Synthesis of 10-Ethyl Flavin: A Multistep Synthesis Organic Chemistry

Laboratory Experiment pubs.acs.org/jchemeduc

Synthesis of 10-Ethyl Flavin: A Multistep Synthesis Organic Chemistry Laboratory Experiment for Upper-Division Undergraduate Students Vincent A. Sichula*,† Department of Physical Sciences, Nicholls State University, Thibodaux, Louisiana 70310, United States S Supporting Information *

ABSTRACT: A multistep synthesis of 10-ethyl flavin was developed as an organic chemistry laboratory experiment for upper-division undergraduate students. Students synthesize 10ethyl flavin as a bright yellow solid via a five-step sequence. The experiment introduces students to various hands-on experimental organic synthetic techniques, such as column chromatography, thin layer chromatography (TLC), extraction techniques, and characterizing intermediates and final products by IR and NMR spectroscopy. It also provides an opportunity for students to review important topics such as nucleophilic substitution reactions, hydrolysis of amides, condensation reactions, and reduction of aromatic nitro groups to amines. KEYWORDS: Organic Chemistry, NMR Spectroscopy, IR Spectroscopy, Synthesis, Chromatography, Upper-Division Undergraduate, Laboratory Instruction, Nucleophilic Substitution, Mechanisms of Reactions, Hands-On Learning/Manipulatives he chemistry of flavin compounds has attracted considerable scientific attention over the years because flavin-containing enzymes play important roles in many important biological processes.1 A laboratory experiment for a multistep synthesis of 10-ethyl flavin was adapted and modified to acquaint upper-division undergraduate organic chemistry students with the synthesis of flavin compounds.2−4 The experiment introduces students to various organic synthetic techniques and concepts found in organic research laboratories by performing reactions from the literature. This appears to be the first student laboratory experiment involving the synthesis of 10-ethyl flavin as no similar experiment has appeared in this Journal. The total synthesis of 10-ethyl flavin (7) (Scheme 1) is completed in eight, 4-h laboratory periods in an Intermediate Organic Chemistry class. After completing the overall synthesis, students are required to submit a final report in a style of a scientific journal. Eight students working in teams of two completed the experiment in 4 weeks. Students have conducted this experiment 4 times.

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EXPERIMENTAL OVERVIEW The experiment distinguishes itself from the previously published procedures in several ways (Scheme 1).2−12 The secondary amide 3 is isolated and used in the next synthetic step. Previously, the primary amide 2 was transformed directly into the secondary amine 4 in a single synthetic step by alkylating 2 using ethyl iodide in dimethylformamide (DMF) and removing the protecting group at a high refluxing temperature of 154 °C.2,3 The problem with this procedure is that the high temperature leads to the formation of dark residue impurities; isolation of the product is extensive and, therefore, leads to poor yields. It is also difficult to remove DMF from the reaction mixture during workup. Many undergraduate organic chemistry laboratories lack specialized rotary evaporators to remove DMF during workup. Therefore, the alkylation of 2 is done in acetonitrile (ACN), a low boiling point solvent, instead of DMF.7−11 The removal of the trifluoroacetyl protecting group is carried out by the hydrolysis of amide 3 in 6 M NaOH/MeOH (50:50 v/v) at 60 °C.12 This method produces

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PEDAGOGICAL GOALS This laboratory activity exposes students to several techniques and skills that are not well covered in traditional organic chemistry laboratory courses. The pedagogical goals are • To introduce students to multistep synthesis and the synthesis of flavin compounds • To introduce students to experimental organic synthetic techniques found in research laboratories © XXXX American Chemical Society and Division of Chemical Education, Inc.

• To teach students how to analyze and characterize organic compounds using IR and NMR analysis • To teach students how to write a scientific journal article to introduce students to the use of chemistry writing software, such as ChemDraw, and bibliographic software programs, such as endnote and reference manager

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DOI: 10.1021/ed5001087 J. Chem. Educ. XXXX, XXX, XXX−XXX

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Laboratory Experiment

Scheme 1. Synthetic Route for the Preparation of 10-Ethyl Flavin2−4

hydroxide and tin(II) chloride are corrosive. Hydrochloric acid and acetic acid are corrosive and cause severe burns to the skin. Alloxane monohydrate is acutely toxic and, therefore, toxic to liver and kidneys in high doses. Boric acid is toxic if taken internally or inhaled in large doses. Cyclohexane is an irritant and permeator. Dimethyl sulfoxide (DMSO-d6) is a combustible liquid and an irritant. The hazards of the products are unknown. Therefore, students should handle them as hazardous using personal protective equipment. For specific safety instructions for each experiment, please refer to the students’ handouts and MSDS sheets available for free from chemical companies.

a bright orange product with no dark impurities, and the yields are better. The nitro group in 4 is reduced using tin(II) chloride (SnCl2) in ethanol, which are safer reagents. The workup procedure for this method is much quicker and easier, and leads to higher yields.5 The reduction of the nitro group of 4 has previously been achieved in concentrated HCI and tin foil.2,4 The workup procedure for this method is timeconsuming as it requires the removal of the precipitated SnO through filtration that leads to poor yields.

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EXPERIMENT Students work in pairs. Four weeks are required to complete the experiment, meeting two times per week for 4 h each. In the first week in the first lab period, trifluoroacetic anhydride is added to 1 in dichloromethane at 0 °C to prepare 2 as a yellow solid. During the second lab period, students characterize the product using IR and 1H NMR spectra. In the second week in the first lab period, students heat a solution of 2, ethyl iodide, and potassium carbonate in ACN to prepare 3 as an oil. In the second lab period, 3 is isolated and not characterized. In the third week in the first lab period, 3 is refluxed in 6 M sodium hydroxide/methanol solution (50:50, v/v) at 60 °C for 3 h to produce 4. In the second lab period, 4 is isolated as a solid and is subjected to column chromatography in cyclohexane/ dichloromethane (1:1, v/v) to obtain 4 as a bright orange solid. Students collect the IR and 1H NMR spectra of the product. In the fourth week in the first lab period, the nitro group in 4 is reduced in ethanol with SnCl2 to obtain 5 as an oil that is characterized by 1H NMR spectroscopy. In the second lab period, 5 is condensed with alloxane (6) in warm acetic acid to give 10-ethyl flavin (7) as a solid. The solid is recrystallized in methanol, and the final product is characterized by IR and 1 H NMR spectroscopy. The details of the experiment are described in the Supporting Information.

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RESULTS AND DISCUSSION Students started the synthesis of 10-ethyl flavin by protecting 1 using trifluoroacetic anhydride to produce 22,6 to prevent dialkylation of 1. Students obtained 2 with a yield of 63−99% (Table 1). Analysis of both the IR and 1H NMR spectra Table 1. Percentage Yields for Each Compound Obtained by Students Compound

Average Student Yields (%)a

Student Yield Ranges (%)a

Amide 2 Amine 4 Amine 5 Flavin 7

86 66 65 42

63−99 55−73 58−73 24−54

a Percentage yields of 16 groups of students (N = 16) in 4 experimental trials. Four groups of students conducted each experimental trial, and each group comprised 2 students.

confirmed the synthesis of 2; representative student spectra are in the Supporting Information and the chemical assignments in the 1H NMR spectra are given in Figure 1. The second week, students prepared 3 by alkylation of 2 using ethyl iodide in acetonitrile at 60 °C.7−11 The alkylation reaction was achieved in 3 h, although TLC analysis showed the presence of a tiny amount of the starting material. Crude 3 was used in the next synthetic step without purification. The third week, students converted amide 3 to 412 as a solid with a yield of 55−73% (Table 1). Students confirmed the structure of compound 4 with 1H NMR spectroscopy, noting the appearance of methyl (CH3) group at chemical shift 1.22 ppm, the methylene (CH2) group at chemical shift 3.40 ppm, and the NH proton at 6.85 ppm. Their analysis of the IR spectrum of 4 showed the presence of an NH peak and the disappearance of the carbonyl

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HAZARDS All procedures should be carried out under the hood. Students should wear gloves and safety goggles all the time during the experiment. Ethanol, methanol, toluene, acetonitrile, ethyl acetate, and diethyl ether are flammable solvents. Students should be reminded to keep these solvents away from flames. Dichloromethane, chloroform, and ethyl iodide are suspected carcinogens. Trifluoroacetic anhydrides, sodium bicarbonate, potassium carbonate anhydrous, and 4,5-dimethyl-2-nitroaniline are acutely toxic. They cause irritation once in contact with skin and eyes. Methanol is an irritant and a permeator. Sodium B

DOI: 10.1021/ed5001087 J. Chem. Educ. XXXX, XXX, XXX−XXX

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Figure 1. Compounds 2 and 4 1H NMR assignments.

group peak of precursor 2 at 1739 cm−1 (Supporting Information). During the fourth week, students prepared 5 by reduction of the nitro group of 4 in ethanol and SnCl2.5 After isolating 5 in a yield of 58−73% (Table 1), students immediately proceeded with the preparation of 7 by condensation of 5 with 6 and boric acid in warm glacial acetic acid to give a solid.2,3 This transformation is a condensation reaction; students learn about these types of reactions in lectures and are able to apply what they have learned in a lab experiment. During the final lab period, students purified the solid residue by recrystallizing it in methanol to give pure 10ethyl flavin (7) in yields of 24−54% (Table 1). Analysis by 1H NMR spectroscopy of 5 and 7 (Supporting Information) confirmed that the compounds were pure. The students’ assignments and final report demonstrated that the pedagogic goals were met. Students successfully synthesized the flavin compound, correctly characterized the intermediates and final product using IR and NMR spectra, and submitted a final report using ChemDraw and bibliographic writing programs.

this lab for the first time in the laboratory classroom and showing that it can be indeed incorporated into our organic chemistry laboratory course. Their positive feedback was very encouraging. I would like also to acknowledge the physical sciences department at Nicholls State University for the support with the purchase of reagents and chemicals.

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CONCLUSION An experimental procedure for the preparation of 10-ethyl flavin was adopted and developed as an upper-division undergraduate organic chemistry laboratory experiment. The lab introduced students to various hands-on experimental organic synthetic techniques, which will be useful in the students’ futures in chemistry.

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ASSOCIATED CONTENT

S Supporting Information *

Notes for the instructor; handouts for students; list of chemicals and reagents, sample characterization, representative student spectra. This material is available via the Internet at http://pubs.acs.org.

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REFERENCES

(1) (a) Roland, C. T. The Flavins of Milk. J. Chem. Educ. 1936, 13 (10), 481−482. (b) Henderleiter, J. A.; Hyslop, R. M. The Analysis of Riboflavin in Urine Using Fluorescence. J. Chem. Educ. 1996, 73 (6), 563−564. (c) Utecht, R. E. Identification and Quantification of Riboflavin in Vitamin Tablets by Total Luminescence Spectroscopy. J. Chem. Educ. 1993, 70 (8), 673. (d) Hy, Y. J.; Senkbeil, E. G. Fluorometric Analysis of Riboflavin: An Undergraduate Biochemistry Experiment. J. Chem. Educ. 1990, 67 (9), 803−804. (e) Brown, P. R. The Investigation of the Purity and Stability of Nicotinamide and Flavin Coenzymes. J. Chem. Educ. 1976, 53 (2), 98. (f) Gallardo, F.; Galvez, S.; Medina, M. A.; Heredia, A. A Laboratory Exercise for Photochemistry and Photobiology: Production of Hydrogen Peroxide. J. Chem. Educ. 2000, 77 (3), 375−377. (g) Schneider, F. Quantification of coenzyme (FAD) using the apoenzyme of a flavin enzyme, Daminoacid oxidase. Biochem. Educ. 1991, 19 (4), 209−210. (h) Bateman, R. C., Jr; Evans, J. A. Using the Glucose Oxidase/Peroxidase System in Enzyme Kinetics. J. Chem. Educ. 1995, 72 (12), A240− A241. (i) Sumner, J. B. Enzymes the basis of life. J. Chem. Educ. 1952, 29 (3), 114−118. (j) Li, G.; Glusac, K. D. Light-Triggered Proton and Electron Transfer in Flavin Cofactors. J. Phys. Chem. A 2008, 112 (20), 4573−4583. (k) Massey, V. The Chemical and Biological Versatility of Riboflavin. Biochem. Soc. Trans. 2000, 28 (4), 283−296. (l) Gelalcha, F. G. Heterocyclic Hydroperoxides in Selective Oxidations. Chem. Rev. 2007, 107 (7), 3338−3361. (m) Kao, Y. T.; Saxena, C.; He, T. F.; Guo, L. J.; Wang, L. J.; Sancar, A.; Zhong, D. P. Ultrafast Dynamics of Flavins in Five Redox States. J. Am. Chem. Soc. 2008, 130 (39), 13132−13139. (n) Heelis, P. F. The photophysical and photochemical properties of flavins (isoalloxazines). Chem. Soc. Rev. 1982, 11 (1), 15− 39. (o) Massey, V. Activation of Molecular Oxygen by Flavins and Flavoprotein. J. Biol. Chem. 1994, 269 (36), 22459−22462. (p) Ryerson, C. C.; Ballou, D. P.; Walsh, C. Mechanistic studies on cyclohexanone oxygenase. Biochemistry 1982, 21 (11), 2644−2655. (q) Fraaije, M. W.; Kamerbeek, N. M.; Heidekamp, A. J.; Fortin, R.; Janssen, D. B. The Prodrug Activator EtaA from Mycobacterium tuberculosis is a Baeyer-Villiger Monooxygenase. J. Biol. Chem. 2004, 279 (5), 3354−3360. (r) Miller, J. R.; Edmondson, D. E. Structure Activity Relationships in the Oxidation of Para-Substituted Benzylamine Analogues by Recombinant Human Liver Monoamine Oxidase A. Biochemistry 1999, 38 (41), 13670−13683. (s) Ghisla, S.; Thorpe, C.; Massey, V. Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the βhydrogen to the flavin position N(5) as a hydride. Biochemistry 1984, 23 (14), 3154−3161. (t) Sancar, A. Structure and Function of DNA Photolyase. Biochemistry 1994, 33 (1), 2−9. (u) Breslow, R.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address †

Department of Chemistry, Winona State University, Winona, Minnesota 55798. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Special thanks to the students of 2011−2013, Intermediate Organic Chemistry, at Nicholls State University for performing C

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Biomimetic Chemistry and Artificial Enzymes: Catalysis by Design. Acc. Chem. Res. 1995, 28 (3), 146−153. (v) Bruice, T. C.; Main, L.; Smith, S.; Bruice, P. Y. Preequilibrium Complex Formation and Nucleophilic Addition (and its position) as Factors in FlavineCatalyzed Oxidations. J. Am. Chem. Soc. 1971, 93 (26), 7327−7328. (w) Legrand, Y. M.; Gray, M.; Cooke, G.; Rotello, V. M. Model Systems for Flavoenzyme Activity: Relationships between Cofactor Structure, Binding and Redox Properties. J. Am. Chem. Soc. 2003, 125 (51), 15789−15795. (2) Sichula, V.; Hu, Y.; Mirzakulova, E.; Manzer, F. S.; Vyas, S.; Hadad, M. C.; Glusac, D. K. Mechanism of N(5)-Ethyl-flavinium Cation Formation Upon Electrochemical Oxidation of N(5)-Ethyl-4ahydroxyflavin Pseudobase. J. Phys. Chem. B 2010, 114 (29), 9452− 9461. (3) Epple, R.; Wallenborn, E. U.; Carell, T. Investigation of FlavinContaining DNA-Repair Model Compounds. J. Am. Chem. Soc. 1997, 119 (32), 7440−7451. (4) Sichula, V.; Kucheryavy, P.; Khatmullin, R.; Hu, Y.; Mirzakulova, E.; Vyas, S.; Manzer, S. F.; Hadad, C. M.; Glusac, K. D. Electronic Properties of N(5)-Ethyl Flavinium Ion. J. Phys. Chem. A 2010, 114 (46), 12138−12147. (5) Cai, S. X.; Kher, S. M.; Zhou, Z. L.; Ilyin, V.; Espitia, A. S.; Tran, M.; Hawkinson, J. E.; Woodward, M. R.; Weber, E.; Keana, J. F. W. Structure-Activity Relationships of Alkyl- and Alkoxy-Substituted 1,4Dihydroquinoxaline-2,3-diones: Potent and Systemically Active Antagonists for the Glycine Site of the NMDA Receptor. J. Med. Chem. 1997, 40 (5), 730−738. (6) Brown, S. A.; Rizzo, C. A “One-Pot” Phase Transfer Alkylation/ Hydrolysis of o-Nitrotrifluoroacetanilides. A Convenient Route to NALKYL o-Phenylenediamines. Synth. Commun. 1996, 26 (21), 4065− 4080. (7) Lamoureux, G.; Agüero, C. A comparison of several modern alkylating agents. ARKIVOC (Gainesville, FL, U. S.) 2009, 2009 (1), 251−264. (8) Li, C.; Wong, W. T. A convenient method for the preparation of mono N-alkylated cyclams and cyclens in high yields. Tetrahedron Lett. 2002, 43 (17), 3217−3220. (9) Salvatore, R. N.; Yoon, H. C.; Jung, C. K. Synthesis of secondary amines. Tetrahedron 2001, 57 (37), 7785−7811. (10) Seeman, I. J.; Whidby, F. J. The iodomethylation of nicotine. An unusual example of competitive nitrogen alkylation. J. Org. Chem. 1976, 41 (24), 3824−3826. (11) Warwick, G. P. The Mechanism of Action of Alkylating Agents. Cancer Res. 1963, 23 (8), 1315−1333. (12) Vaughn, L. H.; Robbins, D. M. A Rapid Procedure for the Hydrolysis of Amides to Acids. J. Org. Chem. 1975, 40 (8), 1187− 1189.

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DOI: 10.1021/ed5001087 J. Chem. Educ. XXXX, XXX, XXX−XXX