Sequence-encoded layered heteroleptic metalla-[2]catenanes for programmable supramolecular function

Domain: chemistry
Published: 2026-01-12
Editorial outcome: minor_revision

Abstract
Molecular information encoded within supramolecular frameworks offers a powerful paradigm for directing emergent function beyond the genetic code, but systematic investigations into alternative spatial configurations and their consequences remain scarce. Here we use metalla-[2]catenanes to probe sequence–function relationships in layered architectures. By combining two or three size-matched N-heterocyclic carbene ligands with Ag(I) nodes, we selectively construct heteroleptic metalla-[2]catenanes through both direct assembly and supramolecular fusion pathways. X-ray crystallographic analysis unambiguously confirms the targeted sequences, while semiempirical and density functional theory calculations reveal their thermodynamic preference over alternative isomers. Photothermal conversion studies further demonstrate that sequence-specific charge-transfer interactions yield distinct macroscopic responses. Collectively, these results identify heteroleptic metalla-[2]catenanes as a robust model for elucidating how spatial arrangement governs system-level behavior and for advancing molecular coding principles in functional supramolecular design.

Introduction
Molecular information is emerging as a transformative paradigm that extends far beyond the genetic code of nucleic acids1. Information can be encoded within molecular frameworks that have distinct structural and chemical features, redefining how it is stored, processed, and transmitted at the molecular level2,3. In self-assembly, the spatial arrangement of functional motifs acts as a “molecular code” that dictates intermolecular interactions, symmetry, and dynamic response pathways, ultimately directing emergent function4–8. These principles provide a powerful foundation for the bottom-up design of advanced functional materials, with numerous implications for biomimetic manufacturing9,10, energy conversion11, and chiral technologies12,13. Despite these advancements, establishing precise structure–function relationships between the sequence of functional motifs at the molecular level and the macroscopic properties of assembled systems remains a central challenge14–16. Current research on the controllable design of assemblies with specific motif sequences remains limited. To address this issue, recent efforts have focused on the development of multicomponent self-assembly systems incorporating diverse motifs17–19. In metallosupramolecular assemblies, synthetic chemists have achieved nonstatistical precision8,20–22 in multicomponent architectures—overcoming entropy barriers, avoiding self-sorting, and suppressing statistical mixtures—through strategies such as guest templation23,24, geometric complementarity25,26, coordination-site engineering27–31, hierarchical assembly32,33, and kinetic control34–36. Nevertheless, most studies have focused on cavity-centered functions, arising from the anisotropic radial (Fig. 1a, for metallocages) or circular arrangement (Fig. 1b, for metallacycles) of motifs, particularly in recognition37,38 and confined catalysis33,39–42. In contrast, systematic investigations into alternative spatial organizations—such as layered or planar grid arrangements—and their effect on properties (Fig. 1c), including electron transport, optical behavior, and mechanical performance, remain scarce. There is therefore an urgent need to construct self-assembly systems with spatial configurations beyond radial architectures to determine how specific motif arrangements translate into system-level functions.

Building on the previous work with metal N-heterocyclic carbene (NHC) assemblies43–46, we identified tetranuclear metalla-[2]catenane (MCAT) as an ideal platform for this purpose47,48. Unlike conventional metallocages and metallacycles featuring radial arrangements, the four NHC ligands in MCAT use a distinctive stereoscopic layered configuration. Heteroleptic assembly of two or three size-matched NHC ligands with four Ag(I) nodes can theoretically yield MCATs with 8 and 18 possible outcome sequences (Figs. 1d, e), respectively, providing a versatile platform to probe stereoscopic, layered sequence–function relationships.

In this study, we report the selective construction of three heteroleptic MCATs with integrative self-sorting behavior, including two distinct two-component sequences and one three-component sequence. These assemblies were accessed either via direct assembly of NHC ligands with Ag(I) nodes or structural transformation from homoleptic precursors trough a supramolecular fusion strategy. All the structures were unambiguously confirmed by single-crystal X-ray diffraction and high-resolution mass spectrometry. Complementary theoretical calculation studies that combine semiempirical methods and density functional theory (DFT) calculations revealed that the binding energies of the observed MCATs are significantly lower than those of their respective isomers, supporting the thermodynamic preference for the obtained structures. Furthermore, photothermal conversion experiments demonstrated sequence-dependent differences in performance, which can be attributed to charge-transfer interactions between the distinct functional motifs. Collectively, these findings establish heteroleptic MCATs as a versatile platform for probing the relationship between spatial motif arrangement and macroscopic function in multicomponent assemblies. This approach provides new insights into how molecular coding principles can be applied to the design of functional supramolecular systems.

Results
Synthesis and characterization of homoleptic MCATAAAA and MCATBB

Imidazo[1,5-a]pyridinium salts H2A and H2B were prepared in four steps from 2,7-dibromophenazine and 2,6-dibromoanthracene by adjusting the reported procedure49. The complete synthetic route is outlined in the Supplementary Information (Supplementary Figs. 1–26). The imidazo[1,5-a]pyridine-based NHC ligands were strategically chosen to fulfill the structural requirement for optimal interplanar surface-to-surface separation (~6.6 Å), facilitating the directional assembly of mechanically interlocked supramolecular architectures through precise π-system alignment47–49. The homoleptic MCATAAAA was obtained in 93% yield via the reaction of H2A with excess Ag2O in acetonitrile under the exclusion of light at 65 °C for 12 h (Fig. 2, method a). The formation was verified by two-dimensional (2D) NMR spectroscopy and ESI mass spectrometry (Supplementary Figs. 27–32) as follows: Peaks in the 1H NMR spectrum of MCATAAAA (Supplementary Fig. 27) showed distinct shifts and were split into two sets of signals, revealing the presence of two chemically inequivalent di-NHC ligands within the assembly. As expected for AgI–NHC [2]catenane, two di-NHC ligands are located inside the cavity, and the remaining ligands constitute the outer framework. The formation of MCATAAAA was also evident from 13C{1H} NMR spectroscopy showing the two well-resolved sets of carbon signals (Supplementary Fig. 28). The 1H diffusion-ordered spectroscopy (DOSY) NMR spectrum of MCATAAAA showed a single diffusion coefficient (D = 5.01 × 10-10 m2 s-1) for all the aromatic proton signals, suggesting that all the resonances belong to a single assembly (Supplementary Fig. 31). ESI mass measurements also indicated the [2]catenane structure of MCATAAAA, with prominent peaks at m/z = 891.5664 (calcd for [MCAT ‒ 3(PF6)]3+ 891.5541) and 1409.8347 (calcd for [MCAT ‒ 2(PF6)]2+ 1409.8134) (Supplementary Fig. 32), which was consistent with the theoretical distribution.

The single-crystal X-ray diffraction (SCXRD) structure of MCATAAAA is shown in Fig. 3a. The two identical binuclear MRs are interlocked, constituting a Hopf link topology. SCXRD analysis revealed efficient π···π stacking interactions between the four phenazine rings, as inferred from the centroid−centroid separations of 3.4, 3.3, and 3.4 Å between adjacent phenazine planes. These interactions are the main driving forces for the formation of the interlocked layered structure. In addition, there is a crossing angle of ca. 85° between the two “inner” phenazine rings, whereas the “outer” adjacent pairwise phenazine rings are nearly parallel.

However, when H2B (with anthracene replaced by phenazine in H2A) was treated with Ag2O under the same conditions as described for the assembly of MCATAAAA, only the simple binuclear MRBB was isolated rather than an interlocking topology (Fig. 2, method b). The MRBB was identified by NMR spectroscopy and ESI mass (Supplementary Figs. 33–37). The single resonance pattern (rather than the common doublet signals of interlocked topologies47,49) of the 1H (Supplementary Fig. 33) and 13C{1H} (Supplementary Fig. 34) NMR spectra of the resulting assembly suggested a simpler topology. Moreover, the 1H DOSY spectrum (Supplementary Fig. 36) as well as the ESI mass spectrum (Supplementary Fig. 37) also confirm the formation of MRBB (m/z = 630.1871, calcd. for [MRBB ‒ 2(PF6)]2+ 630.1832). These results indicate that compared with purely hydrocarbon-based aromatic groups, heteroaromatic groups have greater advantages in the construction of interlocked topologies50.

Synthesis and characterization of heteroleptic MCATs with two different di-NHC ligands

Inspired by previous work51, we realized that the charge-transfer interaction between two NHC ligands with similar sizes and opposite electronic centers facilitates the formation of heteroleptic AgI-NHC assemblies while inhibiting the formation of homoleptic AgI–NHC assemblies. Thus, we employed a similar strategy to construct two-component MCATs.

First, we attempted to assemble the system by reacting imidazo[1,5-a]pyridinium salts H2A and H2B in a 1:1 molar ratio with a slight excess of Ag2O. Subsequent 1H NMR spectroscopy (Supplementary Fig. 38) revealed that assembly in the presence of two imidazo[1,5-a]pyridinium salts and AgI ions failed to yield a single product. Mass spectrometry (Supplementary Fig. 39) indicated the presence of peaks corresponding not only to MRBB but also to a heteroleptic MCAT, where the ratio of NHC ligands A to B was 3:1. Although we obtained single crystals of MCAT via vapor diffusion, X-ray crystallography confirmed that the structure was only an MCAT formed by the catenation of two MRs (Supplementary Fig. 40). Owing to the similar scattering factors of carbon (C) and nitrogen (N) atoms, the specific arrangement of the ligands could not be definitively assigned. Consequently, we speculated that the products included MR with a mixture of possibly MCATABAA and MCATBAAA. To avoid this issue, we considered introducing the NHC ligand C, which has a similar size but an opposite charge center. This ligand was previously shown to enable the high-yield construction of homoleptic MCATCCCC with AgI (Fig. 2, method c). Considering the electron-donating central core of the proligand H2C, when it is strategically assembled with a di-NHC ligand containing an electron-deficient aromatic scaffold in the presence of Ag2O as a metallic precursor, it may facilitate the creation of a new heteroleptic MACT system. This architecture is anticipated to exhibit increased thermodynamic stability and structural complexity, guided by complementary electronic interactions between the donor–acceptor pair and the directional coordination preferences of the AgI nodes.

To test this hypothesis, heating a mixture of H2A and H2C in a ratio of 1:1 with a slight excess of Ag2O in acetonitrile indeed resulted in the exclusive formation of heteroleptic [2]catenane with a yield of 92% (Fig. 2, method d). With the slow diffusion of diethyl ether into the acetonitrile solution at ambient temperature, yellow block crystals suitable for SCXRD were obtained. Structural analysis of single crystals unambiguously established the composition and molecular structure of this heteroleptic MCAT with a sequence of CAAC (Fig. 3b). In inspection of the structure reveals that every silver(I) ion simultaneously coordinates NHC ligands A and C linearly to create two heteroleptic MRACs, which are further interlocked via mechanical bonds to form heteroleptic MCATCAAC. This arrangement results in extensive π···π stacking between the ligand backbones, which are arranged into pyrene–phenazine–phenazine–pyrene stacks with distances of ca. 3.3–3.4 Å between the mean planes of the stacked rings. Interestingly, the selective formation of this heteroleptic MCAT structure with a donor–acceptor–acceptor–donor stack provides a compelling depiction of intramolecular self-sorting that differs from the commonly accepted self-sorting, in which donor and acceptor aromatic moieties are arranged into complementary alternating stacks that are generally regarded as the most favorable arrangement for providing optimum electronic overlap.

After the solid-state molecular structure of MCATCAAC was determined, relevant NMR spectroscopic experiments performed to further explore its behavior in solution (Supplementary Figs. 41–45). With the 1H NMR spectra of ligand precursors H2A (Fig. 4a) and H2C (Fig. 4c) and the 2D NMR spectra of MCATCAAC, the peaks in the 1H NMR spectrum of MCATCAAC can be clearly identified (Fig. 4d and Supplementary Fig. 41) and are consistent with the characterization of the heteroleptic metalla[2]catenane structure. The 1H NMR spectrum of MCATCAAC (Fig. 4d) shows one set of signals that are equally integrated for each ligand. The total amount of 15 aromatic signals indicated that each ligand maintained its twofold symmetry in the interlocked assembly. Strongly upfield-shifted resonances were observed for the aromatic protons of the phenazine units H9’, H13’ and H10’ (δ = 7.33, 6.74 and 6.66 ppm, respectively), whereas the proton resonances for the pyrene groups H13, H14 and H11 were recorded at δ = 7.81, 7.70 and 7.36 ppm (Fig. 4d), which were attributed to the shielded surroundings inside the cavity of the interlocked architectures. In addition, the 1H DOSY NMR spectrum of MCATCAAC revealed that only one diffusion coefficient D (5.50 × 10-10 m2 s-1) (Supplementary Fig. 45), suggesting that only one stoichiometric group of the assembly formed.

Additionally, the assembly of MCATCAAC in solution was further confirmed by ESI mass data (Fig. 4g and Supplementary Fig. 46). Prominent peaks at m/z = 906.2563 (calcd for [MCATCAAC ‒ 3(PF6)]3+ 906.2271) and 1431.8179 (calcd for [MCATCAAC ‒ 2(PF6)]2+ 1431.8231) were observed, which closely matched the anticipated theoretical distributions.

We subsequently examined the combination of H2B and H2C at a ratio of 1:1 with Ag2O under the same conditions as described for the assembly of MCATCAAC, which resulted in the formation of heteroleptic MCATCBBC with a yield of 90% (Fig. 2, method e). Single crystals of MCATCBBC were obtained by slow diffusion of diethyl ether into an acetonitrile solution of the assembly. SCXRD analysis (Fig. 3c) confirmed that the molecular structure of MCATCBBC closely resembled that of MCATCAAC, where two identical heteroleptic MRBCs interpenetrate to form the heteroleptic MCAT. Moreover, each independent MRBC is composed of di-NHC ligands B and C, as well as two silver ions. Like the MCATCAAC assembly, the pyrene-bridged ligand C was located at the top and bottom, whereas the anthracene-bridged ligand B was located in the middle of the MCATCBBC assembly. Triple typical π···π stacking interactions were also observed between the ligand backbones, which are arranged into pyrene–anthracene–anthracene–pyrene stacks with distances of approximately 3.5 Å between the mean planes of the stacked rings.

Like assembly MCATCAAC, the structure of the heteroleptic MCATCBBC in solution was also investigated (Supplementary Figs. 51–56). Consistent with expectations, the ¹H NMR analysis of MCATCBBC (Fig. 4e and Supplementary Fig. 51) confirmed the equimolar presence of ligand precursors H2B and H2C (1:1 ratio). In addition, strongly upfield-shifted resonances were also observed for the aromatic protons of the anthracene units H9’, H13’, H10’ and H14’ (δ = 6.91, 6.59, 6.52 and 4.95 ppm, respectively), whereas the proton resonances for the pyrene groups H13, H14, H11 and H10 were recorded at δ = 7.77, 7.69, 7.27 and 6.96 ppm, respectively, consistent with the pyrenyl−anthracene−anthracene−pyrenyl stacking mode in MCATCBBC. The 1H DOSY NMR spectrum (Supplementary Fig. 55) of MCATCBBC revealed that all of the aromatic proton signals had a single diffusion constant (D = 5.11 × 10-10 m2 s-1), indicating the presence of only a unique assembly stoichiometry. Moreover, the formation of heteroleptic MCATCBBC in solution was further confirmed by the ESI mass spectrum, which showed a series of peaks at m/z = 904.9167 (calcd for [MCATCBBC ‒ 3(PF6)]3+ 904.9002) and 1429.8345 (calcd for [MCATCBBC ‒ 2(PF6)]2+ 1429.8326), which is consistent with the theoretical isotopic distributions (Fig. 4h and Supplementary Fig. 56).

Considering the labile nature of the Ag–CNHC bond and the efficient assembly of two-component MCATs from NHC ligands and AgI ions, we subsequently performed experiments to investigate whether these heteroleptic systems can be formed by the rearrangement of homoleptic assemblies. Indeed, mixing MCATAAAA and MCATCCCC at a ratio of 1:1 resulted in rapid (few seconds) conversion to heteroleptic MCATCAAC (Fig. 2, method f), as suggested by the 1H NMR spectrum (Supplementary Fig. 47). Interestingly, although the reaction of the proligand H2B with Ag2O produced only the binulear MRBB rather than MCAT, MCATCCCC and MRBB could still be rapidly converted to MCATCBBC when it was mixed in CD3CN at a ratio of 1:2 (Fig. 2, method g), as revealed by 1H NMR spectroscopy (Supplementary Fig. 57). These fusion reactions indicate that heteroleptic MCATCAAC and MCATCBBC, which are selectively formed through integrative self-sorting, represent thermodynamically favorable reaction products.

To investigate whether the structures of MCATCAAC and MCATCBBC varied with respect to the concentration and temperature of the solution, concentration dilution and variable-temperature experiments were subsequently performed. Variable-concentration 1H NMR spectra of MCATCAAC (Supplementary Fig. 48) and MCATCBBC (Supplementary Fig. 58) in CD3CN revealed that only the peak intensities gradually decreased as the concentration was diluted from 6.0 to 0.1 mM, while both the peak count and integral ratios remained largely unchanged. Variable-temperature 1H NMR spectra of MCATCAAC (Supplementary Fig. 49) and MCATCBBC (Supplementary Fig. 59) in CD3CN revealed that the number, shape and chemical shifts of signals changed negligibly over the range of −20~65 °C. These observations indicate that both MCATCAAC and MCATCBBC can be maintained without structural changes in the acetonitrile solution. Furthermore, NMR titration studies of MCATCAAC (Supplementary Fig. 50) and MCATCBBC (Supplementary Fig. 60) revealed that gradually changing the solvent from CD3CN to DMF-d7 resulted exclusively continuous shifts of their characteristic proton resonances. These shifts are consistent with expected solvent effects, indicating that both MCATCAAC and MCATCBBC maintain their structural integrity in the presence of DMF.

Synthesis and characterization of heteroleptic MCAT with three different di-NHC ligands

We have shown the construction of heteroleptic MCATs (with two different di-NHC ligands) either by a one-pot strategy with Ag2O and the corresponding individual di-NHC precursors or via catenane–to–catenane transformations from homoleptic precursors. Notably, in contrast to the assembly from the individual ligands, the conversion of homoleptic assemblies to heteroleptic MCATs can be accomplished in a few seconds at room temperature, presumably because no deprotonation step is required in supramolecular fusion. On the basis of these results, we were interested in whether the structural complexity of the system could be further increased by further fusing two heteroleptic MCATs. We carefully inspected heteroleptic MCATCAAC and MCATCBBC and determined that not only do they have the same pyrene-bridged external ligand, but their internal ligands differ by only two atoms. Considering the similarities in the structure and composition of MCATCAAC and MCATCBBC, we speculated that their fusion can result in heteroleptic MCATs with more sequences. When two heteroleptic MCATCAAC and MCATCBBC were mixed at room temperature at a ratio of 1:1 in CD3CN (Fig. 2, method h), a complex new set of signals could be observed in the NMR spectra (Fig. 4f and Supplementary Figs. 61–64), indicating the formation of a new assembly through self-sorting, denoted MCATCABC. In addition, the 1H DOSY NMR spectrum of the reaction mixture revealed that there was a single diffusion coefficient (D = 5.25 × 10-10 m2 s-1), suggesting that only one stoichiometric group of the assembly formed (Supplementary Fig. 65). The ESI mass data clearly revealed that the product with the formula [Ag4(A)(B)(C)2] formed, and the peaks at m/z = 905.5603 and 1430.8234 could be attributed to the {MCATCABC ‒ 3(PF6)}3+ and {MCATCABC ‒ 2(PF6)}2+ species (Fig. 4i and Supplementary Fig. 66).

Single crystals of the MCATCABC assembly suitable for SCXRD analysis were obtained by slow diffusion of diethyl ether into the solution (VMeCN/VDMSO = 15:1) of the compound. Notably, SCXRD analysis (Fig. 3d) unambiguously confirmed the MCAT structure of MCATCABC, comprising three distinct di-NHC ligands. We note that this is probably the architecture of MCAT with the highest degree of ligand differentiation and precisely defined structure achieved to date. Moreover, the solid-state structure of the MCATCABC assembly revealed that one MRAC consists of phenazine-bridged di-NHC ligand A and pyrene-bridged di-NHC ligand C connected by two silver(I) ions, whereas the other MRBC is supported by ligand C and anthracene-bridged di-NHC ligand B connected by two silver ions. Notably, two pyrene units are located outside the cavity of the two interlocked metallacycles, while anthracene and phenazine units are threaded through the second interlocked metallacycle, resulting in a pyrene–anthracene–phenazine–pyrene parallel arrangement. In the solid structure of MCATCABC, π···π stacking interactions are observed between the pyrene and anthracene groups with an interplanar distance of 3.4 Å, in addition to donor‒acceptor interactions between the phenazine unit and the adjacent pyrene or anthracene group, as inferred from the interring distances of 3.3–3.4 Å. These interactions are the main driving forces for the formation of the interlocked structure.

Further experiments were performed to investigate the stability of heteroleptic MCATCABC relative to those of the homoleptic MCATAAAA, MCATCCCC, and MRBB. A mixture of MCATCCCC (1 equiv), MRBB (2 equiv) and MCATAAAA (2 equiv) was allowed to equilibrate in CD3CN at room temperature (Fig. 2, method i). Analyses of the reaction mixture by 1H NMR were consistent with the presence of MCATCABC as the unique product in solution (Supplementary Fig. 67b). Alternatively, treating the H2A, H2B and H2C ligands at a ratio of 1:1:2 with Ag2O in acetonitrile at 65 °C for 12 h (Fig. 2, method j) resulted in the formation of MCATCABC, as revealed by 1H NMR spectroscopy (Supplementary Fig. 67c). These observations indicate that heteroleptic assembly MCATCABC is the thermodynamically favored reaction product. The stability of the formed heteroleptic MCATCABC was further investigated. Notably, the number, shapes and chemical shifts of the signals changed negligibly in the 1H NMR spectra of MCATCABC either at different concentrations (6.0−0.1 mM) (Supplementary Fig. 68) or at a range of temperatures (−20−65 °C) (Supplementary Fig. 69), revealing the stability of the heteroleptic MCATCABC structure in acetonitrile solution. Moerever, NMR titration studies (Supplementary Fig. 70) indicate that MCATCABC can also maintained without structural changes in the presence of DMF. Furthermore, with the visual molecular dynamics (VMD) program52 and wavefunction software Multifwn 3.853, an independent gradient model based on Hirshfeld partition (IGMH) analysis54,55 revealed three IGMH δginter isosurfaces corresponding to van der Waals interactions within three heteroleptic MCATs (Supplementary Fig. 71). Both pyrene and phenazine moieties contain π electrons (prerequisite for π···π stacking), the interaction itself stems from dispersion forces between parallel π electron clouds, and the nearly flat shape of the δginter isosurfaces between π-conjugated fragments is a hallmark feature of π···π stacking56,57.

Theoretical study

Unlike MCATs that bear a single type of NHC ligand, those incorporating two or more distinct NHC ligands have different component sequences defined by the arrangement of their ligands. Notably, dimerization of either two identical or two different heteroleptic MRs should yield MCATs with numerous possible sequences. However, the above mentioned experimental results revealed only three specific sequences. To elucidate this selective self-assembly, we conducted theoretical investigations employing semiempirical and DFT calculations.

Beginning with MCATCAAC, we focused on its heteroleptic metalla-rectangle building unit, MRAC. As shown in Fig. 5a, statistical analysis indicated that dimerization of two MRAC units could yield not only MCATCAAC but also other sequence isomers (MCATAACC and MCATACCA). Structures of MRAC and three MCATs were optimized via the semiempirical GFN2-xTB method58, followed by DFT calculations for single-point energy calculations via the ωB97X-D hybrid functional59 (Fig. 5c and Supplementary Table 1). The calculated binding energies (EBEs) for the dimerization of the two MRACs to the MCATs were –387.5 kJ mol⁻¹ (for MCATAACC), –306.0 kJ mol⁻¹ (for MCATACCA), and –417.5 kJ mol⁻¹ (for MCATCAAC). Comparisons reveal that MCATCAAC is significantly more stable than MCATAACC and MCATACCA by approximately 30.0 and 111.5 kJ mol⁻¹, respectively. This stability of MCATCAAC is consistent with experimental observations, indicating that the CAAC component sequence is highly favored during the assembly of MCATs. Similarly, the binding energy EBE (–380.0 kJ mol⁻¹) of MCATCBBC was substantially greater than that of MCATBBCC (EBE = –358.0 kJ mol⁻¹) and MCATBCCB (EBE = –290.1 kJ mol⁻¹), confirming the increased stability of the CBBC component sequence isomer that was observed experimentally (Supplementary Fig. 72 and Table 2).

Next, we extended the theoretical analysis to explain the exclusive experimental observation of MCATCABC among possible MCATs incorporating three distinct NHC ligands. Unlike the above mentioned cases, MCATCABC arises from the dimerization of two different heteroleptic metalla-rectangles (MRAC and MRBC). Statistical analysis revealed three other possible sequence isomers, excluding homodimers: MCATABCC, MCATBACC, and MCATACCB (Fig. 5b). Semiempirical optimization and DFT single-point energy calculations (Fig. 5c and Supplementary Table 3) revealed that compared with MCATABCC (EBE = –372.6 kJ mol⁻¹), MCATBACC (EBE = –376.1 kJ mol⁻¹), and MCATACCB (EBE = –298.3 kJ mol⁻¹), MCATCABC has a significantly higher binding energy EBE (–400.3 kJ mol⁻¹). This finding indicates the excellent stability of the CABC component sequence, which is consistent with its exclusive experimental formation.

Near-infrared photothermal conversion study

Although the prevalence of π···π stacking interactions in supramolecular assemblies constitutes a key stabilization mechanism, it further facilitates nonradiative energy migration and photothermal conversion processes. We have demonstrated that different conjugate planes can be integrated into a single discrete supramolecular assembly by a metal‒carbon bond-mediated supramolecular fusion strategy. To reliably determine the effect of different conjugate planes and stacking modes on the photothermal conversion efficiency, the near-infrared photothermal conversion properties of MRBB and five MCATs with different component sequences (AAAA, CCCC, CAAC, CBBC, and CABC) were investigated.

As shown in Figs. 6a, b, the three heteroleptic MCATs experienced different and obvious warming processes. Specifically, a solution of MCATCBBC (1.0 mM in CH3CN, 1.0 mL) experienced a notable increase in temperature from 22.8 to 37.3 °C at 808 nm at 1.5 W cm−2. Intriguingly, a greater temperature increase was detected for MCATCABC (ΔT = 30.1 °C, from 22.4 to 52.5 °C) than for MCATCBBC (ΔT = 14.5 °C, from 22.8 to 37.3 °C). A 39.7% photothermal conversion efficiency was calculated for MCATCBBC (Supplementary Fig. 78), and a 46.5% efficiency was calculated for MCATCABC (Supplementary Fig. 79). Compared with MCATCBBC and MCATCABC, MCATCAAC resulted in greater temperature differences (ΔT = 49.3 °C, from 23.4 to 72.7 °C). According to the equations shown in the ESI, a 79.6% efficiency was calculated for MCATCAAC (Supplementary Fig. 77). Analysis of the solid-state structures reveals that although all three heteroleptic MCATs have the same number of π···π stacking interactions, compared with MCATCBBC, MCATCAAC and MCATCABC exhibit stronger stacking strengths. In addition, in the structure of MCATCAAC, two electron-deficient phenazine skeletons are adjacent to the electron-rich pyrene conjugated groups and arranged in parallel, which is more conducive to electron transport, resulting in a higher photothermal conversion efficiency for MCATCAAC.

In contrast, the solution temperatures of the homoleptic assemblies MCATAAAA, MRBB and MCATCCCC increased by 28.1 °C, 8.6 °C, and 13.9 °C, respectively (Figs. 6a, b and Supplementary Figs. 74–76), which were significantly lower than the temperature increases of the corresponding heteroleptic MCATs. This result further confirms that the near-infrared photothermal conversion performance of the materials can be effectively regulated by changing the ratio and arrangement of different π-conjugated skeletons in the metalla[2]catenane structure. Furthermore, we recorded the electron paramagnetic resonance (EPR) spectra of MRBB and MCATs. The strong signals indicate the presence of unpaired electrons, which is consistent with the charge-transfer interaction in the ground state. After irradiation, the EPR signals of MCATAAAA, MRBB, MCATCCCC, MCATCAAC, MCATCBBC and MCATCABC increased by 12.9, 1.2, 2.4, 16.3, 2.4 and 13.9 times, respectively (Supplementary Figs. 80–85). This observation is consistent with the established conclusion that under 808 nm laser irradiation, the difference in the strong EPR signal with 808 nm laser irradiation of MCATCAAC indicates its excellent NIR photothermal conversion efficiency.

Methods
Synthesis

The ligands H2A, H2B and H2C were synthesized from 2,7-dibromophenazine, 2,6-dibromoanthracene, and 1,6-dibromopyrene, respectively; detailed methods and characterizations of H2A, H2B and H2C are provided in the Supplementary Information. The homoleptic assemblies MCATAAAA, MRBB and MCATCCCC were synthesized using Ag2O and ligands H2A, H2B and H2C, respectively; the heteroleptic assemblies MCATCAAC, MCATCBBC and MCATCABC were synthesized either via direct assembly of NHC ligands with Ag(I) nodes or structural transformation from homoleptic precursors trough a supramolecular fusion strategy; detailed methods and characterizations of MCATAAAA, MRBB, MCATCCCC, MCATCAAC, MCATCBBC and MCATCABC are provided in the Supplementary Information.

X-ray crystallography

Single crystals of MCATAAAA, MCATCAAC, MCATCBBC and MCATCABC suitable for X-ray diffraction were obtained at room temperature. X-ray intensity data for MCATAAAA, MCATCBBC, and MCATCABC were collected at T = 240 K and those for MCATCAAC at T = 180 K on a Bruker D8 Venture system and used Mo Kα radiation (λ = 0.71073 Å). Using Olex260, the structures were resolved with the SHELXT-199761,62 structure solution program using direct methods and refined with the SHELXL63 refinement package using least-squares minimization. All non-hydrogen atoms were refined with anisotropic displacement parameters, and hydrogen atoms were placed in geometrically calculated positions. With these data, the disordered solvent molecules that could not be restrained properly were removed using the SQUEEZE method. The ORTEP-style illustration of four structures can be seen in Supplementary Figs. 86 (for MCATAAAA), 87 (for MCATCAAC), 88 (for MCATCBBC) and 89 (for MCATCABC), with probability ellipsoids. Details of the X-ray crystallographic refinement of MCATAAAA, MCATCAAC, MCATCBBC and MCATCABC can be found in Supplementary Tables 4, 5, 6 and 7, respectively.

Computational details

The computational study using the combination of self-consistent tight-binding (GFN2-xTB)58, and density functional theory (DFT) calculations were performed with the Guassian09 quantum chemistry package64. The geometries of MRs and MCATs were optimized using semi-empirical GFN2-xTB method implemented with D4 model65. The relative stability of feasible supramolecular structures was investigated through single-point calculations using ωB97X-D hybrid functional59 with the 6-31G(d,p)66 basis set for non-metal atoms (C, H, N) atoms and the LANL2DZ67 basis set for metal atoms (Ag). Acetonitrile was used as a solvent for calculations (SMD model)68.

Near-infrared photothermal conversion research

To guarantee same amount of conjugated-π area, the applied molar ratio of the six topologies MCATAAAA/MRBB/MCATCCCC/MCATCAAC/MCATCBBC/MCATCABC was 1:2:1:1:1:1. Compound MCATAAAA (9.33 mg, 0.003 mmol) was added into a solvent of CH3CN (3 mL). After the solid dissolved absolutely, 1.0 mL of this solution was taken into a 2.0 mL sample vessel and put into the bright spot of a laser with 808 nm wavelength at 1.5 W/cm2. Temperature variation of the solution was detected by an infrared camera. Compound MRBB (9.29 mg, 0.006 mmol), compound MCATCCCC (9.59 mg, 0.003 mmol), compound MCATCAAC (9.46 mg, 0.003 mmol), compound MCATCBBC (9.45 mg, 0.003 mmol) and compound MCATCABC (9.45 mg, 0.003 mmol) were detected with the same procedure as compound MCATAAAA.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The article entitled “Sequence-Encoded Layered Heteroleptic Metalla-[2]catenanes for Programmable Supramolecular Function” is well written and well-justified. This study introduces a novel class of supramolecular architecture known as heteroleptic metalla-[2]catenanes (MCATs), designed to investigate how the spatial arrangement of functional motifs influences macroscopic behavior. Unlike traditional radial structures such as metallocages and metallacycles, MCATs feature a layered and stereoscopic configuration, enabling more complex molecular information encoding. Using N-heterocyclic carbene (NHC) ligands combined with silver (Ag(I)) nodes, MCATs were constructed with two or three types of ligands, forming specific sequences through direct assembly or supramolecular transformation. Their structures were confirmed via single-crystal X-ray diffraction and high-resolution mass spectrometry, while theoretical calculations (semiempirical and DFT) demonstrated that the obtained sequences are thermodynamically more stable than alternative isomers. Three heteroleptic MCATs were synthesized: MCATCAAC, MCATCBBC, and MCATCABC, each with different ligand combinations. These were formed through both direct assembly and supramolecular fusion of homoleptic precursors. π···π interactions between ligands played a key role in forming interlocked structures, and a unique intramolecular self-ordering behavior was observed. Spectroscopic studies (NMR, DOSY, ESI-MS) confirmed the formation of well-defined and stable assemblies in solutions, even under varying concentrations and temperatures. Additionally, MCATs were shown to rapidly transform into one another via supramolecular fusion, indicating that heteroleptic structures are thermodynamically favored products. Functionally, the near-infrared (NIR) photothermal conversion efficiency of MCATs was evaluated. Heteroleptic structures exhibited significantly higher efficiency than homoleptic ones. For instance, MCATCAAC showed an efficiency of 79.6%, outperforming MCATCBBC (39.7%) and MCATCABC (46.5%). This is attributed to the arrangement of π-conjugated motifs, which enhance electron transport and energy conversion. Theoretical calculations supported experimental findings, showing that observed sequences (CAAC, CBBC, and CABC) have lower binding energies, indicating greater stability. IGMH analysis confirmed the presence of dispersion interactions between π-conjugated planes, essential for forming interlocked structures.\nComments on the article:\na) In the introduction, when citing different examples of metallocages and metallacycles with NHC ligands, I recommend the following citations for referencing them: Angew. Chem. Int. Ed. 2022, 61, e202208189 and Acc. Chem. Res. 2020, 53, 1401−1413\nb) In the ESI, in the synthesis of compounds 1 and 4, add the solvents or elution solvent to the chromatographic column.\nc) In the synthesis of compounds 3, 6, and 8, the solvent trichloromethane could be replaced by another solvent in their preparation.\nd) In the ESI on line 91, the numbers in the drawing do not match the NMR data, as signal 18 does not exist in the description of the compound and number 14 is missing from the structure.\ne) In the dosy experiments, it is necessary to know the concentration at which the experiment is performed in order to calculate the size. Please indicate the concentrations for each dosy experiment in each ESI figure.\nf) The separation of the three compounds MRBB/MCATABAA/MCATBAAA has been tested using a chromatographic column.\nThe paper is novel and provides a breakthrough in supramolecular chemistry and stereoscopic layered arrangements provide a versatile platform for exploring how spatial molecular coding guides emergent functions.", "reviewer_2": "As we know, π-π stacking aromatic compounds plays an important role to create interesting optoelectronic properties by intermolecular interactions between these π-molecules. However, to achieve structurally well-defined spatial arrangement of functional motifs remains a great challenge. Recently, some groups have reported to obtain this kind of π-stacks through host-guest assembly. And more, these pure supramolecular π-stacks are not enough stable and easy to dissociate as materials especially for actual application due to the weak intermolecular interactions. The authors report to apply tetranuclear metalla-[2]catenane (MCAT) as a versatile platform to achieve layered arrangement of two or three kinds of various functional motifs. More interestingly, using this strategy, several two or three-component four-layer π-stacks are synthesized in one step with high yield. Although the [2]catenane also belong to supramolecules, the interlocked cyclophanes can provide additional stability. Furthermore, they probe sequence–function relationships in layered supramolecular architectures. Overall, I think this manuscript presents an innovative study on the development of structurally well-defined spatial arrangement of various functional motifs. I recommend publication after minor revision.\n1. As for H2B, only the simple binuclear MRBB is formed. Is it possible that the intermolecular interaction between anthracene moieties is much weaker than other two fused polycyclic aromatic hydrocarbons?\n2. As the authors stated, the labile nature of the Ag–CNHC bond help to achieve fusion reaction between MCATs. What substance can cause it to decompose?\n3. In this manuscript, the NIR photothermal conversion properties of these MCATs in CH3CN solution are evaluated and only absorptions from 700 to 900 nm are showed. The authors should show the complete light absorption capability of these interesting π-stacks and provide the absorption spectra from 300 to 900 nm in the supporting information file.", "reviewer_3": "This work builds on previous studies (such as Chem. Eur. J. 2022, 28, e202104617; Angew. Chem. Int. Ed. 63, e202409664 (2024); Angew. Chem. Int. Ed. 62, e202312323 (2023); and J. Am. Chem. Soc. 2023, 145, 18036) focused on homoleptic and heteroleptic metalla[2]catenanes, where organometallic supramolecular architectures result in distinctive layered configurations, in contrast to the radial arrangements of metallocages and metallacycles. While the use of the supramolecular fusion technique to transform homoleptic precursors into heteroleptic assemblies is interesting, it represents an extension of previously reported homoleptic metalla[2]catenanes. Therefore, the novelty of the current work is limited, and it may not be suitable for Nature Communications. Below are a few comments for improving the manuscript:\n1. The synthesis of NHC ligands involves column chromatography in several steps; however, the conditions such as the composition of the eluents and the solvents used, are not specified. This information should be provided for better reproducibility.\n2. There is a typo error in Figure 1. The caption states: “(b) circular arrangement of different functional motifs in metallacycles, (b) radial arrangement of different motifs in metallocages,” which is written twice. This should be corrected for clarity.\n3. The heteroleptic assembly MCATCABC is stable to solvent dilutions and temperature. Given that Ag(I)-NHC complexes are known to undergo transmetallation, this aspect should be explored or at least addressed in the manuscript.\n4. The effect of different solvents such as DMF on heteroleptic assembly is missing whereas it's been well established that similar [2]catenanes homoleptic complexes undergo significant rearrangement (Angew. Chem. Int. Ed. 63, e202409664 (2024))."}, "author_response": "Response to Reviewers\nReviewer #1:\nThe article entitled “Sequence-Encoded Layered Heteroleptic Metalla-[2]catenanes for Programmable\nSupramolecular Function” is well written and well-justified. This study introduces a novel class of\nsupramolecular architecture known as heteroleptic metalla-[2]catenanes (MCATs), designed to investigate how\nthe spatial arrangement of functional motifs influences macroscopic behavior. Unlike traditional radial\nstructures such as metallocages and metallacycles, MCATs feature a layered and stereoscopic configuration,\nenabling more complex molecular information encoding. Using N-heterocyclic carbene (NHC) ligands\ncombined with silver (Ag(I)) nodes, MCATs were constructed with two or three types of ligands, forming\nspecific sequences through direct assembly or supramolecular transformation. Their structures were confirmed\nvia single-crystal X-ray diffraction and high-resolution mass spectrometry, while theoretical calculations\n(semiempirical and DFT) demonstrated that the obtained sequences are thermodynamically more stable than\nalternative isomers. Three heteroleptic MCATs were synthesized: MCAT , MCAT , and MCAT ,\nCAAC CBBC CABC\neach with different ligand combinations. These were formed through both direct assembly and supramolecular\nfusion of homoleptic precursors. π···π interactions between ligands played a key role in forming interlocked\nstructures, and a unique intramolecular self-ordering behavior was observed. Spectroscopic studies (NMR,\nDOSY, ESI-MS) confirmed the formation of well-defined and stable assemblies in solutions, even under\nvarying concentrations and temperatures. Additionally, MCATs were shown to rapidly transform into one\nanother via supramolecular fusion, indicating that heteroleptic structures are thermodynamically favored\nproducts. Functionally, the near-infrared (NIR) photothermal conversion efficiency of MCATs was evaluated.\nHeteroleptic structures exhibited significantly higher efficiency than homoleptic ones. For instance,\nMCAT showed an efficiency of 79.6%, outperforming MCAT (39.7%) and MCAT (46.5%). This\nCAAC CBBC CABC\nis attributed to the arrangement of π-conjugated motifs, which enhance electron transport and energy\nconversion. Theoretical calculations supported experimental findings, showing that observed sequences\n(CAAC, CBBC, and CABC) have lower binding energies, indicating greater stability. IGMH analysis\nconfirmed the presence of dispersion interactions between π-conjugated planes, essential for forming\ninterlocked structures.\nThe paper is novel and provides a breakthrough in supramolecular chemistry and stereoscopic layered\narrangements provide a versatile platform for exploring how spatial molecular coding guides emergent\nfunctions.\nReply: We feel great thanks for your professional review and appreciation of our work.\nComments on the article:\na) In the introduction, when citing different examples of metallocages and metallacycles with NHC ligands, I\nrecommend the following citations for referencing them: Angew. Chem. Int. Ed. 2022, 61, e202208189 and\nAcc. Chem. Res. 2020, 53, 1401−1413.\nResponse: Thank you for this helpful suggestion. The two suggested papers (Angew. Chem. Int. Ed. 2022, 61,\ne202208189 and Acc. Chem. Res. 2020, 53, 1401−1413) have been added as new refs 45 and 46 in the\nintroduction section of our revised manuscript.\nb) In the ESI, in the synthesis of compounds 1 and 4, add the solvents or elution solvent to the chromatographic\ncolumn.\nResponse: Many thanks for your valuable comment. The experimental details for the synthesis of compounds\n1 and 4 have been updated to specify the exact eluent systems used for column chromatography in the revised\nSupplementary Information (Page S3 and S5). The purification of both compounds 1 and 4 were performed\nusing a mixture of dichloromethane and methanol, 95:5 v/v.\nc) In the synthesis of compounds 3, 6, and 8, the solvent trichloromethane could be replaced by another solvent\nin their preparation.\nResponse: Thank you for your thoughtful comment regarding the choice of solvent for the synthesis of\ncompounds 3, 6, and 8. We fully agree that exploring alternative solvents is a valuable aspect of reaction\noptimization. In the specific case of these reactions, the limited solubility of the key starting materials (2, 4,\nand 7) was a critical factor in our solvent selection. Through initial screening, we found that these particular\nreactants exhibited insufficient solubility in many common organic solvents to achieve a reaction concentration\nthat would allow for a practical and efficient transformation. Trichloromethane proved to be the optimal\nsolvent because it provided the necessary, albeit limited, solubility to create a reactive mixture and facilitate\nthe formation of the target compounds.\nd) In the ESI on line 91, the numbers in the drawing do not match the NMR data, as signal 18 does not exist\nin the description of the compound and number 14 is missing from the structure.\nResponse: Thanks for your reminder. The numbers in chemical structure of H A have been corrected.\ne) In the dosy experiments, it is necessary to know the concentration at which the experiment is performed in\norder to calculate the size. Please indicate the concentrations for each dosy experiment in each ESI figure.\nResponse: Many thanks for your essential advice. As suggested, the concentrations of each DOSY experiment\nhave been supplemented in the revised Supplementary Information. Please see Supplementary Figures 21, 26,\n35, 45 and 55.\nf) The separation of the three compounds MR /MCAT /MCAT has been tested using a\nBB ABAA BAAA\nchromatographic column.\nResponse: Thank you for your valuable suggestions. As suggested, we attempted to separate of MR ,\nBB\nMCAT and MCAT using column chromatography. However, attempts to adjust the polarity of the\nABAA BAAA\nmobile phase and the type of stationary phase still failed to achieve the separation of the abovementioned three\ncompounds.\nReviewer #2:\nAs we know, π-π stacking aromatic compounds plays an important role to create interesting optoelectronic\nproperties by intermolecular interactions between these π-molecules. However, to achieve structurally well-\ndefined spatial arrangement of functional motifs remains a great challenge. Recently, some groups have\nreported to obtain this kind of π-stacks through host-guest assembly. And more, these pure supramolecular π-\nstacks are not enough stable and easy to dissociate as materials especially for actual application due to the\nweak intermolecular interactions. The authors report to apply tetranuclear metalla-[2]catenane (MCAT) as a\nversatile platform to achieve layered arrangement of two or three kinds of various functional motifs. More\ninterestingly, using this strategy, several two or three-component four-layer π-stacks are synthesized in one\nstep with high yield. Although the [2]catenane also belong to supramolecules, the interlocked cyclophanes can\nprovide additional stability. Furthermore, they probe sequence–function relationships in layered\nsupramolecular architectures. Overall, I think this manuscript presents an innovative study on the development\nof structurally well-defined spatial arrangement of various functional motifs. I recommend publication after\nminor revision.\nReply: Many thanks for the appreciation of our work and professional comments.\n1. As for H B, only the simple binuclear MR is formed. Is it possible that the intermolecular interaction\nbetween anthracene moieties is much weaker than other two fused polycyclic aromatic hydrocarbons?\nResponse: Thank you for your insightful question. Your suggestion is absolutely correct. Indeed, the formation\nof the simple binuclear MR with the H B ligand is primarily due to the relatively weak intermolecular\nBB 2\ninteractions between its anthracene moieties, especially when compared to other fused polycyclic aromatic\nhydrocarbons like pyrene or heterocyclic systems such as phenazine.\n2. As the authors stated, the labile nature of the Ag–C bond help to achieve fusion reaction between MCATs.\nNHC\nWhat substance can cause it to decompose?\nResponse: Thank you for this insightful question. You are correct that the lability of the Ag–C bond is key\nNHC\nto the process. Additionally, chloride ions can induce the decomposition of interlocked silver MCATs into the\ncorresponding carbene precursors. The driving force for this reaction is the formation of the highly insoluble\nsilver chloride (AgCl) precipitate, which effectively removes Ag⁺ ions from the system and disrupts the Ag–\nC bonds. Taking the assembly MCAT as an example, treatment of assembly MCAT with an\nNHC CABC CABC\nexcess of NH Cl and subsequent anion exchange with NH PF in acetonitrile leads to its disassembly into the\n4 4 6\ncarbene precursors H A, H B and H C. The 1H NMR spectrum of the assembly MCAT after treatment\n2 2 2 CABC\nwith NH Cl/NH PF is completely consistent with that of the mixture of carbene precursors H A, H B and\n4 4 6 2 2\nH C (in a 1:1:2 molar ratio), indicating that the assembly MCAT has been completely decomposed into\nthe carbene precursors H A, H B and H C.\n2 2 2\nFigure R1. Partial 1H NMR spectra (400 MHz, CD3CN) of (a) a mixture of H2A, H2B and H2C (in a 1:1:2 molar ratio), (b) assembly\nMCATCABC after treatment with NH4Cl/NH4PF6, (c) assembly MCATCABC.\n3. In this manuscript, the NIR photothermal conversion properties of these MCATs in CH CN solution are\n3\nevaluated and only absorptions from 700 to 900 nm are showed. The authors should show the complete light\nabsorption capability of these interesting π-stacks and provide the absorption spectra from 300 to 900 nm in\nthe Supplementary Information file.\nResponse: Many thanks for your essential advice. As suggested, the absorption spectra from 300 to 900 nm of\nthese MCATs have been supplemented in the revised SI. Please see the Supplementary Figure 63.\nCAAC\nMCAT\nCABC\nMCAT\nAAAA\nCBBC\ne MCAT\nc CCCC\nn MR\na BB\nb\r\no\ns 50\nb\nA\n0\n300 400 500 600 700 800 900\nλ (nm)\nSupplementary Figure 63. UV−vis absorption spectra (CH3CN, c = 1.0 mM, λ = 300~900 nm) of five MCATs with different\ncomponent sequences (AAAA, CCCC, CAAC, CBBC, and CABC) and MRBB.\nReviewer #3:\nThis work builds on previous studies (such as Chem. Eur. J. 2022, 28, e202104617; Angew. Chem. Int. Ed. 63,\ne202409664 (2024); Angew. Chem. Int. Ed. 62, e202312323 (2023); and J. Am. Chem. Soc. 2023, 145, 18036)\nfocused on homoleptic and heteroleptic metalla[2]catenanes, where organometallic supramolecular\narchitectures result in distinctive layered configurations, in contrast to the radial arrangements of metallocages\nand metallacycles. While the use of the supramolecular fusion technique to transform homoleptic precursors\ninto heteroleptic assemblies is interesting, it represents an extension of previously reported homoleptic\nmetalla[2]catenanes. Therefore, the novelty of the current work is limited, and it may not be suitable for Nature\nCommunications. Below are a few comments for improving the manuscript:\nReply: We appreciate your encouraging comment. We would like to emphasize that, to the best of our\nknowledge, heteroleptic metalla[2]catenanes based on NHC ligands remain relatively rare in the literature.\nTherefore, the present study not only successfully constructs a new type of NHC-based heteroleptic\nmetalla[2]catenane, thereby enriching the design strategies for such intricate supramolecular systems, but more\nimportantly, it demonstrates the potential of the supramolecular fusion approach in accessing architectures\nwith higher structural complexity. This offers new insights and experimental support for the precise assembly\nof such systems. Furthermore, our work provides valuable mechanistic understanding regarding the formation\nof these intricate topological structures. We believe that this study serves as a useful reference for the future\ndesign and construction of more complex supramolecular systems.\n1. The synthesis of NHC ligands involves column chromatography in several steps; however, the conditions\nsuch as the composition of the eluents and the solvents used, are not specified. This information should be\nprovided for better reproducibility.\nResponse: Many thanks for your essential advice to improve the quality of this manuscript. As suggested, the\nexperimental details for the synthesis of compounds 1 and 4 have been updated to specify the exact eluent\nsystems used for column chromatography in the revised Supplementary Information (Page S3 and S5). The\npurification of both compounds 1 and 4 were performed using a mixture of dichloromethane and methanol,\n95:5 v/v.\n2. There is a typo error in Figure 1. The caption states: “(b) circular arrangement of different functional motifs\nin metallacycles, (b) radial arrangement of different motifs in metallocages,” which is written twice. This\nshould be corrected for clarity.\nResponse: We apologize for the typos in our manuscript. The typo has been corrected.\n3. The heteroleptic assembly MCAT is stable to solvent dilutions and temperature. Given that Ag(I)-NHC\nCABC\ncomplexes are known to undergo transmetallation, this aspect should be explored or at least addressed in the\nmanuscript.\nResponse: Many thanks for your valuable advice. As suggested, transmetalation of dicarbene ligands from\nsilver(I) to gold(I) was also performed. However, to date, we have found that it is still not possible to confirm\nthe exact structure of the obtained gold compound through X-ray crystallographic analysis. Consequently, we\nare unable to establish the connection between sequence and function. To avoid providing misleading\ninformation, we recommend temporarily not including this part in the manuscript.\n4. The effect of different solvents such as DMF on heteroleptic assembly is missing whereas it's been well\nestablished that similar [2]catenanes homoleptic complexes undergo significant rearrangement (Angew.\nChem. Int. Ed. 63, e202409664 (2024)).\nResponse: Many thanks for your constructive comment. You have correctly pointed out the potential impact\nof the solvent environment (especially highly polar solvents like DMF) on the stability of heteroligand\nassemblies. We fully agree with this point and have investigated it in our research through NMR titration\nexperiments. The heteroleptic assembly MCAT was first prepared in CD₃CN, to which increments of\nCAAC\nDMF-d were sequentially added. An NMR titration from pure CD₃CN to pure DMF-d showed exclusively\n7 7\ncontinuous shifts of some characteristic proton resonances of MCAT . These signals remained sharp\nCAAC\nthroughout the process, ruling out disassembly or exchange dynamics on the NMR timescale. The observed\nshifts are consistent with expected solvent effects and confirm that the assembly remains intact, demonstrating\nits stability in highly polar solvents like DMF. Using the same method, the stability of MCAT and\nCBBC\nMCAT in the presence of DMF were investigated. The results showed that MCAT and MCAT\nCABC CBBC CABC\ncould also remain stable in the presence of DMF. The 1H NMR spectra of heteroleptic assemblies in different\nratios of CD CN/DMF-d solvent mixtures were provided, please see Supplementary Figure 40 (for\n3 7\nMCAT ), Supplementary Figure 50 (for MCAT ) and Supplementary Figure 60 (for MCAT ).\nCAAC CAAC CAAC\nSupplementary Figure 40. 1H NMR spectra (400 MHz) of MCAT in in different ratios of CD CN/DMF-\nCAAC 3\nd solvent mixtures.\nCBBC 3\nd solvent mixtures.\nCABC 3\nd solvent mixtures.\n7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "All the modifications that have been made to the article are well justified. I therefore consider them to be considered and appropriate for publication in the journal Nature Communications.", "reviewer_2": "I am satisfied with this revision and recommend publication without further revision.", "reviewer_3": "The authors has revised the maniscript to satisfactory level. Overall, I am satisfied with the revisions and believe the manuscript is now ready for publication, pending final editorial checks."}, "author_response": ""}]

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