Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX
pubs.acs.org/jchemeduc
Bioinorganic Laboratory Experiment: Synthesis and Catalytic Activity of a Vanadium Haloperoxidase Model Complex Steven M. Malinak,* Jerald E. Hertzog,† Julia E. Pacilio, and Deborah A. Polvani Department of Chemistry, Washington & Jefferson College, 60 South Lincoln Street, Washington, Pennsylvania 15301, United States
J. Chem. Educ. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 02/17/19. For personal use only.
S Supporting Information *
ABSTRACT: Laboratory experiments that offer interdisciplinary experiences for students are appealing and are increasingly popular additions to undergraduate chemistry curricula. Students can capitalize on their knowledge of multiple areas of chemistry while working through an application, and this fosters the development of progressive problem-solving skills. In this laboratory experiment, students combine the fields of inorganic chemistry with biological chemistry to synthesize and test the catalytic activity of a model of vanadium haloperoxidase, an enzyme naturally produced in certain brown- and red-seaweed species. Students use 1H NMR spectroscopy to monitor the oxidation of thioanisole to methyl phenyl sulfoxide using their prepared vanadium complex as the catalyst. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Hands-On Learning/Manipulatives, Bioinorganic Chemistry, Catalysis, NMR Spectroscopy
P
• how UV−vis spectroscopy can be used to characterize inorganic complexes; • strategies often employed by synthetic inorganic chemists, including recrystallizations using a layering technique and dissolving complexes using crown ethers; and • NMR spectroscopy and how it can be used to monitor the progress of a reaction. Regarding the first point, many enzymes employ an active metal or metal-containing complex. Investigating the structure and reactivity of these active sites can be complicated by the presence of the remaining protein, which tends to constitute the majority of the mass of an enzyme. A synthetic bioinorganic chemist can attempt to synthesize small complexes that approximate the local coordination environment of the metal-containing moiety. The structure, spectroscopic signature, and reactivity can be much more directly probed using these low-molar-mass analogues. Additionally, small changes to the local environment can be more readily accomplished with these synthetic analogues through comparatively simple ligand modification, thereby allowing chemists to explore the impact of coordination environment on reactivity. As the name of the enzyme implies, vanadium haloperoxidase (VHPO) catalyzes the oxidation of halides by hydrogen peroxide, which generates halogenated organic compounds.3
roviding laboratory projects that are interdisciplinary can be of value for numerous reasons. For instance, chemistry laboratories that draw on theory or techniques from biology or biochemistry can help departments meet accreditation standards as defined by the Committee on Professional Training of the American Chemical Society1 and help to illustrate the direct application of chemistry to interesting questions from other fields. This Journal has published numerous examples of lab experiments that focus on the interdisciplinary areas of bioorganic or bioinorganic chemistry.2 Herein, a bioinorganic lab experiment is described that focuses on the synthesis and reactivity of a vanadium complex that models the structure and function of the active site of vanadium haloperoxidase.3 The lab experiment was developed by two independent-study students who modified or provided detailed protocols on the basis of what was reported in the primary literature, and it has been further modified on the basis of work performed by 11 advanced undergraduate students enrolled in our Synthesis Laboratory. Following the protocols outlined below, this experiment can be completed within three to four 3 h lab meetings. There are numerous techniques and concepts that are emphasized in this laboratory experiment. The student learning objectives include demonstrating understanding of • how synthetic analogues of enzyme active sites can be used to more readily explore structural, spectroscopic, and reactivity properties of said active sites; • catalysis; • how IR spectroscopy can be used to characterize inorganic functional groups (such as VO); © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: July 9, 2018 Revised: January 26, 2019
A
DOI: 10.1021/acs.jchemed.8b00543 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
■
HAZARDS Rubber gloves and safety goggles should be worn for all parts of the experiment, and the synthesis should be performed in a fume hood. Potassium hydroxide is corrosive and should be handled with care. Hydrogen peroxide (30%) is a powerful oxidizing agent and should be handled carefully; it is harmful if swallowed and can cause serious eye irritation and burns to the skin. Vanadium(V) oxide and N-(2-hydroxyethyl)iminodiacetic acid and, by extension, product I are skinand eye-irritants and should not be inhaled or swallowed. 18Crown-6 is harmful if swallowed. Ethanol, thioanisole, and deuterated acetonitrile are flammable. Students should always consult Safety Data Sheets for additional information. All chemicals should be disposed of properly in appropriate waste containers.
VHPOs from numerous species are also capable of oxidizing organic sulfides under similar conditions.4 A number of VHPO model complexes have been shown to catalyze both of these processes, including the complex that is the focus of this experiment: K[VO(O2)Hheida)]·H2O (I), where Hheida2− is N-(2-hydroxyethyl)iminodiacetate).5 This pentagonal-bipyramidal complex contains a V(V) center ligated by a terminal oxide; a side-on bound peroxide; and Hheida2−, which is bound through the nitrogen, the hydroxyl group, and the deprotonated oxygens on both carboxylates. The crystal structure5 as well as the spectroscopic5,6 and reactivity5,7 properties of this complex have been reported. The oxidation of thioanisole to methyl phenyl sulfoxide by K[VO(O2)Hheida)]·H2O7 (Scheme 1) has proven to be readily adaptable for use in an advanced undergraduate laboratory.
■
Scheme 1. Oxidation of Thioanisole to Methyl Phenyl Sulfoxide by Hydrogen Peroxide, Catalyzed by [VO(O2)Hheida)]− (I−)
■
Laboratory Experiment
RESULTS AND DISCUSSION
General Practices
This experiment has been conducted for three years in an upper-level laboratory course for advanced undergraduates. Students enrolled in this course work on synthesis and characterization projects for two 3 h laboratory sessions per week throughout the semester (6 h of lab time per week). In general, this course is designed to introduce students to some advanced synthesis techniques. In addition to asking students to complete specific experiments that require these techniques, the course provides a theoretical basis for each of these techniques and stresses the importance of both when and why various synthetic methods are employed. The 11 students who worked individually on this experiment (2016− 2018) completed it in about 2 weeks, which amounts specifically to three or four sessions of meeting time. All students are expected to turn in a lab report for this experiment, wherein they highlight the procedures, show all yield calculations, interpret all spectra obtained (FTIR, UV− vis, and 1H NMR), and use their data to thoroughly discuss the outcomes of their work. In addition, students must complete a postlab exercise that asks them to consider other examples of bioinorganic model complexes through the exploration of the primary literature. Student knowledge is assessed through grading the lab notebook and the responses provided to postlab questions and relevant questions on a comprehensive written final exam. Examples of these latter assessment tools and the results obtained can be found in the Supporting Information (pages S8−S13). The assessment suggests that students were able to meet the objectives of this experiment.
EXPERIMENTAL PROCEDURE
Synthesis of K[VO(O2)Hheida)]·H2O (I)
The procedure for preparing this vanadium complex was modified from that which was published previously.5,6 All reagents used were ACS-reagent-grade from Sigma-Aldrich. Briefly, the synthesis involves adding the H3heida ligand and hydrogen peroxide sequentially to a stirring aqueous solution of potassium metavanadate, the latter of which is prepared in situ, and then layering the aqueous solution with a nonsolvent (ethanol) to induce precipitation of I. Specific details are provided in the Supporting Information (pages S5−S6). Infrared and UV−vis spectroscopy are used to characterize the product. A sample IR spectrum is provided in the Supporting Information (page S19).
Synthesis of K[VO(O2)Hheida)]·H2O (I)
There were a handful of modifications made to the published protocols,5,6 all of which were intended to allow for the successful preparation of I in two 3 h lab sessions. (1) Published procedures call for the adjustment of the pH to 45 or below6 after the addition of the H3heida ligand. Elimination of this step saves time and does not seem to impact the yield or purity of the product. (2) The total stirring time was reduced from overnight to 60 min. (3) Rather than adding minimal ethanol and keeping the solution in a refrigerator for multiple weeks, it was determined that decreasing the amount of solvent
Oxidation of Thioanisole
The detailed protocol for this reaction, which is referred to but not provided in the primary literature,7 can be found in the Supporting Information (page S6). Briefly, stock solutions are prepared of each reagent, aliquots of which are combined in a volumetric flask. A sample of the latter reaction solution is transferred to an NMR tube, and spectra are obtained at predetermined times. All reagents used were ACS-reagentgrade from Sigma-Aldrich. B
DOI: 10.1021/acs.jchemed.8b00543 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
Figure 1. Oxidation of thioanisole (T, δ = 2.5 and 7.3 ppm) to methyl phenyl sulfoxide (MPS, δ = 2.6 and 7.6 ppm) by hydrogen peroxide, catalyzed by complex I, sampled every 4 min beginning after 6 min of reaction time. Note that the oxidation is essentially complete within 22 min.
assigned.5 (There are actually at least four peaks identified in the 610−560 cm−1 region of the IR spectrum, but when 18Oenriched H2O2 was used, and the corresponding isotopic shifts in the spectrum were observed, it was reported5 that the peak at 570 cm−1 was the one corresponding to the vanadiumperoxo. The O−O stretch at 930 cm−1 was also verified through isotopic substitution.5) There are medium-intensity stretches in the 3200−3400 cm−1 range, perhaps indicative of the bound O−H or cocrystallized water. (See the example on page S19 of the Supporting Information; note that it is assumed that the monohydrate potassium salt is obtained on the basis of the elemental analysis and crystal structure originally published. 5 ). The UV−vis spectrum shows absorption at 435 nm (ε = 4 × 102 M−1 cm−1), consistent with what is reported as a peroxo-to-vanadium charge-transfer band (λ = 430 nm, ε = 3 × 102 M−1 cm−1).5 Additional characterization of I was reported5 and includes a crystal structure, elemental analysis, and 13C NMR and 51V NMR spectra, any of which can be employed as dictated by the instructor and available resources.
(H2O) and increasing the amount of nonsolvent (ethanol) provided adequate yield after just 1 week. It is therefore suggested that students use the first 3 h lab session to accomplish the synthesis and solvent layering and the second 3 h session for the isolation and characterization of the product. For the 13 students who performed this synthesis (two independent-study students and 11 students enrolled in the Synthesis Laboratory), the average yield of I was 26%, with a range of 0 to 62%. Three of the 11 Synthesis Laboratory students did not obtain any yield of product, despite the solution presenting the orange-red color typical of I. This is presumably the result of insufficient nonsolvent being added or too much water being used initially, which results in no precipitate. Though not observed, it is also possible that a student could add too much nonsolvent or not layer it carefully, which could cause the product to oil out. To remedy the former situation, students can simply layer more ethanol until cloudiness persists at the interface, return the solution to the refrigerator, and wait for crystals to form. (Because the recrystallization is concentration-dependent, it is recommended that absolute ethanol be used as the nonsolvent in order to minimize the amount of water present.) To remedy the latter situation, students can decant the mother liquor, redissolve the oil in a minimum amount of water, and proceed with the recrystallization using just enough absolute ethanol to produce a persistent cloudiness at the interface. In either case, students will need additional time, which may not always be available (depending on the time allotted to this experiment). In such cases, students who do not obtain I can borrow product from other students, characterize it on their own, and use it for the oxidation reaction, thereby providing the opportunity for all students to obtain the objectives. Characterization of the product is based on IR and UV−vis spectroscopy. Well-resolved IR spectra (which we obtained from KBr pellets, but an ATR accessory could also be used) show two strong, broad CO stretches at 1660 and 1627 cm−1, a strong VO stretch at 977 cm−1, a strong O−O stretch at 930 cm−1, and a medium V−O2 stretch at roughly 570 cm−1, which is consistent with what was identified and
Oxidation of Thioanisole
For 1H NMR experiments, it is reported that (1) the concentration of the vanadium complex used is 250 μM, (2) the ratio of I/H2O2/thioanisole/HPF6 is 1:2000:1000:1, and (3) the order of addition does not impact reactivity as long as the acid is added after the hydrogen peroxide.7 There are no specific protocols provided beyond these. The details offered in the Supporting Information (page S7) can be provided to students. Alternatively, the protocols can serve as a reference to the instructor if having students work through the calculations independently is deemed valuable. The spectra obtained will depend on the NMR instrument used. We have elected to use a benchtop 60 MHz instrument (Nanalysis NMReady 60e with sample warmer) for this experiment for faster throughput. Reference spectra for thioanisole and methyl phenyl sulfoxide were obtained in CD3CN (see the Supporting Information, pages S16−S17). Thioanisole shows the aromatic protons as a broad singlet at 7.3 ppm (unresolved because of the low field strength of the instrument) and the methyl protons at 2.5 ppm, whereas C
DOI: 10.1021/acs.jchemed.8b00543 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Laboratory Experiment
ORCID
methyl phenyl sulfoxide shows both of these signals shifted downfield to 7.6 and 2.6 ppm, respectively. It is therefore easy to follow the progress of the reaction, and no complications arise from the lack of resolution. The NMR instrument used in this experiment requires the prewarming of sample tubes to approximately 30 °C for at least a few minutes so that the sample temperature is comparable to that of the magnet. This presented a practical limitation in that the first spectrum that could be obtained was after approximately 6 min of reaction time (see Figure 1), when the substrate oxidation was already approximately 50% complete (as suggested by relative integrals.) Generally, complete conversion of thioanisole to methyl phenyl sulfoxide in the presence of I is essentially complete within 25 min (see Figure 1). (There is a much slower oxidation of thioanisole observed in the absence of I, although we did not ask students to follow the uncatalyzed reaction.) The large peak that appears between 3.5 and 3.8 ppm has been identified as H2O2 or 18-crown-6, both of which display singlets in this region (see pages S14−S15 of the Supporting Information); the slow migration from 3.8 to 3.5 ppm as the reaction proceeds is similar in all student data and may reflect the loss of hydrogen peroxide as the reaction proceeds. Note that Smith et al. report7 that even after 120 min, the oxidation of thioanisole is only 80% complete. It is not clear why the oxidation proceeds significantly faster under the conditions reported herein, unless it is due to running the reaction at slightly elevated temperature (30 °C), although this cannot be claimed definitively because reaction temperature was not reported in the literature. Regardless, complex I catalyzes the oxidation of up to 1000 equiv of thioanisole in a period that can easily be monitored during a single lab meeting.
Steven M. Malinak: 0000-0002-1145-7255 Present Address †
J.E.H.: Institute for Molecular Engineering and Department of Chemistry, University of Chicago, 5640 South Ellis Avenue, Chicago, Illinois 60637, United States Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors would like to express their appreciation to Washington & Jefferson (W&J) College for the funds to develop this laboratory and to the students enrolled in CHM 380: Synthesis Laboratory at W&J College from 2016 to 2018, whose data are compiled herein.
■
■
CONCLUSION The experiment presented here has been an excellent addition to our Synthesis Laboratory course. Students are exposed to the advantages of synthesizing a model coordination compound to imitate the activity of a complicated natural enzyme. The opportunity to have students consult the primary literature as a starting point was stressed. In addition, opportunities to discuss and participate in more advanced crystallization techniques, catalysis, and spectroscopic characterization are present. Overall, students have been successful in their ability to work on an interdisciplinary bioinorganic application starting from the synthesis of a vanadium complex and ending with a method to test its catalytic activity.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.8b00543.
■
REFERENCES
(1) American Chemical Society Committee on Professional Training. Undergraduate Professional Education in Chemistry: ACS Guidelines and Evaluation Procedures for Bachelor’s Degree Programs; American Chemical Society: Washington, DC, 2015. https://www. acs.org/content/dam/acsorg/about/governance/committees/ training/2015-acs-guidelines-for-bachelors-degree-programs.pdf (accessed Jan 2019). (2) Some examples include (a) Barrett, J.; Spentzos, A.; Works, C. An Advanced Organometallic Lab Experiment with Biological Implications: Synthesis and Characterization of Fe2(μ-S2)(CO)6. J. Chem. Educ. 2015, 92, 719−722. (b) Clark, R. A.; Stock, A. E.; Zovinka, E. P. Metalloproteins as Oxidation Catalysts: Moving Toward “Greener” Chemistry in the Inorganic Chemistry Laboratory. J. Chem. Educ. 2012, 89, 271−275. (c) Nishimura, R. T.; Giammanco, C. H.; Vosburg, D. A. Green, Enzymatic Synthesis of Divanillin and Diapocynin for the Organic, Biochemistry, or Advanced General Chemistry Laboratory. J. Chem. Educ. 2010, 87, 526−527. (d) Vecchio, G.; Lanza, V. Synthesis of Superoxide Dismutase (SOD) Enzyme Mimetics. J. Chem. Educ. 2009, 86, 1419−1421. (e) Howard, D. L.; Tinoco, A. D.; Brudvig, G. W.; Vrettos, J. S.; Allen, B. C. Catalytic Oxygen Evolution by a Bioinorganic Model of the Photosystem II Oxygen-Evolving Complex. J. Chem. Educ. 2005, 82, 791−794. (f) Beckmann, B. A.; Buchman, A.; Pasternack, R. F.; Reinprecht, J. T.; Vogel, G. C. An Advanced Laboratory Experiment in Bioinorganic Chemistry. J. Chem. Educ. 1976, 53, 387−389. (3) For example, see Conte, V.; Coletti, A.; Floris, B.; Licini, G.; Zonta, C. Mechanistic Aspects of Vanadium Catalyzed Oxidations with Peroxides. Coord. Chem. Rev. 2011, 255, 2165−2177 and references therein. . (4) ten Brink, H. B.; Schoemaker, H. E.; Wever, R. Sulfoxidation Mechanism of Vanadium Bromoperoxidase from Ascophyllum nodosumEvidence for Direct Oxygen Transfer Catalysis. Eur. J. Biochem. 2001, 268, 132−138 and references therein. . (5) (a) Colpas, G. J.; Hamstra, B. J.; Kampf, J. W.; Pecoraro, V. L. Functional Models for Vanadium Haloperoxidase: Reactivity and Mechanism of Halide Oxidation. J. Am. Chem. Soc. 1996, 118, 3469− 3478. (b) Colpas, G. J.; Hamstra, B. J.; Kampf, J. W.; Pecoraro, V. L. Functional Models for Vanadium Haloperoxidase: Reactivity and Mechanism of Halide Oxidation. J. Am. Chem. Soc. 1994, 116, 3627− 3628. (6) Sun, L. L.; Hermann, K. E.; Noack, J.; Timpe, O.; Teschner, D.; Hävecker, M.; Trunschke, A.; Schlö gl, R. DFT Studies and Experiments on Biocatalytic Centers: Structure, Vibrations, and Core Excitations of the K[VO(O2)Hheida] Complex. J. Phys. Chem. C 2014, 118, 24611−24622. (7) Smith, T. S., III; Pecoraro, V. L. Oxidation of Organic Sulfides by Vanadium Haloperoxidase Model Complexes. Inorg. Chem. 2002, 41, 6754−6760.
List of all reagents used, safety information, copies of the prelab handout and postlab assignment, grading rubric and assessment data for the laboratory notebook and postlab report, relevant final-exam questions with solutions and assessment data, and examples of relevant spectra obtained by students (PDF, DOCX)
AUTHOR INFORMATION
Corresponding Author
*E-mail: smalinak@washjeff.edu. D
DOI: 10.1021/acs.jchemed.8b00543 J. Chem. Educ. XXXX, XXX, XXX−XXX
