In the Laboratory
Natural Product Total Synthesis in the Organic Laboratory: Total Synthesis of Caffeic Acid Phenethyl Ester (CAPE), A Potent 5-Lipoxygenase Inhibitor from Honeybee Hives Mohamed Touaibia* and Michel Guay D epartement de Chimie et Biochimie, Universit e de Moncton, Nouveau-Brunswick, Canada E1A 3E9 *
[email protected] Owing to the importance of multistep synthesis, we explored several natural product syntheses and found that the three-step procedure for the synthesis of caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE, Figure 1) can be performed with excellent results by undergraduate students. The total synthesis can be conducted in three 4-h lab periods. Some natural product total syntheses have recently appeared in this Journal (1, 2). To our knowledge, no analogous experimental procedure has appeared in this Journal. Interest in the biochemical and biological properties of polyphenols has grown considerably, as epidemiological evidence for their beneficial effects on health continues to increase. Dietary polyphenol antioxidants are reported to have many interesting properties, including anti-inflammatory, vasoprotection, anticancer, and antiobesity effects (3). CAPE, the active polyphenol of propolis from honeybee hives (4), is an intriguing target. It has been reported that CAPE inhibits 5-lipoxygenase (5-LO) activity (5). 5-LO is the key enzyme in the metabolism of arachidonic acid (AA) to leukotriene A4 (LTA4) (6). Further metabolism of LTA4 produces LTB4, a potent chemotactic agent for leukocytes that is thought to be a key component in a variety of diseases (7), including inflammatory bowel disease and atherosclerosis. LTA4 can also be converted to the peptidoleukotrienes LTC4, LTD4, and LTE4, which are implicated in allergic hyperreactivity disorders such as asthma (8). Elevated levels of these LTs, associated with several inflammatory and allergic disorders, have been found in various pathologic tissues (9). Instead of the traditional one-step organic synthesis, the present multistep synthesis can be a valuable learning experiment. This three-step synthesis of CAPE sequence is appropriate for teaching students the importance of protecting groups in natural product synthesis. This is a major concept in organic synthesis and one of the determining factors in the successful realization of multistep synthetic projects (10, 11). In this synthesis, we used the acetyl-protecting group. Among the various protecting groups used for the hydroxyl, acetyl is one of the most common groups because of its easy introduction, stability, and its easy removal. During the present synthesis, circular chromatography, an interesting purification method, is introduced. Circular chromatography combines
Figure 1. CAPE structure.
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the advantages of both preparative thin-layer chromatography (TLC) and column chromatography. With spinning, the sample components migrate through the rotor adsorbent as circular bands and have differing affinities for the adsorbent versus the mobile phase. The transparent lid of the chromatotron allows direct observation of UV absorbing of colored compounds during the purification. Separations occur quickly, usually within 1 h, versus the 2 h for preparative TLC or column chromatography. From a green perspective, circular chromatography has also the advantage of consuming less solvent. With this experiment, students will have the opportunity to increase their autonomy by being responsible of the handling, synthesis, characterization, and purification for all the synthesized molecules.1 Knowledge of characterization techniques, infrared (IR) and nuclear magnetic resonance (NMR, 1H and 13C) spectroscopy and mass spectrometry (MS), learned in previous course or labs is called upon. Students also exert their critical thinking by comparing two different chromatographic techniques (column vs circular). Experimental Overview Synthesis All chemicals are commercially available from Aldrich Chemical Co. Detailed experimental procedures are provided in the supporting information. The three-step synthesis of CAPE is summarized in Scheme 1. In the first step, commercially available caffeic acid, 1, is treated with sodium hydroxide and acetic anhydride at 0 °C. Isolation of the crude product is accomplished by cooling, vacuum filtration, and washing with cold water. Recrystallization of the crude product from ethanol provides pure samples of diacetylcaffeic acid, 2, in good yield. Typical student yields are 75-90%. The ester, 3, was synthesized from 2-phenylethanol with acetylated caffeic acid, 2.2 The conversion of 2 into the corresponding carboxylic chloride was achieved by the Vilsmeier-Haack adduct (12) derived from oxalyl chloride (13)3 and N,N-dimethylformamide (DMF) as catalyst. After removal of solvents, the residue is dissolved in dichloromethane or ethyl acetate, the organic extract is washed with water, brine, and then is dried over MgSO4 for purification by circular or flash chromatography. The obtained reactive adduct differs from the intermediate of the VilsmeierHaack reaction (14), a useful method for introducing formyl and acyl groups in electron-rich aromatic compounds, only insofar as the cation is associated with a chloride ion, rather than a dichlorophosphate ion. The mixed anhydride, obtained after the
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r 2011 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 4 April 2011 10.1021/ed100050z Published on Web 01/19/2011
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In the Laboratory
Scheme 1. The Three-Step Synthesis of CAPE
carboxylic acid attack on the imminium carbon of the Vilsmeier-Haack adduct (13), acylates the released chloride ion to produce the corresponding acid chloride. As a second route, the carboxylic chloride can be obtained via the same Vilsmeier-Haack intermediate by replacing oxalyl chloride by thionyl chloride (SOCl2) (12, 15). For safety considerations, oxalyl chloride/ DMF activation is preferred; this step is achieved at room temperature rather than reflux heating with thionyl chloride/DMF activation. Addition of DMF as catalyst was used to form acid chlorides of a large variety of acids including unsaturated ones. In our hands, activation of 2 with oxalyl chloride or thionyl chloride without DMF was achieved in more than 4 h. As such, this activation step is too long and will not fit in our experiment. To introduce the circular chromatography purification technique, the class is split into two groups, one that purifies the crude 3 by flash column chromatography whereas the other uses circular chromatography (Chromatotrons, model 7924, Harrison Research) to afford the required pure samples of acetylated CAPE, 3, in good yield. Typical student yields are 60-70%. Once the desired products are purified, the data (time and solvent quantity) from the whole class are made available to all students and the instructor asks the students to compare the two techniques. As demonstrated by students' results, purification time and solvent quantity were decreased by circular chromatography compared to flash chromatography. Circular chromatography purification was achieved within 1 h and used only 75 mL of eluent, whereas flash chromatography took 2 h and used more than 150 mL of eluent. Base-induced de-O-acetylation in 3 to afford the CAPE was accomplished with 3 equiv of potassium carbonate in methanol and dichloromethane (Scheme 1). After removal of solvents, a white solid in high purity as determined by NMR spectroscopy was obtained. Student yields for the de-O-acetylation range from 50% to 70%. 474
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Characterization Students characterize CAPE and intermediates 2 and 3 by IR and NMR spectroscopy, MS, and melting point. All compounds generate an interesting set of spectra that provide students with an excellent opportunity to practice and develop their skills at spectral interpretation. The IR spectrum of the acetylated caffeic acid, 2, shows the presence of the OH and carbonyl bonds in the carboxyl group. This is useful because, as a result of rapid proton exchange, the carboxylic acid hydrogens in the 1H NMR spectra often are not observed. This is one of the features of the analysis that students can be asked to explain. The 1H NMR singlet for the methyl hydrogens, with right integration, verifies that the caffeic acid hydroxyls were well protected. The vinyl hydrogen coupling constants are determined by our students. These are based on chemical shifts observed in spectra for their products.4 Coupling constants, J, for the vinyl protons are observed to be 17 Hz. Because J is 11-18 Hz for the trans configuration and 6-15 Hz for the cis configuration, the trans configuration of all products was confirmed. The acetylated caffeic acid, 2, structure was also confirmed by the presence of two alkyl peaks (20.3 and 20.4 ppm) and two ester carbonyl peaks (168.1 and 168.2 ppm) in the 13C NMR spectrum. The high resolution MS spectrum shows the sodium adduct (M þ Na)þ ion. For the intermediate 3, the IR spectrum shows the absence of the OH in the carboxyl group. The obvious pair of 1H NMR triplets and the two alkyl peaks (35 and 65 ppm) in the 13C NMR spectrum verifies that the second esterification was accomplished. Both (M þ Na)þ and (2M þ Na)þ ions are observed in the high resolution MS spectra. The absence of the 1H NMR singlet for the methyl hydrogens, plus the absence of the two alkyl peaks (20 ppm) and ester carbonyls peaks (167-168 ppm) in the 13C NMR spectrum verify that the base-induced de-O-acetylation was successfully accomplished, which confirms the structure of the final compound (CAPE). The high resolution MS spectra shows the protonated ion (M þ H)þ and the sodium adduct (M þ Na)þ. Hazards The organic reactants, products, and solvents are toxic and flammable. Dichloromethane should be handled with particular care as it is anticipated to be a carcinogen. Overexposure may lead to fatigue, weakness, sleepiness, nausea, light-headedness, and irritation of eyes and skin. Methanol and toluene exposure (whether by inhalation or skin contact) can intoxicate and cause damage to the eyes. Oxalyl chloride should be handled with particular care as it is corrosive. Pyridine, sodium hydroxide, and acetic anhydride exposure can cause burns. Potassium carbonate is an irritant. All compounds must be handled in a manner consistent with the information available on their material safety data sheets (MSDS). Hand and eye protection must be worn. All procedures must be conducted in a fume hood. Discussion The proposed procedure gives the organic students an opportunity to investigate a multistep natural product total synthesis. This experiment is suitable for an undergraduate-level organic chemistry lab course. The proposed three-step synthesis of CAPE was executed, with 13 students, in three laboratory periods each lasting 4 h during the last 3 weeks of the second semester of the
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organic laboratory course. This experiment encourages students to think in terms of conversion of carboxylic acids into carboxylic chlorides, retrosynthetic strategy, and the elaboration of key intermediates; examples of protecting groups for several functional groups can be mentioned by the lab instructor. The experiment is particularly interesting if these topics are presented beforehand in the organic lecture. As column chromatography purification is time-consuming, a thorough understanding of alternative options is important for students. This experiment can introduce the students to circular chromatography. In comparison with flash chromatography, students conclude that circular chromatography is faster and requires only a small quantity of solvent. With this experiment, students are introduced to multistep synthesis and biologically active compounds, demonstrating the relationship between organic synthesis and drug discovery. Student feedback has been positive at the completion of the total synthesis. The idea of synthesizing a bioactive molecule with a three-step procedure in more than one laboratory period appealed to the students. Most of the students preferred the circular chromatography purification technique for its shorter purification time and lessened use of solvent. One student commented, “It's less solvent and time consuming and you can monitor the product purification by UV”. If this laboratory is used in a more advanced course, students can fully estimate the 5-LO inhibitory activity for their compound on selected cells. Quantification of 5-LO products can be performed by HPLC with UV detection (16). In addition to the pedagogical advantage, students develop critical-thinking skills and experience the atmosphere of an organic-medicinal chemistry research laboratory. This experiment will increase students' interest in chemistry research. Acknowledgment This research was financially supported by the Universite de Moncton, the New Brunswick Innovation Foundation, and the Medical Research Fund of New Brunswick. We gratefully acknowledge David Barnett (ACRI, Moncton, NB) and Isabelle Rheault (UQAM, QC) for their assistance with the MS analysis. We also thank the students of the 2008-2009 organic chemistry laboratory course for their constructive feedback during the implementation of this experiment. Special thanks to J. Jean-Franc- ois and N. Arya for providing assistance with the preparation of the manuscript. Notes 1. Prior to this three-step synthesis, students in our laboratory had prepared and analyzed another molecule in one step. Thus, they were already familiar with the use of NMR for examination of structural features. 2. We were not able to perform this second step with N,N0 dicyclohexylcarbodiime (DCC); the yields were very poor.
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3. As reported in the literature, this carboxylic acid activation with oxalyl chloride usually lasts 3-5 h (13). In 1 h of activation, the yield of 3 was less than 50%, increasing the reaction time to 2 h gave a yield of 60%. 4. No cis-caffeic acid esters were isolated in our syntheses and 1H NMR spectra showed no evidence of cis isomers.
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Supporting Information Available Experimental procedure; sample characterization data; notes for the student; suggestions for the instructor. This material is available via the Internet at http://pubs.acs.org.
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