In the Laboratory
Isolation of Betulin and Rearrangement to Allobetulin
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A Biomimetic Natural Product Synthesis Brian Green, Michael D. Bentley, Bong Y. Chung, Nicholas G. Lynch, and Bruce L. Jensen* Department of Chemistry, University of Maine, Orono, ME 04469; *
[email protected] The triterpenoids are a collection of over 4000 naturally occurring compounds with 40 skeletal types derived from squalene (1, 2). During the biosynthetic pathway, a series of cyclization and subsequent rearrangement reactions produce a multitude of compounds with diverse structures. To the chemist, the triterpenes have provided elegant examples for structure elucidation, mechanistic and stereochemical studies, and as platforms for chiral syntheses. Despite their wide occurrence in everyday life, this fascinating group of natural products is rarely presented in the undergraduate curriculum. The chemical source for the experiment described herein, the pentacyclic triterpene betulin, literally can be found in your backyard. Betulin (Scheme I) is a pentacyclic triterpene isolated from white birch species (Betula spp.) that are abundant in the northern latitudes of the world. This compound is present in concentrations as high as 30% of the dry weight of bark in a Scandinavian species (B. verrucosa) while the North American white species, paper birch (B. papyrifera) and gray birch (B. populifolia), can have more than 20% of this natural product (3–5). Although its role in the bark is not known, there are several reports of biological activity, including aphid antifeedant activity (6). Its carboxylic acid derivative, betulinic acid (Scheme I), has displayed anticancer activity as an inhibitor of human melanoma (7) and a possible cure for brain cancer (8). In addition, amide derivatives of betulinic acid are known to have anti-HIV properties (9). Betulin has even found its way into some skin creams. Betulin can be easily isolated in 20–22% yield by extraction of dry white birch bark with chloroform. The isolation procedure is remarkably clean and only a trace quantity of lupeol can be seen by TLC analysis of the extract. Subsequent, acidcatalyzed rearrangement of betulin to allobetulin (Scheme I) proceeds by a step-by-step carbocation mechanism identical, in every respect, to the biosynthetic process. In doing this experiment, students have the opportunity to link scientific subject matter to the world around them.
product provides sufficient material for complete spectral analysis by IR, 1H NMR, and 13C NMR. In addition, this experiment exposes students to a number of important techniques found in the organic chemistry laboratory, including extraction, thinlayer chromatography, micro-column flash chromatography, and a complete array of spectroscopic tools for structural analyses. Hazards Chloroform is a volatile solvent, once used as an anesthetic. Chronic exposure to chloroform may lead to liver and kidney damage. Even in low doses, inhalation of chloroform may result in an allergic reaction. p-Toluenesulfonic acid ( p-TSA) is a corrosive non-volatile compound that causes irritation, redness, and burns upon contact with human tissue. These health hazards can be mitigated by working in a well-ventilated hood and by wearing protective gloves and goggles. birch bark CHCl 3 30 min 29
CH2
H
1
26 CH3 C CH3
R
D
28
15
B
A
E
18
9 3
21
19
25
CH3
27
HO
H H3 C
23
CH3
24
p-TSA 60 min
Experimental Overview A two-year curriculum development award by the Camile and Henry Dreyfus Foundation has allowed us to develop 29 laboratory experiments for the undergraduate laboratory program at this university. Fourteen of these experiments utilize natural products—all with unique highly educational NMR, IR, or molecular modeling properties. The isolation and rearrangement of betulin is typical of these types of experiments, which now serve as the backbone of our curriculum. The one-period microscale experiment described herein consists of an isolation procedure for betulin by extraction from birch bark with chloroform followed by purification over a micro-column of silica gel. Betulin is then subjected to acid-catalyzed rearrangement and expansion of its E-ring, in the presence of p-toluenesulfonic acid ( p-TSA), affording allobetulin (Scheme I). The good yield of
20
30
H3C
R = CH2OH, betulin lupeol R = CH3, R = CO2H, betulinic acid refluxing CHCl3 85–95%
CH3
H3C H O
H CH3
H
CH3
CH2
CH3 HO
H H3C
CH3
allobetulin
Scheme I. Isolation of betulin and rearrangement of allobetulin. It’s featured on this issue’s cover. See cover description on page 1891 of this issue for more details.
www.JCE.DivCHED.org • Vol. 84 No. 12 December 2007 • Journal of Chemical Education 1985
In the Laboratory
Results and Discussion
A
4.5
4.0
4.5
3.5
4.0
3.5
3.0
2.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Chemical Shift (ppm)
B
3.8 3.7 3.6 3.5 3.4 3.3 3.2
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Chemical Shift (ppm) Figure 1. 1H NMR spectra of (A) betulin and (B) allobetulin.
H
O
CH3 Me
H
A
3
Me
H
H
Figure 2. Axial and equatorial coupling of C3 axial proton in the A-ring.
29
Me +
30
Me H
20
21 19
+
E CH2OH
28
H 28
:
H
O +
19
H
H
O
H 28
19
– H+ Me
H
Me
18
Me H
Scheme II. Acid-catalyzed rearrangement of E-ring.
30
29
Me
Isolation of Betulin About 500 mg of crude birch bark is cut into small pieces and placed in a 50 mL round-bottomed flask with chloroform (15 mL) and a few small boiling chips. The mixture is refluxed for thirty minutes on a sand bath. At the end of this period, the hot chloroform solution is filtered through a Hirsch funnel, the bark is washed with additional hot chloroform (3 × 10 mL), filtered, and the chloroform extracts combined. Purification of Betulin The chloroform extracts are rapidly passed through a microscale column of silica gel to remove tan impurities and the effluent collected in a clean dry flask. An NMR sample can be obtained by evaporating 5 mL of this solution to dryness using a rotary evaporator and a warm-water bath. The remaining chloroform (30–35 mL) solution is then used for the rearrangement reaction. Betulin (10 mg) is obtained as a white–tan colored solid with a melting point of 248–251 °C. A thin-layer chromatogram is run on a plastic-backed TLC slide using chloroform:ethyl acetate (9:1) as the eluent and the IR and 1H NMR spectra of pure betulin are determined. The NMR solution can be saved and used for the rearrangement reaction. Rearrangement of Betulin to Allobetulin p-Toluenesulfonic acid ( p-TSA) hydrate (120 mg) is dissolved in the remaining chloroform solution of betulin (30–35 mL, containing 55–65 mg of betulin). Water (3 drops) and a microboiling chip are added to the chloroform solution in a 50 mL round-bottomed flask, and the contents vigorously heated under reflux with a water condenser for 60 minutes. After the reflux period, the chloroform solution is cooled to room temperature, washed with 5% aqueous sodium bicarbonate (3 × 25 mL), and dried over anhydrous sodium sulfate. Filtration of the drying agent with a Hirsch funnel and rotary evaporation of the chloroform solution affords allobetulin as a light tan semi-solid. Upon standing, allobetulin crystallizes completely affording 65–70 mg (80–90% yield) with a melting point of 256–262 °C. NMR Analysis The 1H NMR spectra of betulin and allobetulin (Figure 1) demonstrate several characteristic spin–spin coupling patterns that are excellent examples of fundamental NMR phenomena, including diastereotopic protons, long-range coupling, and stereochemical relationships explained by use of the Karplus equation (10). For example, the stereochemistry of the C3 β-alcohol in betulin (Figure 2) is established by the doublet of doublets of the methine proton centered at 3.18 ppm having a large vicinal axial–axial coupling ( J = 10.2 Hz) and a smaller axial–equatorial coupling ( J = 5.5 Hz) (Figure 1A). In addition, an interesting AB pattern is displayed by the diastereotopic protons at C28 (Scheme I), which undergo geminal coupling to give a doublet at 3.80 ppm ( J = 10.8 Hz) and another doublet at 3.33 ppm ( J = 10.8 Hz). The large coupling constant is indicative of the neighboring electronegative alcohol-oxygen atom. The non-equivalent vinylic protons at C29 are displayed as slightly broadened singlets at 4.68 and 4.58 ppm, owing to weak allylic coupling. The allylic methyl group (C30) is found at 1.65 ppm owing to anisotropic deshielding while the remaining
1986 Journal of Chemical Education • Vol. 84 No. 12 December 2007 • www.JCE.DivCHED.org
In the Laboratory
five methyl groups (C23, 24, 25, 26, 27 in Scheme I) are found upfield at 1.0–0.75 ppm. The allylic proton at C19 (Scheme I) is found as an 8-line pattern at 2.38 ppm. Upon acid-catalyzed rearrangement with p-TSA, several chemical transformations in the E-ring take place simultaneously, which, in turn, lead to a highly informative NMR spectrum of allobetulin (Figure 1B). Protonation of the C29 carbon leads to a tertiary carbocation at C20 (Scheme II). Ring expansion then takes place by migration of the C21 methylene group. Capture of the resulting 2o carbocation by the alcohol function from C28 leads to a six-membered E-ring and a five-membered bridging ether ring (11). The diastereotopic protons at C28 remain unaffected by these changes and appear as two doublets centered at 3.77 and 3.44 ppm ( J = 10.0 Hz). However, the C30 allylic methyl group and C29 vinyl hydrogens of betulin are converted into the C20 geminal-dimethyl group of allobetulin. A new highly deshielded singlet at 3.52 ppm that appears in the spectrum is due to the C19 methine proton. Even though this proton does have a vicinal neighbor at C18, the dihedral angle formed by ring expansion and ether formation is 90 degrees. Examination of the Karplus equation predicts that there will be no coupling between these protons (12). The C3 methine doublet of doublets ( J = 10.9 and 5.4 Hz) remains at its original value of 3.20 ppm. Allobetulin is isolated as a high-melting solid, 256–262 °C. Student Evaluation The NMR information provided by the spectra of betulin and allobetulin has allowed us to evaluate student understanding of the topics presented here through several means. We have used reports, problem sets, and examination questions to do this. For example, one can ask how the spin–spin coupling pattern would change if this compound had a C3 α-alcohol function instead of a C3 β-alcohol group (Figure 2). The topic of diastereotopic protons can be approached by first having students identify this type of proton in variety of other molecules and, second, predicting their spin–spin coupling patterns. Good examples of molecules with diastereotopic protons include menthol, 1-phenyl-1-propanol, menthoxyacetic acid, 1-benzyl2-tetralone, terpinol, and 1-benzyl-2-indanone. The unusual 90 degree dihedral angle established between C18 and C19 protons (Scheme II) in allobetulin presents a tremendous opportunity for the student to use molecular modeling software to explore this skeletal feature. Both PC Spartan and PCModel are used in our courses. PCModel has a built-in Karplus equation, which makes this exercise even more graphic. Summary The wide range of educational topics covered in this experiment is scheduled to coincide with lecture discussion of spectroscopic structure determination, following presentations of stereochemistry and carbocation chemistry. Consequently, it can be used in either first- or second-semester organic chemistry laboratory. All of the NMR spectra recorded in this manuscript were obtained on a Varian 200 MHz or 300 MHz instrument, each displaying easily interpreted data.
Acknowledgments The authors wish to thank the National Science Foundation, ILI grant DUE-9352266, for the Varian Gemini 300 NMR spectrometer used in obtaining the data presented in this article. We are extremely grateful to the Camille and Henry Dreyfus Foundation, grant SG-01-011, for the support of UMaine laboratory curriculum development and for purchase of a PerkinElmer Spectrum One B infrared spectrophotometer∙HATR used in our courses. WSupplemental
Material
The complete description of this experiment, including spectra and structures, and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Connolly, J. D.; Hill, R. A. In Methods in Plant Biochemistry; Dey, P. M., Harborne, J. B., Eds.; Academic Press: San Diego, CA, 1991; Vol. 7, Chapter 9, pp 331–359. 2. Harborne, J. B. In Ecological Chemistry and Biochemistry of Plant Terpenoids; Harborne, J. B., Thomas-Barberan, F. A., Eds.; Clarendon Press: Oxford, 1991; pp 399–426. 3. Seshadri, T. R.; Vedanthan, T. N. C. Phytochemistry 1971, 10, 897–898. 4. Batta, A. K.; Rangaswami, S. Phytochemistry 1973, 12, 214– 216. 5. Jaaskelainen, P. Paperi ja Puu 1981, 10, 599–604. 6. Kapoor, V. K.; Chawla, A. S. J. Sci. Ind. Res. 1986, 45, 503–505. 7. (a) Pisha, E.; Chai, H.; Lee, I. S.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.; Pezzuto, J. M. Nat. Med. 1995, 1 (10), 1046–1051. (b) Fuda, S.; Scaffidi, C.; Susin, S. A.; Krammer, P. H.; Kroemer, G.; Peters, M. E.; Debatin, K. M. J. Bio. Chem. 1998, 273 (51), 33942–33948. 8. Fulda, S.; Jeremias, I.; Steiner, H. H.; Pietsch, T.; Debatin, K-M. Int. J. Cancer 1999, 82, 435–441. 9. (a) Evers, M.; Poujade, C.; Soler, C.; Rideill, Y.; James, C.; Leliévre, Y.; Gueguen, J. C.; Reisdorf, D.; Morize, I.; Pauwels, R.; De Clercq, E.; Hénin, Y.; Bousseau, A.; Mayaux, J. F.; Le Pecq, J. B.; Dereu, N. J. Med. Chem. 1996, 39, 1056–1068; (b) Sun, I.; Chen, Ch.-H.; Kashiwada, Y.; Wu, J.-H.; Wang, H.-K.; Lee, K.-H. J. Med. Chem. 2002, 45, 4271–4275. 10. Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identification of Organic Compounds, 7th ed.; John Wiley & Sons, Inc.: New York, 2005; pp 171–172. 11. Reynolds, W. F.; McLean, S.; Poplawski, J. Tetrahedron 1986, 42, 3419–3428. 12. The triterpene saikogenin E (from B. falcatum) has been shown to be formed by way of a similar E-ring carbocationic rearrangement followed by capture of the carbocation by the C28 alcohol function, see Kubota, T.; Hinoh, H. Tetrahedron Lett. 1966, 39, 4725–4728.
www.JCE.DivCHED.org • Vol. 84 No. 12 December 2007 • Journal of Chemical Education 1987
