Laboratory Experiment pubs.acs.org/jchemeduc
Collaborative Student Laboratory Exercise Using FT-IR Spectroscopy for the Kinetics Study of a Biotin Analogue Jhaque Leong, Nathan C. Ackroyd, and Karen Ho* Mount Royal University, 4825 Mount Royal Gate Southwest, Calgary, Alberta T3E 6K6 Canada S Supporting Information *
ABSTRACT: The synthesis of N-methoxycarbonyl-2-imidazolidone, an analogue of biotin, was conducted by organic chemistry students and confirmed using FT-IR and 1H NMR. Spectroscopy students used FT-IR to measure the rate of hydrolysis of the product and determined the rate constant for the reaction using the integrated rate law. From the magnitude of the rate constant, the reaction was found to be activation limited. A student survey was conducted, which showed the laboratory experiment to be a positive learning experience.
KEYWORDS: Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Organic Chemistry, Collaborative/Cooperative Learning, Hands-On Learning/Manipulatives, IR Spectroscopy, Kinetics, Spectroscopy
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Because 2 does not react with carbon dioxide directly,7 Nmethoxycarbonyl-2-imidazolidone, 3, is first synthesized and then hydrolyzed to produce N-carboxy-2-imidazolidone, 4, for studies on the decarboxylation mechanism (Scheme 1).
ab experiments that combine experimental techniques with critical thinking to solve complex problems allow students to gain valuable experience that is applicable to life outside of a teaching lab. The chemical literature includes examples of procedures that use spectroscopic techniques such as NMR,1 UV−vis,2,3 Raman,4 and IR5 to solve problems related to chemical structure and reactivity. Biotin (vitamin B7, Figure 1), 1, is the cofactor involved in enzymatic pathways of carbon dioxide transport.6 Three groups
Scheme 1. (Top) Nucleophilic Addition of 2-Imidazolidone (2) to Methyl Chloroformate Producing NMethoxycarbonyl-2-imidazolidone (3). (Bottom) Saponification of N-Methoxycarbonyl-2-imidazolidone (3) Forming the Carbamate Anion (4) and Methanol
Figure 1. 2-Imidazolidone (2) and biotin (1) have key structural similarity.
compose a biotin molecule: an ureido ring, a fused tetrahydrothiophene ring, and a valeric acid side-chain. Carboxylation and decarboxylation reactions occur through the nitrogen opposite the side-chain, which reversibly adds to CO2 to form a carbamate anion in cells with high CO2 concentration. The structural similarity of the ureido ring with 2-imidazolidone (Figure 1), 2, makes it likely for similar reactions to proceed by the same mechanism. This structure− function relationship permits its use to investigate the mechanisms of biotin catalysis, and there have been many kinetic studies reported using 2 as a biotin analogue.7−10 © XXXX American Chemical Society and Division of Chemical Education, Inc.
The rate constant for the hydrolysis was calculated using eq 1 ln
A
[A]t [A]0 = ([A]0 − [B]0 )kt + ln [B]t [B]0
(1)
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where k is rate constant (L mol−1 s−1) and t is time (s). The results were compared to results obtained by similar experiments.7 To identify the dependency of the addition step in the saponification, students determined whether the reaction is diffusion or activation limited. The analysis and rate study were done in a spectroscopy lab as an illustration of the use of spectroscopy separate from structure determination. The procedures could also be used in an instrumental analysis or physical chemistry course, depending on course offerings. Discussion could then focus more on systematic error or identifying potential reaction mechanisms.
product was determined, and its identity confirmed by FT-IR and 1H NMR spectra. The yield that organic students obtained was 48.7% with the melting point range of 176−179 °C. Further synthetic details are available in the accompanying Supporting Information. Stock solutions of approximately 14.0 M KOH and 12.0 M HCl were prepared in 2 mL Eppendorf tubes, and a 10 mL stock solution of 3 was prepared in a 15 mL centrifuge tube. For rate measurements, the reaction was carried out in separate 2 mL Eppendorf tubes, where 1500 μL of 3 was mixed with 60 μL of 14.0 M KOH. At predetermined time intervals, 100 μL aliquots were removed and added to separate 2 mL Eppendorf tubes containing 5 μL of HCl for quenching. Aliquots for the calibration curve were prepared in 2 mL Eppendorf tubes from the stock solution of 3. A dilute solution of 3 was prepared in an isopropanol and water solution (1:1 v/ v), and sodium acetate was added as the ion source for ESI-MS spectra. A concentrated solution was prepared for 1H NMR spectra by slowly adding chloroform-d (1% TMS v/v) to a small amount of 3 until a solution formed. Further details are available in the accompanying Supporting Information.
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EXPERIMENTAL OVERVIEW In this lab, second-year organic chemistry II students synthesized 3, which was subsequently used by third-year spectroscopy students to measure the rate of saponification using IR spectroscopy. By synthesizing 3, second-year students have the opportunity to observe reactions involving activated carbonyl groups−an important part of organic chemistry. After purification, the product was given to third-year students for analysis and use in a rate study. In the analysis of 3, spectroscopy students are exposed to the second-order effects of strong coupling in 1H NMR spectra. They then used FT-IR to measure the rate of saponification of 3 by following the absorbance change of the carbamate carbonyl signal. By involving multiple courses in the project, the product from one course is used as a reagent in the other, reducing the waste produced by separate experiments. All of the lab periods involved were 3 h long. Organic students required two lab periods. The first included both reaction setup and reflux and could be combined with another procedure. The second included the workup, purification, and analysis of 3. The spectroscopy students needed three lab periods, one for the analysis of the organic students’ product, one to prepare the calibration curve, and one for measuring the rate of hydrolysis. A 1 h demonstration of the required instruments was given during the first lab, and the experiments were completed in groups to allow sufficient instrument time. Both laboratories can run during the same semester with the organic lab scheduled first or the product can be kept for a future semester.
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Data Acquisition
Melting points were measured twice, once to determine the approximate melting point and a second time to determine the range accurately. FT-IR and 1H NMR spectra were obtained by both groups of students. Five FT-IR spectra were obtained for each aliquot in the calibration curve and rate measurements using 20 μL for each spectrum. The results were averaged and plotted with standard deviations.
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HAZARDS Potassium hydroxide, 2-imidazolidone, hydrochloric acid, methyl chloroformate, and chloroform are toxic, as well as skin and eye irritants. In cases of exposure, rinse thoroughly with water. Wear lab coats, gloves, and protective eyewear at all times. Chloroform is a known carcinogen. All synthetic steps should be performed in a fume hood to minimize exposure through inhalation.
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RESULTS AND DISCUSSION Two mass peaks of similar proportions were observed in the ESI-MS, which agree with the singly charged cations of the monomer and dimerized species. The solid-phase FT-IR spectrum shows two carbonyl absorptions at 1750 and 1675 cm−1, which were assigned to the carbonyl groups of the sidechain and ureido ring, respectively. The secondary amide proton is seen at 3325 cm−1 in the pure sample but is not observable in spectra taken from the aliquots due to the overlap with water. Four unique environments are observed in the 1H NMR spectrum, taken on a 60 MHz instrument (see Figure 2). A broad singlet at 6.6 ppm is assigned to the N−H proton based on its characteristic shape and integration. The methyl group is observed at 3.85 ppm by a strong singlet overlapping one of the methylene groups. This assignment was based on the expected chemical shift and the absence of coupling. Between 3.25 and 4.15 ppm, there are two groups of protons with equal relative integrations. From this, they were assigned as the strongly coupled methylene groups. Due to the low field strength available with our instruments, the methylene protons have a relatively small ΔJ/ν ratio. This leads to the observed second-order coupling effects, and the coupling constants were not calculated.
EXPERIMENTAL DETAILS
Chemicals and Equipment
Melting points were determined using a Fisher Scientific melting point apparatus. FT-IR spectra were obtained using a Nicolet 6700 FT-IR spectrometer with Smart Diamond ATR, and 1H NMR spectra were obtained with 60 MHz Varian and Anasazi Instruments NMR spectrometers. A Thermo Scientific LTQ-MS spectrometer was used for ESI-MS spectra. All solvents and chemicals were purchased from Sigma-Aldrich. Sample Preparation
A 250 mL three-neck round-bottom flask was used for the synthesis of 3, and reflux was performed under a fume hood. Methyl chloroformate was added dropwise to a solution of approximately 3 g of 2 in chloroform, and the mixture was refluxed for 40−48 h at 55 °C. The solvent was removed using a rotary evaporator, recrystallized twice from water, and separated by vacuum filtration. Shorter reaction times do not provide complete conversion to product and require additional recrystallization steps. The melting point of the purified B
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Figure 2. 1H NMR spectrum of N-methoxycarbonyl-2-imidazolidone (3). Signal at 1.9 ppm is due to water in the chloroform-d.
Figure 4. Rate of decrease in concentration of N-methoxycarbonyl-2imidazolidone (3) during the hydrolysis. Data points and error bars are calculated from five measurements of each aliquot. Fit was calculated using a function of the form y = a·exp(−bx) + c, a = 0.38, b = −0.0074, c = 0.157.
To measure the rate of the saponification addition step, the decrease in absorbance at 1768 cm−1 was monitored in the aqueous-solution-phase IR, which corresponds to cleavage of the π-bond in the carbamate group. A calibration curve was first constructed by preparing five aliquots of 3 with concentrations ranging from 0.1 to 0.5 M, in 0.1 M increments. A plot of the absorbance as a function of concentration (Figure 3) was used
The second-order rate constant was calculated from the slope of a linear fit for the plot of the integrated rate law as a function of time (Figure 5). The measured change in concentration of
Figure 5. Second-order rate law equation for two reactants as a function of time. Slope is equal to product of the rate constant and difference between initial concentrations of reactants; k = 0.0104 L mol−1 s−1.
Figure 3. Calibration curve for N-methoxycarbonyl-2-imidazolidone (3). Data points and error bars are calculated from five measurements taken from the same aliquot and represent the average and 2σ, respectively. Equation of linear fit: y = 0.0892x + 0.0011, R2 = 0.999.
ester was used to determine the change in concentration of hydroxide, as the reactants are consumed in a 1:1 ratio. The change in hydroxide is calculated using the change in ester concentration, as shown in eq 2
to determine the molar absorptivity constant (ε) in accordance with the Beer−Lambert law, where the slope (0.0892) is equal to ε times the path length (1 cm). The absorbance measurements from each aliquot for the different time intervals were then converted to concentration and plotted as a function of time (Figure 4). Because the reaction proceeds very rapidly, reaching its first half-life in 80 s, an acid quench was used to reduce the error associated with measurements. The use of an acid quench allowed for repeated (5×) measurements at shorter time intervals. Multiple values were then averaged to reduce and provide a measure of experimental error.
[OH]t = [OH]0 − ([ester]0 − [ester]t )
(2)
Including data within the first 150 s, k was found to be 0.0104 L mol−1 s−1. With a k much smaller than the rate of diffusion (109), this indicates a reaction limited by the rate of formation of an activated complex rather than the rate of diffusion. Spectroscopy students completed an online survey about how they felt the lab experience contributed to their learning. The majority (80%) of students responded that the lab helped them develop skills that would allow them to formulate an experiment on their own to solve a problem. Written responses C
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mediates in Biotin-Dependent Carboxylations. J. Am. Chem. Soc. 1996, 118 (50), 12495−12498. (10) Schaeffer, H. J.; Bhargava, P. S. Chemical Reactivity of Models Related to a Proposed CO2∼Biotin Enzyme Complex 1. J. Pharm. Sci. 1964, 53 (2), 137−143.
were mostly positive, and students particularly liked the experience of using the different equipment.
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CONCLUSION The laboratory experiment involves students from second- and third-year courses, which allowed the production of waste to be minimized. Organic students synthesized a compound, identified the structure using FT-IR and 1H NMR spectroscopy, and verified the compound’s purity by measuring its melting point. Spectroscopy students observed the relationship between the content of various chemistry courses, learned a technique for studying a reaction mechanism, and identified saponification as being activation limited. Overall, students found the project to be a beneficial learning experience and to demonstrate the theory learned in class.
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ASSOCIATED CONTENT
* Supporting Information S
Experiment procedure; lab manual guidelines; prelab and postlab questions; and student survey. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Aaron D’Amico and Jessica Eggleton for their contributions to this project. We are grateful for the guidance and support received from the faculty members of the Department of Chemistry and appreciative of the technical support provided by the Laboratory Resource Center and the efforts of the organic chemistry and spectroscopy students at Mount Royal University.
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REFERENCES
(1) Marincean, S.; Smith, S. R.; Fritz, M.; Lee, B. J.; Rizk, Z. NMR Studies of Structure-Reactivity Relationships in Carbonyl Reduction: A Collaborative Advanced Laboratory Experiment. J. Chem. Educ. 2012, 89 (12), 1591−1594. (2) Hamad, M. L. Measuring Tablet Dissolution; An experiment for Teaching Quantitative, Ultraviolet Absorption Spectroscopy. J. Chem. Educ. 2013, 90, 1662−1664. (3) Burgess, A. E.; Davidson, J. C. A Kinetic-Equilibrium Study of a Triiodide Concentration Maximum Formed by the Persulfate-Iodide Reaction. J. Chem. Educ. 2014, 89 (6), 814−816. (4) Nielsen, S. E.; Scaffidi, J. P.; Yezierski, E. J. Detecting Art Forgeries: A Problem-Based Raman Spectroscopy Lab. J. Chem. Educ. 2014, 91 (3), 446−450. (5) Fröbel, S.; Appel, S.; Ganter, C.; Gilch, P. Extracting Picosecond Time Constants from Steady-State Spectroscopy: A Physical Chemistry Experiment. J. Chem. Educ. 2013, 90 (8), 1082−1086. (6) Clarkson, J.; Carey, P. Comparison of the Structures and Vibrational Modes of Carboxybiotin and N-Carboxy-2-imidazolidone. J. Phys. Chem. A 1999, 103 (15), 2851−2856. (7) Caplow, M. Studies of the Mechanism of Biotin Catalysis. J. Am. Chem. Soc. 1967, 89 (17), 4513−4521. (8) Caplow, M.; Yager, M. Studies on the Mechanism of Biotin Catalysis. II. J. Am. Chem. Soc. 1965, 87 (24), 5774−5785. (9) Rahil, J.; You, S.; Kluger, R. Solvent-Accelerated Decarboxylation of N-Carboxy-2-imidazolidone. Implications for Stability of InterD
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