Simultaneous Enantiospecific Recognition of Several β-Blocker

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Simultaneous enantiospecific recognition of several #-blocker enantiomers using molecularly imprinted polymer-based electrochemical sensor Ede Bodoki, Bogdan Cezar Iacob, Adrian Florea, Andreea Elena Bodoki, and Radu Nicolae Oprean Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 Jan 2015 Downloaded from http://pubs.acs.org on January 29, 2015

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Analytical Chemistry

Simultaneous enantiospecific recognition of several β-blocker enantiomers using molecularly imprinted polymer-based electrochemical sensor Bogdan-Cezar Iacob1‡, Ede Bodoki1‡,* Adrian Florea2, Andreea Elena Bodoki3, Radu Oprean1 1

Analytical Chemistry Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 4, Louis Pasteur St., 400349, Cluj-Napoca, Romania 2

Cell and Molecular Biology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 6, Louis Pasteur Street, 400349, Cluj-Napoca, Romania

3

General and Inorganic Chemistry Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 12, Ion Creanga St., 400010, Cluj-Napoca, Romania Corresponding Author * email: [email protected]. Tel.: +40 264 597256/int. 2412 Fax: +40 264 597257

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ABSTRACT: The development of a chiral electrochemical sensor using an electrogenerated molecularly imprinted polymer (MIP)-based ultra-thin film using R(+)-atenolol (ATNL) as a template was reported. The proposed sensor exhibited distinctive enantiospecific oxidation peaks towards the R-antipodes of four β-blocker representatives and additional oxidation peaks common to both enantiomers of each studied β-blocker, allowing thus the simultaneous analysis of all of their enantiomers in a single analysis. The specific preconditioning of the polymer by alternative exposure to aqueous and non-aqueous medium was proven to be essential for the chiral recognition ability of the obtained sensor. The rebinding property of the MIP film was studied by using a well-known redox probe, a change in the morphology and diffusive permeability of the thin polymeric layer in the presence of its template being observed. The applicability of the optimized analytical procedure was demonstrated by the analysis of ATNL’s enantiomers in the range of 1.88 x 10-7 – 1.88 x 10-5 mol/L. The developed polymeric interface is the first reported transductor of a chiral electrochemical sensor able to exhibit simultaneous enantiospecificity towards several β-blocker representatives extensively used in the pharmaceutical and biomedical fields, offering good prospects in the simple, cost-effective and fast assessment of their enantiomeric ratio and total concentration.

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I. Introduction Molecularly imprinted polymers (MIPs) possess quite unique and attractive features placing them at the interface of different science areas, such as polymer and material science, pharmacy, biochemistry and organic chemistry. They are endowed with selectivity comparable to that of affinity biomacromolecules and in addition they show a remarkable stability under various experimental conditions. Due to their unique pre-determined selectivity towards the template molecule, they were extensively employed in the separation techniques as stationary phases.1,2 However, these techniques are time-consuming and require expensive instrumentation, therefore faster and more cost-effective alternatives, such as electrochemical sensors, should be considered. The success in the electrochemical chiral probing of an analyte depends on the material used to modify the sensor’s electroactive surface. There are two main categories relative to the developed chiral electrochemical sensors based on the type of the chiral sensing material. The first group is represented by the naturally occurring chiral interfaces (enzymes, cyclodextrins, alkaloids, etc.), offering a superior selectivity but a poor chemical and thermal stability and with higher costs. These disadvantages are overcame by the representatives of the second category, namely chiral synthetic sensing components such as ligand exchangers, chiral crown ethers, MIPs, etc.3 Even though molecular imprinting has more than 80 years of history, the first reports exploiting the “memory” effect towards a print molecule through electrochemical sensing, appeared only in the early 1990’s.4,5 A boost of interest towards MIP-based electrochemical sensors occurred after 2003, however the number of publications lag far behind the ones employing MIPs in separation techniques. The vast majority of MIP-based electrochemical sensors were developed for the non-chiral analysis of different analytes, and merely around 20 articles report chemosensors designed for the chiral analysis of different small-molecules, like aminoacids3,6-14 or monosaccharides.15 However, enantiospecificity was reported only in the case of aminoacids, and all as the result of a single research group.6-10 The vast majority of pharmaceutical compounds present at least one center of chirality and even though their enantiomers exhibit different pharmacological and pharmacokinetic activities, they are commercialized predominantly as racemic drugs. In case of propranolol (PRNL) for example, the S(-)- enantiomer is 179 more potent than the R(+)- antipode.16 Furthermore, there are many cases when one of the enantiomers causes toxic effects, like the unfortunate birth defects initiated by one of thalidomide‘s enantiomers.17 The majority of the newly marketed drugs contain a chiral active pharmaceutical ingredient. It is more and more evident that the therapeutic benefits/cost ratio favors the manufacture of single-enantiomer drugs. Therefore it is mandatory to develop sensitive, selective

and cost-effective methods of chiral purity assessment applicable from the early stages of drug development, throughout every drug quality control procedure (inprocess and post-production controls). Even though the basic concept of MIP preparation is quite simple, obtaining a strong imprinting effect for electrochemical sensing of a pharmaceutically active compound with a more complicated structure than of an aminoacid, seems to be a more difficult task. Moreover, in the case of natural (proteinogenic) aminoacids the asymmetric carbon is at the molecule’s extremity carrying two functional groups (– NH2 and –COOH) strongly interacting with the commonly used functional monomers, thus easily leading to highly enantioselective imprinted cavities. Therefore, in order to prove the feasibility of MIP-based electrochemical sensors in chiral analysis and especially in the biomedical field, their analytical performance needs to be demonstrated on more complex enantioactive molecules. Here, we propose for the first time a versatile concept of integrating MIPs into chiral electrochemical sensors for the enantiospecific determination of four β-blocker representatives (Figure S-1 in the Supporting Information) using the same sensing unit.

II. Experimental Section 1. Materials Analytical grade standard enantiomers (R(+)/S(-)) of PRNL (99%) and S(-)-atenolol (ATNL) were purchased from Sigma (Steinheim, Germany). R(+)-ATNL was provided from Santa Cruz Biotechnology (Dallas, USA). Pure enantiomers of oxprenolol (OXPRNL) and alprenolol (ALPRNL) were separated by a preparative HPLC method.18 Methacrylic acid (MAA) 99%, pentaerythritol triacrylate (PETRA) and 4,4’-azobis(4-cyanovaleric acid) (ACVA) were purchased from Aldrich (Steinheim, Germany). Tetrabutylammonium hexafluorophosphate (4BA6FPh) was provided from Fluka (Steinheim, Germany). In the case of electrochemical experiments, the working solutions for rebinding consisted of 1μg/mL β-blocker enantiomer in acetonitrile (ACN). Further details concerning the employed reagents and materials can be found in the Supporting Information. 2. Preparation of electrogenerated MIP films The in-situ electrodeposition of non-imprinted and βblocker-imprinted polymer films on the surface of the working CPE was done under potentiodynamic conditions, in a conventional, one compartment electrochemical cell using a platinum wire as auxiliary electrode and a non-aqueous Ag/Ag+ reference electrode. All peak potentials regarding the electropolymerization are referred to this reference electrode. Beforehand, the surface of the CPE (18% (w/w) solid paraffin) was regenerated by polishing to a mirror-like finish on a white clean paper. In a typical electrodeposition, a polymerization mixture consisting of PETRA (20mM) as 3

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cross-linker, MAA (20mM) as functional monomer, ACN (1000 μL) as porogen and template (4mM), with ACVA (1.5mM) as the polymerization initiator, 4BA6FPh (100mM) as supporting electrolyte, was degassed for 5 minutes in an ultrasonic bath. Electropolymerization and cross-linking of the MIP film on the surface of the working electrode was achieved by cyclic voltammetry (CV) using 5 potential cycles from -0.9 to +0.7V at 100mV/s scan rate. A control polymer film, i.e. nonimprinted polymer (NIP), was prepared under the same conditions but in the absence of the template.

3. Characterization of the electrogenerated MIP films Details regarding the electrochemical, spectroscopic and microscopic characterization of the obtained MIP layer are to be found in the Supporting Information. Gravimetric analysis was performed using an electrochemical quartz crystal microbalance (EQCM) module installed alongside the M101 module in an Autolab PGSTAT 302N, driven by Nova 1.10 software, from Metrohm Autolab B.V. (Utrecht, The Netherlands). The AT cut Pt/TiO2 crystals of 6MHz resonant frequency and with a diameter of 1.36cm were from Metrohm Autolab B.V. (Utrecht, The Netherlands). The same reference and counter electrodes were used for electropolymerization as in the case of the chiral electrochemical probing. The impedance response of the EQCM was registered throughout the 5 cycles. Atomic force microscopy (AFM) experiments were carried out in the tapping mode with a Witec alpha 300 system. The silicon cantilevers (Nanosensors) have a typical force constant of 2.8Nm−1 and a resonance frequency around 63kHz. The AFM analyzed surface was of 4µm2. 4. Voltamperometric chiral analysis of β-blockers using the surface-modified MIP-CPEs After careful washing of the MIP-modified electrode in a stirred acidified ACN (10 minutes) for the elimination of the template, the MIP surface conditioning was realized at different pHs (from pH=3.0 to pH=7.0). Sample rebinding was carried out by immersing the activatedMIP-modified electrode in the ACN solution of desired βblocker’s enantiomer. Differential pulse voltammetry (DPV) (initial potential: +500mV; final potential: +1600mV; range: 200µA; rate: 25mV/s; pulse width: 25ms; pulse height: 50mV; ramp height: 5mV) was used for electrode conditioning and oxidation of the bonded βblocker, in 100mM phosphate buffer (adjusted with NH4OH). One typical analysis was represented by the rebinding and the oxidation of one β-blocker enantiomer and a subsequent analysis involved the mechanical removal of the MIP layer by polishing on a white clean paper followed by the electrogeneration of a fresh new MIP layer. In order to decrease experimental variability to maximum and to have a clearer view of the β-blockers’ oxidation peaks, all voltammograms regarding chiral

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probing were normalized by substracting the final DPV (10th DPV scan, representing the background scan) from the 8th DPV scan.

III. Results and discussion 1. Electrochemical behavior of β-blockers Several voltammetric studies of β-blockers on different electrodes have been previously reported.19-23 They undergo an irreversible electrooxidation with the subsequent adsorption of their oxidative product on the electroactive surface of the electrode.21,24 Using a bare CPE, DPVs of the four studied β-blockers show one main oxidation peak in the potential range of +1.08V - +1.42V vs. Ag/AgCl, denoting an anodic shift in the order: PRNL – OXPRNL – ALPRNL – ATNL (Figure 1). Most probably, the observed shift in the oxidation peak is given by the changing electronic density in the vicinity of the electroactive moiety found on the common alkyl side chain of β-blockers, which is influenced by the different aromatic rings and additional functionalities attached to them. The results indicate that β-blockers with even a weak electron-withdrawing functional group on the aromatic ring (such as acetamide in case of ATNL) tends to disfavor the oxidation of the electroactive moiety on the side chain, shifting its oxidation potential towards higher values. However, electron-donating groups linked to the aromatic ring (such as the allyloxy chain in case of OXPRNL) or moieties with positive inductive effect (such as the allyl chain in case of ALPRNL or naphthyl ring in case of PRNL) tend to slightly increase electron density, determining lower oxidation potentials. A single, irreversible oxidation peak involving the transfer of two electrons was previously reported in case of ATNL,19,21 with the generation of 2-[4-(3isopropylamino-2-oxo-propoxy)-phenyl]-acetamide, as oxidation product, on which there is no consensus in the literature yet regarding the involved functional group(s).

Figure 1. Normalized DPVs of 50μg/mL racemic β-blockers in 100mM phosphate buffer pH = 7.0 on bare CPE vs. Ag/AgCl; green line: PRNL, blue line: OXPRNL, black line: ALPRNL, red line: ATNL. DPV parameters: initial potential: +500mV; final potential: +1600mV; rate: 25mV/s; pulse width: 25ms; pulse height: 50mV; ramp height: 5mV.

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The majority of the publications attribute the oxidation peak to the hydroxyl group directly bonded to the chiral center,20-22 whereas a few papers assign it to the secondary amine.25 Unfortunately, none of the published papers rely on experimental data able to confirm one of the hypotheses. Our preliminary investigations using a continuous flow electrochemical cell coupled to Electrospray Ionization-Mass spectrometry bring for the first time proofs regarding the involvement of the secondary amine group in the aforementioned oxidation (Figure S-2 in the Supporting Information).

2. Fabrication and characterization of surfacemodified MIP-CPEs 2.1. Fabrication of the imprinted polymeric film Electropolymerization presents the advantage of achieving simultaneously the generation and tight surface binding of the polymeric film, which considerably shortens the experimental time. The obtained films are physically stable, with a strong adherence onto the electrode material of any shape and size. Furthermore, CV allows the convenient surface-deposition of different polymeric films with varying thickness (down to ultrathin layers) by simply changing the number of potential scan cycles.26,27

Figure 2. Electrodeposition by CV of the non-imprinted (A.) and imprinted (B.) polymeric films (black line) using a Ptcoated quartz crystal and the concomitant electrochemical quartz crystal microbalance analysis (red line) of the forming

polymeric layers. -0.9V - +0.7V vs. Ag/Ag , 100mV/s, 5 cycles; Electrolyte: 100mM 4BA6FPh in ACN.

Although MAA and PETRA generate non-conducting polymers, as long as their layer is kept under a thickness that does not insulate the underlying conductive electrode surface (