Noncovalent Complexes Research Articles

Advancing Cyclic Ion Mobility Mass Spectrometry Methods for Studying Biomolecules: Toward the Conformational Dynamics of Mega Dalton Protein Aggregates.

Native mass spectrometry is a powerful tool for the analysis of noncovalent complexes. When coupled with high-resolution ion mobility, this technique can be used to investigate the conformational changes induced in said complexes by different solution or gas-phase conditions. In this study, we describe how a new-generation high-resolution ion mobility instrument equipped with a cyclic ion mobility cell can be utilized for the analysis of large biomolecular systems, including temperature-induced protein aggregates of masses greater than 1.5 MDa, as well as a 63 kDa oligonucleotide complex. The effects of and the interplay between the voltages applied to the different components of the cyclic ion mobility spectrometry system on ion transmission and arrival time distribution were demonstrated using biomolecules covering the m/z range 2000-10,000. These data were used to establish a theoretical framework for achieving the best separation in the cyclic ion mobility system. Finally, the cyclic ion mobility mass spectrometer was coupled with a temperature-controlled electrospray ionization source to investigate high-mass protein aggregation. This analysis showed that it was possible to continuously monitor the change in abundance for several conformations of MDa aggregates with increasing temperature. This work significantly increases the range of biomolecules that can be analyzed by both cyclic ion mobility and temperature-controlled electrospray ionization mass spectrometry, providing new possibilities for high-resolution ion mobility analysis.

Lipophilic PAMAM Dendrimer: Conceptualization of Targeted Cosmetics and Drug Delivery

Abstract: The structure, properties, synthesis, negligible toxicity, and surface modification of PAMAM (polyamidoamine dendrimers) are all discussed in this review. The properties of supramolecular PAMAM dendrimers in nanopolymer science have shown great progress in delivering medicines. A divergent strategy was used to construct a Generation four (G4.0) PAMAM dendrimer with an ethylenediaminetetraacetic acid core and repeating units of acrylic acid and ethylenediamine. PAMAM dendrimers, have an aminodoamine repeat branching architecture that starts with an ethylene diamine initiator core. A generation [G] is a set of branching steps that follow each other. Drug molecules can be transferred either as covalently bonded to the functional groups on the dendrimer surface or by forming non-covalent complexes with dendrimers. Full generation PAMAM dendrimers are terminated with amine surface [G0, G1, G2, G3, G4], whereas half-generation dendrimers are terminated with carboxylate [G1.5, G2.5, etc]. PAMAM dendrimers appear to have negligible toxicity and immunogenicity, as well as favorable biodistribution-: according to the current study they can improve drug solubility, prevent drug degradation, increase circulation time, and potentially target drugs. According to the characterization study, they exhibit strong lipophilic qualities, allowing them to easily pass the blood-brain barrier. Due to cheaper polydispersity index of dendrimers, they possess greater stability and the void spaces of dendrimers are accessible for drug loading. The existence of a duplet functional group on the dendrimers enables appending vectors, ligands and devices for targed the drug delivery in the body.

The Charge and Phase State of Liposomes Dramatically Affects the Binding of Mannosylated Chitosan

Liposomal complexes with mucoadhesive polymers, e.g., mannosylated chitosan, are considered as prospective antituberculosis drug delivery systems. The properties of such complexes can be critically affected by the charge and phase state of liposomes. The aim of our work was to study the interaction of mannosylated chitosan with liposomes of various compositions and to identify the key patterns of this process. We tracked the interaction by titrating the liposomes with an increasing base-molar excess using the DLS method and ATR-FTIR spectroscopy. Sorption isotherms were obtained using ATR-FTIR spectroscopy and linearized in the Scatchard coordinates to evaluate the dissociation constant (Kdis). The inclusion of cardiolipin (CL) in the lipid composition helps to reduce the Kdis of the complexes by an order of magnitude of 3.8 × 10−4 M and 6.4 × 10−5 M for dipalmitoylphosphatidylcholine (DPPC) and DPPC:CL 80:20 (weight ratio), respectively. Preheating at 37 °C of gel-like anionic liposomes helps to reduce the Kdis to 3.5 × 10−5 M. Anionic liposomes, both in liquid crystal and in the gel-like state, form multipoint non-covalent complexes with chitosan–mannose conjugates due to the partial neutralization of the charges on the surface of the vesicles. Meanwhile, neutral liposomes in both states form unstable heterogeneous complexes, probably due to the predominant sorption of the polymer on the vesicles. Complex formation provides preferable binding with the model mannose-binding receptor concanavalin A and sustained pH-sensitive release of the antituberculosis drug moxifloxacin.

A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.

Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential.

On the Molecular Driving Force of Protein–Protein Association

The amount of water-accessible-surface-area, WASA, buried upon protein–protein association is a good measure of the non-covalent complex stability in water; however, the dependence of the binding Gibbs free energy change upon buried WASA proves to be not trivial. We assign a precise physicochemical role to buried WASA in the thermodynamics of non-covalent association and perform close scrutiny of the contributions favoring and those contrasting protein–protein association. The analysis indicates that the decrease in solvent-excluded volume, an entropic effect, described by means of buried WASA, is the molecular driving force of non-covalent association in water.

Unraveling the Chemistry of High Valent Arylcopper Compounds and Their Roles in Copper-Catalyzed Arene C-H Bond Transformations Using Synthetic Macrocycles.

Recent decades have witnessed a resurgence of the study of copper-catalyzed organic reactions. As the surrogate of noble metal catalysts, copper salts have been shown to exhibit remarkable versatility in activating various C-H bonds enabling the construction of diverse carbon-carbon and carbon-heteroatom bonds. Advantageously, copper salts are also naturally abundant, inexpensive, and less toxic in comparison to precious metals. Despite significant developments in synthesis, the mechanism of copper catalysis remains elusive. Hypothetical pathways such as the two-electron Cu(III)/Cu(I) and Cu(II)/Cu(0) catalytic cycles and the one-electron Cu(II)/Cu(I) catalytic cycle have been invoked to diagram C-H bond transformations because of the formidable challenges to isolate and characterize transient high valent organocopper intermediates. In fact, organocopper chemistry has been dominated for a long time by the acknowledged nucleophilic organocopper(I) compounds. Since the beginning of the new millennium, we have been systematically studying the supramolecular chemistry of heteracalix[n]aromatics. Owing to the ease of their synthesis and selective functionalizations, self-tunable conformation and cavity structures resulting from the interplay of heteroatoms with aromatic subunits, and outstanding properties in molecular recognition and self-assembly, heteracalix[n]aromatics have become a class of privileged synthetic macrocyclic hosts. Our journey to the chemistry of high valent organocopper compounds started with a serendipitous discovery of the facile formation of a stable organocopper compound, which contains astonishingly a Ph-Cu(III) σ-bond under very mild aerobic conditions. When we examined routinely the effect of the macrocyclic structures on noncovalent complexation properties, titration of tetraazacalix[1]arene[3]pyridine with Cu(ClO4)2·6H2O resulted in the precipitation of dark-purple crystals of phenylcopper(III) diperchlorate. Our curiosity about the transformation of an arene C-H bond into an Ar-Cu(III) bond prompted us to conduct an in-depth investigation of the reaction of macrocyclic arenes with copper(II) salts, leading to the isolation of arylcopper(II) compounds which are unprecedented and the missing link in organocopper chemistry. With structurally well-defined organometallics in hand, we have explored extensively the reactivities of both arylcopper(II) and arylcopper(III) compounds, demonstrating their versatility and uniqueness in chemical synthesis. Novel and fascinating arene C-H transformations under copper catalysis have been developed. Using acquired high valent arylcopper compounds as molecular probes, and employing the functionalizations of tetraazacalix[1]arene[3]pyridines as model reactions, we have revealed the diverse mechanisms of copper-promoted arene C-H bond reactions. Elusive reaction pathways of some copper-catalyzed C-X bond activations have also been unraveled. In the meantime, we have also witnessed pleasingly the rapid development of field with the advent of new high valent organocopper compounds. Without any doubt, studies of the synthesis, reactivity, and catalysis of high valent organocopper compounds have been reshaping the field of organocopper chemistry. This Account summarizes our endeavors to explore the chemistry of structurally well-defined arylcopper(II) and arylcopper(III) compounds and the mechanisms of copper-catalyzed arene C-H and C-X bond transformations. We hope this Account will inspire chemists to study thoroughly the fundamentals and the cutting-edge catalysis of high valent organocopper compounds advancing and redefining the discipline of organocopper chemistry.

Molecular simulations of fluoxetine in hydrated lipid bilayers, as well as in aqueous solutions containing β-cyclodextrin

Fluoxetine, which is a well-known antidepressant drug, is studied in hydrated cholesterol-free and cholesterol-containing lipid bilayers through unbiased and biased atomistic molecular dynamics simulations. The latter are conducted for the calculation of the potential of mean force (PMF) of fluoxetine along an axis perpendicular to the two leaflets of the bilayer. The PMF indicates that the drug prefers to reside inside the lipid phase and allows us to calculate important thermodynamic properties, such as the Gibbs energy difference of partitioning from the water to the lipid phase and the Gibbs energy barrier for hopping events between the two leaflets of the bilayer. The results from the biased simulations are in accord with the mass density profiles calculated from the unbiased simulations. Moreover, we estimate the effect of fluoxetine mole fraction on the order parameters of the lipid alkyl chains and on the area per lipid. It is also found that fluoxetine forms a hydrogen bond network with lipids and water molecules penetrating into the lipid phase. In addition, fluoxoetine is studied in detail in aqueous solutions containing β-cyclodextrin. It is observed from unbiased molecular dynamics simulations that the two aforementioned molecules form a noncovalent complex spontaneously and the calculated binding free energy is in agreement with the literature.

Cyclic RGD functionalized PLGA nanoparticles loaded with noncovalent complex of indocyanine green with urokinase for synergistic thrombolysis.

Thrombotic diseases have the characteristics of long latency period, rapid onset, and high mortality rate, which seriously threaten people’s life and health. The aim of this research is to fabricate a novel indocyanine green complex of urokinase (ICG@uPA) and employ the amphiphilic PEG-PLGA polymer to deliver the complex as an enzyme-phototherapeutic synergistic thrombolysis platform. The noncovalent indocyanine green (ICG) complex of urokinase (ICG@uPA) was prepared via supramolecular self-assembly and then encapsulated into cRGD decorated polymeric nanoparticles (cRGD-ICG-uPA NPs) by double-emulsion solvent evaporation method. Then the nanoparticles (NPs) were characterized in terms of particle size, optical properties, in vitro release, etc. The targeting and thrombolytic effect of the nanoparticles were studied both in vitro and in vivo. ICG@uPA and cRGD-ICG-uPA NPs displayed significantly higher photostability and laser energy conversion efficiency than free ICG. Concomitantly, the NPs exhibited selective binding affinity to the activated platelets and specific accumulation in the mouse mesenteric vessel thrombus. Significant thrombolysis was achieved in vivo by photo-assisted synergistic therapy with reduced dose and systemic bleeding risk of uPA. Our results prove that the functional PLGA nanoparticle loaded with the ICG@uPA offers a novel option for effective and safe thrombolytic treatment.

Native mass spectrometry for the investigation of protein structural (dis)order

A central challenge in structural biology is represented by dynamic and heterogeneous systems, as typically represented by proteins in solution, with the extreme case of intrinsically disordered proteins (IDPs) [1–3]. These proteins lack a specific three-dimensional structure and have poorly organized secondary structure. For these reasons, they escape structural characterization by conventional biophysical methods. The investigation of these systems requires description of conformational ensembles, rather than of unique, defined structures or bundles of largely superimposable structures. Mass spectrometry (MS) has become a central tool in this field, offering a variety of complementary approaches to generate structural information on either folded or disordered proteins [4–6]. Two main categories of methods can be recognized. On one side, conformation-dependent reactions (such as cross-linking, covalent labeling, H/D exchange) are exploited to label molecules in solution, followed by the characterization of the labeling products by denaturing MS [7–11]. On the other side, non-denaturing (“native”) MS can be used to directly explore the different conformational components in terms of geometry and structural compactness [12–16]. All these approaches have in common the capability to conjugate protein structure investigation with the peculiar analytical power of MS measurements, offering the possibility of assessing species distributions for folding and binding equilibria and the combination of both. These methods can be combined with characterization of noncovalent complexes [17, 18] and post-translational modifications [19–23]. This review focuses on the application of native MS to protein structure and dynamics investigation, with a general methodological section, followed by examples on specific proteins from our laboratory.

Mechanistic study and synergistic effect on inhibition of α-amylase by structurally similar flavonoids

Natural plant-based flavonoids have the potential effect of reducing postprandial hyperglycaemia, thus they can be used to prevent and control type 2 diabetes mellitus. Here, to explore the antihyperglycemic mechanisms of flavonoids (apigenin, scutellarein, hispidulin and nepetin), their inhibition mechanisms against α-amylase were evaluated using multispectral methods and molecular docking in this study. As a result, nepetin, a competitive inhibitor, exhibited the best inhibitory effect than other three flavonoids, suggesting that adjacent dihydroxyl group on the B-ring played an important role in inhibiting the activity of α-amylase. At the same time, the combination of apigenin and scutellarein/hispidulin/nepetin synergistically improved their inhibitory effects on α-amylase. In addition, fluorescence quenching analysis indicated that the addition of flavonoids could result in the static quenching of α-amylase and the formation of non-covalent complexes. The interaction further led to the alteration of microenvironment and secondary structure of α-amylase, thus reducing the catalytic activity of enzyme. In conclusion, this finding can provide vital insights into the use of flavonoids, especially nepetin, as the potential drug candidates for the treatment of diabetes.

Protective effects and mechanism of amino acids as chokeberry cyanidin and its glycoside protectant under the condition of vitamin C coexistence

In the presence of vitamin C, cyanidin and cyanidin glycosides are degraded during the processing and storage of food products. To solve this issue, we investigated the protective effects and mechanism of action of five amino acids on the stability of cyanidin and its glycosides from chokeberry. The results showed that 0.3% tryptophan most effectively inhibited the degradation of cyanidin and its glycosides in the presence of vitamin C, under ultraviolet, dark, and sucrose-rich conditions. Fluorescence spectrum analysis showed that tryptophan could form noncovalent binding complexes with cyanidin-3-O-galactoside and cyanidin through hydrophobic and electrostatic forces and hydrogen bonds. Molecular docking results showed that the indole structure of tryptophan could form hydrophobic interactions with cyanidin-3-O-galactoside and cyanidin via hydrogen bonding, resulting in greater protection. Therefore, tryptophan could effectively protect cyanidin and its glycosides in cyanidin- and cyanidin glycoside-rich food products.

Demarcating Noncovalent and Covalent Bond Territories: Imine-CO2 Complexes and Cooperative CO2 Capture.

Chemical bond territory is rich with covalently bonded molecules wherein a strong bond is formed by equal or unequal sharing of a quantum of electrons. The noncovalent version of the bonding scenarios expands the chemical bonding territory to a weak domain wherein the interplay of electrostatic and π-effects, dipole-dipole, dipole-induced dipole, and induced dipole-induced dipole interactions, and hydrophobic effects occur. Here we study both the covalent and noncovalent interactive behavior of cyclic and acyclic imine-based functional molecules (XN) with CO2. All parent XN systems preferred the formation of noncovalent (nc) complex XN···CO2, while more saturated such systems (XN') produced both nc and covalent (c) complexes XN'+-(CO2)-. In all such cases, crossover from an nc to c complex is clearly demarcated with the identification of a transition state (ts). The complexes XN'···CO2 and XN'+-(CO2)- are bond stretch isomers, and they define the weak and strong bonding territories, respectively, while the ts appears as the demarcation point of the two territories. Cluster formation of XN with CO2 reinforces the interaction between them, and all become covalent clusters of general formula (XN+-(CO2)-)n. The positive cooperativity associated with the NH···OC hydrogen bond formation between any two XN'+-(CO2)- units strengthened the N-C coordinate covalent bond and led to massive stabilization of the cluster. For instance, the stabilizing interaction between the XN unit with CO2 is increased from 2-7 kcal/mol range in a monomer complex to 14-31 kcal/mol range for the octamer cluster (XN'+-(CO2)-)8. The cooperativity effect compensates for the large reduction in the entropy of cluster formation. Several imine systems showed the exergonic formation of the cluster and are predicted as potential candidates for CO2 capture and conversion.

Inside Back Cover: Mass Spectrometry Detection and Imaging of a Non‐Covalent Protein–Drug Complex in Tissue from Orally Dosed Rats (Angew. Chem. Int. Ed. 36/2022)

Flying elephants and delicate cargo: Electrospray ionization of proteins has been likened to making molecular elephants fly. Helen J. Cooper and co-workers (DOI: 10.1002/anie.202202075) demonstrate mass spectrometry detection and imaging of a non-covalent complex formed in vivo, comprising an endogenous protein and an exogenously administered drug molecule, directly from liver tissue. These methods will provide greater insight into protein–drug complexes in support of drug research and development.

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats.

Here, we demonstrate detection by mass spectrometry of an intact protein-drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein-drug complex comprised fatty acid binding protein 1, FABP1, non-covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non-covalent protein-drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target-drug characterization directly from the physiological environment.

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats.

Here, we demonstrate detection by mass spectrometry of an intact protein–drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein–drug complex comprised fatty acid binding protein 1, FABP1, non‐covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non‐covalent protein–drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target‐drug characterization directly from the physiological environment.

Influence of Association on Binding of Disaccharides to YKL-39 and hHyal-1 Enzymes.

Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.

Interaction mechanism of flavonoids and Tartary buckwheat bran protein: A fluorescence spectroscopic and 3D-QSAR study

Tartary buckwheat bran protein (TBBP) is a valuable by-product of Tartary buckwheat processing. Current studies have indicated that TBBP is a desirable carrier and protector of flavonoids. However, the quantitative structure-affinity relationship of flavonoids with TBBP still remains to be elucidated. Here, by using the fluorescence spectroscopy method to measure the binding constants of 16 flavonoids with TBBP, we employ a combination of the atom-based three dimensional-quantitative structure-affinity relationships (3D-QSAR) and quantum-chemical calculation to explore the underlying binding mechanisms. We show that flavonoids can cause the intrinsic fluorescence quenching and form non-covalent complexes with TBBP driven by hydrogen bonding and van der Waal forces. The atom-based 3D-QSAR model reveals that the rutoside group at the C-7 position of flavones is favorable to enhance the binding constants of flavonoids with TBBP. Quantum-chemical calculations consistently disclose that the rutoside group can intensify interaction forces of flavonoids, thereby strengthening the binding. This work provides theoretical evidence that TBBP has the potential application to be raw materials for develop functional food, and be the delivery carriers of flavonoids.

Intermolecular Interactions in the Formation of Polysaccharide-Gelatin Complexes: A Spectroscopic Study.

Gelatin, due to its gelling and stabilizing properties, is one of the widely used biopolymers in biotechnology, medicine, pharmaceuticals, and the food industry. One way to modify the characteristics of gelatin is molecular modification by forming non-covalent polyelectrolyte complexes with polysaccharides based on the self-organization of supramolecular structures. This review summarizes recent advances in the study of various types and the role of intermolecular interactions in the formation of polysaccharide-gelatin complexes, and conformational changes in gelatin, with the main focus on data obtained by spectroscopic methods: UV, FT-IR, and 1H NMR spectroscopy. In the discussion, the main focus is on the complexing polysaccharides of marine origin-sodium alginate, κ-carrageenan, and chitosan. The prospects for creating polysaccharide-gelatin complexes with desired physicochemical properties are outlined.

Quantifying Biomolecular Interactions Using Slow Mixing Mode (SLOMO) Nanoflow ESI-MS.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful label-free assay for detecting noncovalent biomolecular complexes in vitro and is increasingly used to quantify binding thermochemistry. A common assumption made in ESI-MS affinity measurements is that the relative ion signals of free and bound species quantitatively reflect their relative concentrations in solution. However, this is valid only when the interacting species and their complexes have similar ESI-MS response factors (RFs). For many biomolecular complexes, such as protein–protein interactions, this condition is not satisfied. Existing strategies to correct for nonuniform RFs are generally incompatible with static nanoflow ESI (nanoESI) sources, which are typically used for biomolecular interaction studies, thereby significantly limiting the utility of ESI-MS. Here, we introduce slow mixing mode (SLOMO) nanoESI-MS, a direct technique that allows both the RF and affinity (Kd) for a biomolecular interaction to be determined from a single measurement using static nanoESI. The approach relies on the continuous monitoring of interacting species and their complexes under nonhomogeneous solution conditions. Changes in ion signals of free and bound species as the system approaches or moves away from a steady-state condition allow the relative RFs of the free and bound species to be determined. Combining the relative RF and the relative abundances measured under equilibrium conditions enables the Kd to be calculated. The reliability of SLOMO and its ease of use is demonstrated through affinity measurements performed on peptide–antibiotic, protease–protein inhibitor, and protein oligomerization systems. Finally, affinities measured for the binding of human and bacterial lectins to a nanobody, a viral glycoprotein, and glycolipids displayed within a model membrane highlight the tremendous power and versatility of SLOMO for accurately quantifying a wide range of biomolecular interactions important to human health and disease.

Coupled Cluster Benchmarking of Large Noncovalent Complexes in L7 and S12L as Well as the C60 Dimer, DNA-Ellipticine, and HIV-Indinavir.

In this work, we report the benchmark binding energies of the seven complexes within the L7 data set, six host-guest complexes from the S12L data set, a C60 dimer, the DNA-ellipticine intercalation complex, and the largest system of the study, the HIV-indinavir system, which contained 343 atoms or 139 heavy atoms. The high-quality values reported were obtained via a focal point method that relies on the canonical form of second-order Møller-Plesset theory and the domain-based local pair natural orbital scheme for the coupled cluster with single double and perturbative triple excitations [DLPNO-CCSD(T)] extrapolated to the complete basis set (CBS) limit. The results in this work not only corroborate but also improve upon some previous benchmark values for large noncovalent complexes albeit at a relatively steep cost. Although local CCSD(T) and the largely successful fixed-node diffusion Monte Carlo (FN-DMC) have been shown to generally agree for small- to medium-size systems, a discrepancy in their reported binding energy values arises for large complexes, where the magnitude of the disagreement is a definite cause for concern. For example, the largest deviation in the L7 data set was 2.8 kcal/mol (∼10%) on the low end in C3GC. Such a deviation only grows worse in the S12L set, which showed a difference of up to 10.4 kcal/mol (∼25%) by a conservative estimation in buckycatcher-C60. The DNA-ellipticine complex also generated a disagreement of 4.4 kcal/mol (∼10%) between both state-of-the-art methods. The disagreement between local CCSD(T) and FN-DMC in large noncovalent complexes shows that it is urgently needed to have the canonical CCSD(T), the Monte Carlo CCSD(T), or the full configuration interaction quantum Monte Carlo approaches available to large systems on the hundred-atom scale to solve this dilemma. In addition, the performances of cheaper popular computational methods were assessed for the studied complexes with respect to DLPNO-CCSD(T)/CBS. r2SCAN-3c, B97M-V, and PBE0+D4 work well in large noncovalent complexes in this work, and GFN2-xTB performs well in π-π stacking complexes. B97M-V is the most reliable computationally efficient approach to predicting noncovalent interactions for large complexes, being the only one to have binding errors within the so-called 1 kcal/mol "chemical accuracy". The benchmark interaction energies of these host-guest complexes, molecular materials, and biological systems with electronic and medicinal implications provide crucial reference data for the improvement of current and future lower-cost methods.