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
the .vrtuaraliun
. ~~fluvigeoi~lrI ) a, h ~ w v ;muat t hest tjl~~cc-
tives.
Lucigenin is a powerfully chemiluminescent compound
when reacted with hydrogen peroxide in dilute aqueous, al-
kaline solution^.^ Chemiluminescence occurs when the
product of an exothermic reaction is formed in an electroni-
cally excited state. Since lucigenin, and the major product of
the reaction, N-methylacridone(I1) are both strongly fluo-
rescent, 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
peroxide(III), which cleaves in a forbidden -12 + 21 thermal
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 N-
methylnsridme. The experiment tI11.i i l l u ~ t r a t cnlsuy ~ oithr
ctrnctyx; ,ri~,h,~t,,,.ln~~niirr\. and d t h r \\',~.,d\i,:~rcl-Hc,~i~ndnn
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
I*(&)
OR II*(S,)
--
Then II*@d + I&) -+ II&)
I(%) + hu
II(S,) + h"'
+ ]*(St)
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).
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 ac-
ridone 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.
' For a discussion of Charm in chemistry see Ramette, R. W., J.
CHEM. EDUC., 57, 68, (1980).
McCapra, F. "The Chemistry of Heterocyclic Compounds," Ach-
eson, R. M., (Editw),2nd Ed., Interscience, New York, 1973, p. 615 and
refs. therein.
CH,
1v
CH:$
I
Figure 2. The preparation of lucigenin involving both nucleophilicand eiectro-
philic 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 completed by heating on a steam cone for about 10 min. The solution
(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 crystalliza-
tion.
(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 re-
moved 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)
ethanol1KOH ,A, 393,412,436 nm (r 9,500)
(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
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 or-
ange 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
"
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.