value with other highly responded ion-combined species.

DFT calculations
DFT calculations were run with Gaussian 09 (revision B.01). 6 Geometry optimizations were carried out without symmetry restrictions at the B3LYP level, 7 using the 6-311+G(d,p) basis set. The calculations were performed using chloroform as solvent with the Polarizable Continuum Model (PCM) model. Analytical frequency calculations were used to characterise each stationary point as a minimum. These calculations, carried out at 298.15 K, also allowed for obtaining the thermal and entropic corrections required to calculate Gibbs energy differences.