In the last decade, the contribution of halogen bond (HaB) in liquid phase enantioseparation of halogenated analytes was demonstrated by using polysaccharide-based chiral stationary phases (CSPs) and n-hexane-containing mixtures as mobile phases. On the other hand, it was also shown that polar organic solvents (POSs) like methanol and ethanol weaken or suppress the contribution of HaB to binding and recognition, whereas hydrophobic mechanisms become dominant by using alcohol-based mobile phases. Although some recent studies demonstrated that HaB-based systems exhibit an improved water resistance compared to the analogous hydrogen bond (HB)-based systems, currently no information is available in the literature about the effect of using aqueous organic mobile phases on the enantioseparations of halogenated compounds driven by HaB in normal phase mode. With the aim to gain information about this topic, we investigated the effect of adding water to the mobile phase on the enantioseparation of four hexahalogenated 4,4’-bipyridines by using amylose and cellulose tris(3,5-dimethylphenylcarbamate)-based chiral columns. As a result, chromatographic behaviours dependent on the distinctive halogen atom featuring the analytes were observed. The study confirmed that, when polar organic solvents and aqueous organic mixtures were used as mobile phase, both iodine-dependent and hydrophobic noncovalent interactions contributed to the enantioseparation of iodinated 4,4’-bipyridines.

Computational chemistry has evolved into a powerful and increasingly reliable tool for elucidating chemical phenomena and predicting molecular properties. Nevertheless, accurately modeling enantioselective recognition remains one of the most demanding challenges in separation science. The fundamental obstacle lies in the exceptionally small free-energy differences that distinguish competing enantioselective pathways, typically ranging from only 0.01 to 1 kcal·mol-1. Such subtle energetic variations push current theoretical approaches to their limits, making experimental validation indispensable for assessing and refining computational models. Considering this, we investigated the enantioselective recognition of erythro-mefloquine (MQ) by β-cyclodextrin (β-CD) and heptakis(2,3-di-O-acetyl)-β-cyclodextrin (HDA-β-CD) through an integrated strategy combining capillary electrophoresis (CE), nuclear magnetic resonance (NMR) spectroscopy, and quantum mechanics (QM)-based molecular modeling. This multidisciplinary approach serves a dual purpose: first, to critically evaluate the consistency and complementarity of results obtained from experimental and theoretical methodologies; second, to identify possible existing limitations in our understanding of the molecular mechanisms and noncovalent interactions governing enantioselective recognition. The long-term goal of this work is to establish a robust and systematic framework that integrates orthogonal experimental and computational techniques, providing a more reliable protocol for explaining, validating, and ultimately predicting enantioselective recognition processes.

In the last four decades, computational techniques have become useful tools in liquid-phase enantioseparation for modeling analyte and chiral selector structures, as well as the mobile phase implicitly and explicitly, for exploring mechanisms underlying retention and enantioseparation, and for identifying noncovalent interactions that contribute to analyte-chiral selector binding and recognition. The present chapter briefly summarizes fundamentals and recent advances in the application of molecular modeling for understanding capillary electrophoresis enantioseparations promoted by cyclodextrin-based selectors. A practical example illustrates in detail the calculation and analysis of low-energy structures of single-isomer methylated cyclodextrins and their stereoelectronic properties.

In the present study, an attempt was made to explain enantioselective noncovalent interactions of metaclazepam (MCZ) enantiomers with three native cyclodextrins (CDs) with different cavity size by combined use of capillary electrophoresis (CE), nuclear magnetic resonance (NMR) spectroscopy and quantum-mechanics (QM) calculations. With CE, it was observed that the medium size β-CD binds the enantiomers of MCZ most strongly although with very low enantioselectivity. The affinity of the MCZ enantiomers was lowest towards α-CD. Both α- and β-CDs bind (R)-MCZ more tightly than (S)-MCZ, while the opposite is the case with γ-CD. ROESY experiments in NMR spectroscopy provided an explanation of the affinity strength of MCZ enantiomers towards the studied CDs based on the extent of insertion of the guest molecule into the cavity of CDs. QM calculations indicated the importance of intermolecular hydrogen bonding in selector-selectand interactions, as well as supported experimentally observed enantiomer affinity pattern towards CDs under this study. On the other hand, binding energy values calculated using QM did not always correlate with the binding strengths experimentally observed in CE. In addition, binding energy differences calculated based on QM are at least one order of magnitude higher when compared to the binding energy differences calculated based on the selectivity of enantioseparations in CE. Thus, QM tools need further development (refinement) in order to properly describe fine noncovalent interactions between CDs and their guests.

Background The detection of dispersion forces in enantioselective liquid chromatography (LC) is rather challenging because selectand, selector and mobile phase molecules may participate in multiple types of intra- and intermolecular noncovalent interactions of different strength. In this frame, the LC system can be used as a tool to evaluate the impact of changing the structures of selectand, chiral selector and separation medium, through fast analytical screenings. In the last few years, we studied the enantioseparations of chiral compounds containing the ethynylferrocene moiety as a test probe to identify dispersion forces in LC by using amylose carbamate-based chiral stationary phases (CSPs). Results The results of the study have been reported in a series of three papers. In Part II, we confirmed that the high affinity observed for the second eluted (Rp)-enantiomer of the 1-(iodoethynyl)-3-phenylferrocene toward amylose phenylcarbamate-based selectors could be reasonably based on dispersion forces. In the present Part III of the series, we focus on the impact of changing the 3-aryl substituent of 1-(iodoethynyl)-3-arylferrocenes on the enantioseparation, considering the 1-(iodoethynyl)-3-phenylferrocene as reference for comparison. On this basis, the enantioseparations of seven planar chiral ferrocenes were performed and compared by using amylose-based CSPs. n-Hexane-based mixtures, polar organic solvents and aqueous organic mixtures were used with the aim of evaluating the impact of mobile phases of different polarity on the enantioseparations. The results of the chromatographic analyses confirmed the high and unique affinity of the second eluted (Rp)-enantiomer of the 1-(iodoethynyl)-3-(4-t-butyl)phenylferrocene toward the amylose-based CSPs. Significance By using 1-(iodoethynyl)-3-arylferrocenes as test probes, this study confirmed that dispersion forces may turn steric repulsion into attraction with a strength depending on the structure of the 3-aryl group and on mobile phase polarizability. Furthermore, the possibility to deconvolute hydrophobic and dispersion forces in aqueous mobile phases was also demonstrated.

In the last decade, by integrating experimental and computational analyses, it was demonstrated that halogen bond (HaB) may contribute to binding and enantiorecognition mechanisms underlying the HPLC enantioseparation of halogenated chiral analytes by using cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC)-based chiral columns and n-hexane-based mixtures as mobile phases. When used as a pivotal component of the mobile phase in supercritical fluid chromatography (SFC), carbon dioxide is often considered as an n-hexane-like nonpolar solvent because of its low dielectric constant and zero molecular dipole moment. On the other hand, carbon dioxide may also serve as hydrogen bond (HB) and HaB acceptor due to the presence of nonbonding electrons on the two oxygen atoms, interacting with analyte enantiomers, chiral selectors, and co-solvents. On this basis, we report herein the results of a study aiming at evaluating the impact of using carbon dioxide in SFC in place of n-hexane in HPLC on halogen-dependent enantioseparations by using atropisomeric halogenated 4,4 '-bipyridines as analytes and Lux Cellulose-1 as CDMPC-based chiral column. The experimental investigation was complemented by a computational study performed using (a) quantum mechanics (QM) calculations to map and quantify noncovalent interactions possibly underlying the contact of the analytes with carbon dioxide and with the distinctive pendant groups of the CDMPC and (b) molecular dynamics (MD) simulations to visualize noncovalent interactions acting in the analyte 1/CDMPC chromatographic system in different media. The use of MD simulations to model enantioseparations performed in carbon dioxide-based media was not reported in the literature so far.

Phenylbutanoids, commonly found in various medicinal plants, have attracted significant attention due to their remarkable biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects, as well as for their versatility as starting materials in organic synthesis. Among phenylbutanoids, phenyl-1,3-butadienes represent a unique class of conjugated dienes, characterized by a phenyl (C6H5) group attached to a 1,3-butadiene (-CH=CH-CH=CH2) backbone. In this study, we synthesized the hydroxylated biphenyl 5,5′-di((E)-buta-1,3-dien-1-yl)-2,2′,3,3′-tetramethoxy-1,1′-biphenyl 1, closely related to its corresponding monomer 2, which is known for its broad range of pharmacological activities. The synthesis was carried out using microwave-assisted technologies. The structure of the synthesized compound was confirmed through elemental analysis, 13C-NMR, 1H-NMR, and ESI-MS spectrometry. Furthermore, we computed this novel compound’s conformational energy profile (CEP), evaluating how its energy varies with changes in the dihedral bond angle.

This study investigates the synthesis and photochemical behavior of a series of (E)-1-aryl-1,3-butadienes with different aromatic substituents. Despite their simple structure and straightforward preparation, detailed studies of their photochemical properties, especially UV light-induced (E) to (Z) isomerization, are scarce. Our results demonstrate that these compounds can efficiently undergo photo-triggered geometric changes, highlighting their potential as functional units in photochemical applications. The findings underline the significance of extended conjugation in managing excited-state processes, providing new insights into the dynamics of photoinduced transformations in conjugated diene systems. Additional computational analyses show how geometric modifications influence conformational energies in the synthesized compounds. Overall, these results improve understanding of structure–reactivity relationships and lay the foundation for designing photoresponsive materials based on (E) and (Z)-1-aryl-1,3-butadiene frameworks, with promising applications in photochemistry and materials science.

Carbonic anhydrases (CAs) have emerged as promising drug targets for cancer therapy. In particular, the human (h) CA IX (hCA IX) isoform is expressed in a wide variety of malignancies and appears tightly regulated by micro-environmental hypoxia. Ongoing efforts aim to identify novel classes of selective CA inhibitors (CAIs) by exploring molecular diversity and discovering original chemotypes and pharmacophores. Previously, we identified a new hit compound (TDP1) carrying a trifluorodihydroxypropanone (TDP) motif as an original zinc-binding function (ZBF), which has undergone structural optimization to generate derivatives with selective inhibition profile toward hCA IX. Herein, we report on the synthesis, biological evaluation, X-ray crystallographic analysis, and computational studies of a series of aromatic-substituted TDP derivatives as novel CAI-directed chemotypes. The most potent compounds selectively inhibited hCA IX, with KI values in the submicromolar to high nanomolar range and exhibited significant antiproliferative activity against representative normoxic and hypoxic pancreatic tumor cell lines. Ultrastructural studies indicated for TDPs a possible interference with the mitochondrial function or iron metabolism. Moreover, X-ray crystallography data provided insights into the CA inhibition mechanism, suggesting that these compounds behave similarly to classical CAIs. In summary, this original TDP pharmacophore effectively inhibits human CAs, with relative selectivity towards hCA IX over cytosolic isoforms, thus providing structural insights for the development of a new class of selective anticancer agents.

Multiple factors may impact enantiorecognition ability of CDs, and the validation of computational tools by using suitable experimental data is a critical point. The main advantage of using capillary electrophoresis (CE) for this purpose relies on its higher sensitivity to detect weak noncovalent intermolecular interactions compared to any other technique. In this study, we used CE enantioseparations of tetramisole with β-CD, heptakis(2,3-di-O-methyl)-β-CD, heptakis(2,3-di-O-acetyl)-β-CD and heptakis(2-O-methyl-3-O-acetyl)-β-CD as benchmark separation systems aiming at investigating the molecular bases of these processes by NMR spectroscopy and quantum mechanics (QM) methods. The aim of this study was explaining the subtle differences observed in enantiomer migration order, that means enantiomer affinity pattern of tetramisole toward the used CDs, migration times, and selectivity values. A good correlation between experimental and theoretical data was obtained along with noncovalent interactions patterns fully consistent with the CE outcomes. Significant differences in the enantioselective recognition ability were observed between β-CD and heptakis(2,3-di-O-acetyl)-β-CD in CE, which correlated very well with the recognition model derived from NMR spectroscopy and QM. Importantly, the introduction of dispersion corrections in the used QM model chemistry provided results in better agreement with the experimental observations for the complexes of tetramisole with the acetylated β-CDs.

In the last decade, biological processes involving halogen bond (HaB) as a leading interaction attracted great interest. However, although bound iodine atoms are considered powerful HaB donors, few iodinated new drugs were reported so far. Recently, iodinated 4,4'-bipyridines showed interesting properties as HaB donors in solution and in the solid state. In this paper, a study on the inhibition activity of seven halogenated 4,4'-bipyridines against malignant melanoma (MM) cell proliferation is described. Explorative dose/response proliferation assays were first performed with three 4,4'-bipyridines by using four MM cell lines and the normal BJ fibroblast cell line as control. Among them, the A375 MM cell line was the most sensitive, as determined by MTT assays, which was selected to evaluate the antiproliferative activity of all 4,4'-bipyridines. Significantly, the presence of an electrophilic iodine impacted the biological activity of the corresponding compounds. The 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridine showed significant antiproliferation activity against the A375 cell line, and lower toxicity on BJ fibroblasts. Through in silico studies, the stereoelectronic features of possible sites determining the bioactivity were explored. These results pave the way for the utilization of iodinated 4,4'-bipyridines as templates to design new promising HaB-enabled inhibitors of MM cell proliferation. The antiproliferative activity of seven substituted 3,3',5,5'-tetrachloro-4,4'-bipyridines was evaluated against malignant melanoma (MM) cell lines. The 3,3',5,5'-tetrachloro-2-iodo-4,4'-bipyridine showed high activity on A375, and very low toxicity on control BJ fibroblasts. In silico studies confirmed the highest electrophilic properties for the iodine atoms contained in the most active compounds against MM cell line proliferation. image

N-(3-hydroxyacyl)glycines are compounds of remarkable interest due to their biogenic origin and bioactivity and as precursors of the corresponding 3-acyloxy derivatives which represent an important class of bioactive products of bacterial origin. Commendamide [N-(3-hydroxypalmitoyl)glycine] (1) is a gut microbiota-derived bioactive metabolite that is structurally like endogenous long-chain N-acyl-amino acids belonging to the endocannabinoidome, a complex lipid signaling system involved in several aspects of mammalian physiology and pathology. Thanks to this structural similarity, this compound and its analogues, like the N-(3-hydroxymyristoyl)glycine 2, exert a remarkable bioactivity in mammals, for instance, through activation of G-protein-coupled receptors (GPCRs). N-(3-Hydroxyacyl)glycines are chiral and the availability of their pure enantiomers may bring light to possible enantioselective pathways within the biological processes which these compounds are involved in. A sustainable synthesis of rac-1 and its analogues was recently reported, but asymmetric synthesis and enantioseparation methods to access their pure or enriched enantiomers were not reported so far. In this paper, we report the first direct separation of commendamide enantiomers by using enantioselective high-performance liquid chromatography (HPLC) with polysaccharide-based chiral columns, aqueous-organic mixtures as mobile phases and either electrospray ionization mass spectrometry (ESI-MS) or UV detection. Optimal enantioseparation was obtained by using an amylose tris(3,5-dimethylphenylcarbamate)-based chiral column and acetonitrile/water 60:40 (v/v) (0.1 % acetic acid) as mobile phase. By adopting the same method, the enantioseparation of the analogue 2 was also performed. The molecular bases of the higher retention and selectivity observed for the N-(3-hydroxyacyl)glycine 1 compared to the analogue 2 were explored by computational analysis.

A computational study was performed to unravel mechanisms underlying capillary electrophoresis enantioseparations of daclatasvir and its (R,R,R,R)-enantiomer with native and methylated β-cyclodextrins (β-CDs) as chiral selectors. Considering the enantioseparation results as benchmark, the structures of β-CD and seven methylated β-CDs were optimized by quantum mechanics, and their topography and computed molecular properties were compared. Furthermore, the electron charge density distribution of the macrocycles was also evaluated by calculating the molecular electrostatic potential of pivotal regions of native and methylated β-CDs. The function of hydrogen bonds in the complexation process of daclatasvir and the CDs was derived from quantum mechanics analysis and confirmed by molecular dynamics, as orthogonal computational techniques. The presence of a round-shaped cavity in the CDs used as chiral selector appeared as a necessary requirement for the enantioseparation of daclatasvir and its (R,R,R,R)-enantiomer. In this regard, it was confirmed that the round shape of the CDs is sustained by hydrogen bonds formed between adjacent glucopyranose units and blocking rotation of the linking glycosidic bonds. The presence of hydroxy groups at the 6-position of the glucopyranose units and the concurrent absence of hydroxy groups at the 2-position were evidenced as important factors for enantioseparation of daclatasvir and its enantiomer by methylated β-CDs.

Background: In the first part of our study on possible contribution of dispersion forces in liquid-phase enantioseparations, the enantioseparation of the axially chiral 3,3 '-dibromo-5,5 '-bis-ferrocenylethynyl-4,4 '-bipyridine '-dibromo-5,5 '-bis-ferrocenylethynyl-4,4 '-bipyridine with an amylose tris (3,5-dimethylphenylcarbamate)-based chiral column appeared reasonably consistent with a picture of the enantioselective recognition based on the interplay between hydrogen bond (HB), it-it stacking and dispersion interactions. Results: In the second part of this study, we evaluated the impact of analyte and chiral stationary phase (CSP) structure, mobile phase and temperature on the enantioseparations of planar chiral 1-(iodoethynyl)-3 arylferrocenes (3-aryl = phenyl, 2-naphthyl, 4-methylphenyl, 4-t-butylphenyl) t-butylphenyl) with polysaccharide-based chiral columns. The main aim of the present study was to understand the molecular bases of the high affinity observed for the second eluted (Rp)-enantiomer R p )-enantiomer of some of these analytes toward amylose phenylcarbamate-based selectors when methanol-containing mixtures were used as mobile phases. Significantly, higher affinity of the second eluted (Rp)-enantiomer R p )-enantiomer toward the selector could be also observed for the sterically hindered 1-(iodoethynyl)-3-(4-t-butylphenyl)ferrocene t-butylphenyl)ferrocene (k2 k 2 = 6.21) compared to the smaller 1-(iodoethynyl)-3-(4-methylphenyl) ferrocenes (k2 k 2 = 4.07) as 2.5% methanol was added to the n-hexane-based mobile phase. Significance: This study reasonably showed that the contribution of dispersion forces may explain the unusually large retention of the second eluted enantiomers observed for the enantioseparation of some planar chiral 1(iodoethynyl)-3-arylferrocenes with amylose-based selectors. Based on the obtained results, we can conclude that in liquid-phase enantioseparation steric repulsion can be turned into attraction depending on the features of analyte, selector, and mobile phase.

In this study, the enantioseparation of 14 planar chiral ferrocenes containing halogen atoms, and methyl, iodoethynyl, phenyl, and 2‐naphthyl groups, as substituents, was explored with a cellulose tris(4‐methylbenzoate) (CMB)‐based chiral column under multimodal elution conditions. n ‐Hexane/2‐propanol (2‐PrOH) 95:5 v/v, pure methanol (MeOH), and MeOH/water 90:10 v/v were used as mobile phases (MPs). With CMB, baseline enantioseparations were achieved for nine analytes with separation factors ( α ) ranging from 1.24 to 1.77, whereas only three analytes could be enantioseparated with 1.14 ≤ α ≤ 1.51 on a cellulose tris(3,5‐dimethylphenylcarbamate) (CDMPC)‐based column, used as a reference for comparison, under the same elution conditions. Pendant group–dependent reversal of the enantiomer elution order was observed in several cases by changing CMB to CDMPC. The impact of analyte and chiral stationary phase (CSP) structure, and MP polarity on the enantioseparation, was evaluated. The two cellulose‐based CSPs featured by different pendant groups were also compared in terms of thermodynamics. For this purpose, enthalpy (ΔΔ H °), entropy (ΔΔ S °) and free energy (ΔΔ G °) differences, isoenantioselective temperatures ( T iso ), and enthalpy/entropy ratios ( Q ), associated with the enantioseparations, were derived from van ’t Hoff plots by using n ‐hexane/2‐PrOH 95:5 v/v and methanol/water 90:10 v/v as MPs. With the aim to disclose the functions of the different substituents in mechanisms and noncovalent interactions underlying analyte–selector complex formation at molecular level, electrostatic potential ( V ) analysis and molecular dynamics simulations were used as computational techniques. On this basis, enantioseparations and related mechanisms were investigated by integrating theoretical and experimental data.

Background: Highly ordered chiral secondary structures as well as multiple (tunable) recognition sites are the keys to success of polysaccharide carbamate-based chiral selectors in enantioseparation science. Hydrogen bonds (HBs), dipole-dipole, and & pi;-& pi; interactions are classically considered the most frequent noncovalent interactions underlying enantioselective recognition with these chiral selectors. Very recently, halogen, chalcogen and & pi;-hole bonds were also identified as interactions working in polysaccharide carbamate-based selectors to promote enantiomer distinction. On the contrary, the function of dispersion interactions in this field was not explored so far.Results: The enantioseparation of chiral ferrocenes featuring chiral axis or chiral plane as stereogenic elements was performed by comparing five polysaccharide carbamate-based chiral columns, with the aim to identify enantioseparation outcomes that could be reasonably determined by dispersion forces, making available a reliable experimental data set for future theoretical studies to confirm the heuristic hypothesis. The effects of mobile phase polarity and temperature on the enantioseparation were considered, and potential recognition sites on analytes and selectors were evaluated by electrostatic potential (V) analysis and molecular dynamics (MD). In this first part, the enantioseparation of 3,3 & PRIME;-dibromo-5,5 & PRIME;-bis-ferrocenylethynyl-4,4 & PRIME;-bipyridine bearing two ferrocenylethynyl units linked to an axially chiral core was performed and compared to that of the analyte featuring the same structural motif with two phenyl groups in place of the ferrocenyl moieties. The results of this study showed the superiority of the ferrocenyl compared to the phenyl group, as a structural element favouring enantiodifferentiation.Significance and novelty: Even if dispersion (London) forces have been envisaged acting in liquid-phase enantioseparations, focused studies to explore possible contributions of dispersion forces with polysaccharide carbamate-based selectors are practically missing. This study allowed us to collect experimental information that support the involvement of dispersion forces as contributors to liquid-phase enantioseparation, paving the way to a new picture in this field.

Polymeric permselective films are frequently used for amperometric biosensors to prevent electroactive interference present in the target matrix. Phenylenediamines are the most commonly used for the deposition of shielding polymeric films against interfering species; however, even phenolic monomers have been utilized in the creation of these films for microsensors and biosensors. The purpose of this paper is to evaluate the performances of electrosynthesized polymers, layered by means of constant potential amperometry (CPA), of naturally occurring compound zingerone (ZING) and its dimer dehydrozingerone (ZING DIM), which was obtained by straight oxidative coupling reaction. The polymers showed interesting shielding characteristics against the main interfering species, such as ascorbic acid (AA): actually, polyZING exhibited an AA shielding aptitude comprised between 77.6 and 99.6%, comparable to that obtained with PPD. Moreover, a marked capability of increased monitoring of hydrogen peroxide (HP), when data were compared with bare metal results, was observed. In particular, polyZING showed increases ranging between 55.6 and 85.6%. In the present work, the molecular structures of the obtained polymers have been theorized and docking analyses were performed to understand their peculiar characteristics better. The structures were docked using the Lamarckian genetic algorithm (LGA). Glutamate biosensors based on those polymers were built, and their performances were compared with biosensors based on PPD, which is the most widespread polymer for the construction of amperometric biosensors.

In the last few decades, theoretical and technical advancements in computer facilities and computational techniques have made molecular modeling a useful tool in liquid-phase enantioseparation science for exploring enantioselective recognition mechanisms underlying enantioseparations and for identifying selector–analyte noncovalent interactions that contribute to binding and recognition. Because of the dynamic nature of the chromatographic process, molecular dynamics (MD) simulations are particularly versatile in the visualization of the three-dimensional structure of analytes and selectors and in the unravelling of mechanisms at molecular levels. In this context, MD was also used to explore enantioseparation processes promoted by amylose and cellulose-based selectors, the most popular chiral selectors for liquid-phase enantioselective chromatography. This review presents a systematic analysis of the literature published in this field, with the aim of providing the reader with a comprehensive picture about the state of the art and what is still missing for modeling cellulose benzoates and the phenylcarbamates of amylose and cellulose and related enantioseparations with MD. Furthermore, advancements and outlooks, as well as drawbacks and pitfalls still affecting the applicability of MD in this field, are also discussed. The importance of integrating theoretical and experimental approaches is highlighted as an essential strategy for profiling mechanisms and noncovalent interaction patterns.

The complex formation between daclatasvir and gamma-CD or heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) was studied by isothermal titration calorimetry and molecular modeling. Both techniques supported the predominant formation of a 2:1 complex in case of gamma-CD although a 1:1 complex may be formed to a much lower extent as well. In case of TM-beta-CD the stoichiometry of the complex was exclusively 1:1. Complex formation with gamma-CD did not require dissociation of the daclatasvir dimer, which is present in solution, and resulted in a complex with a binding constant of 1.67.107 M-2. In contrast, formation of the weak TM-beta-CD complex (K = 371 M-1) required dissociation of the daclatasvir dimer. This is in line with the observation that the complex formation in case of gamma-CD is enthalpy-driven, while the process is entropy-driven in case of TM-beta-CD. It is concluded that the plateau observed in capillary electrophoresis is primarily based on the slow dissociation of the daclatasvir-CD complexes caused by steric constrains due to the folded terminal amino acid moieties of daclatasvir exerting a clip effect. In case gamma-CD the thermodynamic stability might contribute to the overall slow dissociation.