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

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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 comprehensive 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 trimethy1 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.
a method of quite general synthetic utility. Provided enereactions and Diels-Alder additions do not figure as menacing 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 thioalkyl[4.51and thioaryl groupd4’, leading to the first sulfursubstituted 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.
1
5
tBu
tBu
tBu
H
H
tBu
H
2
X
=
Me,%;
DCC =
<>-.=.=No
R
I
R
A
K
X
3
R
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
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 temperatures. Most importantly, by using an appropriate sensitizer (tetraphenylporphine, polymer-bound rose bengal,
etc.) and deuterated solvents (chloroform, dichloromethane, 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 peroxymercuration16’, 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 ringopening 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
0 Verlag Chemie GmbH, 6940 Weinheim, 1983
0570-0833/83/0707-0529$02.50/0
529
best studied and mechanistically most understood bioluminescent reactions is firefly bioluminescence [reaction (e)],
R
R
8
I
Me
Me
x
R
SiMe,
R
R
OSiMe,
03
*
R
R
-
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 aperoxylactone has yet been isolated or independently synthesized. Unquestionably, demanding but potentially rewarding work lies ahead on this score.
C H,OH
%No OH'
R
H
R
and the methods limited. In addition, these "high-energy''
compounds are thermally and photolytically labile and undergo acid- and base-catalyzed decomposition, nucleophilic and electrophilic attack, and worst of all, dissociation by traces of transition-metal ions.
Consequently, much pioneering and challenging work
awaits synthetic chemists to improve this unfavorable situation, 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 formaldehyde 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
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 accumulated 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. Dioxetanyl protons usually lie in the chemical shift range 6=4.55.5 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 1,2diols and deoxygenation to the corresponding epoxides by
phosphanes are helpful procedures [reaction (f)], but other
reliable and general chemical transformations need to be
developed.
HO OH
R+
R R
1) LAIHI
0-0
R++R
R R
RBP
-R3P-0
,
R K . ,
R R
(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
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,
PhO
?P h
10
15
16
17
18
19
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 immaterial 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).
1,2-Dioxetanes 3
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].
Dioxetane
Bonds
a
b
c
148
149
147
155
158
149
147.5
149
148
144
149
144
148
150.5
150
144
155
155
148
161
158
150.5
151
155
155.5
Angle
IO’k
21.3
0
11.7
0.8
0
0
15.3
9.6
16.3
1.00f 0.5
8.30k0.5
6.00+ 2.0
Carbonyl Excited
State Products
(Singlets/Triplets)
d
~~
10
15
16
17
18
19
20
21
22
~~~~
144
148
151
143
w
Photophysics
Energy Transfer
~
[a] lo3 k (370.2 K), determined by chemiluminescence measurements under
isothermal conditions [18b].
here the dioxetane ring is essentially planar, but the dihydrodioxin 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 cyclobutene 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.
3. Excitation Yields
We have seen that a characteristic feature of four-membered ring peroxides is their ability to generate electroniAngew. Chem. Int. Ed. Engl. 22 (1983) 829-542
Q
Photochemistry
Direct Photochemical
Sensitized
Photochemical
Lumophore
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 salient features and problems, especially with enzymatically
generated excited states, will be considered here.
53 1
3.1. Luminescence Probes
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 D C 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[251for 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-diphenylanthracene (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].
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 chemilumineschemiexcitation yield (6' for singlets and $T for triplets);
cence yield;
= luminescence yield of excited carbonyl product or lumophore; fluorescence yield (&) for singlets and phosphorescence yield (@ph) for triplets;
@-=energy transfer efficiency of excited carbonyl product to the lumophore
known. This is not a trivial matter, especially for unsymmetrically substituted dioxetanes, which can chemienergize either or both of the possible carbonyl fragments
[reaction (i)], particularly in biological molecules in which
the dioxetane has been postulated as a transient intermediate. If, however, the chemiexcited carbonyl excited state
is known with certainty and its luminescence efficiency
available or determinable, then to determine the desired
q+exc it is only necessary to measure 4Dcby the usual photometric rneth~dd"~.Since under normal conditions the
observed emission is usually fluorescence, this provides direct access to singlet quantum yields (4"). Only under special circumstances can triplet excitation yields (4T)be measured by direct phosphorescence. For example, under degassed conditions the triplet yield of acetone from tetramethyl- 1,2-dioxetane 23 has been reported'201,and the triplet
yield of methylglyoxal from dioxetane 24["] could be determined directly, since such a-dicarbonyl excited states
undergo direct phosphorescence. Significantly, the direct
emission observed in the enzymatic autoxidation of isobutyraldehyde by horseradish peroxidase (HRP) in the presence of a chelating ligand (EDTA, pyrophosphate) has
been identified as derived from triplet acetone produced
from the hypothetical intermediary dioxetane 25["'. HowMe$
Me-&$?
OH
Me
Me
Me
0
Me
23
24
H0-i-r)
H
Me
0
Me
25
I
DPA
DPA
f
hv
H o g
Me-C
8Me
26
H
ever, when the HRP enzyme is replaced by hemin, autoxidation of isobutyraldehyde to triplet excited acetone still
532
'1
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 (6can
) be obtained from eq.
(,)[I9'.
The convenience of these luminescent techniques cannot
be overemphasized. They are simple to carry out, sensitivAngew. 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 assessing excitation yields, particularly in biological system~'~''.
ST
Br
t: 1"
6-
h-
+
1"
Br
!
DBA
DBA
H'
+ 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).
6
29
y+:02HY
Y
28a, X
Bb, X
B C ,
= H. Y = H
= Br, Y = H
x = I.
Y
=
c1
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.However, 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 neutral (Brij-35 and Triton X-100) micellar solutions of chlorophylls all gave excellent results.
Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542
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-phenylethylacetate3lf3'I,and isonicotinic
acid hydrazide 32'351.
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 ~ensitized'~~].
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 carbaldehydes, 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, benzaldehyde, 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. porphines or rubrene, an electron exchange luminescence
(CIEEL) mechanism may be promoted[381.Satisfactory solutions to these analytical problems have not yet been provided.
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;
for triplets); #oboco =yield
#,,,=chemiexcitation yield (@ for singlets and IT
of photoenergized photoproduct; OET= energy transfer efficiency
must be determined either spectroscopically or chromatographically; 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 calculate 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 quantifying the chemical yield of the photoproducts.
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 chromatographic 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. Moreover, 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, HRPcatalyzed 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
P
N
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 estimates indicate that the yield of triplet acetone in this
peroxidase-catal yzed autoxidation is almost 100%.Similarly,
the interconversion of the phytochromes PRand PF is sensitized by enzymatically generated triplet acetone derived
from HRP-catalyzed autoxidation of i~obutyraldehyde'~~~.
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
Numerous other examples
sensitization the aziridine 371401.
of such sensitized and direct chemical titrations of chemienergized carbonyl excited states have been compiled['91.
A
C*3
38
40
,ze
NHAC,M\~
.
Meo
/ OMe
OMe
k H
fi
41
NHA~
/
Me0 \
Me0
f
OMe
42
36
534
35
37
One of the most intriguing and significant studies along
these lines entails sensitized conversion of colchicine 40
Angew. Chem. Inr. Ed. Engl. 22 (1983) 529-542
into b- and y- 4 1 and a-lumicolchicine 42 by triplet acetone generated in the HRP-catalyzed autoxidation of isobutyraldehydecU1. The chemical yield (&,em)
is approximately 7%. What is especially significant is that this photochemical transformation takes place in Colchicum uutumnale 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%.
ks
R2C=0
of steric effects on the rates of thermolysis of 3,3-dialkyland 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.
0-0
A~~I++-A~~I~
Argl?
H
43
Clearly, chemical titration techniques provide a powerful 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.
T~~
S~~
Et Et
Et-t
0-0
0-0
0-0
44
45
46
Trapping experiments would constitute the most unequivocal 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.
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-3methyl- 1,2-dioxetanes 4414'] and 2a,6a-diarylperhydro[1,2]dioxeto[3,4-6][1,4]dioxins 45[491,and the influence
Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542
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 neodymium phosphate laserc5".
535
In the alternative mechanism (n), which involves simultaneous oxygen-oxygen and carbon-carbon bond scission,
dioxetane decomposition occurs concertedly1551(eq. n).
Puckering of the four-membered ring allows optimal alignment 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].
Dioxetane
AH'
[kcal/mol]
50
51
52
25.3f0.3
21.9f0.3
25.4k0.3
As*
Ical/mol/KJ
+ 1.6
-2.1
+ 1.9
AG' (333.2 K) lo4 k (333.2 K)
[kcal/molJ
Is-']
24.8
22.5
24.8
3.8
I10
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 dependence 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 acquire 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 unfortunately necessitates essentially planar four-membered
rings in all three dioxetanes. It is, therefore, not surprising
that the rate constants for their decomposition are almost
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.Crystal structure analysis might provide an answer to this interesting mechanistic problem.
54
53
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 divergence in results of this type as indicated by a recent compilation[601.The rate constants in Table 3 display a pronounced substituent effect, e. g. the parent 1,2-dioxetane
13 decomposes about 75 times faster than the tetramethyl1,2-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 calcul 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 consistent 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
58
10
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
Table 3. Activation parameters of the methyl-substituted 1,2-dioxetanes [a].
~
Dioxetane
13
55
56
57a
57b
1
23
R1
R2
R3
H
H
H
Me
Me
Me
Me
Me
Me
H
H
Me
H
Me
H
H
Me
Me
R5
H
H
H
H
H
Me
Me
Me
H
Me
AH+
[cal/mol-' K - ' J
As'
AGf (343.2 K)
lo4 k (343.2 K)
[cal mol/K]
[kcal/mol]
[s - 'I
19.1 f 1.7
21.5 f0.6
22.5 f 0.2
21.6k0.6
22.1 f 1.0
24.5f 1.5
24.9 & 0.6
- 3 . 0 f 1.0
- 1.420.5
19.9%1.9
21.9+0.7
22.9 f0.3
22.1 t0.7
22.6%1.2
24.8 f I .5
25.1 f 0 . 6
150f10
- 1.3f0.2
- 1.8 f 0 . 8
- 1.6f0.8
- 1.OfO.5
- 0.8 f0.4
85f5
26f3
35f3
27f3
8.6 f0.5
2.0f0.2
[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, spiro[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! Considerable mechanistic work is essential to resolve these problems.
Theoretical studies have provided some progress. The most
recent and sophisticated calculationsr631suggest the diradical mechanism; however, irrespective of their sophistication, 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 accounting 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 dioxe t a n e ~ ~Such
~ ~ l effects
.
might also account for a number of
poorly understood empirical trends in the excitation parameter~"'~.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'651emphasizes 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 biolumine~cence"~~,
especially that of the firefly [reaction (e)].
With the recognition of the phenomenon of chemically in-
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 category 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[681that efficient production
of singlet excited states requires the phenolate form 14a of
the a-peroxylactone 14. In 14a the benzenethiazole group
14 a
(P)
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[691of the dioxetane 59 provides convincing evidence for this hypothesis. Thus, while the unsubstituted 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, O M e , 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 obT
~
+*A,,
--t
D~
+s
~ *
(9)
D = Donor; A = acceptor; k m = effective rate constant of energy transfer;
so = unquenched donor lifetime
( 0)
F1
J.
+
hv
Angew. Chem. Inl. Ed. Engl. 22 (1983) 529-542
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[701for 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 ac537
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
~
*
cernible with 60c and 60d. A static contribution to
quenching in the presence of the enzyme involving enzyme-protected triplet acetone seems to operate.
(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.
x\
60a. X
= H
/
60d, X
=
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,@
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 T2to 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 energetically. However, if triplet acetone is generated enzymatically v i a 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
I
H-C-EH,
COP
H,N@-C-H
H
H
0-61
L-61
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 encounter 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 consequences in the elucidation of photophysical, photochemiAngew. Chem. Int. Ed. Engl. 22 (1983) 529-542
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[751and 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 experiment1771,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 excuses 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%
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 acceptor, 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. However, 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 tripletsinglet energy transfer of chemienergized acetone to 9,lOdibromoanthracene (DBA) in solution and in polymer matrice^^^',^'^. The singlet-singlet energy transfer of chemienergized acetone to ergostatetraenone 62 could also be
studied in an analogous
+
1
62
63
Unconventional energy transfer studies of this type hold
great promise for biologically important acceptors. Even
macromolecules and organelles (chloroplasts) can be selectively 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[641in 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
fireflyfso1arising from the achiral oxyluciferin [reactions (e)
and (p)], while the adamantanone 65 possesses a permanent 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.
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. Possibly “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.
[*] 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).
Angew. Chem. Int. Ed. Engl. 22 (1983) 529-542
67
68
69
539
By analogy, the dioxetane 70 proved useful in elucidating the photomechanistic details of the rearrangement of
Comparison of the
the excited enone 71 to 72 and 73[831.
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.
70
was shown that bioenergized triplet acetone, generated by
HRP-catalyzed autoxidation of isobutyraldehyde, promoted single strand breaks1881and altered the circular dichroisrnl8’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[’”’.
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 semibullvalene 75 was also shown to be a spin-specific triplet processrS4! 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 photodamage 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-dioxetane23, caused
formation of thymine dimers 76 in DNA. Subsequently, it
76
540
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 p h y t o c h r o r n e ~ ~ ~ ~ ~ ,
plant growth hormones involving 3-indoleacetic
the conversion of ergosterol into vitamin D‘97,981,
and the
isomerization of retinalfw1,etc. In addition, the opportunity 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: “Already 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 Professor Frank H . Quina (Universidade de Siio Paulo)for his unfailing and unselfish assistance. Appreciation is extended to
former and present students for their dedicated, diligent, and
stimulating collaboration. Generousfinancial support by the
Financiadora de Estudios e Projetos (Rio de Janeiro), the
Conselho Nacional de Desenvoluimento Cientifico e Tecnologic0 (Brasilia), Fundacao de Ampazo a Pesquisa do Estado de Sao Paulo (Siio Paulo), the Deutsche ForschungsgeAngew. 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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COMMUNICATIONS
(v=2126 cm-'), which under the reaction conditions rapidly loses CO, presumably to give the carbene 5 . The latter
undergoes ring opening to the allene 2, which was collected on a KBr window and characterized by a sharp and
strong infrared absorption at 1886 cm-', persisting at temperatures between 11 K (Ar matrix) and 170 K (neat substance). 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
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 indicates 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 vacuum pyrolysis of bicyclo[3.1.0]hexane-6-carbonylchloride
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 (Germany), March 22-26, 1982. This work was supported by the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
542
0 Verlag Chemie GmbH, 6940 Weinheim. 1983
0:
-
@-cot, ",@C=O
3
5
4
0-
II
H
170 K
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 calculation~[~'.
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.
0570-0833/83/0707-0542 $02.50/0
Angew. Chem. In:. Ed. Engl. 22 (1983) No. 7
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