Structure Elucidation of a Natural Product Roy M. Letcher
University of Hong Kong, Hong Kong
T h o u ~ hstructure elucidation continues to play a major role in modern organic chemistry, student experience is generally confined to elementary qualitative analysis of simple organic compounds, with very few experiments having been reported on the structure elucidation of more complex compounds. This experiment is an attempt to simulate a real-life structure elucidation prohlem through the isolation, characterization, and chemical transformation of an "unknown" naturally occurring monoterpene, with extensive use being made of spectroscopy and aided by hiogenetic considerations. The experimental work is partly based on the recently described' student ex~erimentfor the isolation of (+) - limo~~
~
~
~~~~~~
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classic experiments carried out by Wallach in 1884,"nd from which he concluded (and from whichthe students performing the experiment should conclude) that limonene possesses two double bonds and has one ring. The various steps involved in this experiment are shown below:
I
CITRUS PEEL
In order to achieve the structure elucidation objective, students are, of course, not given the structures shown above, rather they are provided with experimental details using the symbols (A)-(E), with analyses, and with mass and NMR suectra. These appear below in a readily usable form for inkructors, thus 6l;viating the need to re-record this data. Fortunately, this information is not sufficient by itself to enable an un&nblguous structure for (A) to he deduced; before this is possible, the student must first carry out the isolation and transformation (A) (E)and record the all-important UV and IR spectra of (A) and particularly (E). The reactions can be carried out in 9 or 10 hr and generally give satisfactory yields. In order to provide a project element to the experiment, students supply their own orange, lemon, or grapefruit peels, and the various data including yield,
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'
Rothenberger, 0. S., Krasnoff,S. B., and Rolllns, R. B., J. Cnm. Eouc., 57, 741 (1980). Wallach, 0.. Justus Liebigs Ann. Chem., 225, 318 (1884).
rotation, and gas chromatograph trace of the crude limonene from the different sources is compared. Information Given to Students
The oil obtained from the steam distillation of the citrus peel contains a high proportion of (A) hut is not sufficiently pure for structure elucidation studies. Students may wish to confirm this by recording 'H NMR and IR spectra as well as a gas chromatograph trace of the essential oil. The UV spectrum of the crude (A) should, however, he recorded, and a Lassaigne sodium fusion test carried out on a drop of the oil to find if any halogen (Br) is present. (A) is, however, readily isolated pure in the form of its derivatives, the bromine adduct (B) and nitrosyl chloride adduct (C). Since neither (B) nor (C) have spectra which lead themselves to easy structure elucidation, (C) is converted to (E) by a very useful reaction sequence involving firstly, dehydrohalogenation to (D) and then hydrolysis, with (E)providing the necessary data for a ready solution. A final piece of information is provided hy the dehydrogenation of (A). The necessary spectral data etc. for all these compounds appears below: The Bromine Adducf (6) Elemental Composition: (Found: C, 26.35; H, 3.55; Br, 70.1%) Mass Spectrum: rnle 379(5%), 377(15), 375(15), 373(5), 297(50), 293(50), 295(100), 215(30), 213(30), and 133(40). 'H NMR spectrum (see Fig. 1):
182.87 Hz 178.93 Hz 170.18 Hz 141.31 Hz 0.00 Hz
2.040 1.996 1.899 1.577 0.000
1398% 6362% 5371% 482% 732%
The Nifrosyl Chloride Adduct (C) and Transformation to (0 Mass spectrum of (C): mle 203(13%), 201(40), 135(100), 1171301.93i90). Mass spectrum of (D): m/e 165(100%), 148(70), 124(25), 125130). 107(50). T ~ ~ ~ H spectrum N M Rof (D) is shown in Figure 2. The lH NMR spectrum of (E) is shown in Figure 3. Note: (1)The spectrum is unaltered by the addition of D20. (2) Also included are the results of spin decoupling experiments. Mass spectrum of (E): mle 150(40%),and 82(100). The I3C NMR spectra of (E) is shown in Figure 4 for the Volume 60 Number 1 January 1983
79
Figure 1. 'H NMR spectrum of the bromine adduct.
Figure 3. 'H NMR spectrum of compound E
Figure 2. 'H NMR spectrum of compound D
proton noise decoupled spectrum and in Figure 5 for the offresonance spectrum. This data must he supplemented by the I R and UV spectra of (E) obtained from the experimental section. Dehydrogenationof (A)
On heating (A) with sulfur to ZOO0, a new product (F) is formed as an oil exhibiting two strong absorptions in the IR at 1515 and 810 cm-'. Students may wish to carry out this experiment and also record the ' H NMR spectrum of (F) which is also informative. Structure elucidation of (A) should begin with the determination of the molecular formula for (B) from which that of (A) can he deduced along with the number of double bonds and rings in (A). Analysis of the spectral data for (E) should then follow, from which several structures for (E) should emerge. Knowing the reactions involved and having some spectral data of the intermediates, the correspondiny structures for (A) can be drawn. An appreciation of the synthetic utility of nitrosyl chloride olefin adducts, as shown by the sequence (A) (E) should he made. Finally, in order to reduce the possibilities to just one structure, the hiogenesis of monotrrpenes [e.g., (A)] from mevalonic acid should be considered, together with the data from (F).
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Discussion and Rationalization of the Data
The mass spectrum of (B) clearlv shows three bromine atoms and a n b d d m/e for the suspected M+ ion. Students should realize that this is an example of a comuound having no M+ in its mass spectrum, and chat the peaks at m/e 39f 377,375, and 373 (in the ratio 1:3:3:1) aredue to the (M-Br)+ isotope cluster. Consequently, (B) has the formula CloH1&r4, and (A), which contains no bromine, must he C I ~ Hwhich, ~ ~ , 80
Journal of Chemical Education
L Figure 4.
NMR Spectrum (proton noise decoupled) of compound E.
from a double bond equivalent count, must have two olefinic double bonds and one ring. Finally, the UV spectrum of (A) (E< 500 in the A215-250 nm region) shows that the two double bonds are not conjugated. Much information on the structure of (E) is revealed by its 13C NMR spectrum: 6,
Multiplicity
198.2 146.9
5 5
SP%
c
144.0 135.3 110.4 43.2 42.7
d
SP,,
CH
S
SPs. c spa. CH2 spa, CH. spa. CH SPS. CH2 CHa CHs
31.4 20.4 15.5
t t d t 4 4
Assignment CO
This data, among others, shows (E) to he a ketone with the formula C l o ~ ~ 4 0 ; a nfor d which, many structures are possible. The IR ( u 1670 cm-') confirms the presence of the ketone, excludes four- or five-ring ketones, and, furthermore, together 9500) shows it to be with the UV spectrum (A,, 237 nm, E,, a.B-unsaturated. a factor which considerahlv reduces the number of possilhe structures. The ' H NMR iecoupling exneriments reveal that the verv broad olefinic sienal a t 6 6.75 " (1H) is only very weakly coupled to a methyl group (fairly sharp signal a t 6 1.76 (6H)), hut it is strongly coupled to an aliphatic methylene group (6 2.52). This excludes structures with the 3-methyl-2-ene-one moiety leaving only two possible with an structures for (E) viz. 2-methylcyclohex-2-ene-one isopropenyl~substituentat either C-5 or C-6. At this point,
products and their biosynthesis; addition and elimination reactions; measurement and interoretation of IR and UV spectra; interpretation of mass spectra, 13C and lH NMR spectra including spin decoupling. Other interesting features can also he seen in the spectra, such as the retro-Diels-Alder fragmentation in the mass spectrum of (E)and the diastereotopic methylene protons in the 'H NMR of (B), 8 3.88 (2H). Experimental
Figure 5. 13C NMR spectrum (oft-resonance)of compound E
students should deduce the corresoonding struclures for (Dl, (C), and ultimately (A). By doing dhis, t h e synthetic utility of the reaction sequence (Cj -(El should he revealed. A decision between the two possible structures can he made on hiogenetic grounds. From the fact that (A) is a monoterpene (a C10 compound) and that its hiogenesis is expected to follow a prescribed path from mevalonic acid through isopentyl pyrophosphate and geranyl pyrophosphate ( G ) ,the position of substitution should become clear:
For the isolation of (A) and its conversion to (E) via (C), the procedure described by Rothenherger et al.' is adopted. Steam distillation of the macerated peel from 8 medium sized oranges or 11small lemons gives about 4.0 g of oilcontaining (A). The addition ofnitrosyl chloride to 3.0g of crude (A) yields 1.5 g of pure (C) which readily gives (D)(1.0 g) on heating with pyridine. Hydrolysis gives (E) (400 mg) whichis sufficient for both an IR and UV spectrum. In order to weigh (A) and (E)for UV spectra, students are i n t r o ~ duced to the technique of weighing liquids in m.p. capillary tubes.
Brominafion of (A) To 0.5 g of the oil containing (A) dissolved in absolute ethanol (2 ml), a bromine solution (prepared from 0.5 ml of bromine and 5 ml of absolute ethanol) is added dropwise until the soiution color changes to orange. Cooling in an ice bath yields a solid (0.4 gl, which should he recrystallized from ethanol, ultimately giving colorless crystals of (B) m . p 104-105'. Dehydrogenation of (A) On heating a 1:l molar mixture of (A) (0.4 g) and sulfur t o about 200" with a small flame, hydrogen sulfide is given off, and on distilling the product a pale yellow oil is obtained (sufficiently pure far IR and NMR spectra) which runs on the solvent front on silica gel GF TLC plates developed with light petroleum (60-80°). (A1 has a much lower
RF. The isomer 3-isopropenyl-l-methylcyclohexene,incidentally, does not occur in n a t u r e L a s expected. Alternatively, the IR (or lH NMR) spectrum of (F)shows that (A) dehydrogenates to a 1,4-disuhstituted benzene. The experiment illustrates a wide variety of important organic chemistry topics which include: terpenoid natural
NMR spectra were recorded using a Jeol FX 904 Fourier Transform spectrometer for CDCla solutions employing tetramethylsilane as internal standard. 13CNMR spectra were recorded a t 22.53 MHz with an accuracy hetter than 10.05 ppm and 'H NMR were recorded a t 89.6 MHz with an accuracy better than 10.005 ppm. Heilbron. I., and Bunbury, H. M., "Dictionary of Organic Compounds." Eyre and Sponiswoode, London, 1953, p. 1929.
Volume 60
Number 1
January 1983
81
