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Luciferins Bioluminescent Substances.

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ANGEWANDTE CHEMIE
VOLUME 7 . NUMBER 6
J U N E 1368
PAGES 4 0 7 - 4 8 8
Luciferins, Bioluminescent Substances
BY T. GOT0 AND Y.KISHCI”1
The bioluminescence of the American firefly Photinus is due to the reaction of 2-(6hydroxybenzothiazol-2-yl)-A2-1,3-thiazoline-4-carboxylicacid (“firefly luciferin”) with
the enzyme luciferase in the presence of ATP and magnesium ion. In the crustacean
Cypridina, on the other hand, the bioluminescence is due to the reaction of a luciferase
with 8-(3-guanidinopropyl)-6-indol-3-yI-2-(I-methylpropyl)-3,7-dihydroimidazo~l,2-a~pyrazin-3-one (“Cypridina luciferin”). The luciferin in Latia is 1,3,3-trimethyl-2-(4formyloxy-3-methy1-3-butenyl)-l-cyclohexene and that in Renilla is a tryptamine
derivative that has not yet been accurately identified; the luciferins of other luminescent
organisms are not yet known. A review is given of the investigations which have been
carried out on the above tuciferins and the course of the luciferin-luciferase reaction is
examined. Numerous spectral data obtained during the examination of these compounds
are included in the text.
1. The Luciferin-Luciferase Reaction 111
Some eighty years ago, Dubois found that the bioluminescence of the luminescent beetle Pyrophorus is
due to the luciferin-luciferase reaction. Bioluminescence was observed when a hot aqueous extract (“luciferin”) of the insect was mixed with a cold aqueous
extract (“luciferase”) after the initial luminescence had
disappeared. This observation led to numerous studies
on the luciferin-luciferase reaction in many bioluminescent organisms (see Table 1). It is now known that
hot water extracts luciferin and destroys luciferase;
the cold-water extract contains luciferase, and also
luciferin at first, but the latter changes into an inactive
oxidation product on standing in air.
The luciferin-luciferase reaction is the emission of light
as a result of the enzyme (a 1uciferase)-catalyzed oxidation (generally with oxygen) of a substrate (a luciferin). Thus “luciferin” is the name given to a substance having special properties, and is not the name
of one compound or o f a class of compounds. For
example, luciferin from the American firefly Photinus
Table 1. Dates of discovery and names of discoverers of the luciferinluciferase reaction in various luminous organisms.
Pyrophorus (elaterid beetle)
Pholas dactylus (clam)
Photinus (American firefly)
Luciola (Japanese firefly)
Cypridina
(ostracod crustacea)
Pyrocypris
(ostracod crustacea)
Odontosyllis
(marine fireworm)
Systellaspis (shrimp)
Lafia (fresh-water snail)
Achromobacter (luminous
bacteria) Photobacterium
(luminous bacteria)
Heterocarpus (shrimp)
Gonyaulax (dinoflagellate)
Apogon and Parapriacanthus
(fish)
Renilla (sea pansy)
Collybia, Armillaria, and
Omphalia (fungi)
Balanoglossus
(marine euteropneust)
Octochaefus multiporus
(earthworm)
Hoplophorus (shrimp)
1885
1887
19 16
19I7
19 17
Dubois
Dubois
Harvey
Harvey
Harvey
1921
Harvey
1931
I
Harvey
1931
1950
1953
1953
Harvey
Bowden
Strehler
McEIroy
1955
1957
1958
Haneda
Hustings and Sweeney
Haneda and Johnson
1959
1959
Cormier
Airth and McEIroy
1963
1966
1966
I
Dure and Cormier 121
Johnson. Shimomura, and
Haneda [3]
Johnson, Stachel,
Shimomura. and Haneda [4]
[*I Prof. T. Goto and Dr. Y . Kishi
121 L. S. Dure and M.J . Car ier, J. biol. Chemistry 238,790(1963).
Department ofAgricultura1 Chemistry and Chemical Institute
Nagoya University, Nagoya (Japan)
[l] F. H . Johnson and E. H.-C. Sie in W. D. McEIroy and B.
Glass: A symposium on light and life. Johns Hopkins Press,
Baltimore 1961, p. 206.
[3] F. H . Johnson, 0.Shimomura, and Y. Haneda in F. H. Johnson
and Y. Haneda: Bioluminescence in progress. Princeton Univ.
Press, New Jersey 1966, p. 385.
[4] F. H. Johnson, H.-D. Stachel, 0. Shimomura, and Y. Haneda
in [3], p. 523.
Angew. Chem. internat. E d i t .
/ Vol. 7 (1968) / No. 6
407
is completely different from luciferin from the marine
crustacean Cypridina. The luciferases of these organisms are also different.
I
327
535 562
381
2. Isolation and Structure of Firefly Luciferin
The luciferin-luciferase reaction in firefly was discovered by Harvey[51 in 1916. This author first prepared
“luciferin” and “luciferase” solutions from the lanterns
of luminous insects as well as from non-luminous insects. Luciferin and luciferase from luminous insects
emitted light when mixed, as did luciferin from nonluminous insects and luciferase from luminous insects.
Luciferin and luciferase from non-luminous insects or
luciferin from luminous insects and luciferase from
non-luminous insects did not exhibit luminescence
when mixed. The emission of light thus evidently depended on the source of the luciferase.
In 1947, McElroy showed that the “luciferin” obtained
by Harvey was in fact adenosine triphosphate (ATP) [51.
The firefly luciferin was first isolated in 1957 from
Photinuspyralis 161. This substance is stable in oxygenfree alkaline media, but is readily oxidized by oxygen
to dehydroluciferin, a compound that can also be isolated from the firefly. The UV spectrum of the luciferin
indicates a benzothiazole chromophore, and acid hydrolysis affords 6-hydroxybenzothiazole and cysteine.
Consideration of these facts led to the assignment of
structure ( I ) to the luciferin [71 and of structure (2) to
the dehydroluciferin.
/
500
m
550
600
l(nrn-
Fig. 1 . Spectrum of the luciferin ( 1 ) of the American firefly Phortnus.
acid, (-1 base, (- - -) base. A , absorption; F, fluorescence;
L, luminescence.
(-.-.)
I
250
300
348
393
350
400
544
450
h(nm)
500
550
600
+
Fig. 2. Spectrum of dehydroluciferin (2) of the American firefly
Phorinus. (-.-.)
acid, (-) base, (- - -) base. A, absorption;
F, fluorescence.
Though the various species of fireflies differ in the
color of the light emitted (from green to yellow), all
the species examined, including Photinus, Photuris,
Pyrophorus, Diphotus, and Lecontea, contain the luciferin ( I ) [lo]. The luciferin of Luciola cruciata was also
identical with that of Photinus [111.
(I) R
= H, R’= OH
(3) R = H , R ’ = NH2
(4) R = R’= OH
The structures of the compounds were also verified by
total synthesis from ethyl N-Cp-methoxypheny1)oxamate. Only D-( -)-luciferin produced light, and was
therefore identical with the natural compound; L-(+)luciferin was inactive. The UV, fluorescence, and emission spectra of firefly luciferin ( I ) and dehydroluciferin (2) are shown in Figures 1 and 2181.
Of several analogs of luciferin [Q] that have been synthesized and tested for bioluminescence, only “aminoluciferin” (3) and “4-hydroxyluciferin” ( 4 ) gave a
positive reaction, aminoluciferin giving red luminescence.
[5] E. N . Harvey: Bioluminescence. Academic Press, New York
1952.
[6] B. Bitler and M . D. McElroy, Arch. Biochem. Biophysics 72,
358 (1957).
[’TI .E. H. White, F. McCapra, and G. F. Field, J. Amer. chem.
SOC.83, 2402 (1961); 85, 337 (1963).
[8] M . D. MeEIroy and H . H. Seliger, Advances in Enzymology
25, 119 (1963).
[9] E. H. White, H. Worther, G . F. Field, and W. D . MeElroy,
J. org. Chemistry 30, 2344 (1965); E. H . White and H . Worther,
ibid. 31, 1484 (1966); E. H. White, H . Worther, H . H . Seliger, and
W . D. McElroy, J. Amer. chem. SOC.88, 2015 (1966).
408
3. The Luminescence of the Firefly[**I
The luminescence of the firefly requires the presence
of luciferin (LH2), luciferase (E), magnesium ions,
ATP, and oxygen. The first step is a dark reaction
leading to the formation of enzyme-bound luciferyl
adenylate (LH2-AMP) 1131.
LHz
+ ATP + E
E-(LHz-AMP)
+ P-P
As can be seen from Figure 3a, this reaction depends
on the concentrations of luciferin and of ATP. In the
next step the enzyme complex E-(LHz-AMP) reacts
with oxygen and light is emitted; the reaction product
remains bound to the enzyme. Because of this product
inhibition, the reaction leading to the emission of light
also depends on the quantity of luciferase (cf. Fig. 3b).
[lo] H. H. Seliger and W. D . McElroy, Proc. nat. Acad. Sci. USA
52, 75 (1964).
1111 Y. Kishi, T . Goto, S. Inoue, 0. Shimomura, and Y. Hirara,
paper read at the XI. Pacific Science Congress, Tokyo 1966.
[12] W. D. McElroy and H. H . Seliger in [3], p. 427.
I131 W. D. McElroy and A . Green, Arch. Biochem. Biophysics 64,
257 (1956); W.E.Rhodesand W.D.McElroy, Science (Washington)
128, 253 (1958); J. biol. Chemistry 233, 1528 (1958).
Angew. Chem. internat. Edit. 1 Val. 7 (1968) No. 6
Mgl+
lLV,-.'i-,
+
2
4
6
t
0
2
4
HzO
"0"
+
E- L H - A M P
)
1
Lucilerase
0
+ P-P
P
,
2
E-(LH~-AMP)
L H ~i ATP i E
6
(mm-
Fig. 3. Luminescence of the American firefly Photinus.
(a) o n introduction of ATP and (b) on successive introduction of
luciferase. L = integrated light units (arbitrary units).
Scheme 1.
reaction.
Suggested scheme for the Iuciferin (LH+luciferase(E)
Dehydroluciferin (L) was formerly regarded as a direct
product of the reaction leading to light emission. However,
more recent results indicate that dehydroluciferin does not
correspond to the ground state of the excited intermediate,
but is instead an alternative oxidation product, which is not
involved in the emission of light, and cannot be regarded as a
product formed from the excited intermediate.
The active center of firefly (Photinus) luciferase was thought
to consist of two thiol groups. However, contrary to the
belief that the energy source for the bioluminescence would
be the energy-rich phosphate bond, there is no indication of
any rupture of the anhydride bond in luciferyl adenylate and
formation of a n LH2-S-E bond during bioluminescence [12L
Since one mole of luciferin consumes one mole of oxygen [8J, equation (a) cannot be correct. The alternative
equation (b) is also wrong, since it would not provide
the required energy of more than 60 kcal/mole, and
Synthetic firefly luciferyl adenylate exhibits chemiluminescence in the absence of the enzyme when treated with KOH or potassium tert-butoxide in dimethyl
sulfoxide [161.
E-CLHz-AMP)
E-(LH2-AMP)+
+ 1/2 0 2 + E-(L-AMP) + H20 + hv
0 2
+ E-(L-AMP)+
H202+ hv
(a)
(b)
no Hz02 has been detected in the reaction mixture.
Moreover, the fluorescence spectra of dehydroluciferin
and its derivatives do not agree with the bioluminescence spectra. UV spectroscopic studies point to a
possible intermediate having an absorption maximum
at 400-410 nm 1141. Since the bioluminescence quantum
Recently, McCapra et al.
and Seliger et al. [16bl
reported the chemiluminescence of 5,5-dimethylluciferin derivatives, i.e. the phenyl ester (4a) and the
adenylate (4b), respectively, and identified the ketone
(4c) as the reaction product. The fluorescence spectrum of (4c) is superimposable on the chemiluminescence spectrum. The proposed mechanism involves a
peroxide intermediate having a four-membered ring;
similar intermediates occur in the chemiluminescence
0
(4'4
(h),
yield is close to one[lsl, the fluorescence yield of the
intermediate must be almost 100 %.
Synthetic L-luciferin does not exhibit bioluminescence
and does not consume oxygen, but like D-luciferin, it
reacts with ATP to liberate pyrophosphate (P-P) when
treated with ATP, luciferase, and magnesium ions [121.
On the basis of all the known results, it is possible to
formulate Scheme 1.
The reaction would provide more than 100 kcal/mole;
the product L=O has not yet been detected. L and
possibly L=O inhibit the production of light, and can
be removed from the enzyme by the addition of pyrophosphate or of coenzyme A.
[141 H. H . SeCiger and W. D.McEiroy in 131, p. 405.
[151 H . H . Seliger and W. D. McEiroy, Arch. Biochem. Biophysics 88, 136 (1960).
Angew. Chem. internat. Edit. 1 Vol. 7 (1968) No. 6
x
=
OC6H5
(4b), X
+
hV
= Adenylate
of lophine derivatives and of derivatives of acridinecarboxylic acids.
The chemiluminescence of luciferyl adenylate in
DMSO and the fluorescence of the spent chemiluminescence reaction mixtures are identical with the fluorescence of the anion (4c). The product of this chemiluminescence, however, proved to be too unstable to
be isolated. In-vitro bioluminescence of luciferin at
p H 7.5 is yellow-green (565 nm), but in acidic media
(pH 6.0) it becomes red (620 nm). The chemiluminescence of luciferyl adenylate (red emission) corresponds
~-
[161 H. H. Seliger and W. D. McElroy, Science (Washington)
138, 683 (1962).
[16a] F. McCapra, Y . C. Chang, and V . P. Francois, Chem. Commun. 1968, 22.
[16b] T . A. Hopkins, H . H. Seliger., E. H . White, and M. W. Cass,
J. Amer. chem. SOC. 89, 7148 (1967).
409
to the bioluminescence, and little information is available concerning the yellow-green emitting species in
bioluminescence. Seliger et al. suggested the possibility of the dianion of ( 4 4 acting as the emitter.
4. Isolation and Structure of Cypridina Luciferin
The crustacean Cypridina hilgendorfii is abundant
along the coast of Japan. The organism is about 3 mm
long, and emits a strongly luminescent secretion, into
sea water. The luciferin-luciferase reaction of Cypridina was discovered by Harvey in 1917 [51. The luminescent activity is preserved almost indefinitely in completely dry Cypridina, and the luminescence is restored
by moisture. Attempts to isolate the luciferin from
dry Cypridina led at first to a “purified”, but not to a
crystalline luciferin 1171.
The reason for the difficulty encountered in the extraction
and purification of the luciferin is that the substance is
present in dry Cypridina onIy in very small quantities, and is
extremely unstable to oxygen and water. The most successful
of the methods developed was the benzoylation method
described byAnderson [181, in which the luciferin is benzoylated
in butanol extract with a large excess of benzoyl chloride
and without a basic catalyst.
Crystalline luciferin was obtained in 1957 1191 from Anderson’s benzoyl luciferin, which was first purified by chromatography o n cellulose powder. The luciferin crystallized out
from an aqueous-methanolic solution as the hydrochloride
on acidification with concentrated hydrochloric acid.
Since 1917, several workers have proposed partial structures
for luciferin o n the basis of the “purified” product. For
example, the luciferin is claimed t o be a peptone. a phospholipid, a polyhydroxybenzene derivative, a hydroquinonelike compound having a ketohydroxy side chain, a flavoprotein, a pyridine nucleotide, and a chromopolypeptide [201.
Though the U V spectra indicate that “purified” luciferin is practically identical with the crystalline product, acid hydrolysis of the “purified” luciferin gives
about 20 amino acids [201, whereas that of crystalline
luciferin gives only a few [191. Catalytic hydrogenation
gives a compound (hydroluciferin) whose UV spectrum is typical of an indole[191. It has been difficult
to carry out a structural analysis of the luciferin because of the small quantity of material available, and
a structure that seemed plausible at first [211 was later
found to be wrong. Quantitative determination of the
amino acid compositions of luciferin and of hydroluciferin with the aid of the amino acid analyzer [22,241
gave the results shown in Table 2.
[17] F. LTsuji, A. M . Chase, and E. N. Harvey in F. H. Johnson:
The Luminescence of Biological Systems. Amer. Assoc. for the
Advancement of Sci., Washington 1955, p. 127.
I181 R. S. Anderson, J. gen. Physiol. 19, 301 (1935).
[19] 0. Shimomura, T. Goto, and Y. Hirata, Bull. chem. SOC.
Japan 30 929 (1957).
[20] Cf. p. 130 in [17].
[21] Y. Hirata, 0.Shimomura, and S. Eguchi, Tetrahedron No. 5 ,
p. 4 (1959); 0. Shimomura, J. chem. SOC.Japan 81,179 (1960).
[22] S. Eguchi, J. chem. SOC.Japan 84,86 (1963).
[23] Y . Haneda, F. H. Johnson, Y. Masuda, Y. Saiga, 0 . Shimomura, H.-C. Sie, N . Sugiyama, and I. Takatsuki, J. cellular comparat. Physiol. 57, 55 (1961).
[24] Y. Kishi, T. Goto, Y. Hirata, 0.Shimomura, and F. H. Johnson, Tetrahedron Letters 1966, 3427; in [3], p. 89.
Glycine
Arginine [a]
Isoleucine [b]
Ammonia
Vacuum
0 2
Vacuum
0.96
0.62
0.16
0.93
0.38
0.16
0.33
0.03
0.03
0.98
0.40
0.27
I O2
0.12
0.41
0.96
0.41
In 1961, Johnson et al. extracted luciferin directly from
living Cypridinar231. Apart from saving a great deal
of time, this permits an increase in the yield by a factor
of more than ten (about 20 mg of crystalline luciferin
per kg of moist Cypridina).
The complete structure of Cypridina luciferin (11) was
determined chemically in 1966 [241. Enzymatic (luminescence) or non-enzymatic air oxidation of (11) yields
two fluorescent substances, i.e. oxyluciferin (9) and
etioluciferin (5a); (9) can be converted into (5a) by
acid hydrolysis. The structure of etioluciferin (5a) was
established as follows: On hydrolysis with Ba(OH)2 in
the absence of oxygen it gave etioluciferamine, the
composition of which was found by high-resolution
mass spectrometry to be C15H17N5. The etioluciferin
itself has a guanidino group in place of an amino group,
and has the composition C16H19N7. On catalytic hydrogenation it gave a compound (hydroetioluciferin)
having a UV spectrum typical of the indole nucleus;
the pH-dependence of the U V spectrum pointed to a
P-aminomethylindole structure ( B ) . The NMR spectrum of (5a) in conjunction with the amino acid comThe
position led to the partial structures ( A ) and (0).
partial structure (C) was deduced from the pK values
and the diazo coupling reaction of (5a).
There are only two possible complete structures (5a)
and (56) that can be formed from these four partial
structures. (5b) is ruled out by the mass spectrum of
etioluciferarnine, since the peak at m/e = 141 must be
assigned to the radical ion (6).
y
(SO)
(6)
2
R = (CHz),-NH-C=NH, R’= H
m/e = 141
yH2
(56) R = H, R ’ (CHz),-NH-C=NH
Angew. Chem. internat. Edit. / Vol. 7 (1968)
/ Mo. 6
The structure (Sa) was finally confirmed by total synthesis from (7) and (8) 1251, which were treated successively with sodium hydroxide solution, methanolic
KOH, and HZN-C(=NH)-SCH~. The assumptions
concerning the relative positions of the amino and
indole groups on the pyrazine nucleus were confirmed
by the UV and NMR spectrac261.
three forms of the luciferin are best formulated as
( I l a ) , ( I l b ) , and ( I l c ) .
The UV, fluorescence, and emission spectra of Cypridina luciferin and oxyluciferin are shown in Figures
4 and 5.
I
250
On treatment with hydrochloric acid, oxyluciferin (9)
gives a-0x0-P-methylvaleric acid (10) as well as (5a).
The NMR spectrum of (9) contains a sharp OH singlet
signal. It can be seen from Table 2 that the nitrogen
atom that appears as isoleucine does not also form
arginine or glycine. Since the keto acid (lo} must be
derived from the isoleucine part, C-2 of (10) must have
been attached to the primary amino group of (5a).
This leads to the structure (9) for the oxyluciferin.
300
350
, /' /',
\
LO0 450 500
l(nrn)
Fig. 4. Spectrum of Cypridina luciferin (11). (-1 acid, (- --)
A, absorption; F, fluorescence; L, luminescence.
rn
250
300
350
LOO
CHS- C Hz- C H- C O - COOH
I
(10)
cH3
The NMR spectrum of Cypridina luciferin contains
no OH signal. The orange-red solution of luciferin
hydrobromide in methanol is decolorized by anhydrous
HBr, and the UV spectrum of the solution is then very
similar to that of (5a). Luciferin has three PKa values,
i.e. t 2 , 8.3, and >11; it istyellow in basic media. The
LI
(Zlai R'= H
(12) R'= CO-CeH,
+
pk, = 8,3
$3
base.
/
\
L50
500
550
l(nrn)-
Fig. 5. Spectrum of Cypridina oxyluciferin (9). (-) acid, (A. absorption; F fluorescence.
(9)
550
A
/
ii
\
- -)
base.
The benzoylated Cypridina luciferin obtained by Anderson's method has a UV spectrum similar to that of
(Sa) or ( I r a ) , and probably has the structure (12).
The absolute configuration at the carbon atom of the isoleucine portion was determined enzymatically as follows:
Hydrolysis of hydroluciferin gave a mixture of isoleucine and
alloisoleucine; this mixture was treated with L- and D-aminoacid oxidases and the products were examined with an amino
acid analyzer. It was found that Cypridina luciferin is built
up from tryptamine, arginine, and L-isoleucine portions. This
structure explains the unusual result of the amino acid
analysis (Table 2) [27L
Cypridina luciferin has been obtained by total synthesis from
(10) and (5a) (reaction with Pt/HP or Al/Hg followed by
reaction with dicyclohexylcarbodiimide).
The luciferins of the luminous fish Apogon and Parapriacanthus are identical with Cypridina luciferin [281.
5. The Luminescence of Cypridina
R"
As early as 1919, Harvey observed that the in-vitro
luminescence of Cypridina is a first-order reaction
(Fig. 6)[51. Further kinetic studies by Chase and Lorenz
showed that two first-order reactions take place, one
of which produces light while the other is a dark re-
=
H
[25] Y . Kishi, T, Goto, S. Inoue, S. Sugiura, and H. Kishimoro,
Tetrahedron Letters 1966, 3445.
[26] T. Goto and S. Inoue, unpublished.
Angew. Chem. internat. Edit.
1 Vol. 7 (1968) No. 6
1271 Y . Kishi, T. Goto, S. Eguchi, Y . Hirata, E. Watanabe, and T.
Aoyama, Tetrahedron Letters 1966, 3431.
[28J F. H . Johnson, N . Sugiyama, 0. Shimomura, Y. Saiga, and
Y. Haneda, Proc. nat. Acad. Sci. USA 47,486 (1961); Y. Haneda,
F. H . Johnson, and 0. Shimomura in [3], p. 533.
41 1
action "291. According to Johnson et al. 1301, one molecule of luciferin consumes one atom of oxygen during
luminescence; the quantum yield is at least 0.28. Since
the light-producing reaction and the dark reaction gave
the same products, i.e. oxyluciferin (9) and etioluciferin (Sa), the luminescence reaction can be represented by
LH2
+
Enzyme
112 0 2
---+ (9) + (5a) + hv
In air,an orange-redaqueousluciferinsolutionturns red,
and then colorless, owing to autoxidation (Fig. 7) 1191.
4y,
0
@m
20
,
,
LO
60
,
80
t (sec)
,
,
100
120
-
1LO 160'
Fig. 6. Bioluminescence in the system Cypridina luciferinlluciferase.
The left-hand ordinate refers to curve A (L = integrated light units) and
the right-hand ordinate to curve B.
initial red shift points t o the formation of a red
substance, which can be isolated from the reaction
mixture. This red substance emits very little light in
the presence of the enzyme, but the luminous activity
can be restored by chemical reduction; addition of a
reducing agent such as ascorbic acid to the reaction
mixture prevents the formation of the red substance [321.
This phenomenon was observed by Harvey, Anderson,
and others, who regarded it as a reversible oxidation
of the luciferin 151. However, the structure of this substance and its role in the reaction leading to the emission of light are not yet known.
Cypridina luciferin exhibits spontaneous chemiluminescence [331 when dissolved in dimethyl sulfoxide, with
or without a base, in the presence of oxygen, though
the quantum yield of this chemiluminescence is low
(cu. 0.1 5 % of bioluminescence). When diglyme containing acetate buffer is used as the solvent, the quantum
yield of the chemiluminescence of luciferin increases
to more than 10 % of that of bioluminescence and is
then about twice as great as that of luminol chemiluminescence in DMSO with potassium tevt-butoxide [33aJ. The simplest analog (13) of luciferin is also
chemiluminescent in DMSO with acetate buffer and
consumes one mole of oxygen during the reaction [33al.
McCupra and Chung [33bJ studied the chemiluminescence of the 6-phenyl-2,8-dimethyl derivative (14) in
( 1 3 ) , R' = R2 = R3 = H
( 1 4 ) . R' = R3 = CH3, R2
=
C6H,
500
40C
1
300
' 5
c -
Y
2OC
10C
c
Fig. 7. Non-enzymatic oxidation of Cypridina luciferin with air in
phosphate-buffered aqueous solution at pH = 5.6.
(-) freshly prepared solution, (- - -) after 1 day, (. . . .) after 3 days,
(-x-)
after 7 days, (---.) after 52 days.
A similar change is observed in the spectrum of the
bioluminescence in the presence of the enzyme 1313, but
the enzyme-catalyzed reaction is extremely fast. The
1291 A . M . Chase and P. B. Lorenz, J. cellular. cornparat. Physiol.
25, 53 (1945).
[30] F. H. Johnson, 0. Shimomura, Y. Saiga, L . C . Gershman,
G. T. Reynolds, and J . R. Waters, J. cellular. comparat. Physiol.
60, 85 (1962).
1311 A. M . Chase and E. H. Brigham, J. biol. Chemistry 190, 529
(1951).
412
DMSO in the presence of potassium tert-butoxide.
They isolated as the reaction product 2-acetamino-3methyl-5-phenylpyrazine (shown as the anion (1 7)),
whose fluorescence spectrum in the alkaline medium
corresponds to the chemiluminescence spectrum, and
suggested a mechanism involving a four-membered
ring peroxide intermediate (see Section 3). Kinetic
investigations indicate a participation of radical intermediates 133aJ.
Bioluminescence of Cypridina is more dependent on
the structure of the substrate than the above chemiluminescence. Thus, phenyldesindolylluciferin (15) is
very weakly bioluminescent, whereas the bioluminescence activity of norluciferin (14) is comparable with
that of luciferin[33cl. Replacement of the alkyl side
[32] A . M. Chase, J . H . Ball, C. E. Cornelius, and R. J . Lederman
in [I], p. 258.
[331 F. H. Johnson, H.-D. Stachel, E. C.Taylor, and 0. Shimomura in [3], p. 67.
[33a] T. Goto, S. Znoue, and S. Sugiura, unpublished.
[33b] F. McCapra and Y.C.Chnng, Chem. Commun. 1967, 1011.
[33c] D. A. Coviello, S. Inoue, and T. Goto, paper read to the
Chemical Society of Japan in Nagoya (Oct. 17, 1967).
Angew. Chem. internat. Edit. J Yo!. 7 (1968)
f No. 6
chain (R1) of luciferin by other alkyl groups diminishes
the bioluminescence activity moderately, but hydrolysis of the guanidino group of luciferin to the corresponding amino group lowers the activity [25,33dJ considerably.
6. Bacterial Luciferin
In bacterial luminescence, reduced flavin mononucleotide, a n enzyme, oxygen, and a long-chain aliphatic
aldehyde are essential factors for the production of
light. The quantum yield is very low and the reactions
involved are complicated. The aldehyde was initially
regarded as the bacterial luciferinr341, but its role in
the reaction has not yet been definitely established 18-35].
Despite extensive efforts to explain the luminescence,
it is not certain which substance is to be regarded as
the bacterial luciferin.
7. Renilla Luciferin
Renilla luciferin was isolated in a purified form from
Renilla reniformis by Cormier et al. 1361. It is stable in
neutral or basic solution, and in acidic solution it is
converted into “activated luciferin”, which emits light
(Amax = 485 nm) in the presence of luciferase or on
autoxidation (without enzyme), with formation of an
oxidized luciferin. Activated luciferin is also formed
on incubation of luciferin with luciferase, calcium ions,
and adenosine 3‘,5’-diphosphate under anaerobic conditions.
Renilla luciferin is a tryptamine derivative with an
anionic group. Alkaline hydrolysis of the dehydro-
I,
luciferin yields tryptamine. Activation of the luciferin
is accompanied by removal of the anionic group,
which may be sulfate.
Thus the reaction leading to the emission of light again
proceeds in at least two steps. I n the first step, which
is a dark reaction, sulfate is probably transferred from
the luciferin to the adenosine 3’,5’-diphosphate, with
formation of active luciferin. This is enzymatically
oxidized in the second step, and light is emitted. The
absorption and fluorescence spectrum of Renilla luciferin are shown in Figure 8.
8. Odontosyllis Luciferin
The luminescence of Odontosyllis involves a luciferin,
luciferase, and oxygen. McElroy 181 found a fluorescence emission maximum at about 510 nm for partly
purified luciferin; this maximum agrees with that observed during luminescence. Autoxidized luciferin can
be reduced to the active form.
Shimornura et al. [37J recently obtained a purified luciferin from Odontosyllis enopla as a colorless, nonfluorescent substance. After light emission (Amax =
507 nm), a fluorescent product having a fluorescence
maximum at 507 nm was obtained. Attempts to convert this product into an active luciferin with reducing
agents have been unsuccessful. The absorption and
luminescence spectra of Odontosyllis luciferin and of
the luminescent product are shown in Figure 9.
507
362
250
300
350
LOO
l(nm)
250
[*(21181
,)
,
300
350
k(nrn)
.\?\
>‘ . , ,.\
-
400
L50
500
Fig. 8. Spectrum of Renilla luciferin. (-) neutral, (- - -) base,
acid. A, absorption; F, fluorescence.
(-.-.)
[33d] Y. Kishi, Dissertation, Nagoya University, 1966.
[34] W. D . McElroy, J. W. Hastings, V . Sonnenfeld, and J . Coulombre, J. Bacteriol. 67, 402 (1954).
[35] J. W. Hastings, Q. H . Gibson, J . Friedland, and J . Spudich in
[3], p. 151, and further literature cited there.
1361 K . Hori and M . J . Cormier, Biochirn. biophysica Acta 102,
386 (1965); M . J. Cormier, K . Hori, and P . Kreiss in 131, p. 349.
Angew. Chem. internat. Edit. / Vol. 7 (1968) / No. 6
L50
500
550
A
and of the
Fig. 9. Spectrum of Odontosyllis luciferin (neutral) (-)
luminescent product($ (neutral) (---.), neutral (- - -), (--..-..)
neutral. A, absorption; F, fluorescence; L, luminescence.
In the absence of enzyme, Odontosyllis luciferin can
be readily oxidized to a pink substance having absorption maxima at 260, 330, and 520 nm. This change can
be brought about by the addition of iodine. The pink
substance remains unchanged in the presence of oxygen and luciferase.
[37] 0. Shimomura, F. H . Johnson, and Y . Saiga, J. cellular.
cornparat. Physiol. 61, 275 (1963).
413
9. Latia Luciferin
Luciferin has been isolated in an almost pure state from
the fresh-water snail Latia [381. The substance gives an
absorption maximum at 212nm and two IR bands
at 1745 cm-1 (strong) and 1685 cm-1 (weak). Latia
luciferin is fairly volatile, and has a molecular weight
of 236 (determined by mass spectrometry). When the
luciferin is mixed with luciferase solution, a luminescence with an emission maximum at 520nm can be
observed.
10. Fungal Luciferin
Kuwabara and Wassink [39J isolated fungal luciferin
from the luminous fungus Omphalia flavida. In the
presence of luciferase or NaOH and H202 it emits
light. The absorption, fluorescence, and emission spectrum are shown in Figure 10.
490 52L
I A\ : h’.
I \ I
\.
F/
\L
‘.,
Recently, Shimoura and Johnson deduced the structure
of Latia luciferin (18) and its luminescent product
10
300
350
400
450
500
550
h(nrn)
(19)[3*aJ. The product, dihydro-P-ionone, however,
may not be regarded as the emitter of the bioluminescence.
[38] 0. Shimomura, F. H. Johnson, and Y . Haneda in [31, p. 391.
[38a] 0. Shimomura and F. H. Johnson, Biochemistry, in press.
1391 S. Kuwabara and E. C. Wassink in [3], p. 233.
Fig. 10. Spectrum of luciferin from the fungus Omphalia flavida at
= 6.5. A, absorption; F, fluorescence; L, luminescence.
PH
The authors are grateful to Professor Y. Hirata for his
interest in this work.
Received: July 6, 1967; revised: February 19, 1968
[A 620 IE]
German version: Angew. Chem. 80, 417 (1968)
Soot Formation in Premixed Hydrocarbon Flames
BY K. H. HOMANNI*I
The critical fuel-oxygen ratio for soot formation and its dependence on temperature and
pressure are discussed for various hydrocarbons. It is shown in the case of the Bunsen
flame that the shape and structure of the flame front also have a decisive effect on soot
formation. In the reaction zone of fuel-rich hydrocarbon flames, unsaturated compounds
are formed, and the concentration profiles of these products can provide quantitative
information about their importance in soot formation. The Lcmechanism”of soot formation is discussed on the basis of the concentrations of higher hydrocarbons and the
variation of the number and size of the soot particles. The effects of electric fields and
foreign additives on soot formation have been given consideration.
1. Introduction
The familiar yellow light of a candle flame is due to the
continuous thermal radiation of solid particles of soot,
whereas the bluish light of an oxygen-rich hydrocarbon
flame is mainly due to the emission from molecules.
Interest in soot particles as a source of light has been
rather pushed into the background by the importance
of soot formation in other fields. Large quantities of
soot are used as a filler in the rubber industry. Fine
[*] Dr. K. H. Homann
Institut fur Physikalische Chemie der Universitiit
34 Gottingen, Biirgerstrasse 50 (Germany)
414
carbon blacks with high blackening powers are finding
growing use in paints and lacquers. On the other hand,
soot formation is very undesirable in many combustion
processes, such as in diesel engines or in gas turbines,
since soot particles are difficult to burn, and are partly
deposited as a hard coating or leave [the motor together with the exhaust gases to pollute the atmosphere.
In industrial firing systems, soot is desirable in the
flame because of the radiation of heat, but not in the
cool exhaust gases.
Thermal processes yield various types of carbon-like
deposits, ranging from very hard, brittle, and sometimes vitreous substances to the loose, oily gas blacks.
Angew. Chem. internat. Edit, 1 Val. 7 (1968) 1 No. 6
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