Waldemar Adam University of Puerto Rico Rio Piedras, Puerto Rico 00931
I
I
Biological Light a-Peroxylates as bioluminescent 'intermediates
"Biological light," the emission of electromagnetic radiation bv livine matter. is one of the marvels of nature that hasFascinated and hspired man throughout history. The first recorded observation of this s ~ e c t a c u l a ruhenomenon (in reference to the firefly) dates hack almost 3,500 years to China. Also Aristotle was enchanted by the magical glow exhibited by certain plants and reported on it some 2,400 years ago. During the last 500 years all sorts of scientists. ohilosoohers.. .nhvsicians. ooets. and maeicians, and s u c h dikinguished s c h o i a i as ~ e s c a r t i s , Bovle. Bacon. and Shakesueare. have wondered about the mist& behind this "cold fire," which is most frequently blue in color, hut shades of green, yellow, orange, and red are observed as well. The haphazard distribution of bioluminescent species in the animal and plant world is most succinctly described hy the late E. N. Harvey, professor of biology a t Princeton University, who dedicated a lifetime to the study of this phenomenon
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Imagine the names of all the forms of life written an a huge blaekboard-the scores of phyla, classes, and orders descending from there to the hundreds of thousands of plant and animal species. Then imagine a man with a handful of wet sand standing off from the board. He throws the sand at it. Where a grain sticks to a name, that species becomes bioluminescent-where not, no hiolurnineseenee. The capacity really seems as disorganized and inexplicable as that. Thus, in animal life, where bioluminescence is most abundant, especially in salt waters, bioluminescent marine organisms range from microorganisms (dinoflaggelates) to vertebrates (fish), which are actually the highest order of life exhibiting this phenomenon, with ample examples of sponges, worms, crustaceans, snails, clams, and shrimps in between. While numerous bioluminescent saltwater oreanisms are known. oddlv enoueh only one freshwater species, the New zealand limpet, has been observed to date. Among the terrestrial organisms that are hioluminescent we find centipedes, worms, and insects, hut not higher orders such a s amphibians, reptiles, birds, and mammals. In plant life only bacteria and fungi are hioluminescent. There must be some interestine evolutionarv reasons for such random occurrence 01' h~oluminescentspecies in rhr. fauna and flora of our planet. Although there exists grear divereenre of oninions. W. 1). ?vlcElrm and ti. ti. Selirer ( I ) proposed the intriguing of ~oohns~ o ~ k i University ns theory that the conversion of chemical energy into light was essential as a survival mechanism. Thus, initially when l ~ f estarted on this planet, maybe some four billion years ago, the earth's atmosphere consisted predominantly
of hydrogen and was devoid of free oxygen. The primeval f o m s of life necessarily had to be anaerobic, not depending on free oxygen in their metabolism. With time the intense ultraviolet radiation from the sun degraded the abundant water into elemental oxygen and hydrogen, the latter, being much lighter, diffused into the outer spheres, thereby enriching the atmosphere of our planet with free oxygen. As the molecular oxygen content in the ocean waters began to rise, the anaerobes were threatened with annihilation by oxidation. For many species this undoubtedly must have been the fate, but some learned to cope with the free oxygen by binding it back into substances, water or carbon dioxide for example. If the large quantities of energy liberated in these oxidations had been degraded into heat, the usual manifestation of chemical change, the organisms would have been doomed by being burned up. Instead, the available chemical energy was utilized to promote organic substances within the organism to emit "biological light." For example, in the firefly this conversion is nearly 100% efficient with less than 1% of heat, hence "cold fire." Of course, subsequent generations in the evolutionary ladder learned to incorporate free oxygen into their metabolic cycle, becoming completely dependent on it in sustaining their existence. Once orimitive oreanisms had adanted to chaneed at" mospheric conditions, higher life forms employed "biological linht" to uerform imoortant hioloeical functions. The morebbvious'utilizatio~of hiolumin~scencein the preservation of species entail attracting of mates, luring of prey, and hiding from predators. For example, inhabitants of temperate and tropical climates may have witnessed the delightful mating dance of the firefly on warm sommer nights. While the male signals a t random his intentions in yellow flashes, the female communicates her desire, answering each call by flashing a t fixed time intervals. Not only does this intriguing signaling ritual serve as a sex lure, but it also permits distinguishing among the many thousands of firefly species since each has a particular periodicity. Still more bizarre is the mating ritual of the fireworm habitating the Bahama waters. About one
This article is based an colloquia delivered by the author at the Universities of Zurich, Clausthal-Zellerfcld, Konstanz, Giessen, Frankfurt, Wageningen, Groningen, Leiden, asd Wiirzburg while on sabbatical at the University of Zurich as a Guggenheim fellow in 1972-73. Part of it appeared originally in German in Chernie in unser Zeit. 7 161, 182 (1973). 138
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Figure : Angler f s t t
Figure 3. Amino acid composition of Cypridina luciferin
Figure 2 . Hatchet f s h
hour past sunset a few days after a full moon, the female attracts the male by radiating intensely. Male and female engage in a frantic chase, enveloped in a hright luminous cloud, ejecting sperms and eggs for fertilization. The angler fish (Fig. 1) has its light organ located at the tip of a tentacle, extending forward fmm its forehead. To attract its prey, the light organ is activated and the lured victim is motioned without resistance into the trap for immediate consumption. As a means of camouflage against predators, numerous organisms have utilized "hiological light." An ingeneous case is the hatchet fish (Fig. 2). This small creature, about the size of two thumbnails, has its light organ (photophores) along the side and belly surfaces. When activated, an intense bluish glow is emitted downward and softer tones sidewards, blending it beautifully into its surroundings, which are diffusely lit by penetrating sun rays, making the fish invisible towards predators especially from below. No doubt, mother nature must have developed hioluminescence for other more suhtle beneficial purposes; however, little is known. It is still anyone's guess why certain bacteria and fungi must radiate "hiological light." Defining the secret of hioluminescence a t the molecular level traces hack as far as the 17th century when R. Boyle in England noted that fungi would only radiate light in the presence of molecular oxygen. Later a t the beginning of the 18th century R. Rbamur in France observed that powdered. dried. hioluminescent orzanisms emitted lieht bn addition of water, showing that the phenomenon c o k d be repmduced in uitro. The fundamental scientific work was executed by R. Duhois in France towards the end of the 19th century. By studying marine and terrestrial s~ecies.he was able to demonstrate that "hioloeical lieht" is the consequence of chemical change. From the organism he extracted a substrate, which he coined luciferin (fmm lucifer meaning light hearer in Latin), and the enzyme, that he called luciferase. On mixing of the luciferin and luciferase in the presence of free oxygen, he could mimic a t will the natural bioluminescence radiated by the organism in uiuo. The oxidation (shown in eqn. (1)) forms oxyluciferin as the oxidized product. Some 50 years later
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luciferin + luciferase -a+ oxyluciferin + hu (1) E. N. Harvey intensively extended Duhois' (2) pioneering work and we are indebted to him for much that we know about this hiological phenomenon. However, the unlocking of the chemistry behind "hiological light" has come forth only during the last decade as a result of the joint efforts of biochemists and organic chemists all over the world. Among the principal contributors in this area we find W. D. McElrov. H. H. Selieer. and E . H . White of Johns Hopkins ~ni;ersity, J. W. ' ~ a s t i n g sof Harvard Universitv. F. H. Johnson and 0. Shimomura of Princeton ~niversit;; M. J. Cormier and K . Hori of the University of Georgia, (all from the USA), F. McCapra of the University of Sussex (England), and T. Goto and Y. Kishi of Nagoya University (Japan). At the University of Puerto
Rico we have been concerned with the synthesis, isolation, and characterization of authentic a-peroxylactones (I),
since they were postulated about five years ago as the energy carriers in the generation of "hiological light." The purpose of this article, therefore, is to focus on (1) the involvement of n-~eroxvlactonesin hioluminescence, (2) the synthetic challenge inherent in preparing such thermally labile materials, and (3) the mechanism of light production. The a-Peroxylactone Mechanism
The first luciferin for which the chemical structure is known is that of the firefly Photinus Pyralis, shown to be the henzothiazol derivative ( H a ) (2). In fact, synthetic luciferin (IIa) could he substituted for the natural one if the D-isomer was used. Two other luciferins whose structures are now well established are (IIb) for the crustacean
aH:r H
N H NH ~ N H *
I
H
HO
/IIRJ
illb)
HOmc1
Cypridina Hilgendorfii, investigated by Y. Kishi and T. Goto (3) and (IIc) for the sea pansy Renilla Reformis, investigated by K. Hori and M. J. Cormier (4). The structural analogy of the latter two, except for minor peripheral alterations, is quite evident and hints a t some common hiosyntbetic path. In fact, as shown in Figure 3 the Cyprldina luciferin (IIb) can be considered as being comprised of the three modified amino acids, tryptophane, arginine, and isolucine. With this important hunch F. McCapra and M . Ruth (5) succeeded in preparing the model luciferin (IId), (the synthetic strategy is exhibited in eqn. (2)) and demonstrated its hioluminescent activity. Except for the Photinus luciferin (IIa), the only henzothiazol derivative known so far that is hioluminescent, it is quite likely that tripeptides serve as hiosynthetic intermediates for many marine luciferins. Volume 52, Number 3, March 1975
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139
tUd1
For the three luciferins (IIa-IIc) discussed here i t has been demonstrated that the oxidation requires equimolar amounts of molecular oxygen, producing, stoichiometrically, carbon dioxide and the respective oxyluciferin. Structure (IIIa) has been characterized as the light emitting oxyluciferin from the Photinus luciferin (6) and (IIIb) from the Cypridina luciferin (7), while (IIIe) has been inferred from the Renilla luciferin. To date no structural information is available on the respective luciferases.
ing to 40-60'C decomposed with chemiluminescence arising from excited acetone. The luminescence was.actually quite feahle since acetone has a very low fluorescence efficiency ( @ F = 0.001-0.01); but in the presence of fluorescers such as mhrene (Va), 9,lO-diphenylanthracene (Vb), or 9,lO-dihromoanthracene (Vc) the luninescence yields could he augmented several 1000-fold due to energy transph ph
Ph
+
bh bh (Va)
bh IVbI
We1
8,
fer. Similarly M. M. Rauhut of American Cyanamid (9) observed that the perhydrolysis of oxalates in the presence of fluorescers (Va-Vc) afforded bright chemiluminescence with a quantum efficiency of 0.23 (per 100 molecules reacted, 23 photons are produced) and suggested the carbon dioxide dimer (VI) as the energizing intermediate ( e m (7)).
* &;Cr,NHYNH.
LXKf
NH
HO
t nib1
tllla1
T H
JJGCrn imei
Closer inspection of the luciferin substrates (IIa-IIe) and oxyluciferin products (IIIa-IIIe) in this oxidative decarhoxylation, reveals that each substrate has a hase-labile hydrogen and activated carhoxyl group (in the firefly the carboxylic acid group is activated by replacing the hydroxyl group with adenylate, AMP for short, which is also performed by the luciferase), while the product contains a carhonyl group. Consequently, the luciferinluciferase oxidation (eqn. (1)) can he specified more precisely by the general reaction shown in eqn. (3), illustrated for the Photinus in eqn. (4) and Cypridina and Renilla in eqn. ( 5 ) , using truncated structures in order to emphasize the essential features.
This reaction is the most efficient chemiluminescent process known so far and has been developed into a commercially available product under the trade name "Coolite," serving as an emergency light source. Finally, F. McCapra (10) suggested the a-peroxylactone (Id) as the energy source in the perhydrolysis of the acridine derivative (VII) into excited acridone (VIII), with a quantum efficiency of -10% (eqn. (8)) M~
r
Me
1
(W11
(Id)
Once the function of such four-membered ring cyclic peroxides as energizers in chemiluminescence was recognized, the involvement of a-peroxylactones (I) as intermediates in the oxidative decarboxylation of luciferins (IIaIIc) was a logical extension. Thus, F. McCapra, et al. (11) and independently E . H. White, et al. (12) suggested the general a-peroxylactone mechanism, involving, respectively, (Ia-Ic) for the Photinus, Cypndina, and Renilla hioluminescence, shown in eqn. (9) and illustrated for the Cypridina luciferin in eqo. (10).
11lb.r)
tnhel
Although eqn. (3) gives important stoichiometric and structural information for the oxidative decarhoxylation in the bioluminescence of luciferins, it does not hear out any mechanistic details nor reasons and insights why this chemical transformation is luminescent. A decisive contribution on this problem was made by K. R. Kopecky and C. Mumford of the University of Alberta (Canada) who showed that the authentic, stable 1,2-dioxetane (IV), readily prepared by base-catalyzed cyclization of the ap~ropriate0-hromo hydroperoxide (eqn. ( 6 ) ) ,(8) on warm-
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/ Journal of Chemical Education
route is still viable. It would indeed be surprising that such structurally analogous luciferins as ( I D ) and (IIc) would be so divergent in their mechanism of oxidation. In any case, the involvement of a-peroxylactone (Ib) as energizer in the Cypridina bioluminescence is established. The future shall see whether the a-peroxylactone mechanism shall be reconfirmed for the Photinus and Renilla. It shall he interesting to see whether in cases still mechanistically nebulous, namely the bioluminescent bacteria (161, this mechanism is operative, as has been very recently postulated (17). Synthesis of n-Peroxylactones
Unaware of the connection of a-peroxylactones with "biological light," we had already commenced our synthetic work on these novel cyclic peroxides in 1966, motivated strictly by chemical curiosity. Of course, once we realized the potential involvement of these thermally labile substances in bioluminescence (18% our total commitment to this synthetic challenge was precipitated in 1968. Initially our strategy projected the three potential routes in eqn. (12), which are (a) the 0-lactone route, (h) the a-lactone route, and (c) the cycloaddition route.
(Ibl
imb) (101
Shortly after the postulation of the a-peroxylactone mechanism for the luciferin bioluminescence, it was suhmitted to scrutiny, utilizing oxygen-18 labelling experiments. As demonstrated in eqn. (111, the a-peroxylactone
IN)
mute (cyclic intermediate) predicts that 50% of the oxygen-18 label of the molecular oxygen should be deposited in carbon dioxide. Alternatively, the intervention of the acyclic intermediate (IX), a mechanism that was quite popular prior to the existence of 1,2-dioxetanes, dictates that carbon dioxide is unlabelled, unless oxygen-18 water is used. In vivo and in uitro experiments on the firefly (13) and sea pansy (14) illustrated that no oxygen-18 labelled carbon dioxide was formed when labelled oxygen was used. In contrast, similar experiments on Cypridina (15) confirmed the n-peroxylactone route since the carbon dioxide was about 50% oxygen-18 labelled. In the latter case the danger of complete isotopic exchange of labelled carbon dioxide in aqueous media was demonstrated when very small amounts of luciferin were oxidized. Since this has been the case in the sea pansy experiments in view of the limited supply of luciferin (IIc), the a-peroxylactone
In the 0-lactone route (a), modelled after the classical 0-lactone synthesis involving internal nucleophilic displacement of a &halogen by the carboxylate ion on base treatment of @-haloacids (19),we hoped that the a-hromo peracid (X) could afford the desired a-peroxylactone (I) via hase-catalyzed dehydmbromination. The necessary abromo peracid (X)was readily prepared from the respective a-bromo acid on submission to hydrogen peroxide in methanesulfonic acid. However, when (X) was treated with base, even at low temperatures molecular oxygen was liberated with regeneration of the a-bromo acid. We were aware of the fact that generally peracids deoxygenate into the corresponding acids and molecular oxygen (20), but we had hoped that internal nucleophilic displacement by the percarboxylate anion would outdo the decomposition. In the a-lactone route (h) we contemplated dehydratively cyclizing a-hydroperoxy acids (XI), using any of the cyclants that proved successful in the direct conversion of 0-hydroxy acids into 0-lactones (21). Unfortunately no examples of a-hydropemxy acids (XI) were known at the time. Anticipating their acid and base triggered Grob fragmentations (22) shown in eqn. (13) 0
it was clear to us that any success in preparing derivatives of (XI) was dictated by mild neutral conditions. ConseVolume 52, Number 3. March 1975
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quently, we decided to investigate the perhydrolysis of rulactones (XII), since in their dipolar form they would add hydrogen peroxide the desired way (eqn.14).
we anticipated that disilyl ketene acetals (XIV) should on photo-oxygenation and subsequent methanolysis lead to the long searched for a-hydroperoxy acid (XI), as shown in eqn. (18). o
n
mn
cxn]
(XIII)
Again, a t the time of our work no a-lactones (XII) had been reported (23), and in view of their elusive nature, we decided to generate the ru-lactones in situ by photodecarboxylation of malonyl peroxides (XIII) (24) in the presence of hydrogen peroxide at low temperatures (eqn. (14)). As e'xpected the desired ru-hydroperoxy acid (XI) was obtained (-609'~ yield); but during photodecarboxylation significant quantities (-20% yield) of the corresponding a-hydroxy acid were produced which made the purification of 1x1) difficult. To circumvent this drawback, we prepared the stable a-lactone bis(trifluoromethy1)acetolactone (XIIa). a gaseous substance a t room temperature (25), in order to pinnit hydrogen peroxide trapping posterior to photodecarboxylation of malonyl peroxide (XIIIa). Ironically, as shown in eqn. (15), we had destabilized the dipolar open form to such an extent that (XIIa) was obliged to act in itsgenuine cyclic form promoting nucleophilic substitution at the carhonyl group. We abandoned the ru-lactone approach.
U
(Xb)
(ma)
The cycloaddition approach (c) was motivated by the fact that singlet oxygen adds to activated olefins to afford l,2-dioxetanes (26). We tried this directly on a numher of ketenes (eqn. (16))
d
n*
R =l e B u
11)
R=CF,
hut they failed to react (27). Next we hoped to prepare 1,2-dioxetanes (IV), functionalized a t the 3-p.os~t~on; but in the case of thioalkyl groups (X = RS-, cf. eqn. (12)). even at Dm. . . . the singlet oxvaen adducts fragmented . Ice temperatuies (28jr In view of the observation (29) that trimethylsilyl gmups can participate in ene-reactions with singlet oxygen (eqn. 17))
Figure 4. a-Peroxylactone chemiluminescence.
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Journal ofChemical Education
Much to our satisfaction, the sequence of reactions in eqn. (18) could he .executed in essentially quantitative yields for (XIa), where R = t-Bu and R' = H, at last enabling us to perform the cyclization (XI) (I), as shown in eqn. .(I91(30).
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Best results were obtained when dicyclohexyl carhodiimide (DCC) in carbon tetrachloride at low temperatures was used as cyclant. When the carbon tetrachloride solutions were stored in the freezer, they were stable for periods of weeks; but on warming to room temperature, they decomposed within 10-30 min, liberating carbon dioxide with chemiluminescence. Of course, the self-luminescence (Fig. 4) was quite weak; but the activated luminescence in the Dresence of fluorescers (Va-Vc) rivalled the heautv of the natural phenomenon. We thus had succeeded m our quest to synthesize "hiological light" chemically. The Mechanism of Excited State Generation With the demonstration that a-peroxylactones decay with chemiluminescence, there can he little doubt now that they serve as energizers in luciferin bioluminescence. The obvious and important question which arises is how these substances are capable of playing the role of promoting the conversion of chemical energy into light energy. Especially noteworthy is the efficiency of this process in the production of "hiological light." For example, as we have mentioned already, for the firefly bioluminescence the yellow 562 nm emission ( -50 kcal) is nearly 100% efficient (Fig. 5), with less than 1% of heat production. This is truly remarkable since even in our most efficient light bulbs barely 20% of the electrical energy is converted into light; the rest, 8070, is wasted as heat. Of course, not all bioluminescent organisms are as effective as the firefly in this impressive energy conversion. Thus, the Cypridina
I r e 5 F r e f l y bolumtnescence
Figure 6 C y p r d i n a biolumnescence
system produces blue 465 nm radiation (-62 kcal) a t about 3090 efficiency (Fig. 6), while the sea pansy generates 509 nm emission (-56 kcal) with only 4% efficiency. As previously stated (eqn. (I)), the most efficient chemiluminescent system is Coolite (Fig. I ) , -23% efficient, while a-peroxylactone (Ib) affords less than 1% excited acetone, emitting a t 425 nm (-67 kcal). Since considerable quantities of energy must be made available by the a-peroxylactones to energize the carhonyl ~ r o d u c t s(-60-85 kcal minimallv). - . it was of im~ortance Lo assess the kinetics and thermodynamics of the &peroxvlactone decarhoxvlation. With respect to the kinetics, the decarboxylati& followed strictly the first-order rate law, providing the activation parameters AH$ = 18.8 kcal/mole, AS$ = -8.9 gibbs/mole, and AG$ (300°K) = 21.5 kcal/mole (30) for the t-butyl case (Ia). Unfortunately, no thermodynamic parameters were available to calculate the heat of reaction for the decarboxylation of (Ia); but simple thermochemical calculations using group additivity values and reasonable strain energies for the fourmembered peroxide cycle indicate that -80 kcal are set free in the decarboxylation (31). The thermal stability of a-peroxylactones is indeed remarkable, particularly if it is realized that this molecule stores -100 kcal/mole on thermal activation. As shown in Figure 8, more than sufficient energy is available to energize acetone in its singlet or triplet state, but not quite enough for carbon dioxide. We could consider a-peroxylactones as kinetically stable exiplexes of ketone and carbon dioxide. That only the ketone is electronically excited is corroborated by the fact that its fluorescence spectrum matches the chemiluminescence spectrum. Although more than e n o u ~ henergy is bound in a-peroxylactones to generate excited carbonyl products during their decarboxylation, it was desirable to establish the quantum efficiency of this process. In this connection it is of interest to mention that for the related 1,2-dioxetanes A. P. Schaap and T. Wilson (32) demonstrated that triplet n,r* states were produced essentially quantitatively, while N. J. Turro and P. Lechtken (33) showed that these triplets were fabricated directly from the 1,2-dioxetane, rather than by intersystem crossing of a singlet excited recurso or. Bv means of ~hotochemicaltransformations promoted h y t h e dioxetanes, activated chemiluminescence involving energy transfer to 9,10-dimethylanthracene (a singlet state quencher) and 9,lO-dihromoanthracene (a triplet quencher), and direct measurement of fluorescence and ph&phorescence quantum yields, Turro et al. established that the triplet-singlet ratio was @T/$S = 125 for n,a* excited acetone from 1,2-dioxetane, with a quantum efficiency of nearly 50% for triplets. Similar activated chemiluminescence measurements for the a-peroxylactone (Ib) gave a ratio @T/@S= 115 25 (341, with a triplet acetone yield of only 590 (eqn. 20).
Thus, the singlet yield of carbonyl pmduct from a-peroxylactones in chemiluminescence is very low (-0.0590 for acetone) compared to the bioluminescence (-100% for firefly oxyluciferin). This is clearly of biological significance in those cases in which high singlet yields of excited products are required. In this context it appears relevant to synthesize authentic a-peroxylactones of natural luciferins, or a t least of closely related model substrates, in order to compare their absolute efficiency of excited state production, as well as the relative proportion of singlet and triplet excited products with those of the enzyme-catalyzed oxidative decarboxylations. It is not impmbable that in the less efficient bioluminescent cases triplet excited states are manufactured efficiently to energize "dark" biological processes. In such systems the spectacular phenomenon of "biological light," a manifestation of singlet excited states, might merely constitute inevitable side reactions. The direct thermal generation of triplet excited state products 3p* from singlet ground state reactants 'Ro no doubt is a rare event in chemistry in view of spin conservation. Thus, the four-membered ring cyclic peroxides constitute a unique opportunity to speculate on the factors that promote such spin forbidden processes. One
*
Figure 8. Energetics of decarboxylation of n-peroxylactones
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,
mechanism, suggested by W. H. Richardson and H. E. O'Neal (31), involves diradicals (eqn. (21)) and is supported by thermochemical calculations. Fragmentation of the peroxide bond leads initially to the sindet diradical a singlet via kt, which can decarboxylate "ia k , excited product or recyclize via kc back to the a-peroxylactone. The singlet-triplet energy gap in diradicals is expected to be small ( = / n,> and off-diagonal elements such as (a, / hv I nr) become (a, I nl) in the spin-orbit coupling matrix. In view of the effective overlap a, and n, orbitals such off-diagonal matrix elebetween ~. ments are non-zero and lead to a finite probability for the spin flip essential in the S(a,a*) = T (&a*) transition.
+
Spin-orbit coupling for the a-peroxylactone molecule is illustrated in Figure 12. At the left, a one-center interaction on the left-hand oxygen of the peroxide bond exerts a torque and the resulting motion of electronic charge with accompanying spin inversion leads to triplet excited ketone and ground state carbon dioxide products. Be this as it may, in the peroxylactone molecule there must he an incentive to generate such a spin decoupling torque on the electrons. It is significant to observe (Fig. 12) that vibrations leading to puckering of the four-membered ring in the a-peroxylactone, which a t the same time represents
so X X Figure 9. State correlation of dioxetane decomposition.
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/ Journal of Chemical Education
Figure 10. Energy level diagram for T (n.u*jspin flip.
e
S(T,T*]
Figure 11. Spin-Orbit coupling in the carbony1 gmup.
the motion of the atoms to form the activated complex for the allowed 2s-2a concerted cyclorevenion, induce precisely the required torque for spin flip. Thus, vibrationally induced spin-orbit coupling in a-peroxylactone affords triplet excited ketone product. Of course, an essential thermodynamic constraint is that sufficient energy is made available during the decarboxylation. Undoubtedly the proportion of singlet and triplet excited products will depend on the exothermicity of the process and the efficiencies of fluorescence and phosphorescence of the excited products involved. And the Future?
Despite the intensive activity on the elucidation of "biological light" during the last century, we have barely scratched the surface and much remains to be divulged on the biological, biochemical, organic, and physical aspects of this captivating phenomenon. For the biologists the unravelling of the ecological and biogenetic factors shall be a great challenge, as well as the understanding of how and why bioluminescence is used in specific marine and terrestrial s~ecies.The biochemists. on whom the weinht of responsibility no doubt must fall; have the important task of characterizing the enzvmatic functions and bioswthetic pathways. he organic chemist unquestionably must contribute more vigorously in the identification of the luciferins and their oxidation mechanism, and synthesize large stocks of these materials for investigation since isolation from natural sources is tedious. For exam~le. 100,000fireflies must be processed to obtain 50 mg of pure luciferin: vet the svnthetic material is readilv available in large quantities a t t h e exorbitant price of $1000 per gram. The physical chemists and physicists, who have been minimally engaged so far, must help in shedding light on the still dark problem of energy conversion in bioluminescence. Especially intriguing appears to be the possibility that excited states are utilized in "dark" biological reactions, recently alluded to by G. Cilento of Sao Paulo University (Brazil) (41) and E . White and colleagues of Johns Hopkins University (42). No doubt it will require the coordinated efforts of all branches of natural science to derive some direct benefit for humanity from "biological light," besides answering man's curiosity on the secret behind this fantastic spectacle of nature. Already some important strides have been made. Besides the obvious utilization as emergency light sources, as for example the Coolite product, it has been observed that Aequorea bioluminescence (a jellyfish) is triggered by ultra small quantities of calcium ions. In radioactive fallout some active strontium ions are always produced and bioluminescence might be useful in detecting harmful nuclear radiation. On the other hand, firefly luciferin biolumincescence requires adenosyl triphosphate (ATP), an essential component of solar life. Thus, firefly luciferin might serve for detection of life on other planets. More tangibly, certain bacterial diseases increase the ATP content; firefly luciferin shall prove of great convenience in detecting such diseases in the very early stages. Even cancer might he combatted more expediently since cancerous cells have been found to diminish the light efficiency of the bioluminescence of firefly extracts. Also drug addiction and pollution, two current problems of tremendous social impact, may become amenable to swifter policing because certain bioluminescent bacteria could he tailored for such purposes. In short, the future of bioluminescence is great! Acknowledgment
The contributions on the part of my undergraduate, graduate, and postdoctoral students in our own work on this subject are gratefully appreciated. Much of our work was financially assisted by the National Science Foundation, the Petroleum Research Fund, the Research Corpo-
F i g u r e 12. Vibrationally i n d u c e d spin-orbit c o u p l i n g in a-peroxylactones.
ration, and the University of Puerto Rico. Research fellowships from the A. P. Sloan Foundation and J. S. Guggenheim Memorial Foundation are particularly recognized. Literature Cited ( I ) Seligrr. H. H., and MeElmy. W. 0 . "Light: Physical and Biological Action." Academic Press, London. 1965. 12) Whito,E. W.,McCapra, F., and Fieid, G.F., J A m e r Chem. Soc., 85,337119631. (3) Kishi, Y . . Goto, T . . Hirafa. Y . . Shimomura, 0 , and Johnson. F. H., Tstrahedmn Lett.. 8427 (1966): Kishi. Y.. Goto, T.. Eguchi. S., Hirata. Y.. Wsfanahe. E.. and Aoyema. T.. Tefrohedmn Lett.. 3437 (19681: Kishi. Y.. G o b . T . , Inone. S.. sugiars,s., and ~ i r h i m o r o H.. , ~ e t m h e d m n ~ e t rN46 . , 119661. 14; Hori, K.. andCormier. M.J..Pmc. Not. Aced Sei. U.S.A., 70, 120119731. ( 5 ) McCapra. F., andRoth. M . J C . S. C h r m Commun., 89411972). (6) Suauki. N.,andGoul. T . . J C . S. C h e m Commun, 2021 IlS11). (7; Goto, T . , Inone, S., and Sudura, S., J . C . S. Chem. Commun., 3873 11968). Goto, T . . inone. S.. Sugiure, S.. Nirhikawe. K.. hobo, M., and Abe. Y.. J . C. S. C h e m Commun, 403511968), (8) Kopeeky, K. R., and Mumford,C., C0n.J Chem., 47.709(19631. 19) Rsuhut. M.M.,Areountr Chem. Re$.. 1.80l19691. (10) McCapra.F..PureAppl. Ch#m.. 24.611 l1970i. (111 McCapra, F., Chang. Y. C.. and Rancois. V. P., J. C . S . C h p m Commun.. 22 (1968); MeCspm, F.,andChsnb,Y. C..J. C . S. C h e m Cammun., 1011 119671. (12) Hopkins, T . A,, Seliger, H. H., White, E. H., and Cali. M. W., J . Amer Chem. S a c . 89.7148l1967). (131 Deluca. M., and Demp*y. M. E.. Biochem. Biaphys. Re*. Commun.. 40. 117
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