Analytica Chimica Acta 713 (2012) 103– 110

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Analytica Chimica Acta

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Direct affinity screening chromatography–mass spectrometry assay foridentification of antibacterial agents from natural product sources

Kevin A. Schuga,!, Evelyn Wanga, Sijia Shena, Sunaina Raob, Stephanie M. Smithb,Laura Huntb, Laura D. Mydlarzb,!!

a Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USAb Department of Biology, The University of Texas at Arlington, Arlington, TX, USA

a r t i c l e i n f o

Article history:Received 26 September 2011Received in revised form16 November 2011Accepted 16 November 2011Available online 25 November 2011

Keywords:Natural productsNoncovalent interactionsElectrospray ionizationDynamic titrationPseudoplexaura porosa

a b s t r a c t

A direct affinity screening – mass spectrometry assay, coupled to liquid chromatography, is presented as atool for natural product drug discovery. Using the assay, fractionated extracts from a Caribbean gorgoniancoral were shown to contain a new chemical entity (NCE) which binds to a mimic of the Gram positivebacterial cell wall (lysine–d-alanine–d-alanine). Conditions for observation of a specific noncovalentcomplex between the NCE and the target mimic using electrospray ionization-mass spectrometry werevalidated in a series of positive and negative control experiments, which featured flow injection analysis-based titrations. While the structural identity of the NCE could not be determined due to limited samplequantities, this work provides proof-of-principle for such an approach to potentially accelerate drugdiscovery from natural product sources.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Electrospray ionization (ESI) is well known as a soft-ionizationsource for mass spectrometry (MS). It has been used extensivelyfor the interrogation of noncovalent interactions in a wide varietyof small molecule and macromolecule-based molecular recogni-tion systems [1–3]. In this context, ESI-MS is widely recognizedfor enabling fast and sensitive analysis, as well as direct access tointeraction stoichiometry, without the need for labeling or immo-bilization strategies. The ability to handle complex mixtures andextract quantitative information is further enhanced by couplingESI-MS with techniques, such as flow injection analysis (FIA) andhigh performance liquid chromatography (HPLC) [4].

The use of ESI-MS as a versatile detection tool in a variety ofdirect or indirect affinity screening approaches has been broadlytermed affinity selection-mass spectrometry (AS-MS) [5]. DirectAS-MS refers to an experimental set-up where an ionic noncovalentcomplex of interest is transferred intact into the mass spectrom-eter for direct analysis and manipulation. Different approaches

! Corresponding author at: 700 Planetarium Pl.; Box 19065; Arlington, TX 76019-0065, USA. Tel.: +1 817 272 3541; fax: +1 817 272 3808.!! Corresponding author at: 501 S. Nedderman Dr.; Box 19498; Arlington, TX

76019-0498, USA. Tel.: +1 817 272 0397; fax: +1 817 272 2855.E-mail addresses: kschug@uta.edu (K.A. Schug), mydlarz@uta.edu (L.D. Mydlarz).

including competitive binding, titration, host–guest screening, andgas phase tandem mass spectrometry can be used in this context[3]. In contrast, indirect AS-MS methods involve, initially, the iso-lation of molecules of interest from a complex matrix by selectiveaffinity extraction, followed by the liberation of these moleculesand their detection using mass spectrometry, perhaps in conjunc-tion with HPLC separations. Overall, the various direct and indirectmethods are characterized by different advantages and limitations.While indirect methods may be more amenable to high through-put screening, direct methods provide better confidence in thespecificity of the observed interaction and allow their further inter-rogation using full scan and tandem mass spectrometry techniques.Both direct and indirect approaches have found significant use indrug discovery programs, in industry and in academia [5,6].

An active area of drug discovery, in general, is the pursuitof new chemical entities (NCEs) for treating infections causedby bacteria. Drug-resistant bacteria, including the Gram positivevancomycin-resistant Enterococci (VRE) and methicillin-resistantStaphylococcus aureus (MRSA) bacteria, have been identified as highpriority pathogens due to an increased incidence of infection andthe lack of available drugs for effective treatment [7]. The resultof such infections are prolonged hospital stays, increased heathcare costs, and the use of secondary and tertiary treatment optionswhich may be less effective and more toxic than preferential ones.Yet, discovery of NCEs appears to be impeded by the economically-driven abandonment of antimicrobial drug discovery efforts by

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2011.11.038

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large pharmaceutical companies [8,9]. In fact, only two new classesof antibiotics have been approved for use in the past 20 years.Thus, a pressing need has arisen for new methods to rapidly iden-tify NCEs as antibiotic lead compounds against these pathogenicorganisms.

A common mode of antibacterial activity for treating Grampositive bacteria is the inhibition of cell wall synthesis [10].There is a wealth of literature that describes the interactionsbetween well-known antibiotics, such as macrocyclic glycopep-tides, and specific small peptide target motifs of Gram positivebacterial cell walls [11–14]. Work performed using direct AS-MS has also contributed significantly to this body of knowledge[15–17]. The binding of macrocyclic glycopeptide antibiotics todepsipeptide mimics of intermediate peptidoglycan cell-surface-synthesis structures in these studies has validated models forcontinuing such work. During peptidoglycan synthesis in normalGram positive bacteria, ligase enzymes facilitate the presenta-tion of terminal l-lysine–d-alanine–d-alanine (Kaa) motifs whichprovide coupling and crosslinking points for the constructionof the peptidoglycan cell wall, as well as sites for inhibi-tion by some antibiotics. Furthermore, there is good correlationbetween the results of target models showing binding of di-and tripeptides models (N-terminal and !-Lys blocked andunblocked variants) with antibiotics such as vancomycin, risto-cetin, and teicoplanin and in vitro bacterial growth inhibitiondata.

Pharmaceutical companies once relied heavily on the discoveryof new chemical entities (NCEs) from terrestrial and marine nat-ural product sources [18,19]. More recently, efforts have shiftedaway from the natural products realm to combinatorial syn-thesis and automated screening technologies, citing better costeffectiveness for these approaches [20]. Traditional methods of“grind-and-find” discovery rely heavily on poorly selective andresource intensive bioassay-guided fractionation schemes thathave significant drawbacks. Still, the fact that more than half ofapproved pharmaceuticals can be linked directly to some naturalsource, or a derivative thereof, emphasizes a significant sup-ply of chemical diversity which is unlikely to have been fullyexplored [21]. The development of new methods which can moredirectly identify promising NCEs have and will continue to benecessary to sustain a resurgence of research into this field[22].

In this work, we report proof-of-principle for a direct affin-ity on-line chromatography–mass spectrometry screening assaywhich can be used to screen natural product extracts for the pres-ence of NCEs. A series of positive and negative control experimentsdemonstrated the potential to detect antibacterial compoundswhich inhibit cell wall synthesis by an AS-MS technique termeddynamic titration [4,23]. To apply this technique to the discovery ofNCEs, we screened crude extracts from a range of Caribbean coralsfor antibacterial activity against human and marine pathogenicbacteria. As a result of these screens one Caribbean gorgoniancoral, Pseudoplexaura porosa, showed promising data and was tar-geted to apply the AS-MS assay. Data from the HPLC–ESI-MS assaycorrelated with results of standard growth inhibition assays atdifferent levels of fractionation. The results indicated the pres-ence of a previously unreported compound, identified by selectivecomplexation in the HPLC–ESI-MS assay and by accurate mass anal-ysis in an ion trap-time-of-flight-mass spectrometer (IT-TOF-MS),in a fraction which has been shown to significantly inhibit thegrowth of normal and drug-resistant bacterial strains. Thus, thereported direct AS-MS assay was demonstrated to be a viable way tobypass or enhance traditionally time-consuming bioassay-guidedfractionation approaches and facilitate identification of potentialNCEs from natural product sources earlier in the drug discoveryworkflow.

2. Experimental procedures

2.1. Dynamic titration control experiments

A flow injection analysis AS-MS titration analysis was performedto confirm the ability of the instrument to detect noncova-lent complexes between a series of macrocyclic glycopeptidesantibiotics and the bacterial cell wall target mimic, Acetyl-lysine(acetate)–d-alanine–d-alanine (Ac2Kaa; monoisotopicmass = 372.200885 Da) (Bachem, Torrance, CA). The macrocyclicglycopeptides, vancomycin, ristocetin, teicoplanin, and teicoplaninaglycone (Advanced Separation Technologies, Whippany, NJ) areconsidered positive controls based on their interactions withGram positive peptidoglycan cell wall synthesis intermediates. Asnegative controls, antibiotics which should not bind to the targetmimic due to their known ribosomal inhibition activity, chloram-phenicol, gentamycin, neomycin, spectinomycin, and tetracycline(Duchefa Biochemie, Haarlem, The Netherlands) were evaluated.To perform dynamic titration, the host antibiotic was delivered in acontinuous flow by a syringe pump (in 100% water + 5 mM ammo-nium acetate; 30 "L min"1) at a constant concentration (20 "M).A known amount of guest depsipeptide Ac2Kaa (1.00 # 10"9 mol)was injected (2 "L injection) into the flowing stream. The mixtureof host and guest was then passed through a 200 "L volume ofblue PEEK tubing and allowed to band-broaden prior to reachinga conventional electrospray source interfaced with a LCQ DecaXP ion trap mass spectrometer (Thermo Fisher Scientific, WestPalm Beach, FL). The data from the formed temporal compositionalgradient of guest and host were extracted from the Xcalibur soft-ware (Thermo Fisher Scientific) and analyzed. For systems where ahost–guest complex could be observed, a dissociation constant forthe interaction was determined using an in-house-built softwareprogram. Specific details of the software, including the use of amodified Gaussian distribution function to fit the data, have beendescribed elsewhere [4,23]. LC–MS grade water was from Burdick& Jackson (Muskegon, MI) and ammonium acetate (NH4OAc) wasfrom Sigma–Aldrich (St. Louis, MO). ESI-MS source conditionswere optimized separately for robust observation of complexformation between vancomycin (10 "M) and Ac2Kaa (10 "M) bydirect infusion.

2.2. Sample collection, preparation, and fractionation

The gorgonian coral P. porosa was collected using SCUBA fromthe Looe Key Reef research site in the Florida Keys, USA in Julyof 2008 under the specifications of a Florida Fish and WildlifeConservation Commission nonresident saltwater fishing license.Gorgonian fragments (5 cm each from 5 individual coral colonies)were collected at depths of 5–10 m. All specimens were identifiedby L.R. Hunt, L.D. Mydlarz, and E. Bartels. Coral fragments were flashfrozen in liquid nitrogen and shipped on dry ice to the University ofTexas at Arlington, and stored at "80 $C until use. At the Universityof Texas at Arlington, samples from multiple coral colonies werepooled, lyophilized on a VirTis Benchtop K lyophilizer (The VirTisCompany, Gardiner, NY) and ground in a mortar and pestle to afine powder. 100% ethanol (Decon Labs, Inc., King of Prussia, PA)was added at a ratio of 10 mL to every 0.2 g of homogenized coral.Extracts were then transferred to pre-weighed vials, evaporated todryness under nitrogen, and final weights determined. All sampleswere diluted to a stock concentration of 100 mg mL"1 and stored at"20 $C until further analysis.

The extract was reconstituted in 20% aqueous ethanol and sub-jected first to reversed phase solid phase extraction (SPE). A stepfractionation/elution procedure was used to reduce the complex-ity of the sample and obtain samples for testing. SPE was carriedout using Bakerbond ODSII C18 (500 mg sorbent, 6 mL solvent

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capacity) (J.T. Baker, Phillipsburg, NJ) reversed phase pre-packedextraction cartridges. The cartridges were first conditioned with10 mL of methanol and then equilibrated with 10 mL of 99/10.5% acetic acid/methanol. The reconstituted extract was splitinto six portions and applied to six separate SPE cartridges toavoid column overload. The sample was loaded and fractions werecollected following sequential elution in 5 mL aliquots by 99/1,75/25, 50/50, 25/75, and 0/100 0.5% acetic acid/methanol (v/v),followed by 10 mL (2 # 5 mL) of 100% n-propanol. Aligned frac-tions were recombined following collection. Methanol (MeOH)and glacial acetic acid were from J.T. Baker. n-Propanol (nPrOH)was from Burdick & Jackson. Each fraction was evaporated todryness under nitrogen and/or by lyophilization (depending onorganic solvent content) and dry weight was recorded. The solu-tions were then reconstituted for analysis by the growth inhibitionassay and the HPLC–ESI-MS direct affinity assay, as describedbelow.

Semi-preparative HPLC was used to further fractionate frac-tion 9 to track the presence of a potential NCE identified by theAS-MS assay. A Finnigan Spectrasystem HPLC and UV6000LP photo-diode array detector (Thermo-Fisher Scientific, Inc., Waltham, MA)connected on-line to a Foxy, Jr. fraction collector (Teledyne-Isco,Inc., Lincoln, NE) was used to fractionate the sample in discreteelution time segments. A 30-min gradient mobile phase com-position (0–3 min, hold 75:25 mobile phase A:mobile phase B;3–18 min, linear gradient to 1:99 A:B; 18–30 min, hold at 99% B;where mobile phase A was 100% water and mobile phase B was90% acetonitrile + 10% isopropanol) was used in conjunction witha Luna C18 (10 mm i.d. # 150 mm L, 10 "m dp) at a flow rate of3 mL min"1 to obtain 30 fractions in 1 min segments. Fractionswere evaporated to dryness by lyophilization and dry weight wasrecorded. Fractions were analyzed for the m/z ion signature of theNCE by HPLC–ESI-MS, and fractions containing the compound ofinterest were also subjected to the growth inhibition and AS-MSassays.

All samples and fractions were contained in amber vials to min-imize the exposure of any potentially photo-labile compounds tolight. All dry and/or reconstituted samples were stored at "20 $C,until they were used.

2.3. Growth inhibition assay

Bacterial growth inhibition activity was assessed using a bac-teria turbidity assay and conducted in sterile 96-well flat-bottommicrotiter plates (Greiner Bio-one, Monroe, NC). Ethanol extractsof P. porosa were diluted in nutrient media Difco marine broth (Bec-ton, Dikinson and Co, Le Pont de Claix, FR) or Luria Broth (LB, Miller,Novagen, Merck KGaA, Darmstadt, Germany), depending on thebacteria being tested, for a final concentration of 250 "g mL"1 perwell. All strains were either purchased from the American TypeCulture Collection (ATCC, Manassas, VA) and ATCC strain numbersare included, or received as generous gifts from K. Ritchie (MoteMarine Laboratory) and GenBank accession numbers are given forreference. Human pathogenic Gram positive bacteria included inthe study were Enterococcus faecalis (ATCC #29212), vancomycin-resistant Enterococcus faecium (VRE; ATC #700221), S.. aureus (ATCC#29213), Methicillin-resistant S. aureus (MRSA; ATCC #43300), andBacillus subtilis (ATCC #6051). Human pathogenic Gram negativebacteria were Escherichia coli (ATCC #10536) and Pseudomonasaeruginosa (PAO-PR1; ATCC #39018). Marine pathogenic Gramnegative bacteria were Vibrio alginolyticus (GenBank accession#X744690) and Serratia marcescens (PDL100; ATCC #BAA-632).All bacteria strains were handled in a biological safety cabinetand in accordance with the University’s Biosafety Level 2 policies.Overnight bacteria cultures, in exponential growth, were diluted toan optical density (OD600nm) of 0.2 and added to wells containing

extracts and fresh broth for a final assay volume of 200 "L per welland a concentration of 250 "g mL"1 extract, with a maximum of0.25% ethanol in each well. Assays were run in triplicate wells andexperiments contained bacteria with extract, along with the follow-ing controls: bacteria only, bacteria + positive control (commercialantibiotic), or bacteria + ethanol (to control for ethanol effects).Assays were run at appropriate temperatures for the select bacteria(37 $C for human pathogenic strains and 29 $C for marine strains).The plates were gently mixed, read over a period of 5 h at OD600with a Biotek Synergy 2 spectrophotometer, and analyzed usingGEN5 software (Bio-Tek Inc, Winooski, VT).

Growth inhibition of bacteria cultures was calculated by com-paring the growth rate (GR) of bacteria with coral extracts tobacteria with ethanol controls. The GR was calculated with thefollowing formula: 3.3log (tf/ti))/n), where, tf is final OD600, tiis initial OD600, and n = tf " ti. The linear portion of logarithmicgrowth was used to select the initial (t = 1 h) and final hour(t = 2 or 3 h) and was kept standard between runs within eachbacteria species. Percent growth inhibition was determined bycalculating the difference in GR of wells with P. porosa extractsto bacterial growth without extracts and only ethanol as vehiclecontrol.

2.4. Direct affinity screening by HPLC–ESI-MS

The direct affinity screening HPLC–ESI-MS assay was performedon a Surveyor HPLC (pump and autosampler; Thermo Fisher Sci-entific) coupled to the LCQ Deca XP ion trap mass spectrometerequipped with a conventional ESI source. An external syringe pump(KD Scientific, Holliston, MA) was placed in line with the solventflow to complete the experimental set-up shown in Fig. 1. Coralfractions taken from SPE were diluted 10-fold in 50/50 acetoni-trile/water + 5 mM NH4OAc for injection. The samples (25 "L) wereinjected into the HPLC, operated at 40 "L min"1 flow rate with agradient program starting at 75/25 v/v of mobile phase A/mobilephase B (mobile phase A was water + 5 mM NH4OAc + 0.5% HOAc;mobile phase component B was acetonitrile + 5 mM NH4OAc)(held for 3 min) and increasing to 1/99 A/B in 10 min, with asubsequent hold at this composition for 15 min, and then re-equilibration back to 75/25 A/B, prior to the next injection. TheHPLC column was a Tosoh TSKgel C18 (1.0 # 50 mm, 3 "m dp)(Tosoh Bioscience LLC, King of Prussia, PA). The syringe pump wasconsistently operated at 10 "L min"1 for all experiments, joinedinto the chromatographic flow path with a zero dead volumet-junction, to provide a total flow of 50 "L min"1 into the ESIsource.

The samples were first injected with no affinity ligand (instead,50/50 acetonitrile/water) added post-column (“Run 1” in Fig. 1) toascertain the major ion signals produced from the sample. Majorion signals were recorded by manually searching the total ionchromatogram for signals having an intensity above 5 # 105 ioncounts. After Run 1, the sample was injected again, but with 30 "MAc2Kaa (dissolved in 50/50 acetonitrile/water) added post-columnfrom the syringe pump (“Run 2” in Fig. 1). A manual calculation ofexpected complexes to be observed based on the sum of massesof the dominant signals from Run 1 with the mass of the ligand(372 Da) were manually searched in Run 2 data to ascertain thepresence of formed ionic complexes. The properties of compoundsidentified, which bound to the affinity ligand, were further inter-rogated by high resolution ESI-MS and tandem mass spectrometryas described below.

2.5. High resolution HPLC–ESI-MS and MSn

To aid in the characterization of sample components, identi-fied by the HPLC–ESI-MS screening assay as potential antibacterial

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Fig. 1. Experimental set-up for a direct AS-MS assay to identify compounds in coral extract which bind to a Gram positive cell wall synthesis target mimic.

agents, relevant fractions were also analyzed using HPLC–ESI-MSon a hybrid ion trap-time-of-flight-mass spectrometer (LCMS-ITTOF) coupled to a Prominence HPLC (Shimadzu Scientific Instru-ments, Inc., Columbia, MD) to obtain high resolution (R %10,000FWHM), mass accurate (<5 ppm error), full scan and tandem massspectrometry fragmentation information. Reversed phase HPLCwas carried out on a Shimpack XR-ODS C18 column (2.0 mmi.d. # 100 mm L, 2.2 "m dp) (Shimadzu). Fractions were reconsti-tuted to 0.2 mg mL"1 and 20 "L was injected by autosampler. Agradient separation was used where mobile phase A was the sameas above and mobile phase B was composed of 90/10 acetoni-trile/isopropyl alcohol. The flow rate was 250 "L min"1.

Compounds eluted from the column entered the ESI source,which was operated in the positive ionization mode. Three acqui-sition events were incorporated in a repeating duty cycle to firstcollect full-scan data, followed by MS/MS, and then MS3, in asoftware-automated data-dependent fashion. Thus, observed ionswere isolated and fragmented to obtain qualitative informationthroughout the chromatographic run. Priority was given to thoseions indicated by the HPLC–ESI-MS screening assay to have formeda stable complex with the affinity ligand. To aid in the assign-ment of possible elemental formulae for the compounds, FragmentPredictor software (Shimadzu) was used. This software uses highmass accuracy full scan and tandem mass spectrometric data, aswell as observed isotope abundances to match possible elemen-tal formulae to the ions of interest. The software also accountsfor standard rules [24] in assignment of elemental formulae basedon the nitrogen rule, double bond equivalence, and mass accuracytolerances.

3. Results and discussion

Identifying novel NCEs with antibacterial properties from com-plex mixtures of natural product extracts requires innovativemethods, which provide results in a more expedited fashion com-pared to traditional “grind and find” methodologies. To accomplishthis, a direct affinity on-line chromatography–AS-MS screeningassay, capable of handling complex mixtures from natural prod-uct extracts, was developed and a series of validation control andapplication experiments were performed. The specificity of ESI-MS for monitoring selective complexation between Ac2Kaa andselected antibacterial compounds (both positive and negative con-trols) was demonstrated using a series of quantitative bindingdeterminations performed by dynamic titration [4,23]. The basisof this approach is the use of flow injection analysis to create aprecisely dispersed zone of the guest compound (e.g. Ac2Kaa) inthe presence of a constant concentration of host (e.g. vancomycin),whereby, if complex formation is observed in the mass spectra,an appropriate model can be applied to determine the magnitude

of binding affinity between the host and guest. The multi-pointtitration in a continuous flow is facilitated by the temporal vari-ation of guest concentration (a “peak”) in the presence of host,as the mixture enters the mass spectrometer. A clear distinctionbetween commercial antibacterial compounds which were sup-posed to bind Ac2Kaa, versus those which were not, allowed usto next evaluate post-column addition of the affinity ligand toHPLC separations of coral extract fractions, to identify compoundsin the mixture which might also exhibit antibacterial activity.Once such a compound was identified, it was further interro-gated by high resolution HPLC–ESI-MS. Accompanying the resultsof the AS-MS assay, a traditional spectroscopic growth inhibi-tion assay was used to track the antibacterial activity of fractionscontaining the potential NCE. Described in detail below, all ofthe results were consistent with the identification of a poten-tially new antibacterial agent against normal and drug-resistantGram positive bacteria from the Caribbean gorgonian coral, P.porosa.

3.1. Dynamic titration control experiments

The experimental design for dynamic titration control exper-iments (Fig. 2A) includes its application for monitoring theformation of complexes between commercial antibacterial com-pounds and Ac2Kaa (Fig. 2B) and the calculation of binding affinity(dissociation constants) using an in-house-built software program.The program fits a modified Gaussian function to the flow injectionanalysis data to determine the concentration of guest at each pointon the curve. The signal of the complex recorded at each point for avariable concentration of guest and a constant concentration of hostgenerates a titration profile from which a dissociation constant canbe determined by fitting the data with a 1:1 solution phase bindingmodel [4,23].

Results of the control/validation experiments using commer-cially available antibiotic compounds are presented in Table 1. Fivecompounds known to inhibit bacterial growth through ribosomalinhibition were used as negative controls. No complex formationwas observed with the target ligand. Four macrocyclic antibiotics,known to inhibit cell wall synthesis in Gram positive bacteriawere used as positive controls. Significant complex formation wasobserved with the target mimic, and in each case, a dissocia-tion constant was also extracted. Values of dissociation constantsobtained for vancomycin and ristocetin were in good agreementwith those reported in the literature, determined using both stan-dard solution phase and traditional mass spectrometric bindingconstant determination techniques (%1 "M for vancomycin, and%2 "M for ristocetin, binding Ac2Kaa under similar solution phaseconditions [11–17]. These control experiments indicate that opti-mized ESI-MS instrumental parameters used in this experiment

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Fig. 2. Determination of binding constants by dynamic titration. (A.) A known amount of guest (N0,G) was injected in a continuous flow of the host compound (c0,H) andallowed to band broaden prior to entering the ESI source and mass spectrometer. (B.) Extracted ion chromatograms for the ionic complex formed between host and guestwere clearly visualized in three discrete injections over a period of approximately 30 min. (C.) Data for the complex normalized to that of the free host ion response were inputinto a software program which simultaneous fit a modified Gaussian function and a 1:1 host–guest binding model to determine the dissociation constant for the interactionsystem.

preserved the highly specific binding interactions between cell wallsynthesis inhibitors and Gram positive bacterial cell wall synthesistarget mimics, while potential false positives were minimized.

3.2. Broad-spectrum antibacterial activity of P. porosa crudeextracts

The gorgonian coral P. porosa has been investigated for poten-tial compounds with antitumor properties, mainly small moleculediterpenoids [25]. The potential for antibacterial properties havebeen limited to examining crude extracts against marine bacte-ria and marine fungi [26,27]. In this study, 100% ethanol extractsof the lyophilized corals were tested for their inhibition of growthagainst a suite of Gram positive and Gram negative bacteria, mainly

human and marine pathogens (Fig. 3). P. porosa extracts inhibitedthe growth of Gram positive human bacteria species more signif-icantly than marine strains, with activity against B. subtilis and S.aureus the highest. The inhibitory activity of the extracts againstEnteroccocus species were moderate, but significantly higher invancomycin-resistent Enterococcus, making E. faecalis and VRE goodtarget strains for continued assays after fractionation of the crudeextract since limited compound supply did not permit the use ofall strains.

3.3. Targeted fractionation and AS-MS of P. porosa extracts

A combination of SPE and HPLC fractionation procedures inconjunction with the direct AS-MS screening assay were used to

Table 1Positive and negative ESI-MS binding specificity validation experiments.

Antibiotic Inhibition mechanism Complex with Ac2Kaa Kd ± S.E. ("M) (n = 3)a

Chloramphenicol Ribosomal function No N/AGentamycin Ribosomal function No N/ANeomycin Ribosomal function No N/ASpectinomycin Ribosomal function No N/ATetracycline Ribosomal function No N/ARistocetin Cell wall synthesis Yes 6.8 ± 0.9Teicoplanin Cell wall synthesis Yes 0.4 ± 0.1Teicoplanin aglcyone Cell wall synthesis Yes 6.0 ± 0.8Vancomycin Cell wall synthesis Yes 2.8 ± 1.5

a In the flow injection analysis format, where complexes are formed, a dissociation constant can be calculated based on dynamic titration methodology [4,23].

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Fig. 3. Broad spectrum antibacterial activity of crude P. porosa extracts against Gram positive and Gram negative bacterial strains. Inset: P. porosa.

identify components in the crude extract which exhibited antibac-terial activity against Gram positive bacteria. First, the residue ofthe crude extract was reconstituted in 20% aqueous ethanol andsubjected to SPE. Eleven fractions were collected by increasing themobile phase strength in a step-wise fashion, as described in Sec-tion 2. Each fraction was either dried under N2(gas) or lyophilized(depending on water content of the eluent), and then reconstitutedin 100% ethanol to test for antibacterial activity against E. fae-calis and vancomycin-resistent Enterococcus. As mentioned above,growth inhibition assays were restricted to these bacterial speciesdue to limited sample quantity and because their inhibition wasthe most relevant to the incorporation of the Ac2Kaa target mimicligand in the AS-MS assay. The fractions each had different antibac-terial activity (Fig. 4). Fractions 1–4 were collected during thesample loading stage, and since no significant antibacterial activitywas recorded for each of these fractions, only fraction 1 is shownin Fig. 4. Fraction 9 exhibited the highest activity against E. faecalis(%70% inhibition), significantly greater than that recorded for thecrude extract. Significant inhibition of VRE was also recorded forthis fraction, although slightly less than the crude extract and ofthe vancomycin-sensitive strain. For initial evaluation and applica-tion of the AS-MS assay, the complete crude extract was deemedto be too complex to start with initially. Therefore, fraction 9 was

Fig. 4. Antibacterial activity, measured by percent growth inhibition, of crude andfractionated P. porosa extracts against E. faecalis and vancomycin-resistant Entero-coccus (VRE).

targeted for application of the AS-MS assay, as it exhibited thehighest antibacterial activity.

Fraction 9 was diluted 10-fold in water for injection into theHPLC system. For the first run, a blank 50/50 acetonitrile mixturewas added post-column instead of the Ac2Kaa ligand to deter-mine the m/z values associated with the major ion signals observedfrom the separation of the fraction. Expected values that might berecorded if 1:1 complex formation with the Ac2Kaa ligand occurredfor each of the observed m/z values in the first run (m/z +372) weremanually calculated. The diluted fraction was then injected a sec-ond time with the target mimic ligand added post-column and thepresence of signals for potential complex ions were monitored inthe ion chromatogram.

During the 20–22 min region of the ion chromatogram, a pos-sible complex was observed (m/z 1320–1328) between the targetmimic ligand and an envelope of signals with m/z 948–952, whichhad been observed in the first run (Fig. 5). The analysis wasrepeated several times and was shown to be consistent. Addi-tionally, collision-induced fragmentation of ions assigned as thenoncovalent complex returned the appropriate signals consistentwith those observed in Fig. 5A (m/z %950). No other complexes wereobserved between other components, represented as MS ion sig-nals, and the target ligand. The identification of a compound whichmay be responsible for the observed antibacterial activity of frac-tion 9, based on a m/z signature and specific binding to Ac2Kaain the AS-MS assay, provided a handle for further tracking of thecompound of interest in subsequent purification steps.

To demonstrate that the compound could be tracked using theAS-MS assay, but understanding that very limited sample quan-tities were available following SPE fractionation, an additionalround of fractionation was performed by semi-preparative HPLC.Approximately 1 mg of dry material was obtained from late elut-ing fractions. Upon reconstitution and analysis by HPLC–ESI-MS,fractions 25–28 contained the MS ion signature of the compoundof interest. Due to very limited amounts, these four fractions alongwith fractions 23 and 24 (as controls) were subjected to the antibac-terial growth inhibition assay against only one bacteria (E. faecalis)and with a concentration of 150 "g mL"1 instead of the typical250 "g mL"1. Of the fractions, fraction 25 exhibited the highestrelative inhibition with 41.2 ± 2.7% inhibition of growth. This wassignificantly higher than fraction 23 and 24 which did not con-tain the MS ion signature (15.8 ± 0.5% and 9.8 ± 1.6%, respectively).Fractions 26 through 28 did demonstrate some inhibitory effects

K.A. Schug et al. / Analytica Chimica Acta 713 (2012) 103– 110 109

Fig. 5. Components present in an ion envelope from P. porosa SPE fraction 9 (top and middle panel in (A.)) form a noncovalent complex with Ac2Kaa (middle panel in (B.))during direct AS-MS. The bottom panels of (A.) and (B.) (as controls) indicate that the signals of interest are confined to a discrete time frame.

against E. faecalis that was less than fraction 25 (range of 26.8 ± 2.6to 33.2 ± 2.8%) further supporting the HPLC–ESI-MS data that thesefractions did contain less of the signal. Since fraction 25 had thehighest relative inhibition of growth, it was then subjected to theAS-MS assay, and the results confirmed that a specific complexwith Ac2Kaa was observed, as before, in the expected elution time-frame. The fraction was not fully purified by the HPLC fractionation,as evidenced by additional impurities, which eluted through thechromatographic run (data not shown). The amount of materialremaining following HPLC fractionation, antibacterial assay, andAS-MS assay was insufficient for further processing. Nevertheless,the AS-MS provided consistent data through two rounds of fraction-ation and these results provided proof-of-principle for the viabilityof such an assay for directed tracking of the potential NCE.

3.4. High resolution HPLC–ESI-MS

Purity and quantity of material obtained from fractionationof the P. porosa extract precluded use of spectroscopic structuraldetermination tools, such as NMR, to determine the identity of theNCE of interest. Therefore high resolution mass spectrometry andtandem mass spectrometry was used to gain additional insight.Fraction 9 from the SPE fractionation was injected into a HPLC–ESI-ion trap–time-of-flight-MS instrument. The analysis indicated thatthe envelope of signals for the NCE observed during the AS-MSassay could be separated into two discrete compounds (Fig. 6).Each compound exhibited an interesting and atypical mass spec-tral pattern. The first peak was characterized by dominant ionsat m/z = 948.5652 and 953.5196 (!m/z = 4.9544), and the secondpeak was characterized by dominant ions at m/z = 950.5842 and955.5393 (!m/z = 4.9551). A circa 5 mass unit difference in thesignals is difficult to explain in terms of the make-up of a singlecompound, especially since each signal was characterized by itsown reasonable isotope distribution and identical retention times.Moreover, a closer inspection of the data related to complex forma-tion with Ac2Kaa from the AS-MS assay (shown in Fig. 5B) indicatedthat noncovalent binding occurred with the 950/955 m/z compound(major complex ion signals were observed at m/z 1322 and 1327;!m/z = 372). Thus, the two peaks shown in Fig. 6A seem to berelated, perhaps simply differing by degree of saturation some-where in the molecule, however only one of the compounds bindsto a significant degree with the target ligand.

Tandem mass spectrometry was applied to the 950/955 ion sig-nals, in an attempt to obtain further information which might be

used for elemental formula prediction. MS/MS of m/z 950.5829yielded three dominant product ion signals at m/z 677.3233(!m/z = 273.2596), 515.2595 (!m/z = 435.3234), and 405.1342(!m/z = 545.4487). MS/MS of m/z 955.5377 also yielded three dom-inant product ions at m/z 679.3242 (!m/z = 276.2135), 517.2723(!m/z = 438.2654), and 405.1313 (!m/z = 550.4064), as well asthree minor product ion signals at m/z 677.3127, 515.2556, and347.0890. Clearly, similar fragmentation patterns can be envi-sioned, and again, there seems to be minor differences simply inthe degree of saturation involved (circa 2 m/z unit shifts). Both m/z950.5829 and 955.5377 yielded a product ion of 405.13, indicatingthat the structural unit which corresponds to this fragment is likelyto be identical in each ion.

Using the high mass accuracy, fragmentation, and isotope ratiodata, Formula Predictor software could be used to predict pos-sible elemental formulae. However, given the large mass of thecompound of interest, the software produced many (>40 formulaewith mass accuracy error <10 ppm) reasonably ranked possibil-ities (restricting atoms in the compound to only C, H, N, andO). The top four ranked elemental formulae for m/z 955.5377were C33H47N14O18 (score 99.03; ppm difference = "0.10; double

Fig. 6. High mass accuracy MS on an HPLC–ESI-IT-TOF-MS instrument. Chromatog-raphy resolved two compounds (A.) in the ion envelope of interest, each of whichhad similar signal character. The mass spectrum for the first peak is shown in (B.)and that for the second peak is shown in. (C.)

110 K.A. Schug et al. / Analytica Chimica Acta 713 (2012) 103– 110

bond equivalents (DBE) = 4.0), C34H70N18O14 (score 98.58, ppm dif-ference = "1.57; DBE = 9.0), C37H78N8O20 (score = 95.17, ppm dif-ference = "2.93, DBE = 3.0), and C38H74N12O16 (score = 91.50, ppmdifference = "4.40, DBE = 8.0). Searches based on accurate mass andthese elemental formulae in the Dictionary of Natural Productsreturned no hits. Similar searches in SciFinder Scholar returnedsome similarly defined peptidic-based structures but no closematches. In the end, without a pure sample of sufficient quantityto apply a larger suite of structural and elemental determinationmethods, such as NMR, IR, X-ray, and thermogravimetric analy-sis, it is virtually impossible to discern the identity of a circa 1000molecular weight compound based on mass spectrometry alone.Unfortunately, the additional purification steps needed were pre-cluded by the size of the coral sample available for extraction.Future attempts will be made beginning with a larger quantity ofraw material.

4. Conclusions

This work demonstrated the potential of a new directchromatography–AS-MS assay for identification of antibacterialNCEs from natural product extracts. The assay was validatedby a quantitative dynamic titration approach, which served todemonstrate the optimal settings of the instrument for discrim-inating specific binding events. The assay performed against anatural product extract incorporated a well-characterized targetmimic (Ac2Kaa) as a probe for compounds that bind to the cellwalls of Gram positive bacteria to inhibit biosynthesis and bacte-rial growth. Thus, the assay could be used in direct purificationprocesses and provide some insight into the potential biologi-cal mechanism of compounds in the extracts and fractions. Themethodology/approach was supported by the successful identifi-cation of a novel NCE with broad-spectrum antibacterial activityin ethanol extracts from the Caribbean gorgonian coral P. porosa.Antibacterial activity was preserved and enhanced through a seriesof fractionation steps where focus was placed predominantly onthose fractions which contained a compound shown to bind withthe target mimic ligand by AS-MS. Even though this particular studywas hampered by limited material, this approach can increase theefficiency of the process related to identifying NCEs from naturalproduct sources by being able to analyze complex mixtures andutilizing far less material than traditional bioassay-guided frac-tion. Our lab is currently working on expanding synergistic affinityextraction media to increase selectivity in the extraction steps [28].In the present study, a target mimic ligand relevant to probingantibacterial activity against Gram positive bacteria was incorpo-rated in the assay, but in principle, this approach could be used in

any context where active compounds from complex mixtures canbe delineated based on their propensity for binding a target ligandusing ESI-MS.

Acknowledgements

KAS, EW, and SS acknowledge support from the NationalScience Foundation (CHE-0846310), the U.T. System NSF LouisStokes Alliance for Minority Participation (LSAMP) program, andthe Shimadzu Equipment Grants for Research program. LDMacknowledges funding from the National Science Foundation (OCE-0849799), UTA start-up funds and Research Enhancement Programsupport for LH, SMS and SR.

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