Four-Membered Ring Peroxides as Excited State Equivalents: A New Dimension in Bioorganic Chemistry

Four-Membered Ring Peroxides as Excited State Equivalents: A New Dimension in Bioorganic Chemistry

By Waldemar Adam* and Giuseppe Cilento

Dedicated to Professor Giinther 0. Schenck on the occasion of his 70th birthday

Consideration of chemiluminescent and bioluminescent processes invariably brings to mind the four-membered ring peroxides, 1,2-dioxetanes and a-peroxylactones. Nevertheless, while chemiluminescence and bioluminescence are well established fields of scientific study, isolable and characterized “high-energy’’ molecules such as the 1,2-dioxetanes and a-peroxylactones are of relatively recent vintage, about a decade. These intriguing compounds produce electronically excited products which manifest themselves by light emission during their thermal decay; more importantly, these transformations are involved in luminescent as well as dark enzymatic processes. It is not our intention to present a compre- hensive overview in this progress report, but rather to focus on those aspects which require further study.

1. Synthesis

The isolation of the first stable l,Z-dioxetane, the trime- thy1 derivative 3, prepared by the Kopecky sequence [reaction (a)], was reported in 1969[31, while the first a-peroxylactone (1,2-dioxetanone), the tert-butyl derivative 2, was obtained in 1972 [reaction (b)][’]. Since then, well over a hundred dioxetanes 3 and about a dozen a-peroxylactones 4 have been isolated and characterized.

1

tBu tBu H

tBu H

tBu

X = Me,%; DCC = < > - . = . = N o

R A

H 2

I K X R R

3 4

Besides reactions (a) and (b), which have become established classical routes, photooxygenation has proved to be

[*I Prof. Dr. W. Adam Institut fur Organische Chemie der Universitat Am Hubland, D-8700 Wiirzburg (Germany) and Departamento de Quimica, Universidad de F’uerto Rico Rio Piedras, PR 0093 1 (USA) Prof. Dr. G. Cilento Departamento de Bioquimica, Instituto de Quimica Universidade de S2o Paulo, C.P. 20.780, S2o Paulo (Brazil)

Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542 0 Verlag Chemie GmbH,

a method of quite general synthetic utility. Provided ene- reactions and Diels-Alder additions do not figure as men- acing side reactions, photosensitized singlet oxygenation of olefins and ketenes [reaction (c)] constitutes a convenient and facile approach to the four-membered cyclic peroxides 3 and 4[31. Substituents used include alkyl, aryl, alkoxy, aryloxy, dialkylamino, and recently even thio- alkyl[4.51 and thioaryl groupd4’, leading to the first sulfur- substituted 1,2-dioxetanes 5 and 6, respectively. In 5 and 6 the spiroadamantane substitution stabilizes the dioxetane ring sufficiently to allow spectroscopic characterization of these labile species.

5 6

The advantages of the singlet oxygenation method [reaction (c)] over the classical Kopecky route [reaction (a)] are the usually higher yields, mild conditions, and low temper- atures. Most importantly, by using an appropriate sensi- tizer (tetraphenylporphine, polymer-bound rose bengal, etc.) and deuterated solvents (chloroform, dichlorome- thane, acetone, etc.) very labile dioxetanes and a-peroxylactones can be detected by ’H- or 13C-NMR spectroscopy without requiring isolation.

Methods of more limited scope include peroxymercura- tion16’, pero~yhalogenation[~’, silica gel-catalyzed rearrangement of endo-peroxides[8’, electrochemical oxygenation~‘~’, and ozonation of vinylsilanes~’O1. A method of promising synthetic potential entails base-catalyzed ring- opening of epoxides[”l, leading to hydroxy-substituted dioxetanes 12. The functionalized dioxetanes 7, 8, 11, and 12 should provide opportunities of preparing derivatives bound to biomolecules such as sugars, steroids, fatty acids, proteins, pyrimidines, purines, etc., which should be interesting substrates for biological testing.

This summarizes the repertoire of synthetic methods for 1,2-dioxetanes and a-peroxylactones. As is typical for the preparation of four-membered rings, the yields are low

6940 Weinheim, 1983 0570-0833/83/0707-0529 $02.50/0 529

R R

8

I

M e M e

OSiMe,

* 0 3 R SiMe,

R R

R R x C H,OH

%No OH' - H

R R

and the methods limited. In addition, these "high-energy'' compounds are thermally and photolytically labile and undergo acid- and base-catalyzed decomposition, nucleo- philic and electrophilic attack, and worst of all, dissocia- tion by traces of transition-metal ions.

Consequently, much pioneering and challenging work awaits synthetic chemists to improve this unfavorable situ- ation, especially if biologically reIevant four-membered ring peroxides are to be prepared. But even simple species for mechanistic scrutiny, for example the parent dioxetane 13, present a formidable synthetic challenge. Its transitory existence is claimed on the basis of the observed formalde- hyde chemiluminescence in the gas phase singlet oxygenation of ethylene"']. Attempts to prepare isolable quantities by low-temperature photosensitized singlet oxygenation of a solution of ethylene in CFCl, failed. Not even traces of peroxidic material was detected. However, after persistent efforts, ca. 10 mg of the parent 1,2-dioxetane 13 was recently isolated via the classical Kopecky route. The observed chemiluminescence on thermal decomposition and the characteristic NMR spectra support this success[131.

2. Characterization

The most characteristic property of four-membered ring peroxides is their ability to emit light upon decomposition [reaction (d)]. In chemistry this is referred to as chemiluminescence and in biology as bioluminescence. One of the

530

best studied and mechanistically most understood bioluminescent reactions is firefly bioluminescence [reaction (e)],

in which luciferin is converted enzymatically by luciferase and ATP into the corresponding carbonyl product (oxyluciferin), COz, and light via the intermediary a-peroxylactone 14[14]. However, although all dioxetanes and a-peroxylactones emit light, even if often only rather feebly, the corollary is far from true that all luminescent chemical or biological autoxidations involve four-membered ring peroxides. While in chemistry it is, at least in principle, possible to isolate the suspected dioxetane and a-peroxylactone, or synthesize them independently, in biology the evidence is indirect and circumstantial. To the best of our knowledge no genuine biologically derived dioxetane or a- peroxylactone has yet been isolated or independently synthesized. Unquestionably, demanding but potentially re- warding work lies ahead on this score.

14

J.

Even if the four-membered ring peroxides in question can be prepared and isolated, what criteria other than light emission [reaction (d)] can be employed for their definitive characterization? In view of the large volume of accumu- lated data['51, spectroscopic identification is convenient and reliable. The carbonyl frequency at ca. 1870 cm-' is particularly characteristic of a-peroxylactones, whereas for dioxetanes NMR spectroscopy is usually helpful. Dioxeta- nyl protons usually lie in the chemical shift range 6=4.5- 5.5 and the ring carbon atoms between 6= 80 and 110.

For their chemical identification several criteria can be useful. Of course, besides light emission on thermolysis, the products are usually the expected carbonyl fragments. Reduction of dioxetanes by LiAIHl to the respective 1,2- diols and deoxygenation to the corresponding epoxides by phosphanes are helpful procedures [reaction (f)], but other reliable and general chemical transformations need to be developed.

R+ R++R - R 3 P - 0 R K . , ( f )

By far the most rigorous method, providing important structural parameters such as bond lengths and bond angles, is X-ray structure analysis. However, its general use is limited. On the one hand, the dioxetane must be crystalline and have good reflecting properties; on the other, since dioxetanes are radiation sensitive, the crystals must survive X-ray exposure. Despite these limitations, considerable progress has been made along these lines. The first X-ray structures reported concern the "superstable" dioxetanes

HO OH 1) LAIHI 0-0 RBP ,

R R R R R R

Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542

and 15[”], derived from 2,2-biadamantylidene and the sterically hindered cyclobutadiene, respectively. More recently we have added the dioxetanes 16-21 and even the bisdioxetane 22 to the list[’81. The latter compound is interesting in that the two dioxetane rings are arranged anti to another and are considerably puckered, but the dioxane ring is planar. This is particularly surprising in view of the dioxetanes 17-19, in which the reverse applies. Thus,

cally excited carbonyl products, e. g. aldehydes, ketones, esters, carboxylic acids, and anhydrides. In this sense we consider these “high-energy” molecules as excited state equivalents, since they serve as chemical or even biological precursors to excited states. Such chemienergized excited states derived from four-membered ring peroxides behave identically to their photoenergized equivalents in their photophysical and photochemical properties. Thus, among the known photophysical phenomena, excited states of this type display direct chemiluminescence (DC) in the form of fluorescence (singlet states) and phosphorescence (triplet states), or undergo energy transfer chemiluminescence (ETC) with an appropriate luminescing photoacceptor (lumophore), which subsequently either fluoresces or phosphoresces (Scheme 1). In this respect it is completely im- material whether the four-membered ring peroxide is of chemical or biological origin, except that in the latter cases such emissions are termed bioluminescence. The chemienergized carbonyl product can undergo direct photochemical transformations such as rearrangements, isomerizations, fragmentations, or cycloadditions, but can also sensitize such photochemical transformations via energy transfer to an appropriate photoactive acceptor (Scheme 1).

P h O

? P h

16 10 15

17 18 19

1,2-Dioxetanes 3

Carbonyl Excited State Products

(Singlets/Triplets)

22

Table I . Bond lengths [pm] and twist angles [“I in dioxetane rings, and rate constants for decomposition [s-’1 of the annelated dioxetanes 17-19 la].

Diox- Bonds Angle IO’k etane a b c d ~~

10 15 16 17 18 19 20 21 22

21.3 0

11.7 0.8 1.00f 0.5 0 8.30k0.5 0 6.00+ 2.0

15.3 9.6

16.3

148 147.5 155 149 149 155 147 148 148 151 155 144 161 158 144 158 149 149 150.5 144 148 151 150.5 148 155 143 150 144 155.5

Photophysics w Photochemistry Q Energy Transfer

Lumophore

Direct Photo- Sensitized chemical Photochemical ~~~~ ~

[a] lo3 k (370.2 K), determined by chemiluminescence measurements under isothermal conditions [18b].

here the dioxetane ring is essentially planar, but the dihy- drodioxin ring boat-like. The greatest degree of puckering is observed in 10; this arises to a large extent from steric interactions of the adamantyl groups (see Section 4). Of course, 15 is necessarily planar in view of the fused cyclo- butene ring. In the future one can expect to see intensified activity in acquiring structural data of this type, particularly since the few examples discussed here do not allow Q

priori predictions to be made concerning the degree of dioxetane ring puckering. It must be stressed at this point that no X-ray structure analyses of a-peroxylactones have been reported so far since stable a-peroxylactones of good crystalline quality have not yet been isolated.

Fluorescence; Phosphorescence Rearrangements ; Isomerizations; Fragmentations; Cycloadditions

Scheme 1

One of the central analytical problems concerns the determination of the excitation parameters, i . e. the efficiency of singlet (4’) and triplet (43 excited state generation during decomposition of 1,2-dioxetanes or a-peroxylactones. Specifically, for mechanistic reasons it is desirable to know the total chemiexcitation efficiency (q5T +@) and the efficiency of spin state selection (4T/q5S). Of course, for this purpose either the quantum yield of the photophysical processes can be measured or the chemical yield of the photochemical reactions determined (Scheme 1). These methods have been described in detail[’91, and only the sa- lient features and problems, especially with enzymatically generated excited states, will be considered here.

53 1

3. Excitation Yields

We have seen that a characteristic feature of four-membered ring peroxides is their ability to generate electroni-


3.1. Luminescence Probes

For luminescence techniques, by means of which the quantum yield is experimentally established, the key mathematical relationships for direct (DC) and energy transfer (ETC) chemiluminescence are given in equations (g) and (h), respectively. In the case of DC, the chief prerequisite is that the identity of the excited carbonyl product is

QDC =direct chemiluminescence yield; @mC= energy transfer chemiluminescence yield; )...= chemiexcitation yield (6' for singlets and $T for triplets);

= luminescence yield of excited carbonyl product or lumophore; fluorescence yield (&) for singlets and phosphorescence yield (@ph) for triplets; @-=energy transfer efficiency of excited carbonyl product to the lumophore

known. This is not a trivial matter, especially for unsym- metrically substituted dioxetanes, which can chemienergize either or both of the possible carbonyl fragments [reaction (i)], particularly in biological molecules in which

the dioxetane has been postulated as a transient intermediate. If, however, the chemiexcited carbonyl excited state is known with certainty and its luminescence efficiency available or determinable, then to determine the desired q+exc it is only necessary to measure 4Dc by the usual photometric rneth~dd"~. Since under normal conditions the observed emission is usually fluorescence, this provides direct access to singlet quantum yields (4"). Only under special circumstances can triplet excitation yields (4T) be measured by direct phosphorescence. For example, under de- gassed conditions the triplet yield of acetone from tetramethyl- 1,2-dioxetane 23 has been reported'201, and the triplet yield of methylglyoxal from dioxetane 24["] could be determined directly, since such a-dicarbonyl excited states undergo direct phosphorescence. Significantly, the direct emission observed in the enzymatic autoxidation of isobutyraldehyde by horseradish peroxidase (HRP) in the presence of a chelating ligand (EDTA, pyrophosphate) has been identified as derived from triplet acetone produced from the hypothetical intermediary dioxetane 25["'. How-

'1

Me$ Me-&$? O H H0-i-r) H H o g

M e Me 0 M e 0 Me-C M e Me Me 8 Me

23 24 25 26

H

ever, when the HRP enzyme is replaced by hemin, autoxidation of isobutyraldehyde to triplet excited acetone still

occurs, except that its phosphorescence can no longer be observed directly["! Consequently, it was postulated that in the HRP-catalyzed autoxidation the triplet acetone was screened by the enzyme against quenching by oxygen, but in the hemin-catalyzed autoxidation of isobutyraldehyde it was not. An intermediate case is the HRP-catalyzed autoxidation of methyl 2-methylacetoacetate, which affords triplet biacetyl, presumably via the dioxetane 26, as indicated by its directly observable phosphorescence[241. Here, the triplet biacetyl is also not screened by the enzyme against quenching by oxygen; but since, compared to triplet acetone, triplet biacetyl phosphoresces quite efficiently, its phosphorescence can be observed directly near oxygen completion. These unusual results are of considerable biological significance.

In general, it can be said that DC is a convenient method, but has limited scope. The most serious disadvantages are that triplet yields can seldom be determined, the excited-state product must be known, and the fluorescence and phosphorescence yields must be sufficiently large to enable quantitative determinati~n"~].

Energy transfer chemiluminescence (ETC) has by now established itself as the most popular technique for the determination of excitation yields. It was pioneered by the Russian school[251 for monitoring the excited carbonyl products responsible in the chemiluminescence accompanying hydrocarbon autoxidation. With the proper choice of lumophore to which the excitation energy of the chemienergized carbonyl excited state is transferred, singlet (4') and triplet (4T) yields can be obtained photometrically'261.

For studying singlet excited states 9,lO-diphenylanthra- cene (DPA) is frequently used as a lumophore; its fluorescence is sensitized via singlet-singlet energy transfer by the chemienergized carbonyl product. In practice the DPA quantum yield (@ED$) is measured at infinite DPA concentrations under conditions where Stern-Volmer kinetics apply (4:;;'" = 1.00), and since the fluorescence yield of DPA is known, the singlet quantum yield (4") can be calculated from eq. (h)[l9].

I D P A

D P A f hv

9,lO-Dibromoanthracene (DBA) has been widely used to study triplet excited states. Through spin-orbital coupling the bromine atoms catalyze the spin-forbidden triplet-singlet energy transfer from the chemienergized triplet carbonyl product to DBA, thereby sensitizing its fluorescence. Here the DBA quantum yield (#gg) is determined at infinite DBA concentration using Stern-Volmer kinetics (@",BA=0.25), and since the fluorescence yield of DBA is known, the triplet yield (6) can be obtained from eq. (,)[I9'.

The convenience of these luminescent techniques cannot be overemphasized. They are simple to carry out, sensitiv-

532 Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542

ity is merely limited by the quality of the photometric equipment on hand, and most importantly the exact nature of the chemienergized excited states involved in the energy transfer need not to be known. It should, therefore, not be surprising that such methods are extensively used in as- sessing excitation yields, particularly in biological system~'~''.

B r

t: 1" + h- 6- ST B r 1"

! DBA

DBA + hv

For this purpose water-soluble lumophores have been developed, such as sodium 2-anthracenesulfonate (AS) and sodium 9,1O-dibromo-2-anthracenesulfonate (DBAS). Thus, in the HRP-catalyzed autoxidation of isobutyraldehyde, generation of triplet acetone was confirmed by showing that fluorescence of DBAS, but not of AS, could be sensitized'221. In addition, fluorescence of 2 8 a - ~ ' ~ ~ ' and flavins 291291 could be stimulated effectively by triplet acetone generated from HRP-catalyzed autoxidation of isobutyraldehyde (28a : fluorescein; 28b : eosin; 2% : rose bengal).

Y y+:02H Y

28a, X = H. Y = H B b , X = Br , Y = H

Y = c1 B C , x = I.

6 29

Similarly, the lumophores 28 and 29 could be sensitized by HRP-catalyzed autoxidation of straight-chain aldehydes, which presumably occurs via triplet aldehydes derived from intermediary dioxetanols such as 2S301. Howev- er, triplet excited 3-indolecarbaldehyde, produced in the HRP-catalyzed autoxidation of 3-indolacetic acid via the postulated a-peroxylactone 2713'', could not be detected with flavins. When this enzymatic autoxidation was camed out in the presence of micelle-solubilized chlorophylls, these lumophores fluoresced strongly, thus indicating that electronically excited 3-indolecarboxaldehyde occurs as an inte~mediate'~~'. Cationic (CTAB), anionic (SDS), and neu- tral (Brij-35 and Triton X-100) micellar solutions of chlorophylls all gave excellent results.

Thus, this novel detection system holds great promise for the search of enzymatically generated triplet carbonyl products via peroxidase-catalyzed autoxidation of appropriate substrates, provided a chemically induced electron exchange luminescence (CIEEL) process can be excluded. For example, using micellar solutions of chlorophylls it was possible to detect triplet carbonyl products in the HRP-catalyzed autoxidation of phenylacetaldehyde 30'331, ethyl 2-formyl-2-phenylethylacetate 3lf3'I, and isonicotinic acid hydrazide 32'351.

H' 30 31 32

Even complex biomolecules can serve as lumophores to monitor enzyme-generated triplet excited carbonyl products. Thus, in the HRP-catalyzed autoxidation of 3-indoleacetic acid in the presence of tRNAPhe, emission from the 4-thiouridine moiety 33 could be ~ensit ized'~~]. In a control

A

OH OH

experiment it was shown that photoenergized 3-indolecarboxaldehyde sensitized the same emission. When tRNAPh' is replaced by yeast tRNA, which lacks the thiouridine group, no sensitization is observed. More recently it has been found that triplet acetone, triplet straight-chain car- baldehydes, and triplet 3-indolecarbaldehyde, all generated from the appropriate substrate via HRP-catalyzed autoxidation, elicit red emission from intact chloroplasts[371. Processes other than energy transfer could be excluded.

After having presented the advantages of energy-transfer chemiluminescence (ETC) to detect and quantitate chemically and/or biologically generated excited states, its limitations should now also be pointed out. Clearly, in order to be efficient, the excitation energy of the lumophore must be low enough to cause exothermic energy transfer. For simple carbonyl excited states, such as acetone, benzalde- hyde, methyl benzoate, etc., the choice of an appropriate lumophore is not difficult, because all of these species have relatively high excitation energies. Even DPA and DBA or AS and DBAS suffice, although their singlet excitation energies are as high as ca. 70 kcal/mol. However, for extensively conjugated carbonyl excited states whose singlet excitation energies lie below 50 kcal/mol and triplet excitation energies below 30 kcal/mol, the choice of appropriate lumophore is problematic. If lumophores with sufficiently low excitation energies are used, e. g. por- phines or rubrene, an electron exchange luminescence (CIEEL) mechanism may be promoted[381. Satisfactory solutions to these analytical problems have not yet been provided.

Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542 533

3.2. Chemical Titrations

As outlined in Scheme 1, in this type of detection system the chemienergized excited state undergoes a known photochemical transformation either directly or via energy transfer. The mathematical relationships for the determination of the excited state yields are given by eqs. 0') and (k). In practice the chemical yield ($chemf of photoproduct

#=-hem = direct or sensitized yield of chemienergized photoproduct; #,,,=chemiexcitation yield (@ for singlets and IT for triplets); #oboco =yield of photoenergized photoproduct; OET= energy transfer efficiency

must be determined either spectroscopically or chromato- graphically; the photochemical yield is usually known or can be determined readily by the usual photomechanistic techniques for photochemical reactions. For sensitized photochemical reactions, the energy transfer efficiency is determined at infinite concentration of photoacceptor under Stern-Volmer kinetics ($ET= 1.0). All the necessary experimental data is, therefore, available to calcu- late deXc either from eq. (j) or (k). If the direct or sensitized photochemical transformation is singlet-state specific, singlet yields (@) are obtained, but if it is triplet-state specific, then triplet yields (@) are obtained. Such photochemical determination of excitation yields is commonly referred to as "chemical titration" of chemienergized excited states.

The now classical exampre of sensitized chemical titration by energy transfer is shown in reaction (1). Thus, singlet and triplet excited acetone react with maleonitrile to give the oxetane 34 and f~maronitrile"~'. Hence, 4' and dT can be determined in one experiment from eq. (k) by quan- tifying the chemical yield of the photoproducts.

A more recent spin state-specific chemical titrant of chemienergized singlet and triplet excited carbonyl products is the azo compound 35, which on singlet sensitization gives the tetracyclic hydrocarbon 36, and on triplet sensitization the aziridine 371401. Numerous other examples of such sensitized and direct chemical titrations of chemienergized carbonyl excited states have been compiled['91.

A P N

36 35 37

The major advantage of these chemical titrations is that no special photometric equipment is necessary to measure quantum yields. Instead, the chemical yield of photoproduct is determined by standard spectroscopic and chro- matographic techniques. Furthermore, one has at one's disposal a wide range of photochemical transformations. Again, sensitized chemicaI titrations are more convenient than the direct, since in the former the exact nature of the chemienergized excited state need not to be known. More- over, in direct chemical titrations for each specific case a specific dioxetane or a-peroxylactone needs to be synthesized as an excited state equivalent. In sensitized chemical titrations, however, only a suitable photoacceptor needs to be sought, whose excitation energy, of course, must lie below that of the chemienergized excited state so that energy transfer is efficient. It is not surprising, therefore, that sensitized chemical titrations of this type have been used to monitor enzymatically generated excited states. For example, the 7% yield of isopropyl alcohol found when the HRP-catalyzed autoxidation of isobutyraldehyde is carried out in ethanol has been attributed to reduction of the intermediary triplet acetone by

In the meantime a number of such dark photobiological transformations have been reported'*''. For example, HRP- catalyzed autoxidation of isobutyraldehyde in the presence of chloropromazine 38 affords the sulfoxide 39 and the radical cation of 381411. It is known that chloropromazine is

0

CH3 38

C*3

39

photooxidized via triplet sensitizationi4z1, implicating triplet-triplet energy transfer from enzymatically produced triplet acetone. In this respect DBAS serves as inhibitor, presumably by quenching triplet acetone. Quantitative es- timates indicate that the yield of triplet acetone in this peroxidase-catal yzed autoxidation is almost 100%. Similarly, the interconversion of the phytochromes PR and PF is sensitized by enzymatically generated triplet acetone derived from HRP-catalyzed autoxidation of i~obutyraldehyde'~~~.

40 41

N H A C , M \ ~ ,ze N H A ~

k H fi . /

/ OMe M e 0 \ f Meo OMe M e 0 OMe

42

One of the most intriguing and significant studies along these lines entails sensitized conversion of colchicine 40

534 Angew. Chem. Inr. Ed. Engl. 22 (1983) 529-542

into b- and y- 41 and a-lumicolchicine 42 by triplet acetone generated in the HRP-catalyzed autoxidation of iso- butyraldehydecU1. The chemical yield (&,em) is approximately 7%. What is especially significant is that this photochemical transformation takes place in Colchicum uutum- nale L.. even in parts not exposed to light[451. It is, therefore, conceivable that in the living organism triplet excited states are produced endogenously, which sensitize the colchicine-lumicolchicine conversion by energy transfer. The fact that a-lumicolchicine 42, a photodimer of P-lumicolchicine 41, is produced under biological conditions in the dark has important implications for dimerization of thymine mediated by endogenously generated excited states. In this connection a photoadduct between lysozyme and riboflavin has recently been isolated from the HRP-catalyzed autoxidation of isobutyraldehyde[&]; formation of the Patemo-Biichi adduct 43 between enzymatically generated triplet 3-indolecarbaldehyde and the uridine group of tRNA has been demonstrated[36b1. From this chemical titration it was estimated that the triplet excitation yield (@T) of 3-indolecarbaldehyde in the HRP-catalyzed autoxidation of 3-indoleacetic acid approaches cu. 20%.

H 43

Clearly, chemical titration techniques provide a power- ful and convenient tool for the detection and quantitation of enzymatically generated excited states allegedly derived from intermediary 1,2-dioxetanes and a-peroxylactones. Together with the luminescent probes, they provide a wide spectrum of generally applicable analytical methods. The future should witness further intensified activity in this still virgin territory of bioorganic chemistry.

4. Mechanistic Incognita

To account for the thermal stability of dioxetanes and a - peroxylactones, and to explain their ability to generate electronically excited states, a thorough understanding of the mechanism of their thermal decomposition is essential. It is, therefore, hardly surprising that since the discovery of dioxetanes intensive efforts have been expended on this perplexing mechanistic pr~blem"~]. Two extreme mechanistic views have persisted over the last decade, each backed up by convincing experimental data. Based on thermokinetic calculations[471, the diradical mechanism (m) was proposed as the best explanation of the thermal stability of dioxetanes (eq.m). Furthermore, the high propensity of triplet state generation, i. e. the (Tn,n*) state of the carbonyl product, can be accounted for by imposing the kinetic conditions k,,,> k, and kT> kdiSc.

Recently, evidence to support the diradical mechanism has been presented which uses Hammett correlations of substituent effects on the decomposition rates of 3-aryl-3- methyl- 1,2-dioxetanes 4414'] and 2a,6a-diarylperhy- dro[1,2]dioxeto[3,4-6][1,4]dioxins 45[491, and the influence

S~~ T~~

ks R2C=0

of steric effects on the rates of thermolysis of 3,3-dialkyl- and trialkyl-1,2-dioxetanes~so1. However, one of the biggest drawbacks of the diradical mechanism is its failure to account for the unusual stability of the tetraethyl- 1,2-dioxetane 46[5'1.

Et Et Et-t

0-0 A ~ ~ I + + - A ~ ~ I ~

0-0 0-0 Argl?

0-0

44 45 46

Trapping experiments would constitute the most une- quivocal proof for the involvement of diradical intermediates such as 47 in the thermolysis of 1,2-dioxetanes. However, although no positive results have been reported yet, an interesting observation concerns the formation of acetophenone and propene in the thermolysis of the dioxetane derivative 48. The diradical 49, which fragments via the transition state 49 ', was proposed as precursor of the observed product[521.

41 48 49 49*

Neither has it been possible so far to detect dioxetane diradicals spectroscopically. For example, multiphoton infrared excitation of tetramethyl-1,2-dioxetane in the gas phase did not result in detection of diradical intermediates, thus limiting their lifetimes to less than 5 d3]. Still more convincing evidence that such diradicals, if indeed formed, must be extremely short-lived (less than 10 ps) is provided by the fact that they were not even detected by picosecond spectroscopy in the 264 nm pulsed photolysis of tetramethyldioxetane in acetonitrile using a mode-locked neodym- ium phosphate laserc5".


In the alternative mechanism (n), which involves simul- taneous oxygen-oxygen and carbon-carbon bond scission, dioxetane decomposition occurs concertedly1551 (eq. n). Puckering of the four-membered ring allows optimal align- ment of the hatched orbitals shown in the formulae to form an n,x* excited state of the carbonyl product. To explain the high yield of triplet excited product it has been proposed that enhanced spin-orbital coupling occurs during puckering[561.

Recent evidence in favor of this decomposition mode is provided by the series of dioxetanes 50-52L571: the activation parameters (Table 2) clearly show that the fused six-

Table 2. Activation parameters of the bicyclic 1,2-dioxetanes 50-52 [a].

Diox- AH' As* AG' (333.2 K) lo4 k (333.2 K) etane [kcal/mol] Ical/mol/KJ [kcal/molJ Is- ' ]

50 25 .3f0 .3 + 1.6 24.8 3.8 51 21 .9f0 .3 -2.1 22.5 I10 52 25.4k0.3 + 1.9 24.8 4.0

[a] Determined by chemiluminescence measurements under isothermal conditions [%].

membered ring dioxetane 51 is considerably more thermally unstable than the homologues 50 and 52[581. This de- pendence of the thermal stability on ring size can hardly be explained in terms of the diradical mechanism (m). It was

that in dioxetane 51 the four-membered ring is puckered and thus already partway along the decomposition coordinate of the concerted mechanism (n), whereas in dioxetanes 50 and 52 the four-membered rings can be planar, and thus require greater thermal activation to ac- quire the twisted transition state. Examination of Dreiding models corroborates this proposal.

To test this hypothesis, the set of crystalline dioxetanes 17-19 were prepared and their activation parameters and crystal structures determined"sb! As the structural data show (Table I), the dihydrobenzodioxin annelation unfor- tunately necessitates essentially planar four-membered rings in all three dioxetanes. It is, therefore, not surprising that the rate constants for their decomposition are almost

Table 3. Activation parameters of the methyl-substituted 1,2-dioxetanes [a].

the same (Table 1). Challenging work lies ahead to synthesize a set of crystalline dioxetanes with fixed puckered or planar geometries, e . g . of type 53 and 54. It is known that 53 is considerably more thermally labile than 54[591. Crys- tal structure analysis might provide an answer to this interesting mechanistic problem.

53 54

Clearly, experimental evidence has been amassed both for and against the diradical and concerted To resolve this dichotomy, a number of studies on the thermal stability and excitation parameters have recently been performed. For example, the complete set of methylated 1,2-dioxetanes 1, 13, 23, and 55-57 have been prepared and their activation parameters determined (Table 3)[l31. The principal intention here was to test the substituent effect of one particular alkyl group, in this case methyl, by measuring the activation parameters under identical conditions in the same laboratory, and thus avoid the great diver- gence in results of this type as indicated by a recent compilation[601. The rate constants in Table 3 display a pro- nounced substituent effect, e. g. the parent 1,2-dioxetane 13 decomposes about 75 times faster than the tetramethyl- 1,2-dioxetane 23 at 343.2 "K, and the change in activation enthalpy (AH') per methyl substituent is ca. 1 kcal/mol. While this significant substituent effect provides evidence in favor of the concerted mechanism, thermokinetic calcu- l a t i o n ~ ' ~ ~ ' assuming a diradical decomposition pathway also predict a monotonic increase in the thermal stability of 1,2-dioxetanes on increasing methylation.

It is also interesting to mention that the efficiency of chemienergization increases with increasing methylation, the increase being mainly in the triplet yield. This is con- sistent with the empirical trend that the triplet yield (#T) increases with the activation energyf6".

A persisting problem is to provide an explanation for the unusual thermal stability of the 1,2-dioxetane 10 with

10 58

two spiroadamantane moieties. Even a single spiroadamantane moiety significantly stabilizes such dioxetanes against t h e ~ o l y s i ~ [ ~ * ~ ~ ~ . Because of the rigid geometry of

Diox- etane

13 55 56 57a 57b 1

23

R1

H Me Me Me Me Me Me

R2 R3 R5

H H Me H H Me Me

H H H Me H Me Me

H H H H Me H Me

A H + [cal/mol-' K - ' J

19.1 f 1.7 21.5 f 0.6 22.5 f 0.2 21.6k0.6 22.1 f 1.0 24.5f 1.5 24.9 & 0.6

As' [cal mol/K]

- 3 . 0 f 1.0 - 1.420.5 - 1.3f0 .2 - 1.8 f0 .8 - 1.6f0 .8 - 1 . O f O . 5 - 0.8 f 0.4

AGf (343.2 K) [kcal/mol]

19.9% 1.9 21.9+0.7 22.9 f 0.3 22.1 t0.7 22.6% 1.2 24.8 f I .5 25.1 f 0 . 6

~

lo4 k (343.2 K) [s - 'I

150f10 85f5 2 6 f 3 3 5 f 3 2 7 f 3

8.6 f 0.5 2 . 0 f 0 . 2

[a] Determined by chemiluminescence measurements under isothermal conditions 1131.

536 Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542

the adamantyl group, the four equatorial methylene H atoms in 10 are interlocked[l6].

One consequence of this is that 10 has one of the most puckered dioxetane rings (torsional angle cu. 21.3 "). The interlocking of these H atoms prevents further twisting of the four-membered ring, and the diradical mechanism, therefore, seems to be the more plausible. In this context it would be important to investigate the, as yet unknown, spi- ro[adamantanedioxetane] 58, since in this molecule such stabilization arising from interlocking of H atoms is not possible.

It should be evident that a mechanistic explanation for even such a straightforward molecular property as the thermal stability of these intriguing dioxetanes has not been satisfactorily solved experimentally. The dichotomy of diradical and concerted pathways persists! Consider- able mechanistic work is essential to resolve these problems. Theoretical studies have provided some progress. The most recent and sophisticated calculationsr631 suggest the diradical mechanism; however, irrespective of their sophistica- tion, all quantum-mechanical methods used predict a puckered transition state, both for the concerted as well as for diradical decomposition[601. Consequently, theoreticans also have plenty of food for thought. Apart from account- ing for the thermal stability of these "high-energy'' compounds, a still more significant problem is to provide an explanation for the efficiency of generation of excited states in terms of their structures. A novel and provocative hypothesis is that an exciplex between the two carbonyl fragments is formed as the initial excited-state species in the decomposition of 1,2-dio~etanes[~~]. In fact, exciplex emission has been observed for indole-substituted diox- e t a n e ~ ~ ~ ~ l . Such effects might also account for a number of poorly understood empirical trends in the excitation pa- rameter~"'~. Clearly, molecular spectroscopists should also get involved here.

Still more perplexing is the unusual ability of dioxetanes to chemienergize preferentially triplet excited carbonyl products. Of significance and importance in this context is the still poorly understood phenomenon of energy-surface crossings. A qualitative treatment'651 emphasizes the potential of such a view, and more intensive action along these lines appears mandatory.

Mechanistic work on the decomposition of four-membered ring peroxides of biological importance is even more difficult because compounds of this type are too labile to be isolated. Nevertheless, significant progress has been made in understanding the mechanism of luciferin biolu- mine~cence"~~, especially that of the firefly [reaction (e)]. With the recognition of the phenomenon of chemically in-

J. F1 + hv

duced electron exchange chemiluminescence (CIEEL) between peroxides and easily oxidized fluore~cers[~~], model studies confirmed that the catalyzed luminescent decomposition of a-peroxylactones belonged to this novel cate- gory of chemiluminescence reactions [reaction (o)]'~~]. This then enabled the high quantum yield (cu. 9ovo) of singlet excited states observed in luciferin bioluminescence to be rationalized. Thus, reaction (p) represents an intramolecular version of the model process (o) [~~] . Significant in this context is the long-known fact[681 that efficient production of singlet excited states requires the phenolate form 14a of the a-peroxylactone 14. In 14a the benzenethiazole group

(P) 14 a

behaves as the electron donor and th'e a-peroxylactone moiety as the electron acceptor; however, since this takes place intramolecularly, electronically excited oxyluciferin is produced efficiently after decarboxylation. In fact, a recent model study[691 of the dioxetane 59 provides convincing evidence for this hypothesis. Thus, while the unsubsti- tuted dioxetane 59 (X = H) gave a large triplet yield of excited product, the phenolate derivate 59 (X=Oe) gave predominantly a singlet excited product. Clearly, intramolecular CIEEL must also operate here analogously to the a-peroxylactone 14 of firefly luciferin. In the future other examples of such intramolecular electron exchange chemiluminescence will surely be discovered.

59, X = H, OH, OMe, Oe

Another area of biological investigation in which important progress has been made are energy-transfer phenomena involving enzymatically generated excited states[*''. Only some of the recent highlights will be presented here. For example, in normal triplet-singlet energy transfer involving free collisions [eq. (q)], the Stern-Volmer constant [eq. (r)] for triplet acetone quenching should not exceed that ob-

T ~ * + A,, --t D~ + s ~ * (9)

D= Donor; A = acceptor; km= effective rate constant of energy transfer; so = unquenched donor lifetime

( 0) served for molecular oxygen, i.e. Ksv is ca. 2 x lop3 to 5 x lo3 L mol-'. Frequently, however, KsvL lo4 L mol-I. Since the Forster criteria[701 for long-range triplet-singlet energy transfer are not met in all cases, one may infer from these high Ksv values that the acceptor is very near the ac-

537 Angew. Chem. Inl. Ed. Engl. 22 (1983) 529-542

tive site prior to excitation by the donor. This proximity, therefore, facilitates triplet-triplet energy transfer [eq. (s)]. This mechanism may even apply when the Forster overlap

T ~ * + A, --t D~ + T ~ * (s)

criterion is met. Such a case appears to be the quenching of enzymatically generated acetone phosphorescence by the xanthene dyes 28””. When the integrated sensitized dye emissions and the fluorescence yields are taken into account, the relative efficiencies of populating the singlet excited state of the dye are found to be 7 : 15 : 100, respectively, for fluorescein, eosin, and rose bengal. The significantly greater S, yield with rose bengal most probably arises because the triplet energy of the acetone donor is first transferred to an upper triplet state of the dye acceptor: the iodine atoms (heavy atom effect) in the rose bengal favor formation of its singlet excited state uia enhanced intersystem-crossing [eq. (t)]. If only the traditional Forster mechanism [eq. (q)] were to operate, fluorescein would have had the highest relative efficiency.

Evidence in support of this hypothesis is provided by the chemienergized fluorescence of DBA by triplet acetone generated thermally from tetrarnethyldioxetane. By analogy to eq. (t), an upper triplet state (T2) of DBA is first produced via allowed triplet-triplet energy transfer; subsequently, the intramolecular heavy atom effect enhances intersystem-crossing from the T2 to the Sl state[711. This mechanism appears also to apply to the energy transfer from enzymatically generated triplet acetone to DBAS as acceptor. In this context it is of interest to mention that energy transfer to AS occurs as efficiently as to DBAS; however, no fluorescence is observed with AS since it lacks the bromine atoms of DBAS to promote intersystem-crossing of the T2 to the S, state. Instead, internal conversion of the T2 to the T, state of AS is favored; the TI state, however, does not emit.

A further interesting case of energy transfer concerns the tyrosine derivatives 60. Thus, when triplet acetone is chemienergized by thermolysis of tetramethyldioxetane in the presence of these tyrosines, the Stern-Volmer quenching plots (monitored by DBAS) are linear‘721. Except for the parent tyrosine, 60a, the quenching rate constants approach those operating under diffusion control conditions. Presumably the energies of the T2 states are too high, so that the T, states of the tyrosines 60b-d are energized directly. The intramolecular heavy atom effect cannot, therefore, be observed experimentally for these quenchers, since the energy transfer process of eq. (t) is not accessible ener- getically. However, if triplet acetone is generated enzymatically via HRP-catalyzed autoxidation of isobutyraldehyde, the Stern-Volmer quenching plots for the parent tyrosine 60a and its dichloro derivative 60b are almost linear, whereas for the dibromo 60c and especially for the diiodo 60d derivatives, the corresponding plots have a marked upward curvature. A heavy atom effect is also dis-

538

cernible with 60c and 60d. A static contribution to quenching in the presence of the enzyme involving enzyme-protected triplet acetone seems to operate.

x\ 60a. X = H

60d, X = I / X

The protective role of the enzyme is most convincingly demonstrated in the quenching of enzymatically generated acetone phosphorescence by D- and L-tryptophan 61[731. Thus, when D-tryptophan is present, competitive quenching by L-tryptophan is very inefficient; however, when the converse applies, quenching by D-tryptophan is still observed. This chiral discrimination precludes the possibility that triplet acetone and the quencher exist freely in solution under these conditions, since triplet acetone generated by thermolysis of tetramethyldioxetane is quenched equally efficiently by D- and by L-tryptophan. The chiral discrimination by the enzymatically generated triplet acetone probably involves a collisional complex of the quencher and the active site of the enzyme prior to energy transfer.

CO,@ COP

H-C-EH, H,N@-C-H

H H

0-61 L - 6 1

Several features about these unusual results with enzymatically generated triplet acetone still remain unsolved. For example, complete quenching by quinones can be observed even at concentrations much lower than that of the enzyme[741. Under these conditions the quencher can en- counter at most one acetone triplet during the lifetime of the latter (ca. lo-’ s). Quenching by long range energy transfer must, therefore, operate. In this respect the Forster overlap criterium is fulfilled for some, but not all quinones. Quenching by electron transfer or charge transfer interaction is unlikely because both triplet acetone and quinones are electron acceptors.

A further important unresolved question concerns how the excited species transfers energy to organelles such as chloroplasts and to acceptors in micelles. When the triplet energy donor is shielded by the protecting enzyme, energy transfer may require the intermediacy of exposed groups of the macromolecule or of the organelle. For micelles the acceptor presumably resides at or near the micellar surface. No doubt, interesting results should be forthcoming in this fascinating bioorganic discipline.

5. Photological Divertissement

The fact that 1,2-dioxetanes and a-peroxylactones are masked electronically excited states, defined here as “excited state equivalents”, clearly has important conse- quences in the elucidation of photophysical, photochemi-


cal, and photobiological phenomena’‘]. As implied in Scheme 1, chemically and biologically photoenergized excited states behave in the same way as excited states generated by the action of light. Clearly, chemi- and bioenergization provide fascinating opportunities to study photological problems in an unconventional manner, also referred to as photochemistry[751 and p h o t ~ b i o l o g y [ ~ ~ ~ in the dark. The fundamental difference, however, in these two approaches is that photoenergized generation of excited states competes with photoabsorption by the substrate, whereas chemi- and bioenergization entail straightforward conventional techniques, with the proviso, however, that the four-membered ring peroxides must first be synthesized. This is frequently not a trivial task and presumably has restricted more frequent use of these “high-energy” molecules. Nevertheless, the preparation of the stable dioxetane 10 is an undergraduate laboratory experi- ment1771, and the synthesis of the more labile tetramethyldioxetane 23 can be mastered by a proficient laboratory technician in a few days. Consequently, there are no ex- cuses for not employing these interesting molecules.

As a divertissement, a number of photophysical, photochemical, and photobiological problems will be presented in which 1,2-dioxetanes and a-peroxylactones have been employed as “excited state equivalents”. This selection should serve to encourage more intensive activity in this promising area. In this context a number of problems will be outlined that could possibly be tackled with the aid of four-membered ring peroxides.

x r x 1%

62 + 1

63

Unconventional energy transfer studies of this type hold great promise for biologically important acceptors. Even macromolecules and organelles (chloroplasts) can be selec- tively energized by four-membered ring peroxides. The feasibility of such studies has already been demonstrated with enzymatically generated excited but authentic dioxetanes should prove more convenient.

With the observation of exciplex emission[641 in the direct chemiluminescence of the indolyldioxetanes 63 and the suggestion that exciplex formation might be quite general in dioxetane decompositior11*~~, a number of stimulating problems present themselves. For example, the optically active dioxetane 64 causes circularly polarized fluorescence of the chiral adamantanone 65[791. In fact, circularly polarized bioluminescence has been observed for the fireflyfso1 arising from the achiral oxyluciferin [reactions (e) and (p)], while the adamantanone 65 possesses a perma- nent chiral center. A chiral excited state of the oxyluciferin arising from exciplex formation might possibly be the cause of circularly polarized bioluminescence in the firefly. Consequently, it would be of interest to synthesize an optically active a-peroxylactone 66, or for that matter a 1,2-dioxetane, which subsequently leads to an achiral product, but whose intervening chiral excited state (presumably an exciplex) might emit polarized chemiluminescence. An interesting luminescent probe for elucidating excited state geometries thus presents itself.

In photophysics, dioxetanes lend themselves particularly well to energy transfer studies. By means of photoenergization it would be difficult to transfer excitation energy from a weakly absorbing donor to a strongly absorbing accept- or, especially if the absorption bands extensively overlap. A specific example is given in eq. (u), in which an excited carbonyl compound (weakly absorbing donor) transfers its excitation energy to the 9,lO-disubstituted anthracene (strongly absorbing acceptor) (X = Ph: DPA; X = Br: DBA). Clearly, with conventional photoenergization all the light is preferentially absorbed by anthracene. Howev- er, by heating the appropriate dioxetane in the presence of the anthracene acceptor the desired excited carbonyl product is generated via chemienergization, and competitive photoabsorption problems are, therefore, circumvented. Hence, important photophysical data, which were previously difficult to obtain, can be acquired for the triplet- singlet energy transfer of chemienergized acetone to 9,lO- dibromoanthracene (DBA) in solution and in polymer ma- trice^^^',^'^. The singlet-singlet energy transfer of chemienergized acetone to ergostatetraenone 62 could also be studied in an analogous

[*] No collective term exists in the dictionary for photobiology, photochemistry, and photophysics, so that we shall define the term photology, as the science (logia) of light (phos).

64 65 66

Bisdioxetanes, for example the benzoic acid anhydride dimer 22@’], should be excellent precursors for the thermal generation of upper excited states, since each dioxetane ring represents the equivalent of a trapped photon. Possi- bly “biphotonic” processes might be included in such unconventional studies.

In photochemistry, use of chemienergization to tackle mechanistic problems has been surprisingly rare. The value of dioxetanes is clearly evident in the decomposition of 67, where they chemienergize the formation of enone 69 via the 3n,n* excited state of dienone 68le2’ . In this manner a long standing photomechanistic problem of whether n,n* or n,x* triplet states are involved in dienone rearrangement could be resolved.

67 68 69


By analogy, the dioxetane 70 proved useful in elucidating the photomechanistic details of the rearrangement of the excited enone 71 to 72 and 73[831. Comparison of the yields of the 1,3-acyl shift product 72 with those of the oxa-di-x-methane (ODPM) product 73 of the chemi- and photoenergized enone 71 leads to the conclusion that a n,n* triplet is the principal precursor for the I,l-acyl shift and a n,x* triplet for the ODPM process.

was shown that bioenergized triplet acetone, generated by HRP-catalyzed autoxidation of isobutyraldehyde, promoted single strand breaks1881 and altered the circular di- chroisrnl8’l in DNA. As supporting evidence that enzymatically produced triplet acetone was the damaging species, is the fact that such damage can, in part, be inhibited by DBAS. That a metabolic system can generate excited states in situ from intermediary dioxetanes has recently been demonstrated in the degradation of benzo[a]pyrene by liver microsomes. The accompanying weak chemiluminescence was attributed to the intervening dioxetane 77[’”’.

70 71

OH

77

Me‘

72 73

In this exploitation of chemienergization the specific excited-state equivalents, dioxetanes 67 and 70, had to be synthesized. Of course, this is not always a simple task as already mentioned in Section 1. It is considerably more convenient to sensitize a particular photochemical reaction by energy transfer via a chemienergized carbonyl excited state as donor. For example, using tetramethyl-1,2-dioxetane 23, a clean source of triplet acetone, it could be confirmed that the rearrangement of azoalkane 35 to aziridene 37 is a spin-specific triplet reaction‘?]. Similarly, using chemienergized triplet acetone the di-n-methane rearrangement of dibenzobarrelene 74 to afford semibullval- ene 75 was also shown to be a spin-specific triplet proc- essrS4! In the future such photochemical applications of dioxetanes should certainly become more frequent.

74 75

For the application of four-membered ring peroxides to photobiological problems a rather extensive compilation of the possibilities was made right at the beginning of this field[75’851. Significant progress was achieved once enzymatic generation of electronically excited triplet states was established[271. A number of problems have been solved, but many still remain.

Thus, considerable attention has been devoted to photo- damage of DNA by thymine dimerization in view of its connection with carcinogenesis1861. A key experiment[871 was the demonstration that chemienergized triplet acetone, generated from the tetramethyl-1,2-dioxetane 23, caused formation of thymine dimers 76 in DNA. Subsequently, it

76

If chemi- and bioenergized triplet excited states can lead, via energy transfer, to thymine dimerization in DNA, the photochemical reversion of such damage should, in principle, also be reasible in this manner. Indeed, model studies reveal that photoenergized excited indolesly’l and q u i n o n e ~ ~ ~ ~ ~ , species that can be generated enzymatically in the dark, induce splitting of thymine dimers 76.

Another area of great challenge and potential is photoaffinity labeling1941. For example, the carbonyl functions of hormonal ligands, e.g. steroidal ketones, could be masked in the form of dioxetanes or a-peroxylactones. After complexation with the receptor, decomposition of the dioxetane would release the electronically excited ligand, which would become permanently bound to the receptor, thereby revealing the specific binding site. The advantage of such “photoaffinity labeling in the dark” is that it could be performed in vivo in aqueous systems.

Stimulating problems of bioorganic significance in which “excited state equivalents” should prove of interest concern photoregulation mediated by phy tochro rne~~~~~ , plant growth hormones involving 3-indoleacetic the conversion of ergosterol into vitamin D‘97,981, and the isomerization of retinalfw1, etc. In addition, the opportu- nity of creating a radically new line of chemotherapeutic agents presents itself.

We shall close with the following citation110oJ, which best describes our contention that the fun has just begun: “Al- ready today preparative photochemistry plays its own role within the methods of chemistry in view of the special possibilities it offers. However, its real importance arises from the abundant interdisciplinary connections, through which chemical problems caused by biological radiation become especially typical”.

7he authors wish to express their deep gratitude to Profes- sor Frank H . Quina (Universidade de Siio Paulo) for his un- failing and unselfish assistance. Appreciation is extended to former and present students for their dedicated, diligent, and stimulating collaboration. Generous financial support by the Financiadora de Estudios e Projetos (Rio de Janeiro), the Conselho Nacional de Desenvoluimento Cientifico e Tecno- logic0 (Brasilia), Fundacao de Ampazo a Pesquisa do Es- tado de Sao Paulo (Siio Paulo), the Deutsche Forschungsge-

540 Angew. Chem. In:. Ed. Engl. 22 (1983) 529-542

meinschaft, the Fonds der Chemischen Industrie, the Fritz Thyssen Stiftung, the Alexander von Humboldt Stiftung, and the Volkswagenwerk Stiftung is acknowledged. The latter deserves special emphasis since it presently sponsors the collaborative efforts of the Brazilian and German groups.

Received: March 25, 1983 [A 459 IE] German version: Angew. Chem. 95 (1983) 525

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(v=2126 cm-'), which under the reaction conditions rap- idly loses CO, presumably to give the carbene 5 . The latter undergoes ring opening to the allene 2, which was col- lected on a KBr window and characterized by a sharp and strong infrared absorption at 1886 cm-', persisting at tem- peratures between 11 K (Ar matrix) and 170 K (neat sub- stance). The absorption disappeared at the latter temperature with concomitant formation of the dimer 6, which has previously been isolated during a preparation of 5 in solution by an alternative route13'. 6 was isolated by preparative gas chromatography and identified by direct comparison with an authentic sample.

1,2-Cyclohexadiene** By Curt Wentnrp*, Gerhard Gross, Andre Maquestiau, and Robert Flammang

@-cot, ", @C=O 0: - 3 4 5

The structure of 1,2-cyclohexadiene has been a matter of controversy. Calculations and experiments have suggested a planar zwitterionic or diradicaloid ground state l1']. However, it was recently shown in elegant trapping experiments that the species is chiral in solution at room temperature, but that optical activity is lost at 80 'C[*]. This indi- cates that the trapped species is the nonplanar allene 2 and that at 80°C it either interconverts with the planar structure 1 or racemizes via a planar transition state.

* signifies O/O or 0

We now wish to report the direct spectroscopic observation of 2, which conclusively proves that the ground state of 1,2-cyclohexadiene is an allene. 2 was generated by vac- uum pyrolysis of bicyclo[3.1.0]hexane-6-carbonyl chloride 3 at 800°C/10-4 torr. This leads initially to the ketene 4

[*I Prof. Dr. C. Wentrup, G . Gross Fachbereich Chemie der Universitgt Lahnberge, D-3550 Marburg (Germany) Prof. Dr. A. Maquestiau, Dr. R. Flammang Laboratoire de Chimie Organique, Universite d'Etat B-7000 Mons (Belgium)

[**I Presented, in part, at the Chemiedozententagung in Kaiserslautem (Ger- many), March 22-26, 1982. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

II H 170 K

/ \ 0 - 0.5

2 6

The identity of 2 was also ascertained by pyrolyzing 3 in a reactor placed immediately before the ion source of a Varian MAT 311A mass spectrometer. The CID-MIKE mass spectrum demonstrated the formation of an ion (m/z = 80) whose intensity increased with increasing temperature. At the same time, the intensities of peaks due to the starting material 3 diminished.

Confirmation of the reaction pathway 3 -+ 2 may also be obtained from the analogous formation of allene by pyrolysis of cyclopropanecarbonyl chloride, as observed by IR and mass spectrometry in the present study and previously by photoelectron ~pectroscopy~~~.

The observation of an allenic absorption for 2 at 1886 cm-' clearly identifies this molecule as a ground state allene. Ring strain and an obvious deviation from linearity causes a shift by approximately 70 cm-' from the normal position of the C=C stretching vibration in allenes. This conclusion is also supported by very recent ab initio calcu- lation~[~'.

Received: February 22, 1983 [ Z 283 IE] German version: Angew. Chem. 95 (1983) 551

CAS Registry numbers: 2, 14847-23-5; 3, 85763-23-1 ; 4, 85763-24-2; 6, 28229-15-4.

542 0 Verlag Chemie GmbH, 6940 Weinheim. 1983 0570-0833/83/0707-0542 $02.50/0 Angew. Chem. In:. Ed. Engl. 22 (1983) No. 7

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