Electrospray ionization tandem mass spectrometric studies to probe the interaction of Cu(II) with amoxicillin

Chinese Chemical Letters 25 (2014) 39–45

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Original article

Electrospray ionization tandem mass spectrometric studies to probe the interaction of Cu(II) with amoxicillin Ramaiyan Sekar a, Suresh Kumar Kailasa b, Yuan-Chin Chen a,c, Hui-Fen Wu a,d,e,f,* a

Department of Chemistry, National Sun Yat Sen University, Kaohsiung, 804 Depatment of Chemistry, S. V. National Institute of Technology, Surat, 395007, India c Department of Chemistry, Tamkang University, New Taipei d School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 806 e Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung, 804 f Center for Nanoscience and Nonotechnoloty, National Sun Yat-Sen University, Kaohsiung, 804 b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 June 2013 Received in revised form 6 September 2013 Accepted 24 September 2013 Available online 8 November 2013

This study describes the application of electrospray ionization mass spectrometry (ESI-MS) to investigate copper ion interaction with amoxicillin. ESI mass spectra of Cu–amoxicillin complexes show complex ions at m/z 828, 792, 753, 731, 428, 388 and 366 corresponding to [63Cu+(2A-H)+2H2O]+, [63Cu+(2A-H)]+, [2A+Na]+, [2A+H]+, [63Cu+(A-H)]+, [A+Na]+ and [A+H]+ (where A = amoxicillin). Based on the observed m/z values of Cu–amoxicillin complex ions, it is found that the Cu–amoxicillin ratios are 1:1 and 1:2, and the copper ions exhibited three feasible coordination numbers (2, 4 and 6) with amoxicillin complexes. The structures and coordination numbers of copper–amoxicillin complex ions were probed from their collisionally activated dissociation (CAD) spectra. Based on these results, it is confirmed that the copper ions could form stable tetrahedral and octahedral complexes with amoxicillin. This study validates the applicability of ESI-MS for probing copper–amoxicillin complex ions. ß 2013 Hui-Fen Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Amoxicillin Copper ion ESI-MS

1. Introduction Antibiotics are produced by microorganisms which can kill or prevent the reproduction of bacteria. Antibiotics selectively inhibit protein synthesis and disrupt the cell walls of bacteria. For example, penicillins effectively damaged the cell walls of certain bacteria, but not the cell membranes of animals. Amoxicillin (2S, 5R, 6R)-6-[(R)-( )-2-amino-2-(p-hydroxyphenyl)acetamido]-3,3dimethyl-7-oxo-4-thia-1-azabi-cyclo[3.2.0] heptane-2-carboxylic acid, C16H19N3O5S, MW 365.1 Da) is an important antibiotic which belongs to a class of b-lactam antibiotics. It is also chemically known as D-( )-a-amino-p-hydroxybenyzyl penicillin and derived from the basic penicillin nucleus of 6-aminopenicillanic acid. It shows strong anti-bactericidal activity by inhibiting bacteria protein synthesis on the ribosome which causes misinterpretation of the genetic codes [1]. It is well known that cooper-antibiotics complexes play vital role in various enzymatic redox reactions [2,3]. In addition, these complexes can inhibit metal binding sites in enzymes of bacteria by metal complexes and their subsequent penetration into lipid membranes. Amoxicillin shows antibacterial

* Corresponding author at: Department of Chemistry, National Sun Yat Sen University, Kaohsiung 804. E-mail address: [email protected] (H.-F. Wu).

activity on various bacteria, including H. influenzae, N. gonorrhoea, E. coli, Pneumococci, Streptococci and certain strains of Staphylococci. In this connection, several hypotheses have illustrated the interactions of metal ions with various antibiotics, including amoxicillin [4,5]. It is also observed that metal ions-antibiotics noncovalent interactions can be influenced in the stereochemistry of the drug [6]. Furthermore, in vitro cholinesterase inhibitory activities have been studied using Cu(II) complexes derived from 2-(diphenylmethylene)hydrazinecarbothioamide derivatives [7]. Furthermore, metalloproteins play an important role in various biochemical pathways [8]. Recently, various analytical techniques, such as spectrometry [9,10], polarimetry [11], capillary electrophoresis [12], 1H NMR and potentiometry [13] have been used to study copper–amoxicillin and other metals–drug complexes. Therefore, it is necessary to study metal–amoxicillin complexes structures for the understanding of their activity and reaction mechanism. Electrospray ionization mass spectrometry has been widely used for the characterization of a wide variety of inorganic complexes in gas phase [14]. ESI-MS is an extremely important tool for studying of molecular weights and structures of metal complexes [15–18]. In recent years, many investigators have used ESI-MS to probe the transition metals complexes and metals noncovalent interactions with biomolecules [19–25]. Moreover, our group also demonstrated the application of ESI-MS to the investigation of the hydrophobic peptide (gramicidin A) and drug

1001-8417/$ – see front matter ß 2013 Hui-Fen Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.10.012

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R. Sekar et al. / Chinese Chemical Letters 25 (2014) 39–45

(kojic acid) interactions with various ions (Ca2+/Mg2+/Zn2+, Cl and Fe3+) [26,27]. Turecek et al., investigated the gas phase dissociation behavior of several ternary complexes of copper with amino acids [28]. Lu and Guo evaluated the stereochemistry of ligands in zinc(II)-L-tryptophan-borneol multimeric cluster ions [29] and metal–proton complexes of di-o-benzoyl-tartaric acid dibutyl ester and L-tryptophan in the gas phase by ESI-MS [30]. Meanwhile, Brodbelt and co-workers reported reactions of three transition metals (Ni, Cu, Co) with three crown ethers and four acyclic ethers (glymes) by ESI-MS [31]. Chu et al., described the fragmentation chemistry of Cu-amines and -oligopeptide complexes by ESI-MS [32]. The success of these earlier works led us to probe copper–amoxicillin complex ions by ESI-MS. Moreover, there were no reports on the investigation of copper–amoxicillin complexes by ESI-MS. Therefore, we have undertaken ESI-MS

studies to probe Cu–amoxicillin complexes and to evaluate fragmentation chemistry of complexes by collisionally activated dissociation. 2. Experimental Amoxycillin, methanol (HPLC grade) and CuCl2 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Milli-Q (Millipore, Milford, MA, USA) ultrapure water was used in all of the experiments. All reagents were of analytical grade and used directly without further purification. ESI-MS experiments were performed on a Finnigan MAT ion trap mass spectrometer (Finnigan LCQ-Advantages, San Jose, CA, USA). A micro syringe pump (Harvard Apparatus, Edenbridge, Great Britain) was used for injecting sample solutions into the

Fig. 1. ESI mass spectra of amoxicillin (5.0 mg/mL) using (a) 100% water, (b) MeOH–H2O at 2:1 (v/v) system and (c) CAD spectrum of [2M+H]+ ion at collision energy 25 eV.

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mass spectrometer. All the mass spectra were obtained in positive ion mode. The ion trap analyzer was operated at a pressure of 1.5  10 5 Torr. Capillary desolvation temperature 250 8C, ESI spray needle voltage 5000 V, a tube lens offset voltage 80 V and capillary voltage 10 V were used in this study. Each mass spectrum represented an average of 10 individual scans. Stock solution of amoxicillin was prepared in MeOH:H2O (2:1, v/v). The copper complex was prepared using CuCl2. In the preparation of copper–amoxicillin complex, amoxicillin was dissolved in methanol and added to CuCl2 in water under constant stirring for 1–30 min at room temperature, keeping the copper– amoxicillin mole ratio at 5:1. The pH of the Cu–amoxicillin complex solution was adjusted by adding 0.01 mol/L of formic acid or 0.01 mol/L of NaOH. The formed complexes were dissolved in MeOH:H2O (1:1, v/v) to obtain a final concentration of 10 mmol/L. This solution was directly injected into ESI-MS. 3. Results and discussion Prior to the investigation of copper complexation with amoxicillin, protonated, sodiated, potassiated and MeOH adducts and gas-phase reactions of amoxicillin with MeOH were explored to establish benchmarks for the identification of complex ions. It is well know that amoxicillin can form different adduct ions, such as protonated, sodiated and methanol-adduct ions under ESI conditions by using H2O, MeOH, MeOH:H2O and MeOH-ammonium acetate buffer as solvents [33]. In ESI, the addition of modifiers (ACN or MeOH) play a critical role in the generation of gas-phase complex ions by rapid desolvation of solvent molecules [34]. In this connection, we studied ESI mass spectra of amoxicillin (5.0 mg/mL) in the positive ion mode using pure water (100%) and MeOH:H2O (2:1, v/v) as solvents (Fig. 1a and 1b). Fig. 1a displays the ESI mass spectrum of amoxicillin using pure water (where A = amoxicillin). The ions at m/z 366 and 731 corresponded to [A+H]+ and [2A+H]+, in addition another intense peak at m/z 349 is attributed to the loss of ammonia from amoxicillin [A+H NH3]+, respectively. Next, we also studied that ESI mass spectrum of amoxicillin (5.0 mg/mL) using MeOH:H2O (2:1, v/v) (Fig. 1b). In this spectrum, amoxicillin exhibited ions at m/z 731, 398, 366 and 349 which corresponded to [2A+H]+, [A+CH3OH+H]+, [A+H]+ and [A+H NH3]+, respectively. The observed ion at m/z 398 confirmed the formation of the MeOH adduct with amoxicillin by gas-phase reactions [33] with these reactions shown in Fig. 2. Notably, the signal intensity of the dimer +H3N

O S NH N O

HO

MW 365.10

HO

O

m/z 366.0

O

+H3N

S

H+ NH

O S

N

NH

O HO

O

m/z 349.0

O

O

HN OCH3 HO

HO

O

m/z 398.0

Fig. 2. Scheme for the fragmentation of amoxicillin and MeOH gas phase reaction with amoxicillin.

Fig. 3. ESI mass spectrum of amoxicillin (5.0 mg/mL) using MeOH:H2O (1:1, v/v) system in the presence of sodium acetate buffer.

ion of amoxicillin was increased by using MeOH:H2O (2:1, v/v). Furthermore, the CAD spectrum of the dimer ion of amoxicillin was shown in Fig. 1. The major ions observed during the dissociation of [2A+H]+ ions at m/z 714, 572, 366 and 349 corresponded to [2A+H NH3]+, [2A C6H10NO2S+H]+, [A+H]+ and [A+H NH3]+, respectively. The dimer structure of amoxicillin and its possible fragmented structure can be found in Fig. 1c. We also studied the ESI mass spectrum of amoxicillin (5.0 mg/mL) using MeOH:H2O (2:1, v/v) in the presence of sodium acetate buffer (Fig. 3). The ions at m/z 366, 388, 731, 753, 769 and 1140 corresponding to [A+H]+, [A+Na]+, [2A+H]+, [2A+Na]+, [2A+K]+ and [3A H+2Na]+, respectively. In this mass spectrum, the sodiated dimer ion of amoxicillin showed higher intensity than the other ions of amoxicillin. In general, the Cu2+ ion is known to form tetrahedral and octahedral complexes with a variety of ligands. Since, amoxicillin contains acidic and basic groups which can strongly interact with metal species. In addition, it also contains a number of potential donor atoms, such as N, O and S, which can act as anchors for the metal ions. Amoxicillin, as the ligand in the present study, is expected to be bidentate in nature with two donating groups, i.e., the oxygen of carboxylic group and the oxygen of b-lactam (Fig. 4). To support this assumption, ESI mass spectra (Fig. 4) were recorded for Cu(II)-amoxicillin ions at a molar ratio of 5:1 at various pHs (3.0, 6.0 and 9.0) at MeOH:H2O (2:1, v/v) with the complex ion structures inserted in Fig. 4a–c. It can be noticed that ESI mass spectra of Cu–amoxicillin complexes showed complex ions with higher intensity at pH 6.0, including the complex ions at m/z 428, 792 and 828 which corresponded to [63Cu+(A-H)]+, [63Cu+(2A-H)]+ and [63Cu+(2A-H)+2H2O]+, respectively. These results indicated that ESI effectively generated Cu– amoxicillin complex ions with high signal intensities at pH 6.0 (Fig. 4b). During complex formation, the color of CuCl2 solution changed from blue to algae green without turbidity. It is well known that the central metal ions are solvated with solvent molecules and these solvated metal complex ions are easily observed in ESI-MS [34]. To support this assumption, we assigned the possible structures for the mass peak at m/z 828, which corresponded to solvated copper–amoxicillin complex ions, and attributed to [63Cu+(2A-H)+2H2O]+. In addition, the complex ion at m/z 792 as [63Cu+(2A-H)]+ suggested the elimination of two water molecules from the complex ion of [63Cu+(2A-H)+2H2O]+ at m/z 828. The ESI mass spectrum of copper–amoxicillin complex showed a dominant ion at m/z 828 ([63Cu+(2A-H)+2H2O]+) at pH 3.0. Fig. 4b indicates that ESI mass spectrum of Cu–amoxicillin complex at pH 6.0 exhibit ions at m/z 792 and 428 that were

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Fig. 4. ESI mass spectra of Cu(II)–amoxicillin complexes (Cu:amoxicillin, 1:5) at (a) pH 3.0, (b) pH 6.0 and (c) 9.0.

generated with maximum relative intensities (52% and 42%), and assigned as [63Cu+(2A-H)]+ and [63Cu+(A-H)]+. The observed signal intensities of complex ions were entirely different from the mass spectrum of Cu–amoxicillin complex at pH 3.0 (Fig. 4a). Fig. 4c shows the ESI mass spectrum of Cu–amoxicillin complex at pH 9.0. This result indicates that the signal intensity of the dimer of the amoxicillin ion (at m/z 731) was decreased under basic condition, which is due to the high affinity of Na+ ions towards the carboxylate ion. These changes can cause an increase in the repulsive forces between amoxicillin molecules [35]. Therefore, the mass peaks at m/z 792 ([63Cu+(2A-H)]+) and 828 ([63Cu+(2AH)+2H2O]+) were suppressed in the ESI mass spectrum of Cu(II)– amoxicillin complexes at pH 9.0 (Fig. 4c). Furthermore, these results confirmed that the observed complex ions at m/z 428 and 792 and 828 corresponded to 1:1 and 1:2 ratios of

copper:amoxicillin. Amoxicillin is assumed to be a bidentate ligand and the complex ions are deduced to have tetrahedral and octahedral geometry with coordination numbers, i.e., 2, 4 and 6. The possible structures of complex ions are inserted in Fig. 4. It can be noticed that the oxidation state of copper ion is reduced from Cu2+ to Cu+ during ESI-MS detection processes. We suspected that this type of oxidation reaction is generally observed in ESI by ionmolecule reactions between central metal ion and the reacting ligand [36,37]. Therefore, we believe that the oxidation state of the formed Cu–amoxicillin complex ion is reduced from Cu2+ to Cu+ during the gas-phase ion-molecule reactions at the electrospray needle. Although amoxicillin can interact with Cu(II) ions, it has strong tendency to produce amoxicillin dimeric ions with higher intensity than the Cu(II)–amoxicillin complex ions. This is due to the

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Fig. 5. ESI mass spectra of Cu(II)-amoxicillin complexes (Cu:amoxicillin, 1:5) at stirring time (a) 1 min, (b) 15 min and (c) 30 min.

stability of the dimeric ion of amoxicillin in gas phase. We also studied the effect of stirring time on the formation of Cu(II)– amoxicillin complexes at different time intervals (from 1 min to 30 min) at pH 6.0 (Fig. 5). It can be noticed that the Cu(II)– amoxicillin complex ions intensities (at m/z 428 and 792) were gradually increased by increasing stirring time. Based on these results, the maximum signal intensities were observed at a stirring time of 15 min (Fig. 5b). Furthermore, the CAD spectrum of the copper–amoxicillin complex ion ([63Cu+(2A-H)+2H2O]+) is shown in Fig. 6. The CAD of complex ion (at m/z 828) as [63Cu+(2AH)+2H2O]+ gives major fragment ions at m/z 792, 633 and 463, which corresponded to [63Cu+(2A-H)+H]+, [63Cu+(2AC8H9NO2)]+ and [63Cu+(A-H)+2H2O]+, respectively. These fragmented ions were formed through the loss of two H2O (36 Da), loss of C8H9NO2 (152 Da) and loss of one amoxicillin molecule (365 Da) from the complex ion [63Cu+(2A-H)+2H2O]+ (Fig. 6). We assume that the mass peak at m/z 633 was generated by the loss of

one 2-amino-2-(4-hydroxyphenyl)-acetyl-amino molecule from [63Cu+(2A-H)]+ with possible fragmentation shown by dotted lines in the inserted structure in Fig. 6. Importantly, characteristic isotope peaks of 65Cu(II)–amoxicillin complex ions were also observed in the mass spectra (Fig. 5). The signal intensities of Cu(II)–amoxicillin is relatively lower than the signal intensities of protonated monomer and dimer ions of amoxicillin. Based on the observed complex ions and dissociation phenomenon of complex ions, the dissociation of Cu(II)–amoxicillin complex ions in ESI-MS depends on the oxidation state of the metal atom before and after fragmentation, and on the attachment of the copper ions with amoxicillin. The copper–amoxicillin complex ions are preferably dissociated to yield fragment ions in which the oxidation state of copper is reduced (Cu2+ to Cu+). The obtained ESI MS data provided good evidence for the investigation of Cu– amoxicillin complex ions and possible information on the structures of Cu–amoxicillin ions.

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Fig. 6. CAD spectrum of [63Cu+(2M-2H)+2H2O+H]+ ion at collision energy at 25 eV.

4. Conclusion In this study, we explored the potential application of ESI-MS for probing the interactions of Cu(II) ions with amoxicillin. The ESIMS technique provided useful information on the structures of Cu(II)–amoxicillin complexes. We also studied the effect of pH on the formation of Cu(II)–amoxicillin complexes. The observed ESI mass spectra permitted us to assign the possible structures of Cu(II)–amoxicillin complexes and provided useful information on the assignment of copper/amoxicillin ratios, complex geometry and on copper coordination numbers. Moreover, in the Cu– amoxicillin complexes, the copper ions exhibited two oxidation states in the CAD of Cu–amoxicillin complex ion. Since the Cu– amoxicillin complex ions preferably dissociated to yield fragmented ions where the oxidation state of Cu2+ was reduced to Cu+, this result presented here may be applicable to the investigation of copper-bound complex ions of antibiotics and in situ experiments between metal ions-antibiotics. Acknowledgment We thank National Science Council (NSC) for financial support. References [1] N.S. Egorov, Antibiotics: A Scientific Approach Translated from Russian by A. Rosinkin, 1st ed., MIR Publishers, Moscow, 1985. [2] G. Mukherjee, T. Ghosh, Metal ion interaction with penicillins. Part VII: mixedligand complex formation of cobalt(II), nickel(II), copper(II), and zinc(II) with amplicillin and nucleic bases, J. Inorg. Biochem. 59 (1995) 827–833. [3] A.J. Welch, S.K. Chapman, The Chemistry of the Copper and Zinc Triads, Royal Society of Chemistry, Cambridge, 1993. [4] P.J. Niebergall, D.A. Hussar, W.A. Cressman, E.T. Sugita, J.T. Doluisio, Metal binding tendencies of various antibiotics, J. Pharm. Pharmacol. 18 (1966) 729–738. [5] G.V. Fazakerley, G.E. Jackson, Metal-ion interaction with penicillins: kinetics of complexation of nickel(II), J. Pharm. Sci. 66 (1977) 533–535. [6] H. Kozlowski, T. Kowalik-Jankowska, M. Jezowska-Bojczuk, Chemical and biological aspects of Cu2+ interactions with peptides and aminoglycosides, Coord. Chem. Rev. 249 (2005) 2323–2334. [7] Y.C. Chan, A.S.M. Ali, M. Khairuddean, et al., Synthesis and molecular modeling study of Cu(II) complexes derived from 2-(diphenylmethylene)hydrazinecarbothioamide derivatives with cholinesterase inhibitory activities, Chin. Chem. Lett. 24 (2013) 609–612. [8] M.N. Hughes, Coordination compounds in biology, in: G. Wilkinson, R.D. Gillard, J.A. McCleverty (Eds.), Comprehensive Coordination Chemistry, Pergamon Press, Oxford, 1987, pp. 541–545.

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