Synthesizing Novel Anthraquinone Natural Product-like Compounds

Apr 6, 2012 - Synthesizing Novel Anthraquinone Natural Product-like Compounds To Investigate Protein–Ligand Interactions in Both an in Vitro and in ...
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Synthesizing Novel Anthraquinone Natural Product-like Compounds To Investigate Protein−Ligand Interactions in Both an in Vitro and in Vivo Assay: An Integrated Research-Based Third-Year Chemical Biology Laboratory Course Nancy McKenzie,* James McNulty,* David McLeod,† Meghan McFadden,† and Naresh Balachandran Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, L8S 4M1, Canada S Supporting Information *

ABSTRACT: A new undergraduate program in chemical biology was launched in 2008 to provide a unique learning experience for those students interested in this interdisciplinary science. An innovative undergraduate chemical biology laboratory course at the third-year level was developed as a key component of the curriculum. The laboratory course introduces students to some of the fundamental aspects of chemical biology through a series of integrated, research-based laboratory experiments, which illustrate how different areas of the discipline are interconnected. The 12-week stand-alone laboratory course consists of a weekly 1-h lecture and a 4-h laboratory period. During the course, students synthesize a novel anthraquinone natural product-like compound, purify a protein, and perform both an in vitro and an in vivo biological assay to investigate protein−ligand interactions with these materials. Two iterations of the course have been completed with class sizes of 21 (2010) and 11 (2011) students. Because of the modular nature of the course (i.e., organic, biological, and analytical sections), it is widely applicable to a range of upper-level chemistry, biology, molecular biology, and biochemistry undergraduate courses.

KEYWORDS: Upper-Division Undergraduate, Biochemistry, Curriculum, Laboratory Instruction, Organic Chemistry, Bioanalytical Chemistry, Fluorescence Spectroscopy, NMR Spectroscopy, Proteins/Peptides, Synthesis

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hypothesized that TetR-like proteins that regulate antibiotic export mechanisms in antibiotic producing bacteria may serve as reservoirs for resistance genes in pathogenic bacteria.7 ActR interacts with actinorhodin, as well as biosynthetic actinorhodin precursor molecules (Figure 1A), to control the transcription of an actinorhodin efflux pump.7 Upon ligand binding, ActR undergoes a conformational change that prevents it from DNA binding, which leads to the activation of the resistance gene. This mechanism mediates self-resistance to the endogenously produced antibiotic. ActR has also been shown to interact with a variety of synthetic anthraquinone molecules (Figure 1B), indicating ligand-binding plasticity. The overall goal of the laboratory course is to further investigate the diversity of the ActR ligand binding site by synthesizing novel 1,4-dihydroanthracene-9,10-diones. Gaining insight into the

ntegrated research-based undergraduate laboratories develop critical-thinking skills, and enhance student learning and interest in scientific research.1−6 The third-year chemical biology laboratory course at McMaster University is an entirely integrated research-based course that was developed around the ActR protein, a regulator of antibiotic export genes in the bacterium Streptomyces coelicolor.7 S. coelicolor produces the blue pigmented type II polyketide antibiotic actinorhodin (Figure 1A).8 The export of actinorhodin is regulated by ActR, a TetR-like transcriptional repressor.7 TetR-like proteins belong to a large family of transcriptional regulators that are widespread in bacteria.9 The major function of these proteins is the regulation of antibiotic resistance. Antibiotic resistance in clinically relevant pathogens is now widespread, and drug efflux is a common mechanism of multidrug resistance.10 Horizontal transfer of resistance genes among bacteria is well documented, and it has been © 2012 American Chemical Society and Division of Chemical Education, Inc.

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Figure 1. (A) Actinorhodin and the biosynthetic precursor (S)-DNPA. (B) Anthraquinone and synthetic derivatives that interact with ActR.7

structure−activity relationship (SAR) analysis; and (iv) to foster a collaborative approach to working in the laboratory. The organic methodology involved three distinct synthetic steps (Scheme 1). Reaction I was an alkylation reaction of a reactive allyl bromide with triphenylphosphine to give a phosphonium salt. Reaction II involved the in situ generation of the derived ylide using the strong base lithium diisopropylamide (LDA). The derived ylide was trapped with one of four different 4-substituted benzaldehydes in a Wittig olefination process yielding one of four substituted-1,3butadiene products. The choice of aldehyde did not appear to be restricted in any way. Reaction III involved a Lewis acid promoted Diels−Alder cycloaddition reaction of the resulting 1,3-diene with 1,4-naphthoquinone (dienophile) to yield one of the desired structural analogs. The biological section involved isolating the actR plasmid from a cloning strain of E. coli using a QIAprep Spin Miniprep Kit from Qiagen. The DNA was quantified and subjected to restriction digestion analysis to verify the identity of the plasmid. The actR plasmid was transformed by the heat-shock method into an expression strain of E. coli and the ActR protein was purified using immobilized metal affinity chromatography. In the remainder of the course, the anthraquinone derivatives were analyzed for their ability to interact with ActR by both an in vitro fluorescence-based 8-anilino-1-naphthalenesulfonic acid (ANS) competitive displacement assay and an in vivo E. colibased bioluminescence assay. ANS is a fluorescent molecule that is extremely sensitive to changes in the polarity of its environment and can be used as a probe to investigate protein− ligand interactions.7,12−14 Upon binding to a ligand-binding site on a protein, the quantum yield of ANS increases resulting in an increase in fluorescence emission intensity and a blue-shift in the emission maximum wavelength. ANS was shown to interact relatively weakly with ActR7 and the published procedure was adapted for use in an undergraduate laboratory with a microplate reader. A ligand that competitively binds to ActR and displaces ANS will result in a decrease in fluorescence emission intensity and a red-shift in the emission maximum wavelength. A fully autonomous E. coli-based biosensor has been established to investigate small molecule−ActR interactions in vivo.11 The biosensor strain contains the pOactlux plasmid (plux) in which the luxCDABE operon from Photorhabdus luminescens is under the control of a synthetic promoter consisting of −10 and −35 elements flanking a known binding site for ActR, and a pactR plasmid that constitutively expresses

diversity of the ligand binding domain in ActR and other TetRlike proteins is important to understanding antibiotic resistance in clinically relevant pathogens if antibiotic-producing organisms do indeed serve as reservoirs for resistance mechanisms. We chose to develop a laboratory course based on the ActR system for the following reasons: (i) novel organic molecules related to ActR ligands can be synthesized using organic synthesis procedures based on fundamentally important organic reactions (alkylation reaction, Wittig olefination, Diels−Alder cycloaddition); (ii) the ActR protein can be overexpressed and purified from Escherichia coli in a single chromatographic step; (iii) the ActR protein is stable and can be stored for several weeks at 4 °C; (iv) in vitro and in vivo assays have already been developed to investigate protein−ligand interactions;7,11 (v) the ActR project is current and relevant and would expose students to a variety of interesting experiments, techniques, and topics (Table 1); and (vi) the ActR project would provide students with an opportunity to work on a novel, chemical biology integrated research-based project. This laboratory course is suitable for upper-level undergraduate students who have taken chemistry (organic, analytical, and biophysical) and biochemistry (proteins and nucleic acids) courses, and who have had previous organic and analytical laboratory experience. Our students do not have any microbiology laboratory experience prior to this course, which does not seem to hinder them.

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COURSE DESCRIPTION The course was divided into three sections: organic, biological, and analytical. Although the course was presented as a 12-week course, the modularity of the experiments provide flexibility in adapting the course at institutions that have fewer weeks devoted to laboratory instruction. In the organic section, students independently synthesized one of four novel structural analogues of the monomer of actinorhodin (Scheme 1). The students pooled their crude, final product and purified it together using gel permeation chromatography. Four groups of students synthesized the same compound and purified it together for the several reasons: (i) each student synthesized only a small quantity of material and some of the product was lost during the two purification steps; (ii) the synthesis section spanned four weeks of the course, and if a student failed to synthesize their product, they were still able to move forward and obtain a product to test and characterize; (iii) the results of the in vitro and in vivo assays were posted online, so students shared their results for 744

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NMR spectrometer

TLC; extraction; strong base/anhydrous reaction; inert atmosphere; silica gel chromatography TLC; extraction; silica and size exclusion chromatography

Part C: Investigating the Biological Effect of the Anthraquinone Derivatives in an in Vitro Fluorescence Assay and an in Vivo E. coli-Based Bioluminescence Assay Weeks 10−11: In Fluorescence microplate reader Fluorescence spectroscopy Fluorescence-based displacement assay; methods to analyze ligand binding in vitro; fluorescence vitro fluoresemission scans; determination of dissociation constants; Hammett substituents and semilog plots cence assay Week 12: In vivo Incubated shaker; UV−vis and lumi- Sterile technique; luminescence and absorbance spectroscopy Luminescence (e.g., chemiluminescence, radioluminescence, bioluminescence); alternative in vivo bioluminescence nescence microplate reader assays that analyze ligand binding; advantages and disadvantages of in vivo versus in vitro assays assay

Recombinant protein expression; protein affinity tags (e.g., His, HA, Flag, GST); IMAC; bacterial cell lysis (e.g., French pressure cell, sonication, enzymatic); protein stability; protein purification under denaturing conditions; buffer exchange and protein concentration (e.g., dialysis, centrifugal filter units)

Minipreps (alkaline lysis, Qiagen kit); E. coli cloning strains (e.g., XL1-Blue); cloning overview (e.g., PCR, ligation, plasmids, restriction digestion); agarose gel electrophoresis; DNA visualization by ethidium bromide staining Bacterial transformation (e.g., heat shock method, electroporation); expression strains [e.g., BL21(DE3)]; plasmid stability and copy number; transformation efficiency Subculturing (E. coli growth characteristics); pET expression system; factors influencing protein expression; small-scale trial expressions; protein solubility (inclusion bodies versus soluble protein)

Diels−Alder mechanism; stereospecificity; Lewis-acid catalysis; Sephadex LH-20 resin and size exclusion chromatography

Wittig mechanism; E/Z stereoselectivity; inert atmosphere

Lecture topics

NMR spectrometer

Techniques Alkylation; ylides (phosphorus ylides); anhydrous reactions

Instrumentation/Equipment

Anthraquinone Natural Product-like Compounds for Structure−Activity Relationship (SAR) Analysis NMR spectrometer Thin-layer chromatography (TLC); crystallization

Part B: Purification of Recombinant ActR Protein for Use in an ANS Displacement Assay with Anthraquinone Derivatives Week 5: Plasmid Incubated shaker; microcentrifuge; Plasmid isolation; DNA quantitation; restriction digestion; agarose gel isolation and UV−vis spectrophotometer; DNA preparation; agarose gel electrophoresis characterization electrophoresis apparatus Week 6: E. coli Microcentrifuge; incubator Sterile technique; LB agar plate preparation; DNA transformation transformation Week 7: SmallIncubated shaker; microcentrifuge; Sterile technique; bacterial growth and induction; buffer preparation; whole scale protein inUV−vis spectrophotometer; procell lysate preparation; SDS-polyacrylamide gel preparation; SDS-PAGE duction tein electrophoresis apparatus Weeks 8−9: ProIncubated shaker; refrigerated tabBacterial chemical lysis; immobilized metal affinity chromatography tein purification letop centrifuge; UV−vis spectro(IMAC; batch purification); dot blot assay; buffer exchange (centrifugal photometer; protein electrophorefilter unit); Bradford protein assay; SDS-polyacrylamide gel preparation; sis apparatus SDS-PAGE

Part A: Synthesis of Week 1: Ylide synthesis Week 2: Wittig reaction Weeks 3−4: Diels−Alder reaction

Laboratory Schedule

Table 1. Laboratory Schedule Overview, Instrumentation, Techniques, and Lecture Topics Covered in the Course

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Scheme 1. Synthetic Route for the Synthesis of Four Differentially Substituted Anthraquinone Natural Product-like Derivatives

laboratory. The use of a fume hood is mandatory for all organic reactions and for column chromatography. E. coli XL1-Blue and BL21(DE3) are classified as biosafety level 1 (BSL-1) organisms and pose low risk to students. All BSL-1 work is to be done by an open flame using standard aseptic technique.

the ActR protein. In the absence of a small molecule, ActR binds to the synthetic promoter and represses expression of the lux operon. Small molecules that can enter E. coli, bind to ActR in vivo, and cause a conformational change, relieve repression of the lux operon, resulting in the production of light. Students shared their in vitro and in vivo data with the class and determined the structure−activity relationships for the anthraquinone derivatives. They determined whether there was an electronic effect and compared the biological activity of the anthraquinone derivatives in vitro and in vivo. The main advantage of using an in vitro system was that the observed effect could be directly attributed to the small molecule interacting with the protein of interest. One major shortcoming of this approach, however, was the issue of “biological validation”. Unlike possible hits from whole cell screens, which have already been “biologically validated” by demonstrating an ability to cross the cell membrane and withstand metabolism and excretion, hits from in vitro screens, more often than not, are unsuitable in a biological context.

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RESULTS AND DISCUSSION The first step toward synthesizing the anthraquinone derivatives involved the synthesis of the ylide precursor, allyltriphenylphosphonium bromide, via an SN2 nucleophilic substitution reaction. The reaction was straightforward and high yielding (78% average student yield) facilitated by the use of a reactive primary allylic halide with a good leaving group and a nucleophilic phosphine. Isolation of the product was simple because the phosphonium salt had poor solubility in diethyl ether whereas the triphenylphosphine was soluble. Although allyltriphenylphosphonium bromide was commercially available, the students synthesized the compound to gain experience in conducting a simple substitution reaction, crystallization, and isolation. The lecture component of the laboratory provided an opportunity to review substitution chemistry and to discuss reactive features of both nucleophiles and electrophiles, as well as the spectral features of the product. The Wittig synthesis of differentially substituted 1-[(E/Z)buta-1,3-dien-1-yl]-benzene first required the generation of a reactive, semistabilized phosphorus ylide intermediate via deprotonation of allyltriphenylphosphonium bromide, prior to the addition of the aldehyde. The formation of the ylide was inferred by a change in color of the reaction from white to orange upon addition of LDA. Isolation of the product required solvent extraction with diethyl ether to remove the triphenylphosphine oxide, followed by silica gel chromatography to separate the product from the aldehdye. Both the (E)and (Z)-isomers of the diene were obtained because semistabilized ylides generally provide low configurational selectivity.15 The 1H NMR spectra of a representative 2:1 mixture of (E)- and (Z)-alkenes are provided in the Supporting Information. The average student yield for the reaction was 48% and all students had synthesized enough 1,3-diene (∼20 mg) for use in the Diels−Alder reaction. The final step in the synthesis of the anthraquinone derivatives involved a Lewis acid-promoted Diels−Alder

HAZARDS

This set of experiments involves the use of some potentially toxic chemicals including triphenylphosphine, aromatic aldehydes, allyl bromide, and lithium diisopropylamide (LDA); Lewis acids including aluminum chloride, iron(III) chloride, boron trifluoride-diethyletherate; and solvents including dichloromethane (DCM), hexane, ethyl acetate, toluene, tetrahydrofuran (THF), methanol, and chloroform-d (CDCl3). Triphenylphosphine and aromatic aldehydes are irritants and toxic by inhalation. Allyl bromide is a lachrymator, flammable, and a cancer suspect agent. LDA is a strong corrosive base and the THF solution is highly flammable. All Lewis acids are irritants and should also be considered corrosive; aluminum chloride is a teratogen and reproductive hazard, whereas boron trifluoride diethyletherate is flammable. Toluene, methanol, ethyl acetate, hexanes, and diethyl ether are highly flammable while dichloromethane and chloroform-d are toxic and potential carcinogens. Silica gel used for thin-layer (TLC) and column chromatography is toxic by inhalation and nitrogen gas is an inert asphyxiant. Exercise great care when handling these substances. Safety goggles, lab coats, and gloves are to be worn at all times when in the undergraduate 746

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Figure 2. Overexpression and purification of the ActR protein. (A) Small-scale induction of the ActR protein. Whole cell lysates of uninduced (-) and induced (+) cultures resolved by 12% SDS-PAGE and stained with Coomassie Brilliant Blue. (B) Dot blot assay used to analyze fractions from IMAC purification. Fractions containing protein (i.e., flow thru (FT), wash (W), and fractions 17−19) appear as dark circles. (C) Lysate (L), flow thru (FT), wash (W), and purified ActR (P) fractions resolved by 12% SDS-PAGE and stained with Coomassie Brilliant Blue. The ActR protein (asterisk) was purified to near homogeneity. This figure was constructed using student data.

and research-like quality of the course, it is advisable to alter the substituents on the aromatic aldehyde from year to year. There appears to be little restriction on the choice of aldehyde used in the Wittig olefination step, and 1-(4-fluorophenyl)-1,4-dihydroanthracene-9,10-dione and 1-(4-acetamidophenyl)-1,4-dihydroanthracene-9,10-dione have also been successfully synthesized using this method (data not shown). Simple 4-substituted derivatives were chosen to provide experience in the NMR assignments and permit discussion on electronic effects on the reactions and subsequent biological activity. The use of other aldehydes could readily be contemplated to yield structurally diverse 1-aryl-1,3-butadienes.19 Full NMR assignments for the final products are provided in Table 1 in the Supporting Information. These structures provided ample opportunity to discuss the threedimensional conformation of the compounds, enantiotopic versus diasterotopic hydrogens, as well as the differential chemical shifts and coupling data in the lecture component of the course. To test their synthesized compounds in an in vitro ANS displacement assay students first had to overexpress and purify the ActR protein. The actR gene had previously been cloned into the pET-28a(+) expression vector under the control of the IPTG inducible T7/lac promoter.11 Students were randomly supplied with E. coli XL1-Blue (a standard cloning strain) containing either the correct actR expression plasmid (pET28a(+)-actR) or an incorrect plasmid (pET-28a(+)-absA2), and were required to isolate, characterize, and transform the actR plasmid into the E. coli expression strain, BL21(DE3). A Qiagen miniprep kit was used to isolate plasmid DNA from E. coli and the DNA was quantified using both UV absorbance and the band-brightness method. The average student plasmid concentration based on UV absorption was 120 μg/mL (5 μg total DNA), which was sufficient for characterization and transformation. Plasmid DNA was characterized by restriction digestion using enzymes that resulted in two fragments: the pET-28a(+) vector and the gene insert. The pET-28a(+) plasmid (5369 base pairs (bp)) contained either the actR gene (792 bp) or the

reaction in which the differentially substituted 1-[(E/Z)-buta1,3-dien-1-yl]-benzene was reacted with naphthoquinone. The addition of the Lewis acid to the diene and the dienophile resulted in a color change (1-phenyl-, dark green; 1-(4chlorophenyl)-, dark black; 1-(4-nitrophenyl)-, dark brown; 1(4-methoxyphenyl)-, dark green). In addition to the product being formed, TLC analysis revealed the production of side products of varying polarity. Isolation of the product first required solvent extraction with dichloromethane, followed by silica gel chromatography to remove polar side products from the reaction mixture. Students assigned the same derivative pooled their semipure product and performed Sephadex LH-20 gel permeation chromatography to separate the final product from 1,4-naphthoquinone and any remaining side products. The immediate product of the cycloaddition reaction underwent spontaneous aerial oxidation to yield the 1,4-naphthoquinone. The average student yield for the final products was 13%, which resulted in enough compound for the in vivo and in vitro assays. 1H NMR, 13C NMR, and high-resolution electron impact mass spectrometry (HREI MS) data for each of the four differentially substituted 1-(4-Xphenyl)-1,4-dihydroanthracene9,10-diones is reported in Table 1 in the Supporting Information. This experiment provided ample material for the lecture component of the course, such as discussions on the general mechanism for Lewis acid catalysis of the Diels−Alder reaction, cycloaddition and other concerted reactions.16 The spontaneous aerial oxidation of the cycloadduct to the quinone provided an opportunity to discuss keto−enol tautomerism and the easy oxidation of aromatic 1,4-diols to quinones, and the relation of this to biological redox cofactors (such as coenzyme Q10 and FAD). In addition, related Diels−Alder reaction and oxidation sequences,17 as well as asymmetric entries to 1,4naphthoquinones,18 are reported in the literature and may be discussed. Overall, the novel three-step synthetic strategy utilizing a Wittig olefination followed by a Lewis acid-promoted Diels− Alder cycloaddition resulted in the desired products with reasonable purity and sufficient yields. To maintain the novelty 747

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Figure 3. Interaction of ActR with differentially substituted 1-(4-Xphenyl)-1,4,-dihydroanthracene-9,10-dione. (A) Fluorescence emission spectrum of (1) 100 μM ANS alone, (2) 100 μM ANS in the presence of 1 μM ActR and 50 μM 1-(4-nitrophenyl)-1,4-dihydroanthracene-9,10-dione, and (3) 100 μM ANS in the presence of 1 μM ActR. For simplicity, only the spectrum for 4-(nitrophenyl)-1,4,-dihydroanthracene-9,10-dione is shown. This plot was constructed using student data. (B) Hammett analysis of the electronic character and relative binding affinity of differentially substituted 1(Xphenyl)-1,4-dihydroanthracence-9,10-dione. MeO, 1-(4-methoxyphenyl)-; H, 1-phenyl-; Cl, 1-(4-chlorophenyl)-; and NO2, 1-(4-nitrophenyl)-.

absA2 gene (669 bp) and students were required to correctly identify what plasmid they had isolated by agarose gel electrophoresis. The goal of this was to impress upon the students how important it is to initially characterize a plasmid in order to avoid wasting time and effort working with an incorrect plasmid. Students that isolated the incorrect plasmid were required to obtain some of the actR plasmid from a classmate. The actR plasmid was transformed into the E. coli expression strain BL21(DE3). Students were required to come to the lab the next day to analyze their results. Having the students come to the lab outside of the scheduled lab time gave the students a more realistic view of how real research does not fit into a 4-h lab period. Plates can be stored for several weeks at 4 °C, so students that are unable to come to the lab the following day can analyze their results at a later date. Student transformation efficiencies ranged from 100 to 1200 cfu/μg of DNA. Using one of their transformed colonies, students performed a small-scale trial expression of the ActR protein to determine the effect of induction temperature (25 versus 37 °C) on expression. Overall, temperature had a negligible effect on ActR expression as bands of similar intensity were observed for cultures induced at 25 and 37 °C (Figure 2A). To ensure that the purification procedure would be completed in a 4-h lab period, the students were supplied with ActR induced E. coli pellets. Cells were lysed by chemical means and the ActR protein was purified to near homogeneity using a batch IMAC method (Figure 2B,C). Purified ActR was exchanged into a low salt buffer and concentrated using a centrifugal filter unit. The average student ActR concentration, as determined by a Bradford assay, was 15 mg/mL (7 mg total protein), which was sufficient for the in vitro assay. An in vitro ANS displacement assay was used to determine the affinity of the anthraquinone derivatives for the ActR protein. In the first week of the lab, an emission scan of the ANS displacement assay was performed to visually assess whether there was an interaction between ActR and the synthesized derivatives. All four derivatives, to varying degrees, were able to displace ANS from ActR, resulting in a decrease in the fluorescence intensity and a red shift in the maximum emission wavelength (Figure 3A). The visual representation of

the data helped students understand what was happening at the molecular level. Students were required to analyze their emission spectrum and discuss the results with the instructor prior to performing the titrations. This ensured that any systematic errors in the assay would be identified and corrected within the first week of the experiment. Students were also given the opportunity to repeat the emission scan until suitable results were obtained. In the second week of the lab, dissociation constants, Kd, of ActR for each of the derivatives were determined using the ANS displacement assay. Students posted their data online and analyzed the results for all four derivatives. The crystal structure of ActR with (S)-DNPA has been published,20 so students were able to hypothesize how the anthraquinone-like derivatives may bind to ActR. The Kd values of ActR for 1-(4-methoxyphenyl)-, 1-phenyl-, 1-(4-chlorophenyl)-, and 1-(4-nitrophenyl)-1,4-dihydroanthracene-9,10-dione were 0.155 (±0.025) μM, 1.02 (±0.15) μM, 0.998 (±0.15) μM, and 1.70 (±0.34) μM, respectively. These values were generated by a graduate student who worked on lab development. Depending on the level of expertise of the students, the Kd values may fluctuate by 1 to 2 orders of magnitude. Regardless of the actual values, students gain valuable experience determining and analyzing Kd values. The Kd values were used to construct a Hammett semilog plot of relative binding affinity (KdH/KdX) versus the Hammett sigma para (σp) substituent values.21 The σp values were derived from the ionization constants for para-substituted benzoic acids relative to benzoic acid and incorporate both inductive and resonance contributions to electronic effects. Substituent constants can be used for SAR analysis, independent of the nature of the reaction (e.g., enzymatic, ligand binding).22 Hammett analysis suggested that there was a minor electronic effect on the binding of the derivatives to ActR with the methoxy derivative being most active (Figure 3B). An E. coli-based bioluminescence assay11 was used to determine whether the ligands were able to interact with ActR in vivo. In addition to monitoring bioluminescence, students also measured the OD600 value to determine whether the compounds possessed antimicrobial activity against E. coli. The compounds did not inhibit growth (data not shown), 748

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despite having the naphthoquinone core. Naphthoquinone has been reported to inhibit E. coli growth,23 and under our conditions, we have observed inhibition at a final concentration of 150 μM (data not shown). Although the compounds were able to interact with ActR in vitro, they were unable to induce bioluminescence in vivo (data not shown). There may be several possible explanations for this, one of which is that the compounds were unable to cross the Gram-negative cell wall. In addition, it has been shown that some molecules can interact with ActR but not elicit a conformational change.7 If the compounds were functioning in such a manner, then bioluminescence would not be observed, despite an interaction with ActR. To determine whether the compounds are binding to ActR, but not inducing a conformational change, a gel mobility shift assay using a radiolabeled promoter fragment would have to be performed, which is not feasible in an undergraduate laboratory. A lack of an in vivo effect illustrates to the students how results from an in vitro assay can vary from those of an in vivo assay due to the complexity of an in vivo system. Not only is an interaction required between ActR and the compound, but the molecule has to enter the cell and be retained at a concentration high enough to illicit a biological response. Regardless of whether the molecules had any biological effect, exposing the students to both types of assays prepares them for what they would expect to find in an actual research setting.

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ASSOCIATED CONTENT

S Supporting Information *

Prelaboratory manual, detailed student laboratory manuals (which include CAS registry numbers for all chemicals), Table 1, instructor’s notes, grading and assessment, and student feedback on the overall effectiveness of the laboratory. This material is available via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (N.M.) [email protected]; (J.M.) jmcnult@ mcmaster.ca. Author Contributions †

These authors contributed equally to this work.

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ACKNOWLEDGMENTS We are grateful to Justin Nodwell for supplying us with all of the plasmids and strains used in this work. We would like to thank the undergraduate technician Patricia Martin for all of her help and assistance with preparing solutions, reagents, and cultures and for providing all of the CAS registry numbers for the chemicals.

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REFERENCES

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CONCLUSION

This integrated chemical biology laboratory course exposed students to a variety of topics and techniques. Students gained valuable experience in organic synthesis conducting an alkylation, olefination, and a Lewis acid promoted cycloaddition reaction. Experience was gained conducting dry reactions under an inert atmosphere, following reaction progress (thin-layer chromatography) and with product isolation, purification (polar partition and gel permeation chromatography), and structural analysis (NMR) of their products. From a biochemical perspective, experience was gained with both DNA and protein purification and characterization, microbiology, as well as fluorescence and bioluminescence assays. By designing the course around a single protein, and employing a novel synthetic sequence, students were able to experience a research-like environment in an undergraduate laboratory. Student evaluations for the course indicated that they liked that the laboratories were integrated, that everything was relevant, and that the course had a “real-world” purpose. The modularity of the course allows it to be easily adapted to a variety of biology, molecular biology, chemistry, and biochemistry undergraduate laboratory courses. Each of the three parts could be adopted as a series of independent experiments. Furthermore, there are several laboratories that could be omitted, which would allow the course to be condensed into fewer weeks, if necessary. For example, the phosphonium salt is commercially available and week 1 could be omitted. In addition, if the objective was just to expose students to protein purification, then weeks 5−7 could be omitted. Overall, the course presented offers a series of integrated experiments that could be altered to suit a variety of different learning objectives. 749

dx.doi.org/10.1021/ed200417d | J. Chem. Educ. 2012, 89, 743−749