Thiolated Chiral Naphthalene Diimide Derivatives as Effective Selectors of the β‐Blocker Atenolol Enantiomers

Atenolol β‐blocker drug (ATN) to treat hypertension and cardiovascular disorders such as angina pectoris is manufactured commercially in a racemic form, while only its (S)‐enantiomer shows selective β1‐blocking activity. Here, a new axially chiral thiolated naphthalene diimide derivatives (HS‐BIN‐NDI) for the rapid, simple, and sensitive detection of ATN enantiomers is presented. The Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM) experiments show that interactions between a chiral selector and a chiral analyte occur only when their configurations are opposite. The extensive computational simulations support this finding. The association coefficient determined by SPR is high (105‐106 M−1 s−1) and indicates that the interactions are electrostatic. The analytical utility of atropisomeric HS‐BIN‐NDIs is proven, and the voltammetric sensors designed to determine ATN enantiomers are constructed. The developed sensors are characterized by a wide analytical operating range from 5.0·10−3 to 1.0 µm, nanomolar detection limit, high selectivity, and a long lifetime. Several additives like urea, creatinine, glucose, human albumin, and hemoglobin do not affect ATN sensing.

Examples of drugs in clinical use are atenolol, betaxolol, esmolol, metoprolol, nipradilol, oxprenolol, pindolol, and propranolol.Atenolol belongs to the second generation of -blockers.adrenergic receptor-blocking agents (-blockers), competitive antagonists of beta-adrenergic receptors, vary for their selectivity profiles versus −1 receptors expressed mainly in the heart and −2 ones expressed predominantly in different types of smooth muscles (in vessels, skeletal muscles, bronchi, uterus) where they mediate smooth muscle relaxation but in four-times lesser amounts than −1 are also present in the heart. [8]12][13] However, the (S) enantiomer lacks blocking effects on vascular −2 receptors. [13]Atenolol exhibits an increased concentration of the less active (R)-enantiomer due to stereospecific excretion of the (S)-isomer in urine. [14]The needless administration of the non-beta-blocking (R)-enantiomer that makes up 50% of racemate puts the patient at an increased risk of side effects, drug interactions, and loss of cardioselectivity with up-titration of dosing.Therefore, the less interaction potential of (S)-enantiomers compared to (R)-enantiomers further makes it a sensible choice in patients taking CYP2D6 inhibitors or in patients with heart failure or hepatic insufficiency.
Recently, racemic atenolol and propranolol have been used for problematic infantile hemangiomas (IHs), the most common benign vascular endothelial neoplasms, seen more frequently in female infants, in premature infants with a birth weight of less than 1500 g, and twins. [15]They usually appear a few weeks after birth, undergo regression by age four, and do not require any treatment.Still, about one-tenth of cases need to be treated.Unexpectedly, the (R)-enantiomers of atenolol and propranolol were found to inhibit the hemangioma stem cells (HemSCs) vessel formation in vivo.This could be due to lowering cAMP levels and activating the mitogen-activated protein kinase (MAPK) pathway downstream of beta-adrenergic receptors. [16]In vitro, both racemic and (R)atenolol inhibited HemSC to endothelial cell differentiation. [17]s a result, it was suggested that the (R)-enantiomers of atenolol and propranolol could be repurposed to increase the efficiency and lower adverse side effects of the IH treatment. [17]Widespread use of atenolol and possible new applications of single (R)-forms of these potent drugs to treat off-label diseases like problematic infantile hemangiomas, prompted us to optimize our enantioselective sensor to detect both atenolol enantiomers selectively.
Chiral analysis is a constantly growing field of modern chemical analysis, mainly because of its importance, especially in clinical and pharmaceutical applications, biochemistry, and, in recent years, environmental chemistry.[20][21][22][23] A desirable approach to determining individual enantiomers of a given compound is using various types of sensors.The basis of chiral sensors is the so-called chiral selectors, whose role is to selectively interact with a given enantiomer, expressed as the difference in the value of the measured signal.[26] Voltammetric detection of chiral compounds can be accomplished based on the current signal changes of the chiral selector or the chiral analyte.The former approach is used more widely because of its simplicity and high sensitivity.However, a change in the current signal of a chi-ral selector can be triggered by any substance forming a complex with the selector.Sensing based on changes in the current signal of the chiral analyte to be detected provides a considerably more selective approach.The physicochemical properties of atropisomeric naphthalenediimide derivatives make them ideal as chiral selectors for sensitively detecting and determining chiral electroactive analytes.In the present study, we designed simple in-construction (chemisorption process), sensitive, and cheap voltammetric protocols for screening the enantiomers of atenolol in plasma.The recognition process of the ATN stereosiomers is based on the unique properties of axially chiral thiolated derivatives of naphthalene diimide (HS-BIN-NDI) used as a chiral selector.The high affinity of the HS-BIN-NDI selectors to the ATN enantiomers in opposite configurations versus the used selector was proved by applying of square wave voltammetry, surface plasmon resonance, and atomic force microscopy.To our knowledge, for the first time, the direct voltammetric detection of ATN in the appropriate configuration without any amplifiers at the level 300 times lower than the ATN concentration in serum required for producing beta-blockade (0.75-1.88 μm).

Synthesis of HS-BIN-NDI
Both enantiomers of the chiral selector HS-BIN-NDI were prepared in 5 steps (Scheme 1A, see Supporting Information for details).Atropisomeric derivative 1 was obtained by mono-mesylation of commercially available enantiomers of 2,2′diamino-1,1′-binaphthalene (BINAM).N-allyl derivative of naphthaleneimide 2 was prepared by the addition of allylamine to naphthalene dianhydride using microwave irradiation.Monoanhydride 2 was then reacted with N-mesyl BINAM 1 to give naphthalene diimide 3. The thiol group was introduced to the allyl side chain by the radical addition of thioacetic acid and the removal of acetyl by transesterification.
Both enantiomers of metoprolol acid (MA; Scheme 1B) were obtained by hydrolysis of atenolol with hydrochloric acid and desalted on the RPC18 column.

Computations and Modeling
Theoretical considerations consisted of three stages (for a detailed description, see Supplementary Information file): (i) generation of tens of thousands of configurations of the (S)-BIN-NDI complexes with (R)-and (S)-ATN using molecular mechanics and Scheme 1. Scheme of synthesis of chiral selector HS-BIN-NDI (A).Chemical structure of atenolol and metoprolol acid (B).molecular docking routines, [27][28][29][30] (ii) selection of a few hundred structures and quantum chemical reoptimization at the B3LYP/6-31G ** level [31][32][33] (≈1200 basis functions) combined with the Polarizable Continuum Model (PCM) of the water solvent [34] simulating the PBS environment; (iii) selection of a dozen structures for reoptimization with a much larger def2TZVP basis set [35] (≈2300 basis functions) and estimation of the BIN-NDI-ATN counterpoise (CP) corrected interaction energies [36] for the (S)-(R) and (S)-(S) pairs at the B3LYP/def2TZVP level (PCM is not available for CP calculations).All calculations were performed using Gaussian 16 software. [37]Notice that ATN was modeled with the amine protonated, as expected at pH 7.4. [38]At such experimental conditions, the sulfonamide group in BIN-NDI is in its neutral form.

Surface Plasmon Resonance
SPR measurements were performed using a Biacore X100 system (GE Healthcare) from Cytiva (Uppsala, Sweden).Gold sensor chips were cleaned in a mixture of deionized water, 25% ammonia, and 30% hydrogen peroxide in a volume ratio of 5:1:1.The solution was heated to 75°C, and gold sensor chips were immersed in it for 5 min.The sensors were then rinsed first with deionized water, then with 99.8% ethanol, and finally dried with an argon stream.Measurements were carried out in the flow system using 0.01 m PBS buffer (pH 7.4) as a running buffer.Because the components of the Biacore X100 system are not resistant to the majority of organic solvents, the surface of the gold sensor chip was modified with a chiral HS-BIN-NDI selector monolayer (1 mm in DMSO), and the sealing thiol layer (1 mm MCH in EtOH) outside the Biacore X100 system.

Square Wave Voltammetry
Square wave voltammetric (SWV) measurements were performed using an Autolab PGSTAT 12 potentiostat with Eco-Chemie software.The experiments were carried out in a threeelectrode system consisting of (1) a working electrode, which was a gold electrode (ϕ = 1.6 mm), (2) a reference electrode (Ag/AgCl/3 m KCl), and (3) an auxiliary electrode was a platinum plate with an area of ≥ 1 cm 2 .Each time before sensor construction, the surface of the gold electrode was first mechanically cleaned on the wet polishing pad with the addition of aluminum oxide (grain diameter of 1 mm).Residual Al 2 O 3 was removed from the electrode surface with deionized water (Hydrolab, conductivity of ≈0.056 μS×cm −1 ).Then, the gold electrode was cycled in 0.1 m H 2 SO 4 in the potential range of −0.3 -1.5 -−0.3 V versus Ag/AgCl/3 m KCl (v = 50 mV s −1 ) until stable voltammetric curve typical for bare gold electrode was obtained.

Atomic Force Microscopy
AFM-based adhesion measurements were performed with chemically modified tips.Gold-coated NPG probes (Bruker) were gently rinsed with CH 2 Cl 2 /MeOH (1:1 v/v) and then placed in a Petri dish filled with a solution of 11-amino-1-undecanethiol in the same solvent.After ≈1.5 h, the probes were again rinsed with CH 2 Cl 2 /MeOH (1:1 v/v) and left to dry.In the next step, the premodified probes were placed in an aqueous solution containing EDC, NHS, and (S)-or (R)-metoprolol acid (i.e., atenolol with amide moiety replaced by carboxylic group) in 3 mL of water.The coupling reaction was carried out for at least 6 h to complete the attachment of metoprolol acid to free amine groups of tip-bound 11-amino-1-undecanethiol.Further, AFM probes were gently rinsed with water and dried.Finally, they were used to obtain maps of adhesion forces acting between the metoprolol acidmodified tip and the monolayers of (R)-HS-BIN-NDI or (S)-HS-BIN-NDI deposited on the gold surface.Each AFM probe was calibrated using the thermal tune method to obtain the exact value of the spring constant.The curvature radius of the probes was evaluated using tip characterizer samples TGT1 (NT-MDT) and TC1 (BudgetSensors).The adhesion data were collected in Peak Force QNM mode, which can map the nanomechanical properties of the sample.In this mode, the cantilever is modulated along the Z-axis at a certain frequency (usually ≈2 kHz) and default amplitude.At each cycle, the force-distance curve was recorded, and the analysis of the retract curves allows the determination of the adhesion force.

Sensors Design
Introducing a chiral selector on the gold surface was very simple and based on a chemisorption process.To this end, a droplet (voltammetric measurements: 7 μL; AFM and SPR measurements: 100 μL) of a 1 mm solution of a chiral thiolated naphthalene diimide derivative ((R)-HS-BIN-NDI or (S)-HS-BIN-NDI) in DMSO was placed on the gold surface and left under cover overnight.Then, to seal the selector layer and protect the gold surface from non-specific interaction with the analyte (atenolol), the MCH thiol (1 mm EtOH solution, t = 1 h) was used.The sensor prepared in this way was exposed to the analyte solution.

SPR Analysis
The study of selector-analyte interactions in real-time without a labeling step is possible using surface plasmon resonance.The SPR technique provides information on (i) the amount of the analyte in the analyzed sample, (ii) the specificity and selectivity of the formed complex, and (iii) the strength of the interaction of complex components.The binding kinetics can be determined using a technique that provides information about realtime binding, both in the association and dissociation phases.These data provide detailed insight into the strength of binding and stability of the interaction, which is critical for many applications.Sensograms recorded during the interaction of the HS-BIN-NDI selector in configuration (R) or (S) with atenolol also in configuration (R) or (S) in the concentration range from 6.25 to 100 μm are shown in Figure 1.
A significant increase in SPR signal in time, exponential in nature, was observed only when the HS-BIN-NDI selector interacted with the ATN in the opposite configurations.The interaction was negligible when the selector and analyte were in the same configurations.The binding curves recorded for each sample were fitted mathematically with the appropriate interaction model to determine the values of the association (k a ) and dissociation (k d ) rate constants, as well as the values of the equilibrium constants of the dissociation (K d ) and association (K a ) steps determining the strength of the analyte affinity for the appropriate selector.In this case, the 1:1 binding model (one molecule of analyte interacts with only one molecule of the selector) was the best.The obtained values of k a , k d , K d , and K a are shown in Table 1.The interactions between the chiral selector and analyte in the opposite chiral configuration are characterized by a high association rate and relatively low values of dissociation rate.The association rates in the range: 10 5 -10 6 m −1 s −1 indicate the electrostatic character of the interactions. [39,40]Moreover, the high values of the equilibrium constants of the association step (of the order of 10 7 ) and low for the dissociation step (of the order of 10 −8 ) confirm the stability of the formed complexes of the types (R)-HS-BIN-NDI-(S)-ATN and (S)-HS-BIN-NDI-(R)-ATN.

AFM Analysis
The strength of a chiral analyte (ATN) interaction with a chiral selector (HS-BIN-NDI) can also be studied using atomic force microscopy.To anchor atenolol onto the AFM tip surface in a controlled and stable manner, the amide group in the atenolol molecule was converted to a carboxyl group through hydrolysis.The product of the ATN hydrolysis process is known as a metoprolol acid (MA).This modification of the ATN structure did not affect the structure of the stereogenic center.Linker (11-amino-1undecanethiol) was acylated with MA to give again ATN as a secondary amide.The scheme illustrating the concept of the AFMbased adhesion measurements is demonstrated in Figure 2A.It utilizes force spectroscopy methodology, where the forces acting between the tip and the sample are probed as a function of tipsample distance at the nanoscale.Upon physical contact between the tip and the sample, the retraction curves enable an analysis of the adhesion forces affected by the chemical nature and the strength of interactions between tip-bound and surface-bound species.Such an approach is broadly used to quantify adhesion forces between AFM probes and substrates chemically modified with self-assembled monolayers that terminate in distinct functional groups. [41,42]he same methodology was used for direct measurements of the chemical force between chiral molecules. [43]In our case, the AFM tip is chemically modified with 11-amino-1-undecanethiol to which MA molecules are coupled.Hence, the main contribution to the measured forces will come from the interaction be-tween the monolayers of HS-BIN-NDI deposited on gold substrates and MA pendants attached to the AFM tip.Figures 2B-E show histograms illustrating the distribution of the measured adhesion forces between the chemically modified AFM probes bearing MA and the monolayers of chiral selectors immobilized on the gold surface.In all cases, adhesion forces at the nanonewton level can be observed, which proves the interaction between the AFM tip and the sample.However, depending on the chirality of the sampled MA-selector pairs, the forces are significantly different.For pairs of enantiomers with the Table 1.Kinetic parameters obtained from SPR data for the interaction of (S)-and (R)-atenolol with HS-BIN-NDI selector using a 1:1 binding model.

Computational Analysis
For the HS-BIN-NDI-ATN systems, we have no direct structural indications about how the molecules interact with each other or even conformation of HS-BIN-NDI in the solid state.Unlike for NDI functionalized at the N-atoms with the chiral sulfonamide NHSO 2 R moiety (R stands for alkyl), [44] and for atropisomeric NDIs decorated with ─OH, ─OCH 3 , ─NHCH 3 , or ─NO 2 groups, [45] we have not been able to crystallize any atropisomeric derivative functionalized with the chiral sulfonamide group.In such a case, computational modeling of the HS-BIN-NDI-ATN interactions could provide trustable structural indications about the system if the flexibility of the partner molecules can be efficiently analyzed.The two interacting partners are flexible, yet, the BIN-NDI fragment is relatively rigid, and the alkyl group ended with HS plays no role in the HS-BIN-NDI-ATN interactions.Therefore, in HS-BIN-NDI interactions, the conformations of the -NHSO 2 CH 3 moiety play a major role, and for ATN interactions, the conformations of the protonated oxypropanolamine moiety (with the ─NH 2 + , ─OH, and ─O─ proton donor and proton acceptor groups) are primarily relevant.The immense flexibility of the ATN molecule combined with eleven conformers of the (S)-BIN-NDI receptor (Figure 3) caused molecular-mechanics-based docking routines to yield over 220 000 complexes for each ATN enantiomer with the receptor.After removing the redundant structures (RMSD < 0.8 Å), over 350 and over 150 top-scored complexes with (R)-and (S)-ATN, respectively, were reoptimized at the B3LYP/PCM/6-31G ** level assuming a continuous model of the water environment.Modeling of the water medium is necessary to avoid unrealistic folding of the ATN molecule without the presence of the polar solvent surrounding it.23 (R)-and 20 (S)-ATN complexes were recalculated at the higher B3LYP/PCM/def2TZVP level, and the energetic order and interaction energies were estimated and compared with those obtained with the smaller 6-31G ** basis set (Table S1, Supporting Information).We found that there is no significant difference in total energies of the (S)-BIN-NDI-(S)-ATN and (S)-BIN-NDI-(R)-ATN systems (Table S1, Supporting Information).This is because the total energy obtained with the B3LYP method is biased by an error of at least 2.1 kcal mol −1 . [46]Moreover, the total energy obtained in a solvent is subject to an unknown error produced by the PCM cage of the shape varying from complex to complex.Additionally, increasing the basis set qualitatively conserves the complex geometry.However, it modifies the energetic order of the studied complexes, and an entirely unambiguous conclusion cannot be drawn (Table S1, Supporting Information).This unexpected outcome could suggest that based on oneto-one molecule systems, one cannot correctly model the ATN enantiomers' interaction with the (S)-HS-BIN-NDI monolayer at the Au surface.However, it could also imply that selectivity towards the (R)-ATN enantiomer can result from the specificity of the organization of the very (S)-HS-BIN-NDI monolayer on the surface.Still, the interaction energies estimated using both bases sets unequivocally indicate that the complex partners in the most stable (S)-BIN-NDI-(R)-ATN systems interact significantly stronger than those in the most stable (S)-BIN-NDI-(S)-ATN ones (Table S1; Figure S1, Supporting Information).This means that the (R)-ATN molecule in its most stable complex is much more firmly held by the (S)-HS-BIN-NDI monolayer than the (S)-ATN one, which makes the latter easier to dissociate from the surface.

Out of a plethora of intermolecular interactions between the (S)-BIN-NDI-(R)-ATN and (S)-BIN-NDI-(S)-ATN systems,
usually, the most stable are those with the highest number of intermolecular hydrogen bonds.There are two S─O and one C═O electron-donating moiety and one N─H proton-donating moiety in the primary interaction center of BIN-NDI and NH 2 + and OH proton-donor groups in the ATN molecule.Notice that the electron-donating moieties provide two electron pairs with different orientations.The extensive calculations revealed that the most stable (S)-BIN-NDI-(R)-ATN complex also exhibits the strongest interaction between the complex partners (E int = −35.82kcal mol −1 , Figure 4A).It is stabilized by three intermolecular and one intramolecular hydrogen bond (Figure 4A).The most stable (S)-BIN-NDI-(S)-ATN complex is stabilized by two intermolecular and two intramolecular hydrogen bonds: one in the (S)-BIN-NDI and one in (S)-ATN molecule (E int = −32.67kcal mol −1 , Figure 4B).However, it must not be forgotten that for complex stability, the optimal molecule conformations and mutual orientations, and enantiomer configurations are fundamental for obtaining the highest interaction strength (Supporting Information).

Voltammetric Analysis
Voltammetric measurements confirmed the high selectivity of HS-BIN-NDI selectors for discriminating optically active analytes.These measurements were possible by the electroactivity of atenolol.The enantioselectivity of each of the newly synthesized selectors (R)-HS-BIN-NDI and (S)-HS-BIN-NDI was tested based on the changes in the intensity of electrooxidation current signal of the chiral atenolol present in solution in different concentration from the range of 0.005 − 1.0 μm.For both chiral selectors, the measurements were carried out for both (R)-and (S)-atenolol.As shown in Figure 5, with an increase in the concentration of atenolol isomer in the solution with the opposite chiral configuration to the selector covalently immobilized onto the electrode surface, the intensity of the recorded ATN current signal increased.In the identical configuration of atenolol and selector, no changes in the intensity of the current signal of electrooxidation of ATN as a function of its concentration were observed.Based on the recorded SWV curves, the peak current values obtained for the corresponding concentrations of (R)-and (S)-atenolol were determined.The calibration curves, shown in the insets of Figure 5, were plotted.The most stable (S)-BIN-NDI complex with (R)-ATN, which also has the strongest interaction between the complex partners (E int = −35.82kcal mol −1 ) (A), and the most stable complex with (S)-ATN (E int = −32.67kcal mol −1 ) (B).The interaction energies were calculated at the B3LYP/def2TZVP level based on the B3LYP/def2TZVP/PCM(water) geometries; see the Supporting Information file for details.At the experimental conditions with pH 7.4, ATN is in the amine protonated state, while the sulfonamide group in BIN-NDI is in its neutral form.
The regression equations of the plotted dependences (I ATN = f(C ATN ), as well as the analytical parameters: analytical range, the limit of detection (LOD), and the limit of quantification (LOQ) are shown in Table 2.The values of detection and quantification limits were determined according to the equations: where:  is the standard deviation of the response for the lowest measurable concentration, and a is the slope of the calibration curve.Based on the plotted dependences, shown in the insets in Figure 5, it can be unequivocally concluded that the (S)-isomer of the studied naphthalene diimide derivative was selective only  The stability of the proposed sensors (Au/HS-BIN-NDI/MCH) was monitored for 6 months against a constant ATN concentration of 0.1 μm.After the measurement, the sensors were rinsed with a gentle stream of distilled water.Between measurements, the sensors were stored under cover at room temperature.The recorded changes in the intensity of the ATN current signals (see Figure 6A,B) were at the level of 3.5% (RSD), confirming the proposed sensors' high stability.The sensor-to-sensor repeatability was tested using three sensors for each HS-BIN-NDI configuration and for three ATN concentrations chosen from the dynamic range of the calibration curve (see Figure 6C,D).The observed RSDs for sensor-to-sensor repeatability were 2.3% and 2.8% for HS-BIN-NDI selectors in the configuration (S) and (R).
As part of the research, the selectivity of the proposed selectors against urea, creatinine, glucose, human albumin, and hemoglobin was also checked.For this purpose, the selectors immobilized on the gold surface were exposed to the action of various mixtures containing atenolol (0.1 μm) and a one-given interferent.The concentration of the interferent corresponded to its normal blood level: urea (2.5 -6.7 mm), [47] creatinine (0.052 -0.12 mm), [48] glucose (3.5 -5.5 mm), [49] human serum albumin (0.53 -0.75 mm) [50] and hemoglobin (1.64 -2.39 mm). [51]he obtained results in the form of SWV curves are shown in Figure 7A,B.The recorded current signals of atenolol, regardless of the used interferent, practically did not change, both in terms of their position on the potential scale and intensity.Differences in the intensity of the ATN electrooxidation signal did not exceed 4.8%, which suggests that none of the interferents blocked the interaction sites of the selector with the analyte.The correct operation of the selectors was verified by the recovery method against rat plasma.The obtained results presented in Table 3 unquestionably confirmed the high application potential of the proposed sensors.Moreover, the sensitivity of the developed protocols with chiral HS-BIN-NDI selectors enables the detection of ATN in the appropriate configuration at the level 300 times lower than the ATN concentration in serum required for producing beta-blockade (0.75 -1.88 μm).Some of the biotransformation pathways for -blocker drugs in humans are stereoselective. [52]The beta-blocking potency demonstrates only (S)-atenolol, whereas the (R)-enantiomer has no biological activity; [11] so these two ATN enantiomers should be regarded as different drugs.
There is an emergency need for fast and reliable methods to verify the enantiomeric purity of stereoselective drugs.Table 4 summarizes the existing analytical reports focused on atenolol.To date, the separation of the atenolol stereoisomers has only been possible using chromatographic techniques.The voltammetric protocol for determining ATN enantiomers we proposed is the first proposal characterized by such a low detection limit.As a result, it may provide a novel avenue for the specific and ultrasensitive detection of not only atenolol but also a whole group of similar -blockers in real samples.

Conclusion
The importance of chirality and chiral analysis in modern chemistry and chemical technology has various origins.The separa-tion of chiral isomers is crucial in chemistry from both analytical and preparative points of view.The enantiomeric purity of compounds is essential in stereospecific synthesis and the production of pharmaceuticals, pesticides, and some food additives, where only one of the enantiomers exhibits the desired action.A new trend in the determination of chiral compounds is voltammetric techniques.Still, in many cases, the limitations are sensitivity and the inability to unambiguously identify enantiomers and their racemic mixtures due to the proximity of the analytical characteristics of the corresponding voltammograms.
In the present contribution, we designed and synthesized an optically active naphthalene diimide derivative HS-BIN-NDI possessing a donor-acceptor group capable of binding to the gold electrode thanks to the introduction of a thiol chain end.We proposed a simple in-design sensor for the voltammetric detection of the electroactive chiral -blocker atenolol.The sensor's receptor layer is formed only by a monolayer of an axially chiral thiol naphthalene diimide derivative formed by a chemisorption process.Such a thin layer is not a barrier to electron transfer between the electroactive analyte and the electrode surface.Moreover, this way of forming the layer coerces the molecules into the most favorable orientation of the receptor molecules from the point of view of the recognition process.Atomic force microscopy and surface plasmon resonance studies unequivocally confirmed the high affinity of a chiral selector (HS-BIN-NDI) exclusively to a chiral analyte of the opposite configuration.No experimental indications about interactions of very flexible ATN with HS-BIN-NDI caused the necessity of considering over 220000 Table 3. Accuracy of the method for determination of (R)-and (S)-ATN.complexes at the simple molecular-mechanics level.A few hundred top-scored complexes were reoptimized at the B3LYP/PCM/6-31G ** level.Out of a plethora of interactions between the complex partners, we found that the most stable (S)-BIN-NDI-(R)-ATN complex is stabilized by three intermolecular and one intramolecular hydrogen bonds (E int = −35.82kcal mol −1 ).After careful computational analysis, we have come to the conclusion that the difference in (S)-BIN-NDI-(S)-ATN and (S)-BIN-NDI-(R)-ATN interaction must result not from one-to-one interactions but from the specificity of the organization of the (S)-HS-BIN-NDI monolayer on the surface.The detector constructed as an HS-BIN-NDI selector in combination with a voltammetric monolayer provided a simple and reliable platform for enantiospecific detection of atenolol in a multicomponent matrix such as plasma.This study illuminates the principle method of using the surface plasmon resonance technique and designing a suitable self-organized monolayer to develop an efficient chiral detection interface.We suppose that presented method may be used to determine other -blockers with 3-aryloxy-1-amino-2-propanol pharmacophore.Moreover, the studied NDI selectors are electroactive.Therefore, they enable the detection of appropriate enantiomers of both electroactive and non-electroactive analytes.These NDIs can also be used as both selectors and electrochemical visualizers.The extremely low LOD value ≈10 3 times lower than other proposed enantioselective sensors for ATN determination [24,25] and wide analytical range of work allows us to believe that our proposed sensor can find application in the specialized analysis of the broad group of optically active drugs.What is also important is that the applied chiral selectors HS-BIN-NDI allow both qualitative and quantitative analysis.The studies performed using rat plasma proved our proposed tool's high selectivity and functionality in determining atenolol enantiomers in plasma without the need for sample pretreatment.

Figure 3 .
Figure 3.The most stable three out of eleven conformers of the (S)-BIN-NDI receptor stabilized by the N─H─O═C intramolecular hydrogen bond (distances in Å).

Figure 4 .
Figure 4.The most stable (S)-BIN-NDI complex with (R)-ATN, which also has the strongest interaction between the complex partners (E int = −35.82kcal mol −1 ) (A), and the most stable complex with (S)-ATN (E int = −32.67kcal mol −1 ) (B).The interaction energies were calculated at the B3LYP/def2TZVP level based on the B3LYP/def2TZVP/PCM(water) geometries; see the Supporting Information file for details.At the experimental conditions with pH 7.4, ATN is in the amine protonated state, while the sulfonamide group in BIN-NDI is in its neutral form.

Figure 6 .
Figure 6.Results of stability (A,B) and sensor-to-sensor repeatability (C,D) studies performed in 0.01 m PBS buffer for HS-BIN-NDI selectors in the configuration (S) and (R).Bar graphs were constructed based on SWV measurements.

Figure 7 .
Figure 7. SW voltammograms recorded for the immobilized on the gold electrode surface HS-BIN-NDI selectors in the configuration S (A) and R (B) during their interaction with the mixture of appropriate interferent and atenolol in both (R) and (S) configurations.Experimental conditions: 0.01 m PBS buffer, pH 7.4; C HS-BIN-NDI = 1.0 mm; C MCH = 1.0 mM; gold disc electrode (ϕ = 1.6 mm).