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A Precursor Ion Scan Mode-Based Strategy for Fast Screening of Polyether Ionophores by Copper-Induced Gas-Phase Radical Fragmentation Reactions Eduardo J. Crevelin, Bruna Possato, João Luis Callegari Lopes, Norberto Peporine Lopes, and Antônio Eduardo Miller Crotti Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02855 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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Analytical Chemistry

A Precursor Ion Scan ModeMode-Based Strategy for Fast Screening of PolyPolyether Ionophores by CopperCopper-Induced GasGas-Phase Radical Fragmenta Fragmentation Reactions Eduardo J. Crevelin,†,‡ Bruna Possato,‡ João L. C. Lopes,† Norberto P. Lopes,† Antônio E. M. Crotti*,‡ †Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, CEP 14040-903 Ribeirão Preto, SP, Brazil ‡

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, CEP 14040-901 Ribeirão Preto, SP, Brazil KEYWORDS. Ionophores, precursor ion scan, dereplication, mass spectrometry, copper-induced fragmentation

ABSTRACT: The potential of copper(II) to induce gas-phase fragmentation reactions in macrotetrolides, a class of polyether ionophores produced by Streptomyces species, was investigated by accurate-mass electrospray tandem mass spectrometry (ESIMS/MS). Copper(II)/copper(I) transition directly induced production of diagnostic acylium ions with m/z 199, 185, 181, and 167 from α‒cleavages of [macrotetrolides+Cu]2+. A UPLC-ESI-MS/MS methodology based on the precursor ion scan of these acylium ions was developed and successfully used to identify isodinactin (1), trinactin (2), and tetranactin (3) in a crude extract of Streptomyces sp. AMC 23 in the precursor ion scan mode. In addition, copper(II) was also used to induce radical fragmentation reactions in the carboxylic acid polyether ionophore nigericin. The resulting product ions m/z 755 and 585 helped to identify nigericin in a crude extract of Streptomyces sp. Eucal-26 by means of precursor ion scan experiments, demonstrating that copper-induced fragmentation reactions can potentially identify different classes of polyether ionophores rapidly and selectively.

Polyether ionophores comprise a class of natural products that contain carboxylic acid or ester groups as well as tetrahydropyran or tetrahydrofuran rings in their structures. Through their ether and carbonyl oxygen atoms, these ionophores can form electrically neutral and stable complexes with inorganic ions such as alkali and alkaline earth metal cations.1,2 These compounds exhibit a wide range of biological activities including antiparasitic,3 antibacterial,4,5 antifungal,6 antiviral,7 antiproliferative/apoptotic,8,9 and potential antiscarring actions.10 The antibiotic activity of ionophores stems from their strong affinity for alkaline metal ions and from their ability to solubilize and transport metals that disrupt the Na+/K+ ion balance across cell membranes, thereby increasing the osmotic pressure inside the cell and killing the bacterial cell.11,12 Macrotetrolides are a class of ionophores originating from stereospecific tetramerization of the enantiomeric homologues nonactic and homononactic acids.13,14 These compounds display a broad spectrum of biological activities that range from antibacterial,15 antifungal,16 and antitumor actions17 to immunosuppressive effects.18 Over the last two decades, electrospray ionization mass spectrometry (ESI-MS) and its tandem version (ESI-MS/MS) have emerged as the most suitable techniques to identify polyether ionophores and other compounds that do not bear useful chromophore groups in their structures.19,20 ESI-MS detects ionophores mostly as cationized molecules (e.g., [M+Na]+) instead of protonated molecules because they have high affinity for metal ions. In these cases, the cationized molecules are selected as precursor ions in the first stage of MS, which is

followed by activation for fragmentation and consequent production of the corresponding MS/MS spectrum. The ability of ESI to detect metallated molecules has motivated many studies on the use of alkali metal ions such as Li+, Na+, and K+ to enhance the detectability of target compounds21,22, to estimate relative stabilities23-26, to differentiate between closely related isomers27,28, to identify disulfide linkages in peptides29, and to determine molecular masses30. However, interactions between organic compounds and alkali metal cation interactions provide less information as compared to interactions between these compounds and transition metal ions like Co2+, Ni2+, Zn2+, and Cu2+.23 The latter ions are more analytically useful than alkali metal ions due to their specificities for certain functional groups and to their ability to induce specific fragmentation reactions for structural elucidation studies.31 For example, Felder and co-workers studied complexes of cyclam derivatives with divalent transition metal ions and showed that Cu2+ induces ring cleavage reactions via electron transfer from the cyclam nitrogen atoms to the Cu2+ ion.31 The electron transfer creates a radical cation within the macrocycle and induces specific fragmentation reactions such as α-cleavages. Because of its chemical importance in biological systems, copper in both its oxidation states—copper(I) and copper(II)— has been one of the most extensively studied transition metals.32,33 This metal participates in essential life functions such as electron transfer and activation as well as oxygen transport.34 Copper(II) is an open-shell species with configuration [Ar]3d9; its most common coordination numbers vary from four to six.35 This metal cation readily forms complexes

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with oxygen and nitrogen (hard bases) ligands and is considered a borderline hard Lewis acid according to the principle of hard and soft acids and bases (HSAB).36 Moreover, copper(II) can modify its oxidation state and coordination sphere, to induce novel fragmentations that involve redox and radicalbased reactions.35,37 Despite these attractive aspects, the redox properties of copper(II) have rarely been used to elucidate the structure of natural products by ESI-MS/MS.38,39 Considering that (i) copper(II) may induce alternative fragmentation pathways and (ii) information on the fragmentation reactions of [macrotetrolides+Cu]2+ is scarce, in the present study we have used ESI-MS/MS to investigate how copper(II) affects the fragmentation of the macrotetrolides 1-3 (Figure 1). Similarly, we have employed these copper-induced fragmentation reactions to develop a precursor ion scan modebased strategy to identify these compounds in microbial crude extracts. O O

R1

O

O

R4

O

O

R2

O

O

O

O

R3

O

O 1: R1=R2=Me, R3=R4=Et, MW = 764.98 u 2: R1=R2=R4=Et, R3=Me, MW = 779.01 u 3: R1=R2=R3=R4=Et, MW = 793.04 u

Figure 1. Structures of the macrotetrolides isodinactin (1), trinactin (2), and tetranactin (3).

EXPERIMENTAL EXPERIMENTAL METHODS Materials and Reagents. Methanol (CH3OH, HPLC grade) was purchased from Merck (Darmstadt, Germany). Formic acid (FA) and analytical grade copper(II) chloride (CuCl2) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Deionized water (Milli-Q) was used throughout the study. The macrotetrolides were obtained in the form of a mixture of compounds 1-3 from the fermentation broth of Streptomyces sp. AMC 23. The nigericin sodium salt was supplied by Sigma-Aldrich. ESI-QTOF-MS/MS Analysis and Sample Preparation. The copper(II) complexes of macrotetrolides 1-3 and nigericin were obtained by diluting a 80:20 (v/v) CH3OH/H2O solution of these compounds at 1 mg/mL with an aqueous 2 mM copper(II) chloride solution until a final concentration of 1.0 µg/mL of the compounds was achieved. The copper(II) complexes of the macrotetrolides 1-3 were analyzed by electrospray ionization (ESI) mass spectrometry in the positive ion mode on a QTOF II (Micromass, Manchester, UK) mass spectrometer. The ESI interface conditions were as follows: capillary voltage = 3.5 kV, cone voltage = 55 V, source temperature = 150 °C, and desolvation temperature (N2) = 250 °C (mass ranged from m/z 150 to 900). The sample was introduced into the mass spectrometer with the aid of a syringe pump (Harvard, Holliston, MA, USA) operating at a ﬂow rate of 10 µL/min. The tandem mass spectrometry experiments

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(MS/MS) with collision-induced dissociation (CID) were conducted by using argon as collision gas on the doubly charged selected precursor ions ([M+Cu]2+) at collision energy values ranging from 10 to 50 eV. The mass analyzer was calibrated with a 1:1 (v/v) mixture of 0.1% phosphoric acid and CH3OH, to obtain a resolution of approximately 10,000. The accurate masses observed in all the experiments were within 1 x 10-3 Da (from 1 to 7 ppm depending on the mass) of the theoretical masses of the ions. The mass data were processed with the MassLynx V4.1 software. The copper(II) complex of nigericin was analyzed on a Triple TOF 5600+ DualSpray Ion Source AB SCiex (Massachusetts, USA) mass spectrometer. The ESI interface conditions were as follows: ion spray voltage floating = 4.5 kV, declustering potential = 80 V, gas 1 and 2 = 15 psi, and curtain gas = 25 psi. The sample was introduced into the mass spectrometer with the aid of a syringe pump coupled to the instrument operating at a ﬂow rate of 5 µL/min. The MS/MS experiment with CID was carried out by using N2 as collision gas on the selected precursor ion ([M-H+Cu] +) at collision energy values ranging from 10 to 150 V. The mass analyzer was calibrated with a tuning solution that was supplied in the Standards Chemical Kit shipped with the system, to give a resolution of approximately 30,000. The mass data were processed with the Analyst TF software. UPLC-MS analyses. Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) analyses were performed on a Waters ACQUITY UPLC H-Class system coupled to the Xevo® TQ-S tandem quadrupole (Waters Corporation, Milford, MA, USA) mass spectrometer with a Z-spray source operating in the positive mode. A sample (3 µL) of the mixture of macrotetrolides 1-3 was injected into an ACQUITY-BEH C18 column (2.1 × 50 mm, 1.7 µm) from Waters; the mobile phase used for gradient elution consisted of water and methanol at a flow rate of 0.3 mL/min. The gradient elution program started with 80% methanol. Then, methanol was raised to 100% in the following 10 min. Next, the column was eluted with 100% methanol for 5 min and was then returned to the initial conditions (80% methanol) within the following 5 min. The source and operating parameters were optimized as follows: capillary voltage = 3.4 kV, cone voltage = 40 V, source temperature = 150 °C, desolvation temperature (N2) = 300 °C, and desolvation gas flow = 500 L/h (mass range from m/z 400 to 900). The copper(II) complexes of compounds 1-3 were obtained by UPLC-MS analysis with post-column infusion by mixing the effluent with CuCl2 solution at a concentration of 2 mM, whereas the sodiated compounds 1-3 were obtained without post-column infusion. The CuCl2 post-column infusion was accomplished on an IntelliStart Fluidics system built into the instrument. The mass data were processed with the MassLynx V4.1 software. To apply the methodology developed in this work, we carried out a precursor ion scanning experiment using tandem mass spectrometry via UPLC-MS/MS with and without postcolumn infusion of the CuCl2 solution. A sample obtained from Streptomyces sp. AMC 23 cultured in potato dextrose (PD) was analyzed by using an Ascentis Express C18 column (4.6 × 100 mm, 2.7 µm) from Supelco. This column was used in these experiments to demonstrate that, by increasing the particle size and the column interior diameter and length, a chromatographic profile very similar to that for the ACQUITY-BEH C18 column could be obtained. The gradient elution program used for the mixture of the macrotetrolides 1-


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3 started with 80% methanol. The amount of methanol was raised to 100% in the following 8 min. Next, the column was eluted with 100% methanol for 3 min and was then returned to the initial condition (80% methanol) within the following 5 min. In the experiments that used post-column infusion of CuCl2, the precursor ion scans were recorded between 100 and 700 mass units, and the system was operated in the positive ion mode. The collision energy was optimized to 13 eV for the precursor ions of acylium ions with m/z 167, 181, 185, and 199. On the other hand, the precursor ion scans of the sodiated compounds 1-3 were obtained without post-column infusion of CuCl2 and recorded between 100 and 900 mass units. The collision energy was set to 45 eV for the precursor ions with m/z 207, 221, 405, 419, 589, 603, and 617, as previously reported.40 A precursor ion scanning experiment was also performed with the copper(II) complex of nigericin by using UPLCMS/MS with post-column infusion. After that, the developed methodology was applied in a crude extract obtained from Streptomyces sp. Eucal-26 cultured in Czapek medium.41 A sample (10 µL) of the crude extract was injected into an Ascentis Express C18 column (4.6 × 100 mm, 2.7 µm) from Supelco. The gradient elution program started with 80% methanol. The amount of B was raised to 95% in the following 6 min. Next, the column was eluted with 95% methanol for 5 min and was then returned to the initial condition of 80% methanol within the following 5 min. The precursor ion scans were recorded between 200 and 850 mass units, and the system was operated in the positive ion mode. The collision energy was set to 25 eV for the precursor ions with m/z 755 and 585.

RESULTS AND DISCUSSION In the present study, we first aimed to verify whether the redox properties of copper(II) could contribute additional information to the structural elucidation of macrotetrolides as compared to sodium. For this purpose, we examined how copper(II) affected the fragmentation of isodinactin (1). In contrast to other polyether ionophores like monensin and nigericin, isodinactin (1) and related macrotetrolides bear an undissociated carboxyl group. Consequently, the copper(II) ion does not counteract the negative charge to give a singly charged ion. Instead, the copper(II) complex ([M+Cu]2+) of isodinactin (1, MW 764.9) emerges at m/z 414 as a doubly charged ion in the full-scan mass spectrum (data not shown). Figure 2 shows the collision-induced dissociation (CID) spectra of the ions with m/z 414 ([1+Cu]2+) and m/z 787 ([1+Na]+) obtained at 20 eV. The product ions of [1+Na]+ (Figure 2a) resulted from consecutive eliminations of 184 (C10H16O3) and/or 198 (C11H18O3) mass units, produced directly from the precursor ion (m/z 787) via consecutive McLafferty-type rearrangement, as reported previously.40 In contrast, the product ion spectrum of [1+Cu]2+ (Figure 2b) displayed a very different set of ions with even and odd masses. Besides the neutral losses of 184 or 198 mass units, the spectrum also revealed radical losses of 99 or 113 mass units and the product ions of m/z 167 and 181. Moreover, product ions with m/z higher than the m/z of the precursor ion (m/z 414) indicated that copper(II) was reduced to copper(I) after the CID process. This observation attested to the previous observations reported for Monensin A and B.38

Figure 2. (a) MS/MS spectrum of sodiated compound (1) (precursor ion with m/z 787) and (b) MS/MS spectrum of compound (1) with one equivalent of CuCl2 (precursor ion with m/z 414). Proof that copper(II) induces gas-phase fragmentation reactions in isodinactin ([1+Cu]2+) motivated us to investigate how copper(II) impacts the fragmentation of the homologous macrotetrolides trinactin ([2+Cu]2+) and tetranactin ([3+Cu]2+) (Figures S1 and S2). Table S1 lists the accurate-mass data of the main product ions of compounds 1-3 complexed with copper(II) ([M+Cu]2+) and their relative intensities in the corresponding product ion spectrum obtained at 20 eV. We did not include ions with relative intensity lower than 5%. The major fragmentation routes were preceded by opening of the macrotetrolide ring via two competitive mechanisms: a McLafferty-type rearrangement42 (pathways I and III) and a remote hydrogen rearrangement42 (pathways II and IV), presented in Schemes 1 and S3. Considering that two different mechanisms (McLafferty-type rearrangement and remote hydrogen rearrangement) could

Figure 3. a) UPLC-MS profile after the direct addition of copper(II) to the solution of macrotetrolides; b) UPLC-MS profile after the copper(II) post-column infusion experiment (chromatographic peaks 1-3 refer to the copper(II) and sodium complex of macrotetrolides). cleave four C‒O bonds, a total of eight major intermediate



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Q (m/z 199) (Scheme S3). This mechanism resembled the mechanism used to explain the formation of peptide radical cations from Cu(DIEN)(peptide)2+ complexes.43,44 The distonic intermediate ions A1a (Scheme 1) and A3a (Scheme S3) underwent fragmentation by α‒cleavages or remote hydrogen rearrangements. These cleavages resulted in the acylium ions C and E (m/z 167 and/or 181), in accordance to the Odd-Electron Rule.45 Schemes S3, S4 and S5 depict the formation of the other product ions of [macrotetrolides+Cu]2+. The copper-induced gas-phase fragmentation reactions generated a series of product ions that were extremely useful to identify the macrotetrolides 1‒3. Eliminations of 184 and 198 units helped to establish the sequence of nonactic and homononactic acid residues in the macrotetrolides. In addition, copper(II) promoted novel fragmentation pathways as compared to Na+ and induced formation of diagnostic acylium ions

ions could originate from the precursor ion. Here, we will discuss the formation of the most intense product ions considering only four intermediate ions (A1 and A2, Scheme 1; A3 and A4, Scheme S3), as well as the formation of acylium ions (C and P, Scheme 1; E and Q, Scheme S3). We tentatively considered other four intermediate ions (data not shown), but they could not explain the formation of all the most important product ions of [M+Cu]2+ of macrotetrolides. As a result of the redox properties of copper(II), singly charged product ions with higher m/z than the doubly charged precursor ion ([M+Cu]+2) emerged for ionophores 1-3. Reduction of copper(II) to copper(I) in the gas-phase involved homolytic cleavage of the coordinative bond between copper and the carbonyl oxygen. This cleavage can produce the intermediate distonic radical ions A1a (Scheme 1) and A3a (Scheme S3) or form the acylium ions P (m/z 185/199) (Scheme 1) and 2 O R1

3 O

2+

O5

Cu

path I

2+

O

O

HO 8

R2 R3 (I)

A1

O 1O 12 H

H

O

11 O

(II)

O 10

O

8O

A

A2

O

O O

R1

O

+

O R3

O

Cu+

O R1

R2

O O

R3

O

R4

O

O

Cu

O

O

O O

R4 O

6 O7 O

O 9 R3

O

3 O

R1

4 C Cu2+ O OH 5 R2

R4

path II

O

Cu O

6 O7 O

O 9 R3

O O

R4

R2

O 10

R1

O

O

4 O

11 O

2 O

O

O 1O 12 R4

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O

O

HO

O C

N 1,2: m/ z 642 3: m/ z 656

A1a

+ O O R4 O

O

R1

O Cu+

O

O

O

O

O

R3

R2

B 1: m/ z 646 2,3: m/ z 674

O HO

O

O

R2 OH

C 2: m/ z 167 1,3: m/ z 181

P 1: m/ z 185 2,3: m/ z 199

Scheme 1. Proposed mechanism for the formation of distonic ions B and N, and acylium ions C and P.


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that were relatively intense in the product ion spectra. The acylium ion with m/z 199 emerged in the product ion spectra of [1+Cu]2+,[2+Cu]2+, and [3+Cu]2+ (Table 1). On the other hand, the acylium ion with m/z 167 (E for 1 and C for 2) appeared only in the product ion spectra of [1+Cu]2+ and [2+Cu]2+, so its absence could be considered diagnostic for tetronactin (3). The diagnostic acylium ion with m/z 185 (P) arose only for [1+Cu]2+ and distinguished 2 from 1. To verify the potential application of copper(II) to elucidate the structure of macrotetrolides, a mixture of compounds 1-3 was added to CuCl2 and analyzed by UPLC-MS. Based on Figure 3a, the ESI-MS spectra of the peaks corresponding to the macrotetrolides showed that the peak due to the ion [M+Na]+ was much more intense than the peak due to [M+Cu]2+, The higher affinity of macrotetrolides for sodium(I) as compared to copper(II) stemmed from stronger ionic inter-

Figure 4. a) Total ion current chromatogram of an unknown sample obtained from Streptomyces sp. AMC-23. b-e) Extracted ion chromatograms obtained from the precursor ion scans (acylium ions with m/z 181, 199, 185 and 167, respectively). action between the oxygen atoms (hard base) of the macrotetrolides and the sodium cations (hard acid) according to the HSAB principle. To increase the relative intensity of [M+Cu]2+ as compared to [M+Na]+, we employed a postcolumn copper(II) infusion strategy. Post-column methodologies have also been used to enhance the detection of low concentrations of natural products during dereplication studies of complex matrixes such as poliacetylenes.46 As shown in Figure 3b, the peak due to [M+Cu]2+ became more intense than the peak corresponding to [M+Na]+ after post-column infusion of CuCl2.

macrotetrolides 1-3. m/z 167 m/z 181 m/z 185 m/z 199

1 E C P Q

2 C E --P, Q

3 --C, E --P, Q

The precursor ion scan (PIS) technique is a powerful approach to detect and characterize untargeted natural products during qualitative analysis.47 Here, we developed a PIS method to detect and identify the macrotetrolides 1-3 rapidly in an unknown sample of Streptomyces sp. AMC-23 by UPLC-ESIMS/MS. For this purpose, we set the third quadrupole to select the diagnostic acylium ions with m/z 167, 181, 185, and 199 generated in q2 by CID from [M+Cu]2+, which emerged when the post-column infusion of CuCl2 was used. After that, we set the first quadrupole to scan all the precursor ions that produced these acylium ions. Figure 4a illustrates the Full-Scan experiment in the positive ion mode of an unknown sample of a crude extract from Streptomyces sp. AMC-23. To identify the peaks due to macrotetrolides, we first used the PIS mode to monitor the acylium ions with m/z 181 and 199 selectively. These ions were common to the macrotetrolides 1-3 (Table 1). Monitoring of these ions revealed peaks at 8.5, 9.0, and 9.4 min, which indicated that these compounds were indeed macrotetrolides (Figures 4b and 4c). On the other hand, the peak at 8.5 min only emerged upon monitoring of the acylium ion with m/z 185, which is diagnostic for isodinactin (1) (Figure 4d). Finally, monitoring of the acylium ion with m/z 167 resulted only in peaks at 8.5 min and 9.0 min (Figure 4e). Comparison of these data with the data in Table 1 enabled us to identify the peaks at 8.5, 9.0, and 9.4 min as being due to the macrotetrolides isodinactin (1), trinactin (2), and tetranactin (3), respectively. Hence, precursor ion scanning of these copper-induced acylium ions led to detect macrotetrolides 1-3 in a crude extract from Streptomyces sp. AMC-23 selectively. Table 2. Common and diagnostic product ions for the sodiated macrotetrolides 1-3. 1 2 3 m/z 207 + + ‒ m/z 221* + + + m/z 405 + + ‒ m/z 419* + + + m/z 589** + ‒ ‒ m/z 603 + + ‒ m/z 617 ‒ + + +: observed; ‒: not observed; *: common product ions, **: diagnostic product ions. Although copper(II) can induce the formation of diagnostic ions for compounds 1-3, the fragmentation of sodiated 1-3 has also been reported to result in diagnostic product ions.40 The product ions with m/z 221 and m/z 419 were common to sodiated 1-3, whereas the product ion with m/z 589 was diagnostic for 1. The product ions with m/z 207, 405 and 603 only originated from the sodiated macrotetrolides 1 and 2. On the other hand, the ion with m/z 617 was only observed in the product ion spectra of 2 and 3 (Table 2).

Table 1. Diagnostic copper(II)-induced acylium ions for



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To compare the potential application of the diagnostic product ions of [M+Na]+ to identify the macrotetrolides 1-3 by using the PIS strategy, we analyzed the same crude extract of Streptomyces sp. AMC-23 by UPLC-ESI-MS/MS without adding CuCl2 and using the precursor ions listed in Table 2. Figure 5a contains the resulting full-scan chromatogram in the positive ion mode. Then, we performed PIS experiments to monitor the product ions with m/z 221 and m/z 419, which were common to compounds 1-3 (Table 2). The PIS chromatogram obtained by using m/z 221 revealed the presence of four peaks at 8.8 min (m/z 773), 9.3 min (m/z 787), 9.7 min (m/z 801) and 9.9 min (m/z 815) (Figure 5b). These data suggested that compounds 1, 2 and 3 corresponded to the peaks at 9.3, 9.7 and 9.9 min, respectively. However, when we used the ion with m/z 419 in the PIS experiments, the peak at 9.3 min, which could be due to compound 1, did not appear (Figure 5c). In addition, the peaks at 6.9 min (m/z 419) and 10.4 min (m/z 815), which we had not detected when q3 was set to monitor the product ion with m/z 221, emerged. Moreover, the peak at 10.4 min had the same m/z as the peak at 9.9 min. The results of these two PIS experiments were not conclusive because both the peaks at 9.9 min and 10.4 min could be due to compound 3 (m/z 815). Furthermore, monitoring of the product ion with m/z 419 did not detect any peak corresponding to compound 1 (m/z 787), which made additional PIS experiments with the product ions m/z 207, 405, 589, 603 and

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O

O

O

O

O O HO O

OH

+ O- Na

O

Figure 6. Chemical structure of the nigericin sodium salt.

Figure 7. Product ion spectrum of nigericin (precursor ion with m/z 786).

Figure 5. a) Total ion current chromatogram of an unknown sample obtained from Streptomyces sp. AMC-23. b-h) Extracted ion chromatograms obtained from the precursor ion scans (product ions with m/z 221, 419, 207, 405, 589, 603 and 617, respectively).

617 necessary. When we set q3 to monitor the diagnostic product ion m/z 589, we identified the peak at 9.3 min as compound 1. However, PIS experiments involving the product ions m/z 207, 405, 603, and 617 were unexpectedly identical and nonselective, so it was not possible to assign the peaks at 9.7 min and 9.9 min to compounds 2 and 3, respectively, by the PIS experiments only. We had to distinguish these compounds on the basis of their different m/z values. Therefore, the PIS strategy based on the acylium ions produced from [M+Cu]2+ allowed us to identify the macrotetrolides 1-3 for relatively faster (fewer PIS steps) and more selectively as compared to the PIS strategy based on the product ions derived from [M+Na]+. Moreover, despite the additional complexity of the radical ion reactions induced by copper(II) as compared to Na+, the acylium ions originating from [M+Cu]2+ were structurally simpler and highly diagnostic for compounds 1-3. In addition, the post-column infusion of CuCl2 that was used to enhance the intensity of [M+Cu]2+ was a very simple strategy that was not time-consuming. To explore the potential of copper(II) to induce the fragmentation of other polyether ionophores, we extended our study to nigericin (Figure 6), which exhibits antibacterial,48 antifungal,49 and anti-HIV activities,50 among others. As in the case of the macrotetrolides, the product ions spectrum of [nigericin+Cu]2+ (Figure 7) resulted in a series of odd-electron product ions, such as m/z 755 and m/z 585 (Scheme S6), due the ability of copper(II) to undergo reduction to copper(I). Schemes S6, S7 and S8 show the formation of other product ions of [nigericin+Cu]2+. PIS experiments with the ions m/z 755 and m/z 585 helped to detect nigericin in a crude extract of Streptomyces sp. Eucal-26 (Figure 8). These ions were chosen because they provided a relatively intense peak in the product ion spectrum of [nigericin+Cu]2+, and because copper(II) induces their formation. The experiments allowed us to identi-


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fy nigericin selectively as the peak at 9.8 min of the chromatogram, which corresponded to [nigericin+Cu]2 (m/z 786).

Author Contri Contributions The manuscript was written through contributions of all the authors. All the authors have approved the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are grateful to the Brazilian Foundation CNPq for providing fellowship to EJC (grant no. 160035/2011-2). AEMC is grateful to FAPESP for financial support to develop this research (grant no. 2013/20094-0).

REFERENCES

Figure 8. a) Total ion current chromatogram of an unknown sample obtained from Streptomyces sp. Eucal-26. b-c) Extracted ion chromatograms obtained from the precursor ion scans (product ions with m/z 755 and 585, respectively).

CONCLUSIONS We successfully generated copper(II) complexes of the investigated macrotetrolides by ESI-MS by adding copper(II) salt to the solution of ionophores. Accurate-mass electrospray tandem mass spectrometry yielded a different set of product ions with even and odd masses from doubly charged [M+Cu]2+ precursor ions. The gas-phase redox process reduced copper(II) to copper(I) and led to new fragmentation pathways with formation of highly diagnostic acylium ions for macrotetrolides. The PIS strategy based on the diagnostic copper-induced acylium was easily set up to monitor the macrotetrolides 1-3 in a crude extract of Streptomyces sp. AMC 23 in a fast and selective way. Application of the same PIS strategy based on two different copper-induced product ions to monitor nigericin in a crude extract of Streptomyces sp. Eucal-26 was also successful. In addition to the results presented herein, the UPLCESI-MS/MS protocol based on post-column infusion could be useful to identify ionophore metabolites in microbial crude extracts from new derivatives synthesized by genetically engineered systems at early stages.

ASSOCIATED CONTENT Supporting Information Available: The following material is available free of charge via internet at http://pubs.acs.org: Mass spectra of compounds 2 and 3 coordinated with copper(II), formation of the product ions of the copper(II) complexes with macrotetrolides 1-3 and nigericin and ESIQTOF-MS/MS data of copper(II) complexes of compounds 1-3 and nigericin.

AUTHOR AUTHOR INFORMATION Corresponding Au Author * E-mail: [email protected]. Phone: +55 (16) 3315-3747.

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