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How neocarcerand Octacid4 self-assembles with guests into irreversible noncovalent complexes and what accelerates the assembly

How neocarcerand Octacid4 self-assembles with guests into irreversible noncovalent complexes and... ARTICLE https://doi.org/10.1038/s42004-022-00624-4 OPEN How neocarcerand Octacid4 self-assembles with guests into irreversible noncovalent complexes and what accelerates the assembly Yuan-Ping Pang Cram’s supramolecular capsule Octacid4 can irreversibly and noncovalently self-assemble with small-molecule guests at room temperature, but how they self-assemble and what accelerates their assembly remain poorly understood. This article reports 81 distinct Octacid4� guest self- assembly pathways captured in unrestricted, unbiased molecular dynamics simulations. These pathways reveal that the self-assembly was initiated by the guest interaction with the cavity portal exterior of Octacid4 to increase the portal collisions that led to the portal expansion for guest ingress, and completed by the portal contraction caused by the guest docking inside the cavity to impede guest egress. The pathways also reveal that the self-assembly was accelerated by engaging populated host and guest conformations for the exterior interaction to increase the portal collision frequency. These revelations may help explain why the presence of an exterior binding site at the rim of the enzyme active site is a fundamental feature of fast enzymes such as acetylcholinesterase and why small molecules adopt local minimum conformations when binding to proteins. Further, these revelations suggest that irreversible noncovalent complexes with fast assembly rates could be developed—by engaging populated host and guest con- formations for the exterior interactions—for materials technology, data storage and processing, molecular sensing and tagging, and drug therapy. 1 ✉ Computer-Aided Molecular Design Laboratory, Mayo Clinic, Rochester, MN, USA. email: camdl1@icloud.com COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 1 1234567890():,; ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 rreversible bimolecular complexes are highly desirable because is present in the reaction medium for the tethering reaction; the (1) the residence of one molecule inside the cavity of another disassembly of the resulting carcerand complex is not allowed Imolecule is permanent until the breakdown of the cavity- unless the host is broken by excessive heat . When the barrier is containing molecule, (2) the complex formation is one-to-one moderately high, assembly or disassembly requires annealing. For stoichiometric, and (3) for drug therapy, the permanent residence example, a guest can enter or exit the cavity of a host known as and 1:1 stoichiometry confer the cavity-residing molecule with hemicarcerand once the bimolecular system is heated to expand desired high metabolic stability, high potency-to-mass ratio, low- the cavity portal, and the guest remains inside the cavity once the 1–4 acting dose, and low off-target activity . These complexes system is cooled to contract the portal; the disassembly of the 12, 15 generally refer to irreversible covalent complexes that are self- hemicarcerand complex is disallowed without heating . When assembled by two molecules between which there is a covalent the barrier is slightly high, assembly or disassembly can occur bond resulting from the self-assembly process. The desirability of spontaneously and slowly at an ambient temperature. For some irreversible covalent complexes is apparent from the drug action host•guest complexes, the disassembly can be disabled at the 5 6 7 mechanisms of aspirin , penicillin , and sotorasib , all of which ambient temperature by the complexation that subsequently involve an irreversible covalent complex. Notably, sotorasib was heightens the barrier for disassembly. For example, a guest can approved in May 2021 (https://www.fda.gov/news-events/press- enter the cavity of a host known as neocarcerand at room tem- announcements/fda-approves-first-targeted-therapy-lung-cancer- perature and remain in the cavity at room temperature unless the mutation-previously-considered-resistant-drug) as the first-in- portal is opened by heating because the docking of the guest at class personalized treatment for a lung-cancer mutation pre- the cavity induces a host conformational change that conse- viously considered resistant to drug therapy due to sotorasib’s quently closes all cavity portals . G12C unique capability to clinically block the function of KRAS The applications of carcerand and hemicarcerand are however (an enzyme mutant responsible for ~13% non-small-cell lung limited because their complexes cannot be formed in situ and cancers) via irreversible complexation. However, the irreversible adiabatically. Although neocarcerand can form its complexes 13, 14, 16 covalent complexes from in situ cysteine conjugation are limited in situ and adiabatically , the applications of neocarcerand by the infrequent presence of the noncatalytic cysteine in a complexes are also limited due to their slow complexation rates. G12C protein cavity, and inhibiting KRAS offers treatment for only This underscores the need to understand how two molecules self- a subset of cancer patients. A paradigm shift is needed for the assemble into an irreversible noncovalent complex and what design of irreversible bimolecular complexes. accelerates their assembly as these high-level questions hold the In terms of both intrinsic binding from the thermodynamic key to designing irreversible noncovalent bimolecular complexes perspective and constrictive binding from the kinetic with fast complexation rates for broad applications. 8–10 perspective , the irreversible complexes include irreversible To promote irreversible noncovalent bimolecular complex noncovalent complexes that are self-assembled by two molecules design, this article reports 81 distinct pathways of the irreversible between which there is no covalent bond. Here, the intrinsic noncovalent self-assembly of neocarcerand Octacid4 with three binding is the complexation governed by intermolecular inter- known guests —1,4-dioxane (dioxane), p-xylene (xylene), and actions between the two molecules and between the solvent and naphthalene (Fig. 1 and Table S1). These pathways were captured each of the two molecules, while the constrictive binding is the in multiple distinct, independent, unrestricted, unbiased, and complexation controlled by the thermal energy required for the classical isobaric–isothermal molecular dynamics (MD) simula- guest to overcome the steric hindrance from the host during the tions at a high time resolution with an aggregated simulation time exceeding 3.761664 milliseconds at 298–363 K, rendering the assembly or disassembly process, as exemplified below by Cram’s 11 12 supramolecular capsules known as carcerand , hemicarcerand , structural and kinetic information needed to answer the two 13, 14 and neocarcerand . high-level questions and guide the design of irreversible non- The hallmark of the constrictive binding is the heightened covalent complexes that can be formed in situ and adiabatically energy barrier for the assembly or disassembly of a host•guest with desired kinetics for materials technology, data storage and complex. This barrier can make the dissembled or assembled processing, molecular sensing and tagging, and drug therapy. molecules kinetically stable, namely, it takes a long time to assemble the two disassembled molecules or disassemble the two assembled molecules if the barrier for the conversion is heigh- Results tened. When the barrier is extremely high, assembly or dis- The challenge of capturing self-assembly pathways. In view of assembly requires covalent-bond making or breaking, the current state of computational work on guest/ligand-binding 17–20 respectively. For example, a guest can be noncovalently trapped in pathways , unrestricted and unbiased MD simulations of the the cavity of the two bowl-shaped fragments that are rim-to-rim self-assembly of Octacid4 with its known guests are challenging to tethered by linkers (of a host known as carcerand) when the guest perform because the complexation times of Octacid4•guest were Fig. 1 Structure of Octacid4 in complex with a small-molecule guest. a Octacid4� p-xylene. b Octacid4� naphthalene. c Octacid4� 1,4-dioxane. Carbon and oxygen are in blue and red, respectively. The axial or equatorial portal of Octacid4 comprises the C3 and C19 atoms or the O7, O15, C6b, and C17a atoms, respectively. Hydrogen and counter ion are not displayed for clarity. 2 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE estimated to be a few minutes (or hours for the bulkiest guest Octacid4 surrounded with 8 sodium cations, 150 guests, and naphthalene) according to NMR experiments . These com- the Berendsen thermostat. plexation times are many orders of magnitude longer than cur- rent MD simulation times that are on the order of milliseconds. Conformational characterization of the Octacid4� xylene self- To demonstrate the challenge of capturing the Octacid4•guest assembly pathways. Visual inspections of the 40 self-assembly self-assembly pathways, multiple distinct, independent, unrest- pathways of Octacid4•xylene at 298 K revealed that all pathways ricted, unbiased, and classical isobaric–isothermal MD simula- comprised three common steps (Fig. 2 and Videos S1–S6 and tions (hereafter shortened to simulations) were performed using S11–S16). In Step 1, xylene entered the cavity portal exterior the fully deprotonated, apo Octacid4 that was surrounded by space that was confined by two aromatic linkers of the host; as eight neutralizing sodium cations, 10 xylenes to mimic the use of apparent from the representative videos, there were substantial the guest in 10 equivalent excess in the NMR experiments ,60 collisions between xylene and the equatorial portal in the linker NaCl molecules to approximate the ionic strength of the region of Octacid4 that led to the portal expansion for guest 13 21 experimental conditions , and 2359 TIP3P water molecules ingress. In Step 2, xylene passed one methyl group through the to mimic the experimental aqueous solution . Indeed, no equatorial portal, then the phenyl group through the portal with autonomous complexation was observed in any set of 20 the phenyl plane perpendicular to the axial axis (viz., the axis 14,251.6-ns simulations at 298, 340, 363, or 370 K (Table S1) passing two axial portals in the bowl-shaped region), and last the because the simulation time was many orders of magnitude other methyl group through the equatorial portal. In Step 3, shorter than the experimentally estimated complexation time for xylene rotated ~90° to keep its phenyl plane parallel to the axial xylene. axis. This rotation caused the equatorial portal contraction and impeded guest egress, according to the reported structural ana- lyses of apo Octacid4 and the Octacid4•xylene complex . Using the phase-transfer catalyst to capture the self-assembly. According to the reported NMR experiments , the experimen- tally observed self-assembly of Octacid4 with dioxane, xylene, or Kinetic characterization of the Octacid4� xylene self-assembly naphthalene in a sodium borate buffer at pH 9 is mechanistically pathways. A survival analysis of the 40 14,251.6-ns simulations driven by the sodium cation as a phase-transfer catalyst that that all captured the self-assembly of Octacid4•xylene at 298 K chelates with the carboxylates on the Octacid4 surface and con- showed xylene’s mean complexation time to be 1022 ns (95% sequently accumulates the guest on the host surface via the confidence interval: 750–1392 ns). Here the complexation time cation–π interaction or the sodium chelation. The guest accu- was defined as the first time instant at which xylene was inside the mulation on the host surface is similar to immersing the sodium- host cavity and had its phenyl plane parallel to the axial axis of chelated Octacid4 in a neat guest solution. Simulating the latter the host. This xylene orientation was found in the most populated can substantially accelerate the self-assembly according to the law Octacid4•xylene conformation . of mass action, enabling determination of relative complexation To dissect the self-assembly kinetics, the duration of the first times of different guests with Octacid4 for mechanistic insights step is herein termed priming time, and the duration of the last into the self-assembly. Simulating the latter also allows the use of two steps is termed ingression time. These names are used linear-regression analysis to examine the convergency and because during the first step, the host and guest conformations internal consistency of the MD simulations. A goodness of fit(r ) are primed, through self-selection and conformational rearrange- of <0.70 for the natural logarithm plot of the host population ments, for guest ingress, and because during the last two steps, the versus the simulation time indicates problematic simulations. guest enters the host cavity and rotates ~90° to form a complex. This is because the first-order rate (viz., the exponential decay of The priming time was defined as a time period from the the host population over the simulation time) is expected for beginning of the MD simulation to the last time instant at which simulating the self-assembly of Octacid4 with its guest in large the distance between any guest hydrogen atoms and any host excess. methylene hydrogen atoms was greater than 2.6 Å. The Accordingly, 40 simulations were performed using the fully hydrogen–hydrogen distance cutoff (abbreviated as HH cutoff) deprotonated, apo Octacid4 that was surrounded by eight was set at 2.6 Å for the following reasons. Visual inspection of all neutralizing sodium cations and 150 xylenes. Here, the number 40 Octacid4•xylene pathways revealed that xylene was outside of of xylenes was arbitrarily chosen. Gratifyingly, all 40 14,251.6-ns the Octacid4 cavity as long as the center-of-mass distance cutoff simulations with the fully deprotonated, apo Octacid4 with 150 (abbreviated as COM cutoff) for Octacid4 and its guest was ≥7Å. xylenes captured the self-assembly of Octacid4 with xylene at However, xylene could have its terminal methyl group contact the 298 K. Additional simulations were performed using each of the host cavity portal to slightly enter the host cavity portal in some five variations: (1) replacing the fully deprotonated, apo Octacid4 of the 40 Octacid4•xylene pathways at the COM cutoff of 7 or with the octa-anionic Octacid4 possessing five water molecules 8 Å. Xylene could also be relatively away from the portal in some inside the cavity, (2) increasing the number of xylenes to 250, (3) pathways at the COM cutoff of ≥8 Å. Therefore, rather than using replacing 150 xylenes with 150 dioxanes, (4) replacing 150 the COM cutoff, the HH cutoff of 2.6 Å was used to avoid both xylenes with 150 naphthalenes, or (5) changing the Berendsen the methyl group contacting the portal (which consequently thermostat to the Langevin thermostat (Table S1). All these shortens the ingression time) and the guest being away from the simulations captured the self-assembly event. For the simulations portal (which consequently lengthens the ingression time). The with the water-bound Octacid4, all water molecules inside the ingression time was defined as a time period from the last time cavity had a high propensity to interact with the sodium cations instant at which the distance between any guest hydrogen atoms and the carboxylates outside the cavity, and egression of all five and any host methylene hydrogen atoms was greater than 2.6 Å water molecules occurred prior to the ingression of xylene. By to the first time instant at which the guest rotated ~90° inside the contrast, no complexation was observed under the same cavity. The complexation time is now a sum of the priming and conditions if the fully deprotonated, apo Octacid4 surrounded ingression times. by 8 sodium cations was replaced with the fully protonated, apo This dissection reveals that the ingression time (8–934 ps; Octacid4 without any cations (Table S1). All simulations Table S2) is a fraction of the priming time (12–14,199 ns; described hereafter used the fully deprotonated, apo Table S2), and hence the contribution of the ingression time to COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 3 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 Fig. 2 Three common steps of the 40 Octacid4� p-xylene self-assembly pathways at 298 K. Octacid4 and p-xylene are in the stick and stick-and-ball models, respectively. Carbon and oxygen are in orange and red, respectively. Hydrogen, counter ion, and the p-xylenes in the bulk phase are not displayed for clarity. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 19 of the 40 14,251.6-ns simulations at 298 K. The isosurfaces show no repulsion but increasing attraction between the two molecules throughout the pathway. the complexation time is inconsequential. In other words, the 2.6 Å. The use of two different COM cutoffs was because, unlike guest ingress is so fast that the complexation time is determined dioxane that was close enough to the portal at the COM cutoff of primarily by the priming time. This conclusion is independent of 8 Å, naphthalene could have its β-carbon atoms contact the portal the use of the HH cutoff of 2.6 Å because changes of this cutoff at the COM cutoff of 8 Å or be relatively away from the portal in only slightly affect the ingression time that is several orders of some pathways at the COM cutoff of ≥8 Å. Consistent with the magnitude shorter than the priming time. For example, as complexation kinetics of xylene described above, the priming and apparent from Table S2, the priming and complexation times complexation times of dioxane and naphthalene determined from determined from the HH cutoff of 2.6 Å are identical to those the HH cutoff are also identical to those determined from the determined from the COM cutoff of 10 Å, and the average COM cutoff (Table S2), and the average ingression times deter- ingression times from the HH cutoff versus the COM cutoff are mined from the HH cutoff versus COM cutoff are 29/18 versus 95 versus 105 ps. 29/17 ps for dioxane at 298/340 K and 88/670/7790 versus 88/ 668/7790 ps for naphthalene at 298/340/363 K (Table S2). For dioxane, 100 6320-ns simulations captured two self- assembly pathways at 298 K (priming and ingression times using Relative complexation times of different guests with Octacid4. the HH cutoff of 2.6 Å: 3668 and 5681 ns and 6 and 52 ps, Multiple simulations were performed for dioxane or naphthalene respectively; Table S2), and 40 12,640-ns simulations at 340 K under the same simulation conditions as those for xylene. These captured 24 self-assembly pathways (priming and ingression simulations showed that the self-assembly of Octacid4 with times using the HH cutoff of 2.6 Å: 34–2816 ns, 6–50 ps; dioxane or naphthalene was much slower than that of Octa- Table S2). For naphthalene, 100 6320-ns simulations at 298 K cid4•xylene according to the priming time defined using either captured one self-assembly pathway (priming and ingression the HH cutoff of 2.6 Å or the COM cutoff of 8 Å for dioxane and times using the HH cutoff of 2.6 Å: 5721 ns and 88 ps, 10 Å for naphthalene (Table S2). The use of the HH cutoff was respectively; Table S2), 100 6320-ns simulations at 340 K according to the Octacid4•guest-pathway analysis, which revealed captured four self-assembly pathways (priming and ingression that dioxane and naphthalene were close enough (without slightly times using the HH cutoff of 2.6 Å: 1865–4389 ns, and entering the host cavity portal) to the portal at the HH cutoff of 4 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE Fig. 3 Three common steps of the 26 Octacid4� 1,4-dioxane self-assembly pathways at 298 and 340 K. Octacid4 and 1,4-dioxane are in the stick and stick-and-ball models, respectively. Carbon, oxygen, and sodium are in orange, red, and purple, respectively. Hydrogen, counter ion, and the 1,4-dioxanes in the bulk phase are not displayed for clarity, except for the ion that chelates 1,4-dioxane. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 76 of the 100 6320-ns simulations at 298 K. The isosurfaces reveal no repulsion but increasing attraction between the two molecules throughout the pathway. 16–2189 ps, respectively; Table S2), and 100 7900-ns simulations mechanism that is akin to the mechanism for a reported synthetic at 363 K captured ten self-assembly pathways (priming and complex . For naphthalene, the passing of the two head β- ingression times using the HH cutoff of 2.6 Å: 651–7234 ns and carbon atoms, then the α-carbon portion, and finally the tail β- 51–31,035 ps, respectively; Table S2). The ingression times of carbon atoms of the guest through the host portal was completed naphthalene were substantially longer than those of xylene and at the second step (Fig. 4 and Videos S9–S10). dioxane, but these longer ingression times of naphthalene were still a small portion of the naphthalene priming times, confirming that the ingression time is so short that the complexation time is Effect of conformational stability on complexation time.To governed largely by the priming time. understand why the complexation time of xylene is much shorter All pathways of dioxane and naphthalene shared the three than those of dioxane and naphthalene, conformational analyses common steps of the Octacid4•xylene pathways (Figs. 3 and 4), of the 81 pathways were performed and revealed the involvement except for more profound collisions of dioxane or naphthalene of three clusters of the Octacid4 conformations during the with the equatorial portal than those of xylene in the first step Octacid4•xylene self-assembly process at 298 K (Fig. 6). The (Videos S7–S10 and S17–S18) and subtle differences in the most-populated cluster (population: 21/40) had two nearly second step noted as follows. For dioxane, the second step orthogonal aromatic linkers that strongly interacted with xylene involved the passing of the dioxane oxygen through the equatorial according to the intermolecular interactions depicted by the portal during which the oxygen was in the energetically less stable noncovalent interaction gradient isosurfaces (Fig. 6a and half-chair conformation, then the four-methylene portion of Videos S1–S2 and S11–S12), and the assembly involving this dioxane through the portal during which the methylene portion cluster was captured mainly at the early stage of the simulations. was in the energetically stable chair conformation, and last the The less-populated cluster (population: 12/40) had two nearly other dioxane oxygen through the portal during which the oxygen parallel, face-to-face aromatic linkers that moderately interacted was again in the half-chair conformation (Figs. 3 and 5 and with xylene (Fig. 6b and Videos S3–S4 and S13–S14), and the Videos S7 and S17). Interestingly, the Octacid4•dioxane self- complexation involving this cluster was captured at the inter- assembly process followed the mutually induced fitting mediate stage. The least-populated cluster (population: 7/40) had COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 5 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 Fig. 4 Three common steps of the 15 Octacid4� naphthalene self-assembly pathways at 298, 340, and 363 K. Octacid4 and naphthalene are in the stick and stick-and-ball models, respectively. Carbon and oxygen are in orange and red, respectively. Hydrogen, counter ion, and the naphthalene in the bulk phase are not displayed for clarity. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 34 of the 100 6320-ns simulations at 298 K. The isosurfaces reveal no major repulsion but increasing attraction between the two molecules throughout the pathway. two nearly-coplanar aromatic linkers that weakly interacted with xylene (Fig. 6c and Videos S5–S6 and S15–S16), and the com- plexation involving this cluster was captured at the late stage. In contrast to xylene that had the attraction from the two nearly orthogonal aromatic linkers in its top-5 fastest pathways at 298 K (complexation times: 12–41 ns; Table S2; Figs. 2 and 6a), dioxane and naphthalene had the attraction from the two nearly parallel aromatic linkers in their fastest pathways at 298 K (complexation times: 3668 and 5729 ns; Table S2; Figs. 3 and 4). Consistent with the nature of π–π interactions , the Octacid4 conformation with two nearly orthogonal aromatic linkers th corresponded to the 5 most populated conformation of apo Fig. 5 Noncovalent interaction gradient isosurfaces of 1,4-dioxane in Octacid4 in water, but the one with two nearly parallel, face-to- different conformations. a The chair conformation. b The half-chair face aromatic linkers corresponded to none of the top-10 most conformation. Carbon and oxygen are in green and red, respectively. populated conformations of the aqueous apo Octacid4. Hydrogen is not displayed for clarity. The gradient isosurfaces show the These results demonstrate the effect of the host conformational intramolecular interactions using a blue–red scale with blue indicating stability on complexation time. More importantly, the results strong attractions and red indicating strong repulsions. All conformations reveal that the self-assembly of Octacid4 with its guest is governed shown here were obtained directly (no energy minimization) from by the conformational complementarity between the two Simulation 30 of the 100 6320-ns simulations at 298 K. The isosurfaces in molecules not only during the ingression time but also during panels a and b show stronger intramolecular repulsion in the half-chair the priming time, and that the host or guest molecule can adopt, conformation than that of the chair conformation. at a cost of lengthening the complexation time, an unpopulated 6 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE Fig. 6 Three conformational clusters of the 40 Octacid4� p-xylene self-assembly pathways at 298 K. a The most-populated cluster with two nearly orthogonal linkers that strongly attract p-xylene. b The less-populated cluster with two nearly parallel linkers that moderately attract p-xylene. c The least- populated cluster with two nearly-coplanar linkers that weakly attract p-xylene. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulations 17 for a, 7 for b, and 36 for c of the 40 14,251.6-ns simulations at 298 K. conformation to proceed the self-assembly along a repulsion-free path when its populated conformation is not complementary to the conformation of its partner. Opposing effects of the sodium cation on complexation time. According to the conformational analysis of the 43 self-assembly pathways of Octacid4 with dioxane (2 pathways), xylene (40 pathways), and naphthalene (1 pathway) at 298 K, the sodium cation that chelated with the Octacid4 carboxylate either coor- dinated with the guest oxygen atom or formed the cation–π interaction with the guest aromatic ring. However, the chelation with the oxygen atom of the guest that subsequently entered the host cavity was observed in one of the two pathways of dioxane (Table S2); the transient cation–π interaction with the aromatic ring of the cavity-entering guest was observed in only 20 of the 41 self-assembly pathways of xylene and naphthalene (Table S2). These observations indicate that the cavity-entering guest either does not interact at all or does not strongly interact with the carboxylate-chelated sodium cation, so that the cavity-entering guest is not trapped at the host linker region. Instead, the cavity- entering guest interacts with an immobilized guest, which is trapped in the linker region due to its strong interaction with the carboxylate-chelated sodium cation, via the π–π interaction for xylene or naphthalene or the van der Waals interaction for dioxane. Because the π–π interaction and the van der Waals interaction are generally weaker than the cation–π interaction , these weak interactions enable the immobilized guest to usher the cavity-entering guest into the cavity, revealing the role of the sodium cation in shortening the complexation time of Octacid4 Fig. 7 Sodium-restrained Octacid4 conformations. a One pair of linkers with dioxane, xylene, and naphthalene. that form bidendate coordination with the sodium cation. b Two pairs of The conformational analysis of the 43 pathways also identified linkers that form bidendate coordination with the sodium cation. Left: side two small but interesting clusters of Octacid4 conformations that view; Right: top view. were derived a priori from the MD simulations (Fig. 7 and Videos S11–S18). In one cluster with a population of 11/43 minimization and frequency calculations of the two simulation- (Table S2) that was associated mainly with the host conforma- derived conformations with one or two pairs of the bidendate tions with two nearly parallel linkers, the carboxylates from two linkers using the Gaussian 16 program and HF/6-31 G* or nearby linkers of Octacid4 formed a bidentate coordination with B3LYP/6-31 G*, the minimization-derived conformations closely a sodium cation (Fig. 7a and Videos S11–S14 and S17–S18). This resembled the simulation-derived conformations (heavy-atom bidentate coordination rigidified a pair of the bidendate linkers root-mean-square deviations for one or two pairs of the and blocked one of the four equatorial portals of the host. In the bidendate linkers: 0.75 or 0.32 Å for HF/6-31 G* and 0.75 or other cluster with a population of 3/43 (Table S2) that was 0.34 Å for B3LYP/6-31 G*), respectively, and no imaginary associated mainly with the host conformations with two nearly frequencies were found for the minimization-derived conforma- coplanar linkers, all four linkers of Octacid4 were involved in the tions, suggesting that the conformations with one or two pairs of bidentate coordination, resulting in rigidification of two pairs of the bidendate linkers derived a priori from the simulations were the bidendate linkers and blockage of two of the four equatorial local minimal conformations. Because the host conformations portals (Fig. 7b and Videos S15–S16). According to the energy COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 7 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 with the nearly parallel or nearly coplanar linkers were associated with long complexation times as described above, these results indicate that the sodium cation also played a role in lengthening the complexation time of Octacid4 with dioxane, xylene, and naphthalene and that the lengthening role is outweighed by the shortening role of the sodium cation as the populations of the host conformations with the bidendate linkers are lower than those of the host conformations without the bidendate linkers (Table S2). Discussion Internal and external consistencies of the self-assembly path- ways. The 81 self-assembly pathways described above exhibited internal or external consistencies as follows. (1) The natural logarithm of the Octacid4 population versus the simulation time exhibited a linear relationship with r of 0.98 for all 40 simulations that captured the Octacid4•xylene self- assembly, 0.85 for the first 20 of the 40 simulations, and 0.96 for the last 20 of the 40 simulations (Fig. 8). These r values indicate the convergency and internal consistency of the 40 simulations for which the first-order self-assembly rate is expected as explained above. (2) As described above, the simulations using the fully deprotonated Octacid4 under various conditions all captured the self-assembly of Octacid4 with dioxane, xylene, and naphthalene, but no autonomous complexation was observed under the same conditions if the fully deprotonated host was replaced by the fully protonated host. These results are consistent with the use of the Octacid4 solution containing the sodium borate buffer at pH 9 to detect the Octacid4•guest complexation in the NMR experiments and with the phase-transfer catalysis undergirding those NMR experiments. (3) One key finding of the present work is that the guest ingress is so fast that complexation time is determined primarily by the priming time. This finding is consistent with the report that many dense-phase reactions can be considered as gated reactions in that the rate of a local reaction is governed largely by the initial formation of a permissive atomic arrangement (viz., determined mainly by the system priming) within which the local transformation can proceed relatively rapidly . (4) Conformational analyses of all 43 pathways of xylene, dioxane, and naphthalene at 298 K showed that Octacid4 adopted Fig. 8 The exponential decay of the host population over the simulation exclusively a cluster of V-shaped conformations to gulp its guests. time for the Octacid4� p-xylene self-assembly at 298 K. The host These V-shaped conformations have the mean C17a–C6b population and simulation time were obtained from the 40 individual distance (the distance between two diphenoxymethane carbon complexation times of p-xylene listed in Table S2. The linear-regression atoms that control the width of the cavity portal as shown in analysis was performed using the PRISM 5 program. Fig. 1) of 7.0 Å (95% confidence interval: 7.0–7.1 Å) for the entrance portal and the corresponding mean C17a–C6b distance naphthalene (1) at 298 K revealed no repulsion (or only a trace of of 5.6 Å (95% confidence interval: 5.5–5.7 Å) for the opposing repulsion for naphthalene) but increasingly strong attraction portal (Table S3). These mean distances are consistent with the between Octacid4 and its guest in the self-assembly process reported V-shaped conformation proposed for the sliding-door 15 (Figs. 2–4). These observations are consistent with the report that mechanism for the gating of hemicarcerands that are closely the Corey–Pauling–Koltun space-filling models of xylene, diox- related to Octacid4. ane, and naphthalene could be pushed (or pushed with effort for (5) For the self-assembly at 298 K, 12 of the 40 captured naphthalene) through portals of the space-filling model of pathways for xylene and all captured pathways for dioxane and Octacid4 , indicating that a repulsion-free path exists for naphthalene had two nearly parallel linkers that channeled the Octacid4 to self-assemble with xylene, dioxane, and naphthalene. guest into the cavity (Table S2). These linker channels are (7) Most importantly, the capturing of the self-assembly of consistent with the report that an antechamber formed by two Octacid4 with dioxane, xylene, and naphthalene in the simula- parallel linkers of a hemicarcerand played a role in the gating of 15 tions at 298 K is consistent with the Octacid4 complexation with hemicarcerands . these guests detected in the NMR experiments at 298 K . (6) Relative to the noncovalent interaction gradient isosurfaces of dioxane that showed the stronger intramolecular repulsion in the half-chair conformation than that in the chair How Octacid4 self-assembles with its guests and what accel- conformation (Fig. 5), the isosurfaces of the Octacid4 complexes erates their assembly. Collectively, the 81 pathways indicate that in the 43 self-assembly pathways of xylene (40), dioxane (2), and the irreversible noncovalent self-assembly process of Octacid4 8 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE with dioxane, xylene, and naphthalene was (1) initiated by the likely to the extent that the mean complexation time will be guest interaction with the cavity portal exterior of the host to significantly altered. increase the collisions of the portal that led to the portal expan- sion for guest ingress, (2) accelerated by engaging populated Design of irreversible noncovalent complexes with fast conformations of host and guest for the exterior intermolecular assembly rates. Despite the heterogeneity issue as described above, interaction to increase the portal collision frequency, and (3) the realization that the self-assembly initiation by the exterior completed by the portal contraction caused by the guest docking intermolecular interaction and the control of the complexation inside the cavity to impede guest egress. This type of self- time largely by the priming time is an advance and may help assembly is a process of stepwise conformational rearrangements explain why the presence of a cryptic exterior site for substrate or of the two molecules—kinetically starting from the pre-assembly inhibitor binding at the rim of the enzyme-active site is a funda- state (at which the guest interacts with the exterior of the host) to 29–36 mental feature of fast enzymes such as acetylcholinesterase the assembly and post-assembly states (at which the guest and why it is advantageous for the proapoptotic multidomain BAK interacts partially and fully with the interior of the host, respec- 37 protein to have a noncanonical BH3-binding groove that abuts tively)—to gradually increase the attraction between the two the canonical one for constrictive complexation of BAK—due to molecules along a repulsion–free path until the maximal attrac- the steric hindrance from R88 and Y89 on the edge of the cano- tion is reached. The kinetics of this process was governed pri- nical groove —with proapoptotic BH3-only proteins. The reali- marily by the complementarity between the two molecules during zation that the self-assembly acceleration by the adoption of the priming time (on the order of microseconds or longer) rather populated conformations for the exterior intermolecular interac- than the ingression time (on the order of picoseconds to tion is an advance and may help explain why small molecules nanoseconds). reportedly prefer to adopt local minimum conformations when binding to proteins . Further, these realizations suggest that irreversible noncovalent complexes with fast assembly rates could Heterogeneity of the Octacid4� xylene self-assembly pathways. be designed by accounting for the complementarity between guest The 40 Octacid4•xylene self-assembly pathways derived from the and host both of which adopt populated conformations for the converged MD simulations (as evident from Fig. 8) demonstrate exterior intermolecular interactions during the priming time. This the heterogeneity of the self-assembly pathways. As apparent design strategy may facilitate a paradigm shift from irreversible from Table S2, at 298 K, xylene can enter the cavity of the three covalent complex design to irreversible noncovalent complex distinct clusters of the host conformations shown in Fig. 6 and design for materials technology, data storage and processing, Videos S1–S6. In each of these clusters, xylene can also enter the molecular sensing and tagging, and drug therapy—especially the cavity of the host conformations with or without bidendate lin- personalized drug therapies for cancer patients with somatic kers (Videos S1–S6 and S11–S16), indicating that the Octa- G12C 7 mutations other than the KRAS mutation . cid4•xylene pathways can be shortened or lengthened by the sodium cation (Table S2). Methods This heterogeneity is akin to the heterogeneity of protein-folding Molecular dynamics simulation. The fully deprotonated, apo Octacid4 neu- pathways . It points to a limitation of the simulation protocol tralized with 8 sodium ions (or an Octacid4 in a different configuration, such as the used in this work because the protocol captured only the fast self- fully protonated Octacid4, as listed in Table S1) was manually solvated with 150/ 250 copies of a guest (xylene, dioxane, or naphthalene) using PyMOL V1.7.0.3 assembly pathways of Octacid4•dioxane and Octacid4•naphthalene (https://pymol.org) and tLEaP of the AmberTools 16 package (University of at 298 K. This calls for the development of a new simulation California, San Francisco) and then energy-minimized for 100 cycles of steepest- protocol to avoid oversimplification of the self-assembly process descent minimization followed by 900 cycles of conjugate-gradient minimization to such as equating the process to a few fast self-assembly pathways remove close van der Waals contacts using SANDER of the AMBER 11 package (University of California, San Francisco), FF12MClm , and a cutoff of 8.0 Å for for Octacid4•dioxane and Octacid4•naphthalene. As apparent noncovalent interactions. The tLEaP input file for building the fully protonated from the Octacid4•xylene self-assembly pathways, it is not a few Octacid4 and the Cartesian coordinates of the energy-minimized Octacid4•guest complexation times (from the fast pathways) but the mean (in all configurations as listed in Table S1) are provided in Data S1 and S2, complexation time (from the fast, intermediate, and slow path- respectively. The energy-minimized system was slowly heated to 298/340/363/ 370 K in 30 steps under constant temperature and constant volume, and then ways) that offers insight into the delicate balance between the equilibrated for 10 timesteps under constant temperature of 298/340/363/370 K sodium cation’s roles in lengthening and shortening the com- and constant pressure of 1 atm employing isotropic molecule-based scaling. Finally, plexation time of Octacid4•xylene. a set of 20/40/100 distinct, independent, unrestricted, unbiased, and classical Given the known theoretical work on gated reactions and the isobaric–isothermal MD simulations was performed for the resulting system using finding of the present work that the ingression time is so short PMEMD of the AMBER 14/16/18/20 package (University of California, San Francisco), FF12MClm , and a periodic boundary condition at 1 atm and 298/ that the complexation time is governed largely by the priming 340/363/370 K. All simulations used (i) a dielectric constant of 1.0, (ii) the time, it is worth noting the need to avoid overcomplication of the Berendsen coupling algorithm for thermostat and barostat, (iii) the particle mesh self-assembly process, such as simulating the Octacid4•guest self- 42 Ewald method to calculate electrostatic interactions of two atoms at a separation 40, 43 assembly using the octa-anionic Octacid4 possessing five water of >8 Å, (iv) Δt = 1.00 fs of the standard-mass time , (v) the SHAKE-bond- length constraint applied to all bonds involving hydrogen, (vi) a protocol to save molecules inside the cavity or a more complicated host with a the image closest to the middle of the “primary box” to the restart and trajectory variable number of water molecules inside the cavity, for at least files, (vii) a formatted restart file, (viii) the revised alkali-ion parameters , (ix) a two reasons. First, it has not been determined experimentally cutoff of 8.0 Å for noncovalent interactions, (x) a uniform 10-fold reduction in the 40, 43 whether the egression of water molecules that was observed in the atomic masses of the entire simulation system (both solute and solvent) , (xi) NTWX = 100 steps for coordinates’ output, and (xii) default values of all other present work will actually occur under the NMR experiment inputs of PMEMD. conditions once the water-bound Octacid4 is surrounded by a Available in the Supporting Information of Ref. , FF12MClm is a revised layer of water-insoluble xylene or naphthalene. Second, the water AMBER protein forcefield with no parameterization for any Octacid4•guest molecule is much smaller than dioxane, xylene, and naphthalene. complexes . This forcefield is able to (1) capture the experimentally observed exponential decay of the non-native state population of fast-folding proteins over The ingression and egression times of water are hence much simulation time with r > 0.90 and (2) fold these proteins with agreements between shorter than those of the three guests, and binding of water simulated and experimental folding times within factors of 0.6–1.4 . FF12MClm molecules to the host cavity is likely opportunistic rather than was used in this study to investigate the noncovalent self-assembly of small- intrinsic or constrictive. The inclusion of water molecules inside molecule guests with Octacid4 whose aromatic linkers can flip between left‐ and the cavity will affect the Octacid4•guest self-assembly but not right‐handed configurations and usher the guest into the host cavity. This was COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 9 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 because of the effectiveness of FF12MClm in simulating the experimentally 4. Vita, E. 10 years into the resurgence of covalent drugs. Future Med. Chem. 13, observed flipping between left‐ and right‐handed configurations for C14–C38 of 193–210 (2021). bovine pancreatic trypsin inhibitor in solution and because of the need to 5. Vane, J. R. & Botting, R. M. The mechanism of action of aspirin. Thromb. Res. 1/2 compress the simulation time (viz., speed up simulations) by a factor of 10 110, 255–258 (2003). through 10-fold uniform reduction of the system mass. While the hydrogen mass 6. Yocum, R. R., Rasmussen, J. R. & Strominger, J. L. The mechanism of action of repartitioning scheme can also speed up simulations, it was not used in this study penicillin. Penicillin acylates the active site of Bacillus stearothermophilus because it would affect dynamic properties of the system . The forcefield D-alanine carboxypeptidase. J. Biol. Chem. 255, 3977–3986 (1980). parameters for the fully deprotonated Octacid4, dioxane, xylene, and naphthalene 7. Canon, J. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti- are available in the Supplementary Information of Ref. . The forcefield tumour immunity. Nature 575, 217–223 (2019). parameters for the neutral Octacid4 were developed using a published procedure 8. Quan, M. L. C. & Cram, D. J. Constrictive binding of large guests by a and provided in Data S1. The ab initio calculations for developing the fully hemicarcerand containing 4 portals. J. Am. Chem. Soc. 113, 2754–2755 (1991). protonated Octacid4 forcefield parameters were performed using Gaussian 98 9. Warmuth, R. & Yoon, J. Recent highlights in hemicarcerand chemistry. Acc. (Revision A.7; Gaussian, Inc. Wallingford, CT). 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Hemicarcerands permit entrance National Center for Supercomputing Applications. to and egress from their inside phases with high structural recognition and activation free-energies. J. Am. Chem. Soc. 112, 1659–1660 (1990). Survival analysis. The mean complexation time and its 95% confidence interval for 13. Yoon, J. Y. & Cram, D. J. The first water-soluble hermicarceplexes. Chem. the Octacid4•xylene self-assembly at 298 K was obtained from the 40 individual Commun., 10.1039/A607353K (1997). complexation times of xylene listed in Table S2 using the parametric survival function 14. McFerrin, K. G. & Pang, Y.-P. How the water-soluble hemicarcerand [the Surreg() function] implemented in the R survival package Version 3.2.0 . incarcerates guests at room temperature decoded with modular simulations. Commun. Chem. 4, 26 (2021). 15. Houk, K. N., Nakamura, K., Sheu, C. M. & Keating, A. E. Gating as a control Noncovalent interaction gradient isosurface. 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Attribution 4.0 International License, which permits use, sharing, Commun. 492, 135–139 (2017). 46. Feenstra, K. A., Hess, B. & Berendsen, H. J. C. Improving efficiency of large adaptation, distribution and reproduction in any medium or format, as long as you give time-scale molecular dynamics simulations of hydrogen-rich systems. J. appropriate credit to the original author(s) and the source, provide a link to the Creative Comput. Chem. 20, 786–798 (1999). Commons license, and indicate if changes were made. The images or other third party 47. Therneau, T. M. & Grambsch, P. M. Modeling Survival Data: Extending the material in this article are included in the article’s Creative Commons license, unless Cox Model (Springer-Verlag, 2000). indicated otherwise in a credit line to the material. If material is not included in the 48. Contreras-Garcia, J. et al. NCIPLOT: a program for plotting noncovalent article’s Creative Commons license and your intended use is not permitted by statutory interaction regions. J. Chem. Theory Comput. 7, 625–632 (2011). regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements This work was supported by the US Army Research Office (W911NF-16-1-0264) and © The Author(s) 2022 the Mayo Foundation for Medical Education and Research. Responsibility for the COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 11 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Communications Chemistry Springer Journals

How neocarcerand Octacid4 self-assembles with guests into irreversible noncovalent complexes and what accelerates the assembly

Communications Chemistry , Volume 5 (1) – Jan 20, 2022

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ARTICLE https://doi.org/10.1038/s42004-022-00624-4 OPEN How neocarcerand Octacid4 self-assembles with guests into irreversible noncovalent complexes and what accelerates the assembly Yuan-Ping Pang Cram’s supramolecular capsule Octacid4 can irreversibly and noncovalently self-assemble with small-molecule guests at room temperature, but how they self-assemble and what accelerates their assembly remain poorly understood. This article reports 81 distinct Octacid4� guest self- assembly pathways captured in unrestricted, unbiased molecular dynamics simulations. These pathways reveal that the self-assembly was initiated by the guest interaction with the cavity portal exterior of Octacid4 to increase the portal collisions that led to the portal expansion for guest ingress, and completed by the portal contraction caused by the guest docking inside the cavity to impede guest egress. The pathways also reveal that the self-assembly was accelerated by engaging populated host and guest conformations for the exterior interaction to increase the portal collision frequency. These revelations may help explain why the presence of an exterior binding site at the rim of the enzyme active site is a fundamental feature of fast enzymes such as acetylcholinesterase and why small molecules adopt local minimum conformations when binding to proteins. Further, these revelations suggest that irreversible noncovalent complexes with fast assembly rates could be developed—by engaging populated host and guest con- formations for the exterior interactions—for materials technology, data storage and processing, molecular sensing and tagging, and drug therapy. 1 ✉ Computer-Aided Molecular Design Laboratory, Mayo Clinic, Rochester, MN, USA. email: camdl1@icloud.com COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 1 1234567890():,; ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 rreversible bimolecular complexes are highly desirable because is present in the reaction medium for the tethering reaction; the (1) the residence of one molecule inside the cavity of another disassembly of the resulting carcerand complex is not allowed Imolecule is permanent until the breakdown of the cavity- unless the host is broken by excessive heat . When the barrier is containing molecule, (2) the complex formation is one-to-one moderately high, assembly or disassembly requires annealing. For stoichiometric, and (3) for drug therapy, the permanent residence example, a guest can enter or exit the cavity of a host known as and 1:1 stoichiometry confer the cavity-residing molecule with hemicarcerand once the bimolecular system is heated to expand desired high metabolic stability, high potency-to-mass ratio, low- the cavity portal, and the guest remains inside the cavity once the 1–4 acting dose, and low off-target activity . These complexes system is cooled to contract the portal; the disassembly of the 12, 15 generally refer to irreversible covalent complexes that are self- hemicarcerand complex is disallowed without heating . When assembled by two molecules between which there is a covalent the barrier is slightly high, assembly or disassembly can occur bond resulting from the self-assembly process. The desirability of spontaneously and slowly at an ambient temperature. For some irreversible covalent complexes is apparent from the drug action host•guest complexes, the disassembly can be disabled at the 5 6 7 mechanisms of aspirin , penicillin , and sotorasib , all of which ambient temperature by the complexation that subsequently involve an irreversible covalent complex. Notably, sotorasib was heightens the barrier for disassembly. For example, a guest can approved in May 2021 (https://www.fda.gov/news-events/press- enter the cavity of a host known as neocarcerand at room tem- announcements/fda-approves-first-targeted-therapy-lung-cancer- perature and remain in the cavity at room temperature unless the mutation-previously-considered-resistant-drug) as the first-in- portal is opened by heating because the docking of the guest at class personalized treatment for a lung-cancer mutation pre- the cavity induces a host conformational change that conse- viously considered resistant to drug therapy due to sotorasib’s quently closes all cavity portals . G12C unique capability to clinically block the function of KRAS The applications of carcerand and hemicarcerand are however (an enzyme mutant responsible for ~13% non-small-cell lung limited because their complexes cannot be formed in situ and cancers) via irreversible complexation. However, the irreversible adiabatically. Although neocarcerand can form its complexes 13, 14, 16 covalent complexes from in situ cysteine conjugation are limited in situ and adiabatically , the applications of neocarcerand by the infrequent presence of the noncatalytic cysteine in a complexes are also limited due to their slow complexation rates. G12C protein cavity, and inhibiting KRAS offers treatment for only This underscores the need to understand how two molecules self- a subset of cancer patients. A paradigm shift is needed for the assemble into an irreversible noncovalent complex and what design of irreversible bimolecular complexes. accelerates their assembly as these high-level questions hold the In terms of both intrinsic binding from the thermodynamic key to designing irreversible noncovalent bimolecular complexes perspective and constrictive binding from the kinetic with fast complexation rates for broad applications. 8–10 perspective , the irreversible complexes include irreversible To promote irreversible noncovalent bimolecular complex noncovalent complexes that are self-assembled by two molecules design, this article reports 81 distinct pathways of the irreversible between which there is no covalent bond. Here, the intrinsic noncovalent self-assembly of neocarcerand Octacid4 with three binding is the complexation governed by intermolecular inter- known guests —1,4-dioxane (dioxane), p-xylene (xylene), and actions between the two molecules and between the solvent and naphthalene (Fig. 1 and Table S1). These pathways were captured each of the two molecules, while the constrictive binding is the in multiple distinct, independent, unrestricted, unbiased, and complexation controlled by the thermal energy required for the classical isobaric–isothermal molecular dynamics (MD) simula- guest to overcome the steric hindrance from the host during the tions at a high time resolution with an aggregated simulation time exceeding 3.761664 milliseconds at 298–363 K, rendering the assembly or disassembly process, as exemplified below by Cram’s 11 12 supramolecular capsules known as carcerand , hemicarcerand , structural and kinetic information needed to answer the two 13, 14 and neocarcerand . high-level questions and guide the design of irreversible non- The hallmark of the constrictive binding is the heightened covalent complexes that can be formed in situ and adiabatically energy barrier for the assembly or disassembly of a host•guest with desired kinetics for materials technology, data storage and complex. This barrier can make the dissembled or assembled processing, molecular sensing and tagging, and drug therapy. molecules kinetically stable, namely, it takes a long time to assemble the two disassembled molecules or disassemble the two assembled molecules if the barrier for the conversion is heigh- Results tened. When the barrier is extremely high, assembly or dis- The challenge of capturing self-assembly pathways. In view of assembly requires covalent-bond making or breaking, the current state of computational work on guest/ligand-binding 17–20 respectively. For example, a guest can be noncovalently trapped in pathways , unrestricted and unbiased MD simulations of the the cavity of the two bowl-shaped fragments that are rim-to-rim self-assembly of Octacid4 with its known guests are challenging to tethered by linkers (of a host known as carcerand) when the guest perform because the complexation times of Octacid4•guest were Fig. 1 Structure of Octacid4 in complex with a small-molecule guest. a Octacid4� p-xylene. b Octacid4� naphthalene. c Octacid4� 1,4-dioxane. Carbon and oxygen are in blue and red, respectively. The axial or equatorial portal of Octacid4 comprises the C3 and C19 atoms or the O7, O15, C6b, and C17a atoms, respectively. Hydrogen and counter ion are not displayed for clarity. 2 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE estimated to be a few minutes (or hours for the bulkiest guest Octacid4 surrounded with 8 sodium cations, 150 guests, and naphthalene) according to NMR experiments . These com- the Berendsen thermostat. plexation times are many orders of magnitude longer than cur- rent MD simulation times that are on the order of milliseconds. Conformational characterization of the Octacid4� xylene self- To demonstrate the challenge of capturing the Octacid4•guest assembly pathways. Visual inspections of the 40 self-assembly self-assembly pathways, multiple distinct, independent, unrest- pathways of Octacid4•xylene at 298 K revealed that all pathways ricted, unbiased, and classical isobaric–isothermal MD simula- comprised three common steps (Fig. 2 and Videos S1–S6 and tions (hereafter shortened to simulations) were performed using S11–S16). In Step 1, xylene entered the cavity portal exterior the fully deprotonated, apo Octacid4 that was surrounded by space that was confined by two aromatic linkers of the host; as eight neutralizing sodium cations, 10 xylenes to mimic the use of apparent from the representative videos, there were substantial the guest in 10 equivalent excess in the NMR experiments ,60 collisions between xylene and the equatorial portal in the linker NaCl molecules to approximate the ionic strength of the region of Octacid4 that led to the portal expansion for guest 13 21 experimental conditions , and 2359 TIP3P water molecules ingress. In Step 2, xylene passed one methyl group through the to mimic the experimental aqueous solution . Indeed, no equatorial portal, then the phenyl group through the portal with autonomous complexation was observed in any set of 20 the phenyl plane perpendicular to the axial axis (viz., the axis 14,251.6-ns simulations at 298, 340, 363, or 370 K (Table S1) passing two axial portals in the bowl-shaped region), and last the because the simulation time was many orders of magnitude other methyl group through the equatorial portal. In Step 3, shorter than the experimentally estimated complexation time for xylene rotated ~90° to keep its phenyl plane parallel to the axial xylene. axis. This rotation caused the equatorial portal contraction and impeded guest egress, according to the reported structural ana- lyses of apo Octacid4 and the Octacid4•xylene complex . Using the phase-transfer catalyst to capture the self-assembly. According to the reported NMR experiments , the experimen- tally observed self-assembly of Octacid4 with dioxane, xylene, or Kinetic characterization of the Octacid4� xylene self-assembly naphthalene in a sodium borate buffer at pH 9 is mechanistically pathways. A survival analysis of the 40 14,251.6-ns simulations driven by the sodium cation as a phase-transfer catalyst that that all captured the self-assembly of Octacid4•xylene at 298 K chelates with the carboxylates on the Octacid4 surface and con- showed xylene’s mean complexation time to be 1022 ns (95% sequently accumulates the guest on the host surface via the confidence interval: 750–1392 ns). Here the complexation time cation–π interaction or the sodium chelation. The guest accu- was defined as the first time instant at which xylene was inside the mulation on the host surface is similar to immersing the sodium- host cavity and had its phenyl plane parallel to the axial axis of chelated Octacid4 in a neat guest solution. Simulating the latter the host. This xylene orientation was found in the most populated can substantially accelerate the self-assembly according to the law Octacid4•xylene conformation . of mass action, enabling determination of relative complexation To dissect the self-assembly kinetics, the duration of the first times of different guests with Octacid4 for mechanistic insights step is herein termed priming time, and the duration of the last into the self-assembly. Simulating the latter also allows the use of two steps is termed ingression time. These names are used linear-regression analysis to examine the convergency and because during the first step, the host and guest conformations internal consistency of the MD simulations. A goodness of fit(r ) are primed, through self-selection and conformational rearrange- of <0.70 for the natural logarithm plot of the host population ments, for guest ingress, and because during the last two steps, the versus the simulation time indicates problematic simulations. guest enters the host cavity and rotates ~90° to form a complex. This is because the first-order rate (viz., the exponential decay of The priming time was defined as a time period from the the host population over the simulation time) is expected for beginning of the MD simulation to the last time instant at which simulating the self-assembly of Octacid4 with its guest in large the distance between any guest hydrogen atoms and any host excess. methylene hydrogen atoms was greater than 2.6 Å. The Accordingly, 40 simulations were performed using the fully hydrogen–hydrogen distance cutoff (abbreviated as HH cutoff) deprotonated, apo Octacid4 that was surrounded by eight was set at 2.6 Å for the following reasons. Visual inspection of all neutralizing sodium cations and 150 xylenes. Here, the number 40 Octacid4•xylene pathways revealed that xylene was outside of of xylenes was arbitrarily chosen. Gratifyingly, all 40 14,251.6-ns the Octacid4 cavity as long as the center-of-mass distance cutoff simulations with the fully deprotonated, apo Octacid4 with 150 (abbreviated as COM cutoff) for Octacid4 and its guest was ≥7Å. xylenes captured the self-assembly of Octacid4 with xylene at However, xylene could have its terminal methyl group contact the 298 K. Additional simulations were performed using each of the host cavity portal to slightly enter the host cavity portal in some five variations: (1) replacing the fully deprotonated, apo Octacid4 of the 40 Octacid4•xylene pathways at the COM cutoff of 7 or with the octa-anionic Octacid4 possessing five water molecules 8 Å. Xylene could also be relatively away from the portal in some inside the cavity, (2) increasing the number of xylenes to 250, (3) pathways at the COM cutoff of ≥8 Å. Therefore, rather than using replacing 150 xylenes with 150 dioxanes, (4) replacing 150 the COM cutoff, the HH cutoff of 2.6 Å was used to avoid both xylenes with 150 naphthalenes, or (5) changing the Berendsen the methyl group contacting the portal (which consequently thermostat to the Langevin thermostat (Table S1). All these shortens the ingression time) and the guest being away from the simulations captured the self-assembly event. For the simulations portal (which consequently lengthens the ingression time). The with the water-bound Octacid4, all water molecules inside the ingression time was defined as a time period from the last time cavity had a high propensity to interact with the sodium cations instant at which the distance between any guest hydrogen atoms and the carboxylates outside the cavity, and egression of all five and any host methylene hydrogen atoms was greater than 2.6 Å water molecules occurred prior to the ingression of xylene. By to the first time instant at which the guest rotated ~90° inside the contrast, no complexation was observed under the same cavity. The complexation time is now a sum of the priming and conditions if the fully deprotonated, apo Octacid4 surrounded ingression times. by 8 sodium cations was replaced with the fully protonated, apo This dissection reveals that the ingression time (8–934 ps; Octacid4 without any cations (Table S1). All simulations Table S2) is a fraction of the priming time (12–14,199 ns; described hereafter used the fully deprotonated, apo Table S2), and hence the contribution of the ingression time to COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 3 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 Fig. 2 Three common steps of the 40 Octacid4� p-xylene self-assembly pathways at 298 K. Octacid4 and p-xylene are in the stick and stick-and-ball models, respectively. Carbon and oxygen are in orange and red, respectively. Hydrogen, counter ion, and the p-xylenes in the bulk phase are not displayed for clarity. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 19 of the 40 14,251.6-ns simulations at 298 K. The isosurfaces show no repulsion but increasing attraction between the two molecules throughout the pathway. the complexation time is inconsequential. In other words, the 2.6 Å. The use of two different COM cutoffs was because, unlike guest ingress is so fast that the complexation time is determined dioxane that was close enough to the portal at the COM cutoff of primarily by the priming time. This conclusion is independent of 8 Å, naphthalene could have its β-carbon atoms contact the portal the use of the HH cutoff of 2.6 Å because changes of this cutoff at the COM cutoff of 8 Å or be relatively away from the portal in only slightly affect the ingression time that is several orders of some pathways at the COM cutoff of ≥8 Å. Consistent with the magnitude shorter than the priming time. For example, as complexation kinetics of xylene described above, the priming and apparent from Table S2, the priming and complexation times complexation times of dioxane and naphthalene determined from determined from the HH cutoff of 2.6 Å are identical to those the HH cutoff are also identical to those determined from the determined from the COM cutoff of 10 Å, and the average COM cutoff (Table S2), and the average ingression times deter- ingression times from the HH cutoff versus the COM cutoff are mined from the HH cutoff versus COM cutoff are 29/18 versus 95 versus 105 ps. 29/17 ps for dioxane at 298/340 K and 88/670/7790 versus 88/ 668/7790 ps for naphthalene at 298/340/363 K (Table S2). For dioxane, 100 6320-ns simulations captured two self- assembly pathways at 298 K (priming and ingression times using Relative complexation times of different guests with Octacid4. the HH cutoff of 2.6 Å: 3668 and 5681 ns and 6 and 52 ps, Multiple simulations were performed for dioxane or naphthalene respectively; Table S2), and 40 12,640-ns simulations at 340 K under the same simulation conditions as those for xylene. These captured 24 self-assembly pathways (priming and ingression simulations showed that the self-assembly of Octacid4 with times using the HH cutoff of 2.6 Å: 34–2816 ns, 6–50 ps; dioxane or naphthalene was much slower than that of Octa- Table S2). For naphthalene, 100 6320-ns simulations at 298 K cid4•xylene according to the priming time defined using either captured one self-assembly pathway (priming and ingression the HH cutoff of 2.6 Å or the COM cutoff of 8 Å for dioxane and times using the HH cutoff of 2.6 Å: 5721 ns and 88 ps, 10 Å for naphthalene (Table S2). The use of the HH cutoff was respectively; Table S2), 100 6320-ns simulations at 340 K according to the Octacid4•guest-pathway analysis, which revealed captured four self-assembly pathways (priming and ingression that dioxane and naphthalene were close enough (without slightly times using the HH cutoff of 2.6 Å: 1865–4389 ns, and entering the host cavity portal) to the portal at the HH cutoff of 4 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE Fig. 3 Three common steps of the 26 Octacid4� 1,4-dioxane self-assembly pathways at 298 and 340 K. Octacid4 and 1,4-dioxane are in the stick and stick-and-ball models, respectively. Carbon, oxygen, and sodium are in orange, red, and purple, respectively. Hydrogen, counter ion, and the 1,4-dioxanes in the bulk phase are not displayed for clarity, except for the ion that chelates 1,4-dioxane. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 76 of the 100 6320-ns simulations at 298 K. The isosurfaces reveal no repulsion but increasing attraction between the two molecules throughout the pathway. 16–2189 ps, respectively; Table S2), and 100 7900-ns simulations mechanism that is akin to the mechanism for a reported synthetic at 363 K captured ten self-assembly pathways (priming and complex . For naphthalene, the passing of the two head β- ingression times using the HH cutoff of 2.6 Å: 651–7234 ns and carbon atoms, then the α-carbon portion, and finally the tail β- 51–31,035 ps, respectively; Table S2). The ingression times of carbon atoms of the guest through the host portal was completed naphthalene were substantially longer than those of xylene and at the second step (Fig. 4 and Videos S9–S10). dioxane, but these longer ingression times of naphthalene were still a small portion of the naphthalene priming times, confirming that the ingression time is so short that the complexation time is Effect of conformational stability on complexation time.To governed largely by the priming time. understand why the complexation time of xylene is much shorter All pathways of dioxane and naphthalene shared the three than those of dioxane and naphthalene, conformational analyses common steps of the Octacid4•xylene pathways (Figs. 3 and 4), of the 81 pathways were performed and revealed the involvement except for more profound collisions of dioxane or naphthalene of three clusters of the Octacid4 conformations during the with the equatorial portal than those of xylene in the first step Octacid4•xylene self-assembly process at 298 K (Fig. 6). The (Videos S7–S10 and S17–S18) and subtle differences in the most-populated cluster (population: 21/40) had two nearly second step noted as follows. For dioxane, the second step orthogonal aromatic linkers that strongly interacted with xylene involved the passing of the dioxane oxygen through the equatorial according to the intermolecular interactions depicted by the portal during which the oxygen was in the energetically less stable noncovalent interaction gradient isosurfaces (Fig. 6a and half-chair conformation, then the four-methylene portion of Videos S1–S2 and S11–S12), and the assembly involving this dioxane through the portal during which the methylene portion cluster was captured mainly at the early stage of the simulations. was in the energetically stable chair conformation, and last the The less-populated cluster (population: 12/40) had two nearly other dioxane oxygen through the portal during which the oxygen parallel, face-to-face aromatic linkers that moderately interacted was again in the half-chair conformation (Figs. 3 and 5 and with xylene (Fig. 6b and Videos S3–S4 and S13–S14), and the Videos S7 and S17). Interestingly, the Octacid4•dioxane self- complexation involving this cluster was captured at the inter- assembly process followed the mutually induced fitting mediate stage. The least-populated cluster (population: 7/40) had COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 5 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 Fig. 4 Three common steps of the 15 Octacid4� naphthalene self-assembly pathways at 298, 340, and 363 K. Octacid4 and naphthalene are in the stick and stick-and-ball models, respectively. Carbon and oxygen are in orange and red, respectively. Hydrogen, counter ion, and the naphthalene in the bulk phase are not displayed for clarity. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulation 34 of the 100 6320-ns simulations at 298 K. The isosurfaces reveal no major repulsion but increasing attraction between the two molecules throughout the pathway. two nearly-coplanar aromatic linkers that weakly interacted with xylene (Fig. 6c and Videos S5–S6 and S15–S16), and the com- plexation involving this cluster was captured at the late stage. In contrast to xylene that had the attraction from the two nearly orthogonal aromatic linkers in its top-5 fastest pathways at 298 K (complexation times: 12–41 ns; Table S2; Figs. 2 and 6a), dioxane and naphthalene had the attraction from the two nearly parallel aromatic linkers in their fastest pathways at 298 K (complexation times: 3668 and 5729 ns; Table S2; Figs. 3 and 4). Consistent with the nature of π–π interactions , the Octacid4 conformation with two nearly orthogonal aromatic linkers th corresponded to the 5 most populated conformation of apo Fig. 5 Noncovalent interaction gradient isosurfaces of 1,4-dioxane in Octacid4 in water, but the one with two nearly parallel, face-to- different conformations. a The chair conformation. b The half-chair face aromatic linkers corresponded to none of the top-10 most conformation. Carbon and oxygen are in green and red, respectively. populated conformations of the aqueous apo Octacid4. Hydrogen is not displayed for clarity. The gradient isosurfaces show the These results demonstrate the effect of the host conformational intramolecular interactions using a blue–red scale with blue indicating stability on complexation time. More importantly, the results strong attractions and red indicating strong repulsions. All conformations reveal that the self-assembly of Octacid4 with its guest is governed shown here were obtained directly (no energy minimization) from by the conformational complementarity between the two Simulation 30 of the 100 6320-ns simulations at 298 K. The isosurfaces in molecules not only during the ingression time but also during panels a and b show stronger intramolecular repulsion in the half-chair the priming time, and that the host or guest molecule can adopt, conformation than that of the chair conformation. at a cost of lengthening the complexation time, an unpopulated 6 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE Fig. 6 Three conformational clusters of the 40 Octacid4� p-xylene self-assembly pathways at 298 K. a The most-populated cluster with two nearly orthogonal linkers that strongly attract p-xylene. b The less-populated cluster with two nearly parallel linkers that moderately attract p-xylene. c The least- populated cluster with two nearly-coplanar linkers that weakly attract p-xylene. The noncovalent interaction gradient isosurfaces show the intermolecular interactions using a blue–red scale with blue indicating strong attractions and red indicating strong repulsions. All conformations shown here were obtained directly (no energy minimization) from Simulations 17 for a, 7 for b, and 36 for c of the 40 14,251.6-ns simulations at 298 K. conformation to proceed the self-assembly along a repulsion-free path when its populated conformation is not complementary to the conformation of its partner. Opposing effects of the sodium cation on complexation time. According to the conformational analysis of the 43 self-assembly pathways of Octacid4 with dioxane (2 pathways), xylene (40 pathways), and naphthalene (1 pathway) at 298 K, the sodium cation that chelated with the Octacid4 carboxylate either coor- dinated with the guest oxygen atom or formed the cation–π interaction with the guest aromatic ring. However, the chelation with the oxygen atom of the guest that subsequently entered the host cavity was observed in one of the two pathways of dioxane (Table S2); the transient cation–π interaction with the aromatic ring of the cavity-entering guest was observed in only 20 of the 41 self-assembly pathways of xylene and naphthalene (Table S2). These observations indicate that the cavity-entering guest either does not interact at all or does not strongly interact with the carboxylate-chelated sodium cation, so that the cavity-entering guest is not trapped at the host linker region. Instead, the cavity- entering guest interacts with an immobilized guest, which is trapped in the linker region due to its strong interaction with the carboxylate-chelated sodium cation, via the π–π interaction for xylene or naphthalene or the van der Waals interaction for dioxane. Because the π–π interaction and the van der Waals interaction are generally weaker than the cation–π interaction , these weak interactions enable the immobilized guest to usher the cavity-entering guest into the cavity, revealing the role of the sodium cation in shortening the complexation time of Octacid4 Fig. 7 Sodium-restrained Octacid4 conformations. a One pair of linkers with dioxane, xylene, and naphthalene. that form bidendate coordination with the sodium cation. b Two pairs of The conformational analysis of the 43 pathways also identified linkers that form bidendate coordination with the sodium cation. Left: side two small but interesting clusters of Octacid4 conformations that view; Right: top view. were derived a priori from the MD simulations (Fig. 7 and Videos S11–S18). In one cluster with a population of 11/43 minimization and frequency calculations of the two simulation- (Table S2) that was associated mainly with the host conforma- derived conformations with one or two pairs of the bidendate tions with two nearly parallel linkers, the carboxylates from two linkers using the Gaussian 16 program and HF/6-31 G* or nearby linkers of Octacid4 formed a bidentate coordination with B3LYP/6-31 G*, the minimization-derived conformations closely a sodium cation (Fig. 7a and Videos S11–S14 and S17–S18). This resembled the simulation-derived conformations (heavy-atom bidentate coordination rigidified a pair of the bidendate linkers root-mean-square deviations for one or two pairs of the and blocked one of the four equatorial portals of the host. In the bidendate linkers: 0.75 or 0.32 Å for HF/6-31 G* and 0.75 or other cluster with a population of 3/43 (Table S2) that was 0.34 Å for B3LYP/6-31 G*), respectively, and no imaginary associated mainly with the host conformations with two nearly frequencies were found for the minimization-derived conforma- coplanar linkers, all four linkers of Octacid4 were involved in the tions, suggesting that the conformations with one or two pairs of bidentate coordination, resulting in rigidification of two pairs of the bidendate linkers derived a priori from the simulations were the bidendate linkers and blockage of two of the four equatorial local minimal conformations. Because the host conformations portals (Fig. 7b and Videos S15–S16). According to the energy COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 7 ARTICLE COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 with the nearly parallel or nearly coplanar linkers were associated with long complexation times as described above, these results indicate that the sodium cation also played a role in lengthening the complexation time of Octacid4 with dioxane, xylene, and naphthalene and that the lengthening role is outweighed by the shortening role of the sodium cation as the populations of the host conformations with the bidendate linkers are lower than those of the host conformations without the bidendate linkers (Table S2). Discussion Internal and external consistencies of the self-assembly path- ways. The 81 self-assembly pathways described above exhibited internal or external consistencies as follows. (1) The natural logarithm of the Octacid4 population versus the simulation time exhibited a linear relationship with r of 0.98 for all 40 simulations that captured the Octacid4•xylene self- assembly, 0.85 for the first 20 of the 40 simulations, and 0.96 for the last 20 of the 40 simulations (Fig. 8). These r values indicate the convergency and internal consistency of the 40 simulations for which the first-order self-assembly rate is expected as explained above. (2) As described above, the simulations using the fully deprotonated Octacid4 under various conditions all captured the self-assembly of Octacid4 with dioxane, xylene, and naphthalene, but no autonomous complexation was observed under the same conditions if the fully deprotonated host was replaced by the fully protonated host. These results are consistent with the use of the Octacid4 solution containing the sodium borate buffer at pH 9 to detect the Octacid4•guest complexation in the NMR experiments and with the phase-transfer catalysis undergirding those NMR experiments. (3) One key finding of the present work is that the guest ingress is so fast that complexation time is determined primarily by the priming time. This finding is consistent with the report that many dense-phase reactions can be considered as gated reactions in that the rate of a local reaction is governed largely by the initial formation of a permissive atomic arrangement (viz., determined mainly by the system priming) within which the local transformation can proceed relatively rapidly . (4) Conformational analyses of all 43 pathways of xylene, dioxane, and naphthalene at 298 K showed that Octacid4 adopted Fig. 8 The exponential decay of the host population over the simulation exclusively a cluster of V-shaped conformations to gulp its guests. time for the Octacid4� p-xylene self-assembly at 298 K. The host These V-shaped conformations have the mean C17a–C6b population and simulation time were obtained from the 40 individual distance (the distance between two diphenoxymethane carbon complexation times of p-xylene listed in Table S2. The linear-regression atoms that control the width of the cavity portal as shown in analysis was performed using the PRISM 5 program. Fig. 1) of 7.0 Å (95% confidence interval: 7.0–7.1 Å) for the entrance portal and the corresponding mean C17a–C6b distance naphthalene (1) at 298 K revealed no repulsion (or only a trace of of 5.6 Å (95% confidence interval: 5.5–5.7 Å) for the opposing repulsion for naphthalene) but increasingly strong attraction portal (Table S3). These mean distances are consistent with the between Octacid4 and its guest in the self-assembly process reported V-shaped conformation proposed for the sliding-door 15 (Figs. 2–4). These observations are consistent with the report that mechanism for the gating of hemicarcerands that are closely the Corey–Pauling–Koltun space-filling models of xylene, diox- related to Octacid4. ane, and naphthalene could be pushed (or pushed with effort for (5) For the self-assembly at 298 K, 12 of the 40 captured naphthalene) through portals of the space-filling model of pathways for xylene and all captured pathways for dioxane and Octacid4 , indicating that a repulsion-free path exists for naphthalene had two nearly parallel linkers that channeled the Octacid4 to self-assemble with xylene, dioxane, and naphthalene. guest into the cavity (Table S2). These linker channels are (7) Most importantly, the capturing of the self-assembly of consistent with the report that an antechamber formed by two Octacid4 with dioxane, xylene, and naphthalene in the simula- parallel linkers of a hemicarcerand played a role in the gating of 15 tions at 298 K is consistent with the Octacid4 complexation with hemicarcerands . these guests detected in the NMR experiments at 298 K . (6) Relative to the noncovalent interaction gradient isosurfaces of dioxane that showed the stronger intramolecular repulsion in the half-chair conformation than that in the chair How Octacid4 self-assembles with its guests and what accel- conformation (Fig. 5), the isosurfaces of the Octacid4 complexes erates their assembly. Collectively, the 81 pathways indicate that in the 43 self-assembly pathways of xylene (40), dioxane (2), and the irreversible noncovalent self-assembly process of Octacid4 8 COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem COMMUNICATIONS CHEMISTRY | https://doi.org/10.1038/s42004-022-00624-4 ARTICLE with dioxane, xylene, and naphthalene was (1) initiated by the likely to the extent that the mean complexation time will be guest interaction with the cavity portal exterior of the host to significantly altered. increase the collisions of the portal that led to the portal expan- sion for guest ingress, (2) accelerated by engaging populated Design of irreversible noncovalent complexes with fast conformations of host and guest for the exterior intermolecular assembly rates. Despite the heterogeneity issue as described above, interaction to increase the portal collision frequency, and (3) the realization that the self-assembly initiation by the exterior completed by the portal contraction caused by the guest docking intermolecular interaction and the control of the complexation inside the cavity to impede guest egress. This type of self- time largely by the priming time is an advance and may help assembly is a process of stepwise conformational rearrangements explain why the presence of a cryptic exterior site for substrate or of the two molecules—kinetically starting from the pre-assembly inhibitor binding at the rim of the enzyme-active site is a funda- state (at which the guest interacts with the exterior of the host) to 29–36 mental feature of fast enzymes such as acetylcholinesterase the assembly and post-assembly states (at which the guest and why it is advantageous for the proapoptotic multidomain BAK interacts partially and fully with the interior of the host, respec- 37 protein to have a noncanonical BH3-binding groove that abuts tively)—to gradually increase the attraction between the two the canonical one for constrictive complexation of BAK—due to molecules along a repulsion–free path until the maximal attrac- the steric hindrance from R88 and Y89 on the edge of the cano- tion is reached. The kinetics of this process was governed pri- nical groove —with proapoptotic BH3-only proteins. The reali- marily by the complementarity between the two molecules during zation that the self-assembly acceleration by the adoption of the priming time (on the order of microseconds or longer) rather populated conformations for the exterior intermolecular interac- than the ingression time (on the order of picoseconds to tion is an advance and may help explain why small molecules nanoseconds). reportedly prefer to adopt local minimum conformations when binding to proteins . Further, these realizations suggest that irreversible noncovalent complexes with fast assembly rates could Heterogeneity of the Octacid4� xylene self-assembly pathways. be designed by accounting for the complementarity between guest The 40 Octacid4•xylene self-assembly pathways derived from the and host both of which adopt populated conformations for the converged MD simulations (as evident from Fig. 8) demonstrate exterior intermolecular interactions during the priming time. This the heterogeneity of the self-assembly pathways. As apparent design strategy may facilitate a paradigm shift from irreversible from Table S2, at 298 K, xylene can enter the cavity of the three covalent complex design to irreversible noncovalent complex distinct clusters of the host conformations shown in Fig. 6 and design for materials technology, data storage and processing, Videos S1–S6. In each of these clusters, xylene can also enter the molecular sensing and tagging, and drug therapy—especially the cavity of the host conformations with or without bidendate lin- personalized drug therapies for cancer patients with somatic kers (Videos S1–S6 and S11–S16), indicating that the Octa- G12C 7 mutations other than the KRAS mutation . cid4•xylene pathways can be shortened or lengthened by the sodium cation (Table S2). Methods This heterogeneity is akin to the heterogeneity of protein-folding Molecular dynamics simulation. The fully deprotonated, apo Octacid4 neu- pathways . It points to a limitation of the simulation protocol tralized with 8 sodium ions (or an Octacid4 in a different configuration, such as the used in this work because the protocol captured only the fast self- fully protonated Octacid4, as listed in Table S1) was manually solvated with 150/ 250 copies of a guest (xylene, dioxane, or naphthalene) using PyMOL V1.7.0.3 assembly pathways of Octacid4•dioxane and Octacid4•naphthalene (https://pymol.org) and tLEaP of the AmberTools 16 package (University of at 298 K. This calls for the development of a new simulation California, San Francisco) and then energy-minimized for 100 cycles of steepest- protocol to avoid oversimplification of the self-assembly process descent minimization followed by 900 cycles of conjugate-gradient minimization to such as equating the process to a few fast self-assembly pathways remove close van der Waals contacts using SANDER of the AMBER 11 package (University of California, San Francisco), FF12MClm , and a cutoff of 8.0 Å for for Octacid4•dioxane and Octacid4•naphthalene. As apparent noncovalent interactions. The tLEaP input file for building the fully protonated from the Octacid4•xylene self-assembly pathways, it is not a few Octacid4 and the Cartesian coordinates of the energy-minimized Octacid4•guest complexation times (from the fast pathways) but the mean (in all configurations as listed in Table S1) are provided in Data S1 and S2, complexation time (from the fast, intermediate, and slow path- respectively. The energy-minimized system was slowly heated to 298/340/363/ 370 K in 30 steps under constant temperature and constant volume, and then ways) that offers insight into the delicate balance between the equilibrated for 10 timesteps under constant temperature of 298/340/363/370 K sodium cation’s roles in lengthening and shortening the com- and constant pressure of 1 atm employing isotropic molecule-based scaling. Finally, plexation time of Octacid4•xylene. a set of 20/40/100 distinct, independent, unrestricted, unbiased, and classical Given the known theoretical work on gated reactions and the isobaric–isothermal MD simulations was performed for the resulting system using finding of the present work that the ingression time is so short PMEMD of the AMBER 14/16/18/20 package (University of California, San Francisco), FF12MClm , and a periodic boundary condition at 1 atm and 298/ that the complexation time is governed largely by the priming 340/363/370 K. All simulations used (i) a dielectric constant of 1.0, (ii) the time, it is worth noting the need to avoid overcomplication of the Berendsen coupling algorithm for thermostat and barostat, (iii) the particle mesh self-assembly process, such as simulating the Octacid4•guest self- 42 Ewald method to calculate electrostatic interactions of two atoms at a separation 40, 43 assembly using the octa-anionic Octacid4 possessing five water of >8 Å, (iv) Δt = 1.00 fs of the standard-mass time , (v) the SHAKE-bond- length constraint applied to all bonds involving hydrogen, (vi) a protocol to save molecules inside the cavity or a more complicated host with a the image closest to the middle of the “primary box” to the restart and trajectory variable number of water molecules inside the cavity, for at least files, (vii) a formatted restart file, (viii) the revised alkali-ion parameters , (ix) a two reasons. First, it has not been determined experimentally cutoff of 8.0 Å for noncovalent interactions, (x) a uniform 10-fold reduction in the 40, 43 whether the egression of water molecules that was observed in the atomic masses of the entire simulation system (both solute and solvent) , (xi) NTWX = 100 steps for coordinates’ output, and (xii) default values of all other present work will actually occur under the NMR experiment inputs of PMEMD. conditions once the water-bound Octacid4 is surrounded by a Available in the Supporting Information of Ref. , FF12MClm is a revised layer of water-insoluble xylene or naphthalene. Second, the water AMBER protein forcefield with no parameterization for any Octacid4•guest molecule is much smaller than dioxane, xylene, and naphthalene. complexes . This forcefield is able to (1) capture the experimentally observed exponential decay of the non-native state population of fast-folding proteins over The ingression and egression times of water are hence much simulation time with r > 0.90 and (2) fold these proteins with agreements between shorter than those of the three guests, and binding of water simulated and experimental folding times within factors of 0.6–1.4 . FF12MClm molecules to the host cavity is likely opportunistic rather than was used in this study to investigate the noncovalent self-assembly of small- intrinsic or constrictive. The inclusion of water molecules inside molecule guests with Octacid4 whose aromatic linkers can flip between left‐ and the cavity will affect the Octacid4•guest self-assembly but not right‐handed configurations and usher the guest into the host cavity. 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Attribution 4.0 International License, which permits use, sharing, Commun. 492, 135–139 (2017). 46. Feenstra, K. A., Hess, B. & Berendsen, H. J. C. Improving efficiency of large adaptation, distribution and reproduction in any medium or format, as long as you give time-scale molecular dynamics simulations of hydrogen-rich systems. J. appropriate credit to the original author(s) and the source, provide a link to the Creative Comput. Chem. 20, 786–798 (1999). Commons license, and indicate if changes were made. The images or other third party 47. Therneau, T. M. & Grambsch, P. M. Modeling Survival Data: Extending the material in this article are included in the article’s Creative Commons license, unless Cox Model (Springer-Verlag, 2000). indicated otherwise in a credit line to the material. If material is not included in the 48. Contreras-Garcia, J. et al. NCIPLOT: a program for plotting noncovalent article’s Creative Commons license and your intended use is not permitted by statutory interaction regions. J. Chem. Theory Comput. 7, 625–632 (2011). regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements This work was supported by the US Army Research Office (W911NF-16-1-0264) and © The Author(s) 2022 the Mayo Foundation for Medical Education and Research. Responsibility for the COMMUNICATIONS CHEMISTRY | (2022) 5:9 | https://doi.org/10.1038/s42004-022-00624-4 | www.nature.com/commschem 11

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