The Preparation of Lucigenin An Experiment with Charm1 R. G. Amiet Royal Melbourne Institute of Technology, Melbourne, Australia In the search for advanced organic preparative experiments which satisfy many of our criteria, namely, relevance to lecture course material, use of a variety of experimental techniques and J I I I ~ . I ( i~wI ~holding student intvwst. a.6. l l . l ~te~ u n dth:jt I ) a, h ~ w v ;muat t hest tjl~~ccthe . vrtuaraliun ~~fluvigeoi~lr . tives. Lucigenin is a powerfully chemiluminescent compound when reacted with hydrogen peroxide in dilute aqueous, alkaline solution^.^ Chemiluminescence occurs when the product of an exothermic reaction is formed in an electronically excited state. Since lucigenin, and the major product of the reaction, N-methylacridone(I1) are both strongly fluorescent, light emission is observed without the need for an energy acceptor. The most likely mechanism for the reaction is given in Figure 1,where lucigenin is oxidized to the unstable 21 thermal peroxide(III), which cleaves in a forbidden -12 reaction to give electronically excited N-methylacridone. Because the color of the light emitted is that of fluorescent lucigenin, energy transfer occurs in the initial stages, while later the color becomes more bluish, that of fluorescent Nmethylnsridme. The experiment tI11.i i l l u ~ t r a t cnlsuy ~ oithr and d t h r \\',~.,d\i,:~rcl-Hc,~i~ndnn ctrnctyx; ,ri~,h,~t,,,.ln~~niirr\. rules. The preparation of lucigenin as outlined in Figure 2 involves hoth nucleophilic and electrophilic aromatic substitution, nucleophilic aliphatic substitution, reductive coupling, and oxidation reactions. Experimentally, the major techniques involved are steam distillation, decolorization with charcoal, Soxhlet extraction for crystallization, and the use of a dipolar aprotic solvent to enhance ionic reactions. In addition, our students purify the aniline by vacuum distillation. Students have found the experiment to be hoth satisfying
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iluminescent reaction is observed easily in a darkened room and may emit for up to 30 min. Intensity of light emission is enhanced, and duration diminished, by the presence of more or less alcohol in the solutions. A beautiful demonstration of iluoreacenrc. m:y be 4een by aprinklin: ~.ryctnllinelurixf nin "11 tbc w r ~ a c e UI water ,~~nitaintvI in ;I h r ~ beaker e irr.idiate~1 by an ultraviolet lamp. As the lucigenin sinks and dissolves, brilliant yellow-green fluorescent trails are observed. The sequence requires approximately three 4-hr sessions for completion hut may be interrupted a t any convenient stage. Yields are around 80%for each step, and high purities are achieved easily. One potential stumbling block, the methylation of acridone with methyl iodide in ethanol in sealed tubes was overcome by using the potassium salt of acridone in dimethylformamide solution. Methylation then proceeds readily a t room temperature. Acridone is purified hy crystallization from ethanol using a Soxhlet apparatus, as i t is extremely insoluble and difficult to obtain in crystalline form. We have found that it must be crystalline before methylation; if not the yield is greatly diminished.
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For a discussion of Charm in chemistry see Ramette, R. W., J. CHEM. EDUC., 57, 68, (1980). McCapra, F. "The Chemistry of Heterocyclic Compounds," Acheson, R. M., (Editw),2nd Ed., Interscience, New York, 1973, p. 615 and refs. therein.
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]*(St) Then II*@d + I&) -+ II&) I*(&) I(%) + hu OR II*(S,) II(S,) + h"' Figure 1. The mechanism for light emission by reacting lucigenin with hydrogen peroxide in a dilute aqueous, alkaline solution to produce the major product. N-methylacridone (11).
CH,
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1v
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Figure 2. The preparation of lucigenin involving both nucleophilicand eiectrophilic aromatic substitution,nvcleophiiic aliphatic substitution,reductive coupling, and oxidation reactions.
Volume 59
Number 2
February 1982
163
~ x p e r i m e n t a Section l (a) N-Phenylanthranilic acid.3 A 250-ml two-necked flask, fitted with an air-condenser, is charged with o-chloiahenzoic acid (10 g), aniline (40g), anhydram potassium carbonate (10 g), and cupraus oxide (0.25 g) and refluxed for 2 hr using an oil bath at 180-200'. Excess aniline is removed by steam distillation, and decolorizing charcoal (5 g) is added to the brown residual solution. The mixture is boiled for 15 min and filtered while hot. The filtrate is added to a mixture of conc. HCl(10 ml) in water (20 ml), and the precipitated acid is removed by filtration. Yield 10-13 g, m.p. 185O.The acidmay still he colored, hut it is pure enough to proceed without crystallization. (6) Acridone. Crude, dry, N-phenylanthranilic acid is dissolved in conc. HzSOa (2.25 ml of HzSO4 for each gram of acid) and heated on a steam cone for 1hr. The hot green solution is then poured slawlyinto almost boiling water. (CAUTION: Spattering i s minimized by pouring the solution down the walls of the container.) The yellow suspension is boiled for 5 min to hydrolyze any sulfonated acridone and then filtered. The crude acridone is then suspended in a solution of sodium carbonate (8 gin 100 ml of water) boiled for 5 min and removed by filtration; any unreacted acid may be recovered from the filtrate by acidification. The crude aeridone is crystallized using a Soxhlet apparatus from ethanol. Beautiful yellow flakes of aeridone separate from the blue fluorescent solution. Yield about 8090, m.p. 350'. U-V ethanol A,, 381,399 nm (r 10,000) 393,412,436 nm (r 9,500) ethanol1KOH ,A, (c) N-Methylacridone. Acridone (4.1 g) is dissolved in hot ethanolic KOH (1.3 g in 50 ml ethanol) to give a clear yellow solution. The ethanol is removed on a rotary evaporator and the residual hright yellow solid dissolved in dimethylformamide (50 ml). Methyl iodide4 (3.5 g) is slowly added. Some heat is evolved and the reaction is
164
Journal
of Chemical Education
completed by heating on a steam cone for about 10 min. The solution is poured into water and the crude product is removed by filtration. N-Methylacridone crystallizes easily from ethanol as hright yellow needles from hlue fluorescent solutions. Yield 60-90%, m.p. 203'. (d) Lueigenin. T o a refluxing solution of N-methylacridone (1 g) in ethanolic HCl(50 ml ethanol, 10 ml cone. HCI) is added zinc dust (3.2 g) partionwise over 15 min. The mixture is refluxed far afurther 30 min, then added to water (100ml) to give agreen precipitate of the crude his-acridinium compound IV. Without purification the solid is dissolved in dilute HNOl(60 ml, 1M ) and heated on a steam cone for 15 min. The hat solution is filtered, if necessary, and lustrous orange flakes of lueigenin dinitrate separate on cooling the filtrate. Yields 0.6-0.9 g. Dilute aqueous solutions emit a bright yellow-green fluorescence when irradiated with a U-V lamp. ( e ) Observation of ehemilumineseence. Prepare two solutions as follows: Solution A 8.0 g NaOH in 70 ml water 5.0 ml100 volume Hz02 (1.5% wlv, 0.45 M ) 30.0 ml ethanol. Solution B 0.1 g lucigenin in 100 ml water. (Note that these concentrations are not crucial and may he
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the find solutmn w ~ t ha U-V lamp, when mtense hlue fluorescence 1s observed. "Organic Syntheses, Collective Volume 2" Blatt A. H., (EditoO,John Wiley and Sons Inc., New York, 1943, p. 15. CAUTION: Methyl Iodide Is carclnogenb; handle approprlately In a fume hood.
