Laboratory Experiment pubs.acs.org/jchemeduc
The Petasis Reaction: Microscale Synthesis of a Tertiary Amine Antifungal Analog Katherine J. Koroluk, Derek A. Jackson, and Andrew P. Dicks* Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6 S Supporting Information *
ABSTRACT: Students prepare a tertiary amine antifungal analog in an upper-level undergraduate organic laboratory. A microscale Petasis reaction is performed to generate a liquid compound readily characterized via IR and proton NMR spectroscopy. The biological relevance of the product is highlighted, with the tertiary amine scaffold being an important treatment option for resistant bacterial and fungal infections. The procedure allows for postlaboratory discussion of nitrogen as a stereocenter in an ammonium ion, along with principles of combinatorial chemistry and green chemistry. Deliberation of potential mechanisms for the Petasis reaction provides another valuable learning opportunity for students. KEYWORDS: Upper-Division Undergraduate, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives, Amines/Ammonium Compounds, Chirality/Optical Activity, Combinatorial Chemistry, Drugs/Pharmaceuticals, Microscale Lab, NMR Spectroscopy
A
structure. Petasis and Akritopoulou reported synthesis of naftifine and terbinafine, which are members of the allylamine class of antifungals (Figure 1).5 The student product of the
mine preparation is a discussion topic within the lecture component of almost every introductory organic undergraduate course. Surprisingly, however, reports of amine synthesis in the teaching laboratory are relatively uncommon. Ammonia and amine alkylations are known to be problematic as they lead to product mixtures.1 Two reductive amination procedures have been described in this Journal: in the first, a secondary amine was generated,2 and in the second, two heterocyclic tertiary amines were formed.3 The reductive amination of pyruvate esters with sodium triacetoxyborohydride and benzylamine has also been published.4 An alternative approach to tertiary amine synthesis is the lesser-known Petasis reaction. This multicomponent process was first reported in 19935 and is commonly referred to as a “boronic acid Mannich” reaction. The transformation involves combination of a secondary amine, an aldehyde, and a boronic acid to produce a tertiary amine (Scheme 1). The synthetic
Figure 1. Common allylamine antifungal compounds.
Petasis reaction described here (N-benzyl-N-methyl-(E)cinnamylamine, Scheme 2) is structurally related to naftifine (a topical antimycotic)12 and terbinafine (an oral antifungal agent).13 This experiment was incorporated into a third-year undergraduate organic laboratory with a 4.5 h laboratory and two 50 min lectures per week, and a typical enrollment of 25− 45 students. Associated lectures covered concepts of advanced spectroscopy, green chemistry, and combinatorial chemistry, which allowed students to directly connect theoretical principles with their practical experiences.14 The medicinal relevance of the product generated considerable class interest, especially among students studying biological chemistry.
Scheme 1. A General Petasis Reaction
importance of the Petasis reaction is underscored by a recent comprehensive review article.6 Its operative mechanism remains unestablished, with potential intermolecular and intramolecular pathways possible.7−11 A procedurally simple Petasis reaction was developed for an upper-level undergraduate synthetic laboratory. Such an experiment has apparently not been previously reported in the pedagogical chemistry literature. The Petasis reaction is useful in the formation of antifungal compounds that contain a tertiary amine moiety in their © 2012 American Chemical Society and Division of Chemical Education, Inc.
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EXPERIMENTAL OVERVIEW The students performed the synthesis individually. Paraformaldehyde and N-benzylmethylamine (equimolar amounts) in dioxane were refluxed at 90 °C for 10 min. A solution of (E)Published: March 15, 2012 796
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Laboratory Experiment
alkene (965 cm−1) appeared after addition of the styryl moiety to the amine−aldehyde adduct.16
Scheme 2. Student Preparation of N-Benzyl-N-methyl-(E)cinnamylamine
Examination of the Stereocenter
An extremely valuable aspect of the experiment was proton NMR analysis of both the tertiary amine and the corresponding protonated ammonium ion. Students prepared NMR samples of their own liquid product and directly assessed its purity. Most spectra contained small impurity peaks corresponding to dioxane (δ 3.71)17 and N-benzylmethylamine (δ 3.77, 2.45, and 1.78, Figure 2A). However, some students obtained very pure
styrylboronic acid in dioxane was added to the hot reaction mixture, followed by reflux for 90 min. After this time, the reaction solution was acidified and washed with ether. The aqueous layer was then made basic and the product extracted with ether. The organic component was dried with anhydrous potassium carbonate and the solvent removed under vacuum to isolate N-benzyl-N-methyl-(E)-cinnamylamine in yields of 40− 80% (average student yield 60%; lit.5 yield 96%). Students prepared a NMR sample for a teaching assistant to run. Each student then processed the free-induction decay to produce an NMR spectrum. 1H NMR (CDCl3, 400 MHz); δ (ppm): 7.39− 7.20 (10H, m); 6.54 (1H, d, J = 15.9 Hz); 6.31 (1H, dt, J = 15.9, 6.6 Hz); 3.55 (2H, s); 3.19 (2H, dd, J = 6.6, 1.4 Hz); 2.24 (3H, s).
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HAZARDS Sigma-Aldrich MSDS were consulted for all substances used in this experiment.15 Appropriate gloves, safety goggles, and a laboratory coat should be worn at all times. Paraformaldehyde, N-benzylmethylamine, and dioxane are hazardous via skin or eye contact, inhalation, and ingestion. Dioxane and paraformaldehyde are possible carcinogens. N-Benzylmethylamine is corrosive and toxic to mucous membranes. (E)-Styrylboronic acid is irritating to the eyes, respiratory system, and skin. Hydrochloric acid and sodium hydroxide are corrosive to the eyes and skin and are harmful by inhalation and ingestion. Diethyl ether is extremely flammable and is harmful if inhaled, swallowed, or absorbed through skin. The hazards associated with the reactants and solvent are significantly reduced by the very small quantities employed.
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Figure 2. (A) Student 1H NMR spectrum of N-benzyl-N-methyl-(E)cinnamylamine (400 MHz, CDCl3); (B) instructor 1H NMR spectrum of protonated N-benzyl-N-methyl-(E)-cinnamylamine (400 MHz, D2O).
product with virtually no impurities visible. A coupling constant of approximately 16 Hz was apparent between the vinylic proton peaks, a value consistent with that expected for a trans alkene (12−18 Hz).18 Thus, students concluded the double bond stereochemistry had been retained from (E)-styrylboronic acid. Undergraduates rarely encounter the concept of a heteroatomic stereocenter in a practical setting. There are very few references to heteroatoms as stereocenters from a teaching perspective, some of which refer to chirality at sulfur.19,20 A nitrogen atom can act as a stereocenter in configurationally stable ammonium salts, whereas a tertiary amine will generally undergo an umbrella-like inversion of its three substituents and lone pair of electrons.21,22 In addition to obtaining their own product proton NMR spectrum, an instructor spectrum of the protonated ammonium salt was distributed to each student (Figure 2B). Contrasts were easily made between the two spectra, and the effects of generating a stereocenter at the nitrogen atom were apparent. The ammonium ion spectrum clearly showed the new splitting patterns created by diastereotopic protons adjacent to the nitrogen stereocenter. The singlet and doublet of doublet peaks from the methylene protons adjacent to the nitrogen atom in N-benzyl-N-methyl(E)-cinnamylamine were transformed into four separate signals in the ammonium ion spectrum. These peaks appeared at δ
DISCUSSION
Synthesis
During the experiment, acidic and basic workup procedures were performed and the final product was analyzed via IR spectroscopy. Students had to recognize that the acidic workup removed unwanted organic material via washing with ether, whereas the basic workup facilitated tertiary amine product isolation in the ether layer during the subsequent extraction step. When the procedure was first tested in the course, several undergraduates combined the ether layers from both acidic and basic workups. This rendered their product impure due to the presence of unreacted starting material, and emphasized a lack of understanding of the purification steps. Neat IR spectra of the tertiary amine and N-benzylmethylamine starting material were individually acquired. This permitted clear observation of product formation, as the N−H stretch (3300 cm−1) associated with N-benzylmethylamine conspicuously disappeared with product formation. Additionally, a peak derived from a trans 797
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ACKNOWLEDGMENTS The Department of Chemistry, University of Toronto is thanked for financial support via the graduate student Teaching Fellowship Program (D.A.J). A.P.D is grateful to the Faculty of Arts and Science, University of Toronto for a President’s Teaching Award.
4.12, 3.94, 3.66, and 3.54, and showed evidence of both vicinal and geminal couplings. This comparison provided a dramatic visualization of stereochemical effects on an NMR spectrum. Green Aspects
The green aspects of all student reactions are critically analyzed in the course. Recent literature surrounding the Petasis reaction discusses attempts to improve its environmental friendliness, including solvent-free,23 aqueous,24,25 microwave-assisted,26,27 and ionic liquid-accelerated28 procedures. These examples provided a framework to discuss sustainability principles and possible improvements to the laboratory protocol, such as replacing dioxane with a greener solvent. The reaction was performed on a microscale, thereby reducing waste and challenging student practical abilities. The final product consisted of a small quantity of liquid, so a great deal of care was exercised during extraction to prevent mechanical losses. The intrinsic atom economy of a reaction is one measure of process “greenness”.29 The student-run Petasis reaction exhibited a high intrinsic atom economy of 80%, with only 1 equivalent of boric acid produced as a benign byproduct.
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Multicomponent synthetic approaches are extremely important in combinatorial chemistry, with linear two-component procedures requiring more time and effort to synthesize the same products.9,30 Preparation of a wide variety of different compounds with the same core structure provides a library that is screened for pharmaceutical leads. The technique of combinatorial chemistry is a topic students learned in class lectures, with application of the multicomponent Petasis reaction in combinatorial chemistry being a relevant approach introduced through the laboratory. Students consequently appreciated the advantages of multicomponent reactions and their application to the drug discovery process.
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CONCLUSION The novel undergraduate synthesis of a tertiary amine allowed students to gain hands-on appreciation of a functionality that is largely overlooked during laboratory work. The small reaction scale and formation of a liquid tested experimental technique, as diligence was essential to achieve acceptable product recovery. NMR analysis of the protonated product illustrated the spectroscopic effects of introducing a stereocenter into a molecule without altering substituent structures. Recent literature concerning the Petasis reaction promoted a postlaboratory discussion of green synthetic aspects and potential improvements. The experiment connected well with associated advanced lecture topics, which included principles of combinatorial chemistry and multicomponent reactivity. ASSOCIATED CONTENT
S Supporting Information *
Instructions for the students; notes for the instructor; representative product spectra. This material is available via the Internet at http://pubs.acs.org.
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REFERENCES
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Combinatorial Chemistry
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 798
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