Molecular mechanism of secreted amyloid-β precursor protein in binding and modulating GABABR1a

A recent phenomenal study discovered that the extension domain of secreted amyloid-β precursor protein (sAPP) can bind to the intrinsically disordered sushi 1 domain of the γ-aminobutyric acid type B receptor subunit 1a (GABA_BR1a) and modulate its synaptic transmission. The work provided an important structural foundation for the modulation of GABA_BR1a; however, the detailed molecular interaction mechanism, crucial for future drug design, remains elusive. Here, we further investigated the dynamical interactions between sAPP peptides and the natively unstructured sushi 1 domain using all-atom molecular dynamics simulations, for both the 17-residue sAPP peptide (APP 17-mer) and its minimally active 9 residue segment (APP 9-mer). We then explored mutations of the APP 9-mer with rigorous free energy perturbation (FEP) calculations. Our in silico mutagenesis studies revealed key residues (D4, W6, and W7) responsible for the binding with the sushi 1 domain. More importantly, one double mutation based on different vertebrate APP sequences from evolution exhibited a stronger binding (ΔΔG = −1.91 ± 0.66 kcal mol⁻¹), indicating a potentially enhanced GABA_BR1a modulator. These large-scale simulations may provide new insights into the binding mechanism between sAPP and the sushi 1 domain, which could open new avenues in the development of future GABA_BR1a-specific therapeutics.

A recent phenomenal study discovered that the extension domain of secreted amyloid-β precursor protein (sAPP) can bind to the intrinsically disordered sushi 1 domain of the γ-aminobutyric acid type B receptor subunit 1a (GABA_BR1a) and modulate its synaptic transmission.     

3D-visualization of amyloid-β oligomer interactions with lipid membranes by cryo-electron tomography

Amyloid-β (Aβ) assemblies have been shown to bind to lipid bilayers. This can disrupt membrane integrity and cause a loss of cellular homeostasis, that triggers a cascade of events leading to Alzheimer''s disease. However, molecular mechanisms of Aβ cytotoxicity and how the different assembly forms interact with the membrane remain enigmatic. Here we use cryo-electron tomography (cryoET) to obtain three-dimensional nano-scale images of various Aβ assembly types and their interaction with liposomes. Aβ oligomers and curvilinear protofibrils bind extensively to the lipid vesicles, inserting and carpeting the upper-leaflet of the bilayer. Aβ oligomers concentrate at the interface of vesicles and form a network of Aβ-linked liposomes, while crucially, monomeric and fibrillar Aβ have relatively little impact on the membrane. Changes to lipid membrane composition highlight a significant role for GM1-ganglioside in promoting Aβ-membrane interactions. The different effects of Aβ assembly forms observed align with the highlighted cytotoxicity reported for Aβ oligomers. The wide-scale incorporation of Aβ oligomers and curvilinear protofibrils into the lipid bilayer suggests a mechanism by which membrane integrity is lost.

Cryo-electron tomography 3D imaging of amyloid-β oligomers carpeting the surface of lipid bilayers in near native conditions.     

Modulation of αVβ6 integrin in osteoarthritis-related synovitis and the interaction with VTN(381–397 a.a.) competing for TGF-β1 activation

Osteoarthritis is characterized by structural alteration of joints. Fibrosis of the synovial tissue is often detected and considered one of the main causes of joint stiffness and pain. In our earlier proteomic study, increased levels of vitronectin (VTN) fragment (amino acids 381–397) were observed in the serum of osteoarthritis patients. In this work, the affinity of this fragment for integrins and its putative role in TGF-β1 activation were investigated. A competition study determined the interaction of VTN_{(381–397 a.a.)} with α_Vβ₆ integrin. Subsequently, the presence of α_Vβ₆ integrin was substantiated on primary human fibroblast-like synoviocytes (FLSs) by western blot and flow cytometry. By immunohistochemistry, β₆ was detected in synovial membranes, and its expression showed a correlation with tissue fibrosis. Moreover, β₆ expression was increased under TGF-β1 stimulation; hence, a TGF-β bioassay was applied. We observed that α_Vβ₆ could mediate TGF-β1 bioavailability and that VTN_{(381–397 a.a.)} could prevent TGF-β1 activation by interacting with α_Vβ₆ in human FLSs and increased α-SMA. Finally, we analyzed serum samples from healthy controls and patients with osteoarthritis and other rheumatic diseases by nano-LC/Chip MS–MS, confirming the increased expression of VTN_{(381–397 a.a.)} in osteoarthritis as well as in lupus erythematosus and systemic sclerosis. These findings corroborate our previous observations concerning the overexpression of VTN_{(381–397 a.a.)} in osteoarthritis but also in other rheumatic diseases. This fragment interacts with α_Vβ₆ integrin, a receptor whose expression is increased in FLSs from the osteoarthritic synovial membrane and that can mediate the activation of the TGF-β1 precursor in human FLSs.Subject terms: Osteoarthritis, Cell culture     

Cooperative B–H bond activation: dual site borane activation by redox active κ2-N,S-chelated complexes

Cooperative dual site activation of boranes by redox-active 1,3-N,S-chelated ruthenium species, mer-[PR₃{κ²-N,S-(L)}₂Ru{κ¹-S-(L)}], (mer-2a: R = Cy, mer-2b: R = Ph; L = NC₇H₄S₂), generated from the aerial oxidation of borate complexes, [PR₃{κ²-N,S-(L)}Ru{κ³-H,S,S′-BH₂(L)₂}] (trans–mer-1a: R = Cy, trans–mer-1b: R = Ph; L = NC₇H₄S₂), has been investigated. Utilizing the rich electronic behaviour of these 1,3-N,S-chelated ruthenium species, we have established that a combination of redox-active ligands and metal–ligand cooperativity has a big influence on the multisite borane activation. For example, treatment of mer-2a–b with BH₃·THF led to the isolation of fac-[PR₃Ru{κ³-H,S,S′-(NH₂BSBH₂N)(S₂C₇H₄)₂}] (fac-3a: R = Cy and fac-3b: R = Ph) that captured boranes at both sites of the κ²-N,S-chelated ruthenacycles. The core structure of fac-3a and fac-3b consists of two five-membered ruthenacycles [RuBNCS] which are fused by one butterfly moiety [RuB₂S]. Analogous fac-3c, [PPh₃Ru{κ³-H,S,S′-(NH₂BSBH₂N)(SC₅H₄)₂}], can also be synthesized from the reaction of BH₃·THF with [PPh₃{κ²-N,S-(SNC₅H₄)}{κ³-H,S,S′-BH₂(SNH₄C₅)₂}Ru], cis–fac-1c. In stark contrast, when mer-2b was treated with BH₂Mes (Mes = 2,4,6-trimethyl phenyl) it led to the formation of trans- and cis-bis(dihydroborate) complexes [{κ³-S,H,H-(NH₂BMes)Ru(S₂C₇H₄)}₂], (trans-4 and cis-4). Both the complexes have two five-membered [Ru–(H)₂–B–NCS] ruthenacycles with κ²-H–H coordination modes. Density functional theory (DFT) calculations suggest that the activation of boranes across the dual Ru–N site is more facile than the Ru–S one.

Redox-active ruthenium complexes supported by hemilabile κ²-N,S-chelated ruthenacycles undergo unusual dual site B–H bond activation through metal–ligand cooperation with free and bulky boranes.     

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aβo–PrPC binding and downstream pathways

Amyloid β oligomers (Aβo) are the main toxic species in Alzheimer''s disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aβo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aβo binding by a combined ¹⁹F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aβo binding to its receptor PrP^C in HEK293 cells. They reduced the pFyn levels triggered by Aβo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer''s disease pathology, i.e. prion, Aβ and Tau, affecting three different pathways through specific binding to Aβo and are, indeed, promising candidates for further development.

A new approach combining virtual screening, ¹⁹F and STD NMR, and biochemical assays using hiPSC and targetting multiple pathways involving Aβ, PrP^C and Tau provides a more effective strategy for Alzheimer''s disease drug discovery than Aβ only approach.     

Thermodynamics and kinetics of the amyloid-β peptide revealed by Markov state models based on MD data in agreement with experiment

The amlyoid-β peptide (Aβ) is closely linked to the development of Alzheimer''s disease. Molecular dynamics (MD) simulations have become an indispensable tool for studying the behavior of this peptide at the atomistic level. General key aspects of MD simulations are the force field used for modeling the peptide and its environment, which is important for accurate modeling of the system of interest, and the length of the simulations, which determines whether or not equilibrium is reached. In this study we address these points by analyzing 30-μs MD simulations acquired for Aβ40 using seven different force fields. We assess the convergence of these simulations based on the convergence of various structural properties and of NMR and fluorescence spectroscopic observables. Moreover, we calculate Markov state models for the different MD simulations, which provide an unprecedented view of the thermodynamics and kinetics of the amyloid-β peptide. This further allows us to provide answers for pertinent questions, like: which force fields are suitable for modeling Aβ? (a99SB-UCB and a99SB-ILDN/TIP4P-D); what does Aβ peptide really look like? (mostly extended and disordered) and; how long does it take MD simulations of Aβ to attain equilibrium? (at least 20–30 μs). We believe the analyses presented in this study will provide a useful reference guide for important questions relating to the structure and dynamics of Aβ in particular, and by extension other similar disordered proteins.

The convergence of MD simulations is tested using varying measures for the intrinsically disordered amyloid-β peptide (Aβ). Markov state models show that 20–30 μs of MD is needed to reliably reproduce the thermodynamics and kinetics of Aβ.     

Determination of α‐hydroxy acids and their enantiomers in fruit juices by ligand exchange CE with a dual central metal ion system

The content of α‐hydroxy acids and their enantiomers can be used to distinguish authentic and adulterated fruit juices. Here, we investigated the use of ligand exchange CE with two kinds of central metal ion in a BGE for the simultaneous determination of enantiomers of dl ‐malic, dl ‐tartaric and dl ‐isocitric acids, and citric acid. Ligand exchange CE with 100 mM d ‐quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl ‐tartaric acid but not dl ‐malic acid or dl ‐isocitric acid. Addition of 1.8 mM Sc(III) ion to the BGE with 10 mM Cu(II) ion to create a dual central metal ion system permitted the simultaneous determination of these α‐hydroxy acid enantiomers and citric acid. The proposed ligand exchange CE was thus well suited for detecting adulteration of fruit juices.     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Amphiphilic stilbene derivatives attenuate the neurotoxicity of soluble Aβ42 oligomers by controlling their interactions with cell membranes

The misfolded proteins or polypeptides commonly observed in neurodegenerative diseases, including Alzheimer''s disease (AD), are promising drug targets for developing therapeutic agents. To target the amyloid-β (Aβ) peptide plaques and oligomers, the hallmarks of AD, we have developed twelve amphiphilic small molecules with different hydrophobic and hydrophilic fragments. In vitro fluorescence binding assays demonstrate that these amphiphilic compounds show high binding affinity to both Aβ plaques and oligomers, and six of them exhibit selective binding toward Aβ oligomers. These amphiphilic compounds can also label the Aβ species in the brain sections of transgenic AD mice, as shown by immunostaining with an Aβ antibody. Molecular docking studies were performed to obtain structure–affinity relationships. To our delight, four amphiphilic compounds can alleviate the Cu²⁺–Aβ induced toxicity in cell viability assays. In addition, confocal fluorescence imaging studies provide evidence that two compounds, ZY-15-MT and ZY-15-OMe, can disrupt the interactions between Aβ oligomers and human neuroblastoma SH-SY5Y cell membranes. Overall, these studies strongly suggest that developing compounds with amphiphilic properties that target Aβ oligomers and modulate the Aβ oligomer–cell membrane interactions can be an effective strategy for the development of small molecule AD therapeutics.

Amphiphilic compounds with selectivity towards soluble Aβ₄₂ oligomers were developed. Cell imaging studies show the compounds can reduce the interactions between Aβ₄₂ oligomers and SH-SY5Y cell membranes, both in the presence and absence of Cu.     

Enantioseparation of α‐hydroxy acids by chiral ligand exchange CE with a dual central metal ion system

Using two kinds of central metal ions in a background electrolyte, ligand exchange CE was investigated for the simultaneous enantioseparation of dl ‐malic, dl ‐tartaric, and dl ‐isocitric acids. Ligand exchange CE with 100 mM d ‐quinic acid as a chiral selector ligand and 10 mM Cu(II) ion as a central metal ion could enantioseparate dl ‐tartaric acid but not dl ‐malic acid or dl ‐isocitric acid. A dual central metal ion system containing 0.5 mM Al(III) ion in addition to 10 mM Cu(II) ion in the background electrolyte enabled the simultaneous enantioseparation of the three α‐hydroxy acids. These results suggest that the use of a dual central metal ion system can be useful for enantioseparation by ligand exchange CE.     

Estimation of apparent binding constant of complexes of selected acyclic nucleoside phosphonates with β‐cyclodextrin by affinity capillary electrophoresis

Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)‐enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β‐cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)‐enantiomers of ANPs‐based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0–25 mM) of βCD. The apparent binding constants of the complexes of (R,S)‐enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)‐enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of (R,S)‐enantiomers of ANPs with βCD have been found to be relatively weak – their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3–46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3–55.2 L/mol.     

A simple route to syn α-Amino-β-Hydroxy esters by C-2 regioselective opening of a, β-Epoxy esters with metal halides

α,β-Epoxy esters are opened by NaX (X = I, Br) in a regio and stereoselective fashion to β-hydroxy-α-halo esters, which represent suitable precursors of syn α-amino-β-hydroxy esters and β-hydroxy esters.     

Mass spectrometric investigations of α‐ and β‐cyclodextrin complexes with ortho‐, meta‐ and para‐coumaric acids by negative mode electrospray ionization

The mass spectrometric characterization of aqueous solutions of α‐ and β‐cyclodextrins (CDs) and o‐, m‐ and p‐coumaric acids (CAs) by negative ion electrospray ionization (ESI) indicates that the [CD+CA]^? ions were sourced from the inclusion complex present in solution and from the anion attached to CD molecules formed in the spray processes. The anion adducts formed in the spray process contribute significantly to the signal intensity of an ionized inclusion complex thus overestimating the calculated stability constant (K) of solution‐phase complexes by one to two orders of magnitude. The relative intensities of anion adducts in mass spectra depend on the concentration ratio of the anion and the CD in spray droplets, while the relative intensity of the ionized inclusion complex depends on CD and CA concentrations in solutions and the value of K. Ion Mobility Spectrometry Mass Spectrometry [IMS‐MS] measurements show that the collision cross‐section (Ω) values of the [CD+CA]^? or [(CD)₂₊CA]^2? and [CD+CA] complex ions are 5–6% larger than or equal to CD^? or [CD], respectively. Therefore, in the gas phase the anion adducts [CD+CA^?] on cyclodextrin molecules possess the same conformations as the ionized inclusion complexes [CD+CA]^?. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of lactide/ɛ‐caprolactone quasi‐random copolymer by using rationally designed mononuclear aluminum complexes with modified β‐ketiminato ligand

A series of novel aluminum complexes containing bulky aryl‐β‐ketiminato ligands [ArNCH C₁₀H₇C₆H₅O]Al(CH₃)₂ ( 3a , Ar = C₆F₅; 3b , Ar = C₆H₅; 3c , Ar = 2,6‐ⁱPr₂C₆H₃) have been synthesized in high yields. These complexes were identified by ¹H and ¹³C NMR spectroscopy, elemental analysis, and X‐ray structural analysis. All the aluminum complexes could efficiently catalyze the ROP of ɛ‐caprolactone (ɛ‐CL) and Lactide (LA) in a controlled manner. It was found that the steric effect of the ligand has less effect on the ROP of CL, while the polymerization rate of L‐LA was suppressed significantly. More interestingly, this kind of catalysts can promote the random copolymerization of ɛ‐CL and L‐LA. The transesterification side reaction and the polymer composition could be adjusted by modulating the electronic and steric effects of the ligand. In paticular, compound 3c could produce quasi‐random copolymers without transesterification side reactions, as indicated by both the values of the reactivity ratios of the two monomers (r_LA = 1.31; r_CL = 0.99) and the similar average lengths of the caproyl and lactidyl sequences (L_CL = 2.34; L_LA = 2.44). Finally, a drug‐random copolymer conjugates could be easily prepared by using 3c , indicating a potential application of 3c in pharmacutical and biomedical field. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 203–212     

Synthesis of glucosidic derivatives with a spacer arm by reverse hydrolysis using almond β-D-glucosidase

Glucosidase-catalysed synthesis of glucosides with a spacer arm on the anomeric carbon is reported. By using the acceptor as solvent at an elevated temperature, much higher yields of product have been obtained than previously observed.     

Study of isomeric pentacyclic triterpene acids in traditional Chinese medicine of Forsythiae Fructus and their binding constants with β‐cyclodextrin by capillary electrophoresis

In this study, a capillary zone electrophoresis (CZE) method was first developed to identify three microconstituents of isomeric pentacyclic triterpene acids (PTAs including oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA)) in Forsythiae Fructus (FF). The baseline separation of PTAs by CZE were eventually achieved in a background electrolyte (BGE) containing 50.0 mmol/L borax and 0.5 mmol/L β‐cyclodextrin (β‐CD) at pH 9.5 within 13.0 min. Herein, it was not only the compositions of BGE were detail investigated for rapid and good separation, but also the binding ratio and the equilibrium constants (K) for OA, UA and BA with β‐CD was estimated by double reciprocal equation to well understand the separation mechanism. The proposed method allowed the LODs of PTAs were averaged at 1.50 μg/mL with UV detection (at 200 nm). The interday RSD of migration time and peak area were around 2.0 and 4.7% (n = 5), respectively. Thus, the content of PTAs in 19 FF real samples distinguished from maturation stages and geographical areas in China was quantified with the proposed method. Depending on the amount of each PTA in FF, it was demonstrated these microconstituents might benefit to identify their harvested time even qualities.     

Determination of the binding constants of modafinil enantiomers with sulfated β‐cyclodextrin chiral selector by capillary electrophoresis using three different linear plotting methods

Binding constants for the enantiomers of modafinil with the negatively charged chiral selector sulfated‐β‐CD (S‐β‐CD) using CE technique is presented. The calculations of the binding constants employing three different linearization plots (double reciprocal, X‐reciprocal and Y‐reciprocal) were performed from the electrophoretic mobility values of modafinil enantiomers at different concentrations of S‐β‐CD in the BGE. The highest inclusion affinity of the modafinil enantiomers were observed for the S‐enantiomer–S‐β‐CD complex, in agreement with the computational calculations performed previously. Binding constants for each enantiomer–S‐β‐CD complex at different temperatures, as well as thermodynamic parameters for binding, were calculated. Host–guest binding constants using the double reciprocal fit showed better linearity (r²>0.99) at all temperatures studied (15–30°C) and compared with the other two fit methods. The linear van't Hoff (15–30°C) plot obtained indicated that the thermodynamic parameters of complexation were temperature dependent for the enantiomers.     

(±)‐2‐Oxocyclohexanepropionic acid: dual conformational selection and hydrogen bonding in a δ‐keto acid,and a two‐phase technique for crystallizing acids by contact with aqueous solutions of their salts

The asymmetric unit of the title compound, C₉H₁₄O₃, consists of two mol­ecules having conformations that differ by 121.7 (4)° in their rotation about the equatorial substituent bond, so that the side chain extends away from the ring in different directions in the two species. The hydrogen‐bonding mode is acid‐to‐acid dimerization. However, despite the centrosymmetric space group (P), the dimers are asymmetric, formed by pairing mol­ecules of identical chirality but differing conformational type [O⋯O = 2.681 (2) and 2.654 (2) Å, and O—H⋯O = 175 (3) and 176 (3)°]. Two intermolecular C—H⋯O=C close contacts exist, involving the ketone group of one of the mol­ecules. A two‐phase technique is described for slow reforming of crystals of a water‐insoluble acid by contact with an aqueous solution of its water‐soluble salt.     

Enantioseparation of nicotine alkaloids in cigarettes by CE using sulfated β‐CD as a chiral selector and a capillary coated with amino groups

Nicotine (NC) and its related compounds (cotinine (CN), nornicotine (NN), anatabine (AT) and anabasine (AB)) were simultaneously enantioseparated by CE using a capillary with amino groups and sulfated β‐CD as a chiral selector. The optimum running conditions were found to be 30 mM acetate buffer (pH 5.0) containing 8% sulfated β‐CD with an applied voltage of +15 kV at 30°C using direct detection at 260 nm. Using a capillary coated with amino groups, the EOF migrates toward the positive pole. However, when sulfated β‐CD was added to the BGE, it was found that the EOF migrated toward the negative pole due to ionic adsorption of sulfated β‐CD to amino groups on the capillary inner wall. All the cationic analytes migrated as anions, suggesting that they formed stable anionic complexes with sulfated β‐CD. With this system and a simple pretreatment with mini‐cartridges, NC alkaloids in five cigarette samples were enantioseparated. As a result, each of the compounds except for CN was detected. In the case of NC, only (S)‐NC was detected (more than 99.9%), but in the case of NN, AT and AB, the ratios of (S)‐isomer to total isomers were in the ranges 58–70, 81–85 and 59–65%, respectively. On the other hand, only NC was detected in cigarette smoke and the ratio of (S)‐ and (R)‐NCs was 96:4. The amounts of NC alkaloids in cigarettes suggest that the production of (R)‐NC resulted from racemization due to the high temperature/burning of the cigarette.     

Nuclear magnetic resonance to study the interactions acting in the enantiomeric separation of homocysteine by capillary electrophoresis with a dual system of γ‐cyclodextrin and the chiral ionic liquid EtCholNTf2

The enantiomeric separation of 9‐fluorenylmethoxycarbonyl chloride (FMOC)‐homocysteine (Hcy) by CE was investigated using γ‐CD and the chiral ionic liquid (R)‐(1‐hydroxybutan‐2‐yl)(trimethyl)azanium‐bis(trifluoromethanesulfon)imidate (also called (R)‐N,N,N‐trimethyl‐2‐aminobutanol‐bis(trifluoromethane‐sulfon)imidate) (EtCholNTf₂) as chiral selectors. Using 2 mM γ‐CD and 5 mM EtCholNTf₂ in 50 mM borate buffer (pH 9), FMOC‐Hcy enantiomers were separated with a resolution value of 3.8. A reversal in the enantiomer migration order in comparison with the single use of γ‐CD in the separation buffer was obtained. Then, NMR experiments were carried out to elucidate the interactions taking place in the enantiomeric separation of FMOC‐Hcy. NMR analyses highlighted the formation of an inclusion complex since the hydrophobic group of FMOC‐Hcy was inserted into the γ‐CD cavity. Moreover, interactions between EtCholNTf₂ and γ‐CD were also observed, suggesting that the chiral ionic liquid would also enter the cavity of the γ‐CD.