Synthesis of covalent organic pillars as molecular nanotubes with precise length, diameter and chirality

The construction of nanotubes with well-defined structures, although synthetically challenging, offers the prospect of studying novel chemical reactions and transportation within confined spaces, as well as fabricating molecular devices and nanoporous materials. Here we report a discrete molecular nanotube, namely the covalent organic pillar COP-1, synthesized through a [2 + 5] imine condensation reaction involving two penta-aldehyde macrocycles and five phenylenediamine linkers. A pair of enantiomeric nanotubes, obtained in a quantitative and diastereoselective manner, were characterized and resolved readily. NMR spectroscopy, isothermal titration calorimetric and X-ray crystallographic studies revealed that the 2-nm-long and 4.7-Å-wide one-dimensional channel inside COP-1 can accommodate α,ω-disubstituted n-alkyl chains with complementary lengths and electron density distributions. Furthermore, in a length-mismatched host–guest pair, we found that the nonamethylene dibromide thread not only displays a diminished binding constant in solution, but adapts an energetically unfavoured gauche conformation inside COP-1 in the solid state. Single-molecule nanotubes assemble efficiently by linking two penta-aldehyde macrocycles and five phenylenediamine linkers though dynamic covalent imine bonds. These covalent organic pillars, with 2-nm-long and 4.7-Å-wide helical interior channels, show highly selective binding for linear n-alkyl guests with complementary lengths and electronic densities.

The construction of nanotubes with well-defined structures, although synthetically challenging, offers the prospect of studying novel chemical reactions and transportation within confined spaces, as well as fabricating molecular devices and nanoporous materials. Here we report a discrete molecular nanotube, namely the covalent organic pillar COP-1, synthesized through a [2 + 5] imine condensation reaction involving two penta-aldehyde macrocycles and five phenylenediamine linkers. A pair of enantiomeric nanotubes, obtained in a quantitative and diastereoselective manner, were characterized and resolved readily. NMR spectroscopy, isothermal titration calorimetric and X-ray crystallographic studies revealed that the 2-nm-long and 4.7-Å-wide one-dimensional channel inside COP-1 can accommodate α,ω-disubstituted n-alkyl chains with complementary lengths and electron density distributions. Furthermore, in a length-mismatched host-guest pair, we found that the nonamethylene dibromide thread not only displays a diminished binding constant in solution, but adapts an energetically unfavoured gauche conformation inside COP-1 in the solid state. Nanotubes 1-3 , on account of their confined one-dimensional (1D) interior space, are fascinating nanoscale architectures with the potential for a plethora of applications, including molecular recognition and storage [4][5][6][7] , catalysis 8,9 , artificial channels for cross-membrane transportation [10][11][12] , and nano-electronics and mechanical systems [13][14][15] . In addition to carbon nanotubes 16 , the most renowned forms among this type of nanostructure, assorted tubular-shaped nano-assemblies with polymeric nature [17][18][19][20] have also been reported and studied extensively in the past decades. Nonetheless, their lack of structural precision, which renders it difficult to harness both their chemical and physical properties, has been a long-standing bottleneck hindering their further integration into complex molecular systems and functional materials. Atomically precise syntheses leading to discrete nanotubes [21][22][23] of uniform lengths and diameters, as well as one handedness when the target structures are chiral, are crucial for the full exploitation of the anisotropic interior 1D nanochannels. In recent years, although considerable advances in the syntheses of a range of novel macrocycles 24,25 and carbon nanobelts 26 have been made, single-molecule nanotubes with length/diameter ratios greater than unity are still few and far between in the literature.
One straightforward approach to building a discrete tube, be it macro-, micro or nanoscopic, is stacking (Fig. 1a) finite numbers of ring-shaped building blocks so that their cavity space can be extended along the axial direction. On the molecular level, assorted concave macrocyclic compounds (for example, cyclodextrins [27][28][29] , cyclotriveratrylenes 30,31 , resorcinarenes [32][33][34][35] and calixarenes [36][37][38] ) have been employed in this regard to generate single open-ended cavitands and oligomeric tubular capsules with elongated cavities. Similarly, Article https://doi.org/10.1038/s44160-022-00235-w reflux in CHCl 3 for 8 h. By virtue of the reversible nature of the dynamic covalent imine bonds, although numerous partially reacted intermediates were identified ( Supplementary Fig. 10) over the course of the reaction, the targeted [2 + 5] COP-1 with ten newly formed imine linkages was obtained ( Supplementary Fig. 9) exclusively as the most thermodynamically stable species in a quantitative fashion. The resulting COP-1 product is hardly soluble in most common laboratory solvents except chlorinated ones (for example, CHCl 3 and CH 2 Cl 2 , where moderate solubilities of 2.0 and 1.2 mg ml −1 were observed, respectively).
The 1 H NMR spectrum of COP-1 shows (Fig. 1c) a relatively simple pattern because of the overall fivefold symmetry. Notable features of the spectrum include the emergence of an imine peak and the appearance of methylene bridge signals as a virtual quartet, both of which agree with the COP-1 duplex formation. 1 H diffusion-ordered spectroscopy (DOSY) NMR confirmed (Fig. 1d) that all proton signals belong to a single species with a diffusion coefficient on the order of 10 −6 cm 2 s −1 . The formation of the [2 + 5] COP-1 product was also supported (Supplementary Fig. 3) by high-resolution mass spectrometry (HRMS) data.

Stereochemistry, chiral resolution and solid-state structure of COP-1
The inherently chiral p-formyl-T [5] precursor, in principle, undergoes rapid stereochemical inversion 60 similar to that of pillar [5]arenes. Such circumrotations shown by the aromatic units of the macrocycles are put to a halt in the COP-1 duplex because of the presence of the imine covalent linkages. With the help of Pirkle's alcohol-a classic NMR chiral shift reagent-it was concluded ( Supplementary Fig. 15) that the [2 + 5] condensation process between p-formyl-T [5] and p-phenylenediamine undergoes narcissistic chiral self-sorting, leading to the formation of a pair of COP-1 enantiomers instead of a meso structure. By employing a preparative high-performance liquid chromatography (HPLC) system pillararenes 39,40 and paracyclophane 41 derivatives, which can adopt straight pillar-like conformations, could serve naturally as ideal synthons for double open-ended uniform tubes.
To achieve controlled assembly of discrete pillararene superstructures, our research group has made strides in developing improved synthetic protocols for rim-differentiated pillararenes 42,43 . The capability to desymmetrize these macrocyclic scaffolds effectively, coupled with synthetic methodologies to derivatize 44 their distinct upper and lower rims with high selectivity, has already enabled 45-47 the syntheses of tubular pillararene oligomers through non-covalent weak interactions (for example, hydrogen bonds and metal coordination). Next, with the aim of synthesizing discrete covalent organic nanotubes in a similar fashion, we trimmed the alkoxy groups on one side of the rimdifferentiated pillararene rings to produce 48 oligophenolic tiararenes, whose bare C-H functionalized rims allow for the execution of more derivatization schemes. By employing the Duff reaction, multiple aldehyde handles can be grafted onto the tiararene macrocycle for subsequent reactions with amine species to form dynamic covalent imine bonds, which have been used broadly in the construction of assorted covalent organic frameworks [49][50][51][52] , molecular cages 53-55 and knots [56][57][58] , and recently, both discrete and polymeric covalent organic nanotubes 20,23,59 . Following this molecular design strategy, herein we describe the successful construction and resolution of covalent organic pillars (COP-1)-a pair of 2-nm-long and 4.7-Å-wide chiral single-molecule nanotubes-as well as their stereochemistry, solid-state structures and host-guest properties.

Host-guest NMR investigations
The shape and size of COP-1 suggest that, in addition to solvent molecules, this 2-nm-long nanotube could potentially host long linear guests. Nonetheless, no strong interactions between n-alkanes and the hollow channel of COP-1 were observed ( Supplementary Fig. 88) in the preliminary 1 H NMR spectroscopic studies, owing presumably to the lack of energetically favoured interactions between the non-polar paraffin guests and the inner wall of COP-1. Theoretical analyses of the electrostatic potential maps of COP-1 ( Fig. 2d and Supplementary  Fig. 24) show that the channel surface is mostly electron rich except at the edges. Therefore, a series of n-alkyl chains with electron-withdrawing end groups (for example, α,ω-dibromoalkanes and α,ω-alkanediols) was chosen ( Supplementary Fig. 25) as guest candidates by virtue of their complementarity to COP-1 in terms of shape and electron density.
The host-guest complexations between COP-1 and assorted α,ω-disubstituted n-alkanes in CDCl 3 were investigated ( Fig. 3 and Supplementary Section 6) by NMR spectroscopic methods and HRMS. For the α,ω-dibromoalkane series, highly shielded proton signals below 0 ppm on the δ scale, corresponding to the encapsulated n-alkyl chains shielded by COP-1, appeared ( Fig. 3 (left) and Supplementary Section 6.1) from samples containing 1,10-dibromodecane or longer α,ωdibromoalkanes (Br(CH 2 ) n Br (n ≥ 10); up to 1,20-dibromoicosane in our investigations; Supplementary Fig. 29). These high-field 1 H NMR peaks of bound guests, which display the same diffusion coefficients around 10 −6 cm 2 s −1 as those of COP-1 in the DOSY spectra, were assigned (Supplementary Section 6.1) based on the results of ¹H-¹H correlation spectroscopy. These observations indicate that the tubular cavity of COP-1 shows effective binding with α,ω-dibromoalkanes longer than and equal to a certain critical length (n, 10).
In parallel, the investigations into the host-guest interactions between COP-1 and α,ω-alkanediols led ( Fig. 3 (right) and Supplementary Section 6.2) to similar results. The difference is that the proton signals of bound guests in the high-field spectral region could only be identified for [HO(CH 2 ) n OH⊂COP-1] (n ≥ 12) complexes containing 1,12-dodecanediol or longer α,ω-alkanediol threads (up to 1,22-docosanediol in our investigations). The critical length of the α,ωalkanediol guests (n, 12) is two extra methylene units longer than that of the corresponding dibromides. Furthermore, it was found that the heteroatom is only required on one end of the n-alkyl chains, instead of both sides, to provide enough electron density complementarity. Clear high-field 1 H NMR proton peaks of encapsulated mono-substituted 1-bromoalkane and 1-alkanol guests inside COP-1 were observed (Supplementary Section 6.3) from the [Br(CH 2 ) n−1 CH 3 ⊂COP-1] (n ≥ 12; up to 1-bromooctadecane in our investigations) and [HO(CH 2 ) n−1 CH 3 ⊂COP-1] (n ≥ 13; up to 1-tetradecanol in our investigations) complexes.
In the cases of 1,9-dibromononane and 1,11-undecanediol, which are both one methylene unit below the critical lengths in the respective dibromide and diol series, although no 1 H NMR peaks below 0 ppm occur (Fig. 3) 59 and 84) between the COP-1 host and 1,10-dibromodecane and 1,12-dodecanediol guests with the exact critical lengths required to form strong host-guest complexes. In these cases, free and bound guests could be identified clearly in the presence of excess guests, indicating that their threading-dethreading processes are within the slow exchange regime on the NMR time scale. When the amounts of the guests were less than one equivalent of COP-1, no NMR signal corresponding to the free guest could be observed at all in the spectra, and the chemical shifts of those bound guests remained unchanged regardless of their relative concentrations. These findings suggest that the association constants (K a ) of these host-guest systems are beyond the detection limit of the NMR titration method.

Binding constants between COP-1 and α,ω-disubstituted n-alkyl guests
To obtain accurate association constants and deeper insight into this host-guest system, the complexations of α,ω-disubstituted n-alkane guests over and below the critical lengths into the COP-1 nanotube were probed (Supplementary Section 7) by isothermal titration calorimetry (ITC). The K a values between the COP-1 host and the 1,10-dibromodecane and 1,12-dodecanediol guests in CHCl 3 were found (Table 1) to be 1.19 and 2.36 × 10 5 M −1 , respectively, whereas the corresponding binding constants of the 1,11-dibromoundecane and 1,13-tridecanediol guests with one extra methylene group in the main chain are even higher (1.42 × 10 6 and 3.04 × 10 5 M −1 , respectively). These relatively strong binding affinities shown by COP-1 towards these guests longer than and equal to the critical lengths corroborate with their tight binding mode and slow exchange dynamics observed in the NMR titration experiments. It is also noteworthy that these K a values are at least three orders of magnitude larger than those between the per-methylated pillar [5]arene and the same guest species (Supplementary Figs. 104  and 106). The substantially stronger hosting ability displayed by the COP-1 duplex than the individual macrocycle can be attributed to the pre-organization of the tubular structure, the enhanced stabilization provided by the extended inner surface and the rigid conformation without stereochemical inversion, which leads to less entropy loss upon guest binding.
In contrast, the host-guest pairs of COP-1 and α,ω-disubstituted n-alkane guests shorter than the critical lengths show (Table 1) considerably diminished binding constants in CHCl 3 . For example, the K a values of the COP-1 host with the 1,9-dibromononane and 1,11-undecanediol guests drop by one to two orders of magnitude to 2.41 × 10 3 and 3.45 × 10 4 M −1 , respectively. Further analyses of the isothermal titration calorimetry data reveal (Supplementary Tables 5 and 6) that these host-guest pairs have enthalpic gains comparable to those involving guests longer or equal to the critical lengths. At the same time, the more negative entropic terms observed, which lower the binding affinities between COP-1 and these two shorter threads, can be attributed presumably to the greater configurational and/or conformational entropy loss suffered from the formations of mismatched host-guest complexes.
To our surprise, shorter α,ω-disubstituted n-alkyl chains with two fewer methylene units below the critical length (for example, 1,8-dibromooctane and 1,10-dodecanediol) displayed no heat of interaction with COP-1 in their respective ITC experiments, indicating that the complexation thermodynamic parameters become notably different for these shorter guests. Alternatively, by employing the 1 H NMR titration method, it was found (Table 1 and Supplementary  Fig. 100) that the binding constant between COP-1 and 1,8-dibromooctane is 2.40 × 10 3 M −1 , which is on a par with that corresponding to 1,9-dibromononane. In contrast, the 1,10-decanediol guest has (Table 1 and Supplementary Fig. 102) a two orders of magnitude lower K a value (9.43 × 10 2 M −1 ) towards COP-1 compared with that of 1, 11-undecanediol. Overall, the binding constants shown by the COP-1 nanotube vary over a wide range, spanning three orders of magnitude (10 6 -10 3 and 10 5 -10 2 M −1 for the dibromides and diols, respectively), for α,ωdisubstituted n-alkanes differing only by several methylene units.  Fig. 3 | Host-guest interactions between COP-1 and α,ω-  Article https://doi.org/10.1038/s44160-022-00235-w High binding affinities (K a > 10 5 M −1 ) are exclusive for guest threads over and equal to the critical lengths, which presumably correspond to the overall size of the tubular channel of COP-1. This strong length dependence highlights the importance of host-guest complementarity in highly specific molecular recognition events.

Solid-state structures of COP-1 inclusion complexes
Finally, single crystals of the [Br(CH 2 ) 10 Br⊂(M)-COP-1] and [HO(CH 2 ) 12 OH⊂(M)-COP-1] inclusion complexes were obtained (both space group P2 1 2 1 2 1 ; orthorhombic) by slow evaporation of CH 2 Cl 2 and CHCl 3 solutions containing (M)-COP-1 and excess 1,10-dibromodecane and 1,12-dodecanediol, respectively. Their X-ray crystal structures substantiate (Fig. 4a,b and Supplementary Section 4) the threadings of the long linear decamethylene dibromide and dodecamethylene diol guests into the COP-1 tubular channel. In these [2]pseudorotaxanes, the COP-1 tubes adopt similar conformations with only minor variations in terms of void volume from that of the [4•CH 2 Cl 2 ⊂COP-1] structure. The rigid and highly pre-organized COP-1 tubular host allows both the 1,10-dibromodecane and 1,12-dodecanediol guests to adopt fully relaxed anti-periplanar conformations inside, showing torsional angles close to 180° throughout the backbone chains. The electronwithdrawing end groups, as anticipated, position in the centre of the electron-deficient opening areas at both ends of the tubular cavities. The X-ray crystallographic data also help to rationalize the different critical lengths shown by COP-1 when interacting with the dibromide and diol guests. In the case of the [Br(CH 2 ) 10 Br⊂COP-1] inclusion complex, the two large bromine atoms on both ends of the alkyl chain only require a spacer of ten methylene units to reach the electron-deficient edges of COP-1, whereas the 1,12-dodecanediol guest with smaller hydroxy groups needs two extra methylene units to be well stabilized inside the tubular channel of COP-1 in the [HO(CH 2 ) 12 OH⊂COP-1] complex. In contrast, limited by the n-alkyl chain length, it is obvious that shorter α,ω-disubstituted n-alkane guests below the critical lengths cannot fully access the electron-deficient areas at both ends of the tube simultaneously, therefore resulting in weaker binding towards COP-1 in solution. To further elucidate the exact mode of interactions between these length-mismatched host-guest systems, single crystals of the [Br(CH 2 ) 9 Br⊂(P)-COP-1] complex were cured (space group P2 1 2 1 2 1 ; orthorhombic) by slowly evaporating a CHCl 3 solution containing (P)-COP-1 and excess 1,9-dibromononene. In the solid-state structure ( Fig. 4c and Supplementary Figs. 22 and 23), as one would expect, one of the bromine atoms of 1,9-dibromononene sits in the binding pocket at one side of the COP-1 nanotube, whereas the rest of the carbon chain still adopts the extended zig-zag shape. The most striking feature of this X-ray crystal structure is that the remaining bromine, which cannot reach the tube opening, shows (Fig. 4d,e) an energetically unfavoured gauche torsional angle about the third adjacent carbon atom. The energy penalty of this distortion is compensated by a C-Br … π interaction (distance 3.62 Å) with an aromatic ring of the nearby macrocyclic unit, in addition to the stabilization provided by the interactions between the rest of guest and the inner wall of COP-1. This unusual geometry adapted by 1,9-dibromononene inside COP-1 is reminiscent of the folding shown by guests with long n-alkyl chains inside cavitand-based molecular capsules 34 . Our unexpected observation represents a piece of rare crystallographic evidence for such contortion inside the confined space of a synthetic receptor.

Conclusion
In summary, we have successfully devised the synthesis of structurally precise single-molecule nanotubes by dimerizing rim-differentiated macrocycles with multiple covalent linkages. The application of reversible imine bonds, which marshals effective proofreading during the reaction process, avoids mismatch between the ring components and therefore leads to efficient formations of extended nanotubular structures. In particular, we employed penta-aldehyde tiara [5]arene-based macrocycles and ditopic p-phenylenediamine linkers as the building blocks for the construction of a pair of helical COP-1 molecular nanotubes. We showcased that this highly pre-organized host, which possesses a 2-nm-long and 4.7-Å-wide 1D channel with an interior space of ~440 Å 3 , displays specific recognition towards guests with complimentary size and electronic properties.
Our molecular design strategy for building COPs, applicable to [1 n ] paracyclophane and pillar[n]arene derivatives, can also be potentially extended to a broad range of carbon nanorings and nanobelts. We hope that the development of the COPs will expedite the syntheses of discrete molecular nanotubes, or even pure carbon nanotubes, of a variety of sizes and functionalities. Such advancements will undoubtedly stimulate further explorations into molecular behaviours in confined b a c

Chiral resolution of COP-1
HPLC separation was performed on a Shimadzu LC-16A instrument with a Daicel CHIRALPAK IE semi-preparative column (5 μm, ID 10 mm × L 250 mm). The racemic COP-1 product (6.20 mg in 2 ml CHCl 3 ) was filtered with a syringe membrane filter before being injected into the HPLC system. In the mobile phase, the CHCl 3 /MeOH ratio was 7:3, the flow rate was 2.0 ml min −1 and the detection wavelength was 300 nm. For one injection, 2.75 mg (M)-COP-1 and 2.63 mg (P)-COP-1 were collected. After resolution, the solubilities of enantiopure COP-1 in CHCl 3 and CH 2 Cl 2 increased to 4.0 and 1.8 mg ml −1 , respectively.

X-ray crystallography
Single-crystal X-ray diffraction data were collected on XtaLAB Synergy (Dualflex; HyPix) and XtaLAB Synergy R (DW system; HyPix) diffractometers using Cu Kα (λ = 1.54184 Å) micro-focus X-ray sources (PhotonJet (Cu) X-ray source). The raw data were collected and reduced using CrysAlisPro software. The structures were solved by ShelXT with intrinsic phasing and refined on F 2 using full-matrix least-squares methods, with ShelXL and Olex2 used as graphical user interfaces.

Computational methods
The electrostatic potential map was studied using a density functional theory calculation. All calculations were performed with the Gaussian 16 suite of computational programs. The B3LYP functional with the addition of the D3 version of Grimme's dispersion was employed to optimize all stationary points in the gas phase using 6-31G(d) basis sets for all atoms.

ITC
All titrations were conducted using a low-volume Affinity ITC (TA Instruments). Blank titrations in CHCl 3 were performed and subtracted from the corresponding titrations to attenuate the effect of dilution. The data fitting was executed using the NanoAnalyze program.

Data availability
The data that support the findings of this study are available in the Supplementary