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Facile Reduction of 1 2-Dioxetanes by Thiols as Potential Protective Measure against Photochemical Damage of Cellular DNA.

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fide 1,2-dithiane was isolated and small amounts (ca. 3%)
of the dioxetane cleavage products (acetone and hydroxyacetone) were detected by 'H-NMR. Such cleavage products gain greater importance for the simple thiols (Entries
6-9, Table I), although the reactions were conducted at
subambient temperatures, at which thermal decomposition
of the dioxetane is negligible. For example, with thiopheno1 (Entry 9 in Table 1) the extent of dioxetane cleavage
was as much as 50%, even at - IOOT, thus seriously competing with reduction to the triol 2. Similar results were
obtained for tetramethyl-1,2-dioxetane, undergoing quantitative reduction by glutathione to pinacol, but appreciable
cleavage into acetone with thiophenol.
That glutathione is an efficient reagent for the reduction
of peroxides is well e ~ t a b l i s h e d ,but
~ ~ ]that the labile dioxetanes can be so cleanly transformed into vicinal diols is
somewhat astonishing, since in the few reported studies
with divalent sulfur compounds, oxygen transfer prevails.
Thus, the dioxetane is converted into an epoxide and/or a
ketone, while the sulfide is oxidized to the sulfoxide.15'
Furthermore, dialkyl sulfoxylates S(OR)2 are transformed
via intermediary tetraalkylorthosulfites S(OR)4 into dialkyl
sulfites.161These oxygen transfer reactions have been mechanistically interpreted in terms of either a nucleophilic
attack by the sulfide['] on the peroxide linkage or a biphilic
insertion by the dialkylsulfoxylate.[61 Consequently, we
likewise expected oxygenated products rather than disulfides in the reaction with thiols. Indeed, when only equi-
Facile Reduction of 1,2-Dioxetanes by Thiols
as Potential Protective Measure against
Photochemical Damage of Cellular DNA**
By Waldemar Adam. * Bernd Epe. Dietmar Schiffmann,
Franklin Vargas, und Dieter Wild
In recent biological studies"' it was demonstrated that
1 Jdioxetanes are genotoxic. Since these strained fourmembered ring cyclic peroxides are known to be efficient
chemical sources of np*-excited triplet carbonyl products,'" we postulated that the observed D N A damage was
of photochemical origin. However, in view of the quite
moderate photo-genotoxicity displayed by the dioxetanes
studied, we suspected that these labile peroxides were efficiently detoxified in the cell through chemical action. For
example, the living cell guards itself against "oxidative
stress"'" by engaging glutathione, a tripeptide which deactivates reactive oxygen species including peroxides by reduction, itself being oxidized to its disulfide. We now report on the quantitative reduction of dioxetanes to the corresponding vicinal diols by glutathione [Eq. (a)], a reaction
which also takes place with other thiols. The results are
listed in Table I .
HC
,
CH3
I
I
H,C-C-C-CH,OH
I I
HC
,
+
2 R-SH
CH,
I
I
+H,C-C-C-CH,OH
I I
HO
0-0
+
RS-SR
(a)
OH
2
1
Table I . Reaction of I with thiols.
NO.
I.
_.
7
3.
4.
5.
6.
7.
8.
9.
Reaction conditions
T["CI
Thiol [a]
R-SH
Solvent
L-Glutathione
L-Cysteine
L~Penicillamine
rhrro- I .4-Dimercapto2.3-butanediol
I ,3- Propdnedithiol
Thiobenzyl alcohol
Mercaptoacetic Acid
Methyl Mercaptoacetate
Thiophenol
H2O
H20
H2O
MeOH
5
0.16
20
20
10
0.16
0.16
MeOh
MeOH
MeOH
MeOH
MeOH
- 40
- 50
- 40
- 40
- 100
1 [hl
Product yields Imol] [h, cl
Product
balance
["/d
Disulfide
2
0.97
0.98
0.96
0.99
0.96
0.92
0.90
0.99
.-
I
91
95
93
99
24
72
72
72
22
97
89
78
80
50
0 98
0.78
0.56
0.59
0 49
0.93
0.82
0.56
0.58
0.49
0 03
0.18
0.44
0.42
0.5 I
Ketone [d]
~~
-
[a] Stoichiometry 2: I except entries 4 and 5 , for which it was 1: I. [b] 100% conversion; normalized to 1.00 mol: i 2 " % error limits. [c] Determined 'H-NMR
spectroscopically and/or isolated. [d] Acetone and hydroxyacetone formed in equal amounts.
On mixing aqueous solutions of the hydroxydioxetane 1
with glutathione at 5°C in a 1 : 2 stoichiometry, a fast reaction (100% conversion in 10 min, monitored by 'H-NMR)
ensued, leading essentially quantitatively to the glutathione dimer [m.p. 178-180°C ( d e ~ o m p . ) ' ~m.p.obr
~,
178182°C (decomp.)] and the triol 2. Both products were isolated and identified by comparison with authentic materials. Similar results were obtained for cysteine, penicillamine, and threo- I ,4-dimercapto-2,3-butanediol (Entries 2-4
in Table I), except that in the latter case the cyclic disul-
[*I Prof. Dr. W. Adam, DipLChem. F. Vargas
lnstitut fur Organische Chemie der Universitat
Am Huhland, I>-8700 Wiirzhurg (FRG)
Dr. B. Epe. Dr. I>. Schiffmann, Dr. D. Wild
lnstitut fur Toxikologie und Pharmakologie der Universitat
Versbacher Str 9. 0.8700 Wiirzburg (FRG)
[**I This work was supported by the Deutsche Forschungsgemeinschaft
(Sonderforschungshereich No. I72), the Fritz-Thyssen Stiftung, and the
Fonds der Chemischen Industrie. F. Vargas thanks the DAAD for a
doctoral tellowship.
Ailyen,. U w m Irrt. E d Enyl. 27 (1988)
"I.
3
molar amounts of glutathione and dioxetane 1 were used,
the glutathione was converted quantitatively in a fast reaction into its dimer, and in a subsequent slow reaction
(hours) the disulfide was oxidized to its S-monoxide, as
anticipated for an oxygen transfer reaction. With L-methionine (in H 2 0 , 20"C, 20 min, 1 : 1 stoichiometry), 1 was
quantitatively converted into the epoxide 2,3-dimethyl-2,3epoxy-1-butanol, while the methionine itself was converted
into the sulfoxide. In the case of dimethyl sulfide (in
CHC13, - 5"C, 3 h, 1 : 1 stoichiometry) oxygen transfer was
the major reaction (ca. 85%), affording 60% sulfoxide and
12% sulfone as sulfide derived products, and 44% 2.3-dimethyl-2,3-epoxy-l-butanoland 41% of the rearranged, ketone l-hydroxy-3,3-dimethyl-2-butanoneas dioxetane derived products. Dioxetane cleavage into equal amounts of
acetone and hydroxyacetone was observed as secondary
reaction (ca. 15%). A control experiment showed that in
a much slower reaction dimethyl sulfoxide was oxidized
by dioxetane 1 to its sulfone. Clearly, the sulfides behave differently than the thiols in their reaction with dioxetanes.
0 VCH Verlagsgesellscha/i mhH,
0-6940 Weinheim. 1988
0570-0833/R8/0303-0429 S 02.5OJ/O
429
in CDC13 at -30°C exhibited enhanced polarization of
the methylene hydrogens of the acetal linkage. Moreover,
it is of interest to mention that preliminary results with trimethylsilyl phenyl sulfide show that it behaves like a thiol
in that reduction of the malonyl peroxide 3 takes place
affording phenyl disulfide. The analogous reduction by trimethylsilyl phenyl sulfide obtains also for the dioxetane
In this context it was of interest to explore whether other
cyclic peroxides, e.g. dimethylmalonyl peroxide 3, would
exhibit such differentiated chemical behavior towards
thiols and sulfides. The results of the reactions of 3 with
glutathione (GSH) and with dimethyl sulfide are shown
in Scheme 1. The former gave quantitatively glutathione
dimer (GSSG) and malonic acid, a reduction process identical to that of GSH with 1. With dimethyl sulfide, on the
other hand, the major reaction (ca. 62%) was oxygen transfer leading to malonic anhydride"] and dimethyl sulfoxide.
0
1.
In view of these findings, we propose that the reaction
of dioxetanes with divalent sulfur compounds (thiols and
sulfides) are induced by electron transfer, as outlined in
Scheme 2. With the help of such electron transfer, one can
rationalize in a unified manner all three reaction types observed for thiols and sulfides, i.e. reduction for thiols
(X=H, route A), oxygen transfer for sulfides (X=R", route
B), and catalyzed cleavage for both (X=H, R", route C).
Electron transfer involving dioxetanes constitutes the
basis of the CIEEL mechanism[91and also their dark catalytic decomposition by amines"'' is interpreted to proceed
via radical ion pairs. In view of the relatively low oxidation potentials of divalent sulfur compounds,"'] such electron transfer with peroxide oxidants is likely. Although
radical cations of sulfides[81and thiols[12' are well documented, the novel feature in the present report is that such
reactive intermediates can be generated in redox reactions
with peroxides. Work is in progress employing polycyclic
aromatic sulfur compounds as one-electron donors, whose
radical cation chemistry is well established, e.g. chloroprom a ~ i n e . " ~Preliminary
]
results with phenothiazine afford
indeed its radical cation on treatment['41with dioxetane 1
or malonyl peroxide 3 . Significant and relevant for our
purposes is that it evidently also takes place on treatment
of dioxetanes with thiols such as glutathione. That hydrogen abstraction from the thiol may be involved, generating thiyl radicals directly rather than via electron transfer
(Scheme 2), seems to be unlikely, as substantiated by the
finding that trimethylsilyl derivatives of thiols also reduce
dioxetanes affording disulfides. We concede that mechanisms other than electron transfer (Scheme 2) can be proposed to rationalize the present observations and that
4
Scheme I. R=CH,
A mechanistically most revealing minor process (ca. 38%)
gave rise to the insertion product 4. The latter is indicative
of electron transfer chemistry, as observed in the cytochrome P-450-catalyzed dealkylation of sulfides via a-hydroxylation of the intermediary sulfur-centered radical
cation.[81The fortunate happenstance of the present case,
the first example of this type of electron transfer for a
peroxide-sulfide reaction, is that the acetal moiety
(RO-CH,-SR)
survives work-up. Preliminary experiments indicate that such an insertion product as structure
4 is formed also with dioxetane 1, when allowed to react
with thioanisole.
That product 4 is of free radical origin is immediately
clear from preliminary C I D N P experiments, in that 'HNMR monitoring of the malonyl peroxide-sulfide reaction
R R'
I
1
R-C-C-R'
+
X-S-R"
0
it
R"
R R'
I t
'
R-C-C-R'
I
I
R R'
I
/s\
R
I
R-C-C-R'
G
\/
L
0
+
R'
0 00
I
I II
R-C-C-R'
X
=
I
R O
X=H
H. R"
C
A
R R'
I I
+
R-C-C-R'
I
L
i
k
I
OS-R"
@O OH
L
X-S-R"
R R'
I
t
I
I
R -C-C
1
H-S-R"
R"S-SR"
- R'
HO OH
430
0 VCH Verlagsgesellschafr mbH. 0-6540 Weinheim, 1588
0570-0833/88/0303-0430 $ 02.50/0
Scheme 2. Proposed mechanism for the
reaction of dioxetanes with thiols and sultides.
Angew. Chem. Ini. Ed. Engl. 27 (1588) No. 3
more work will be necessary to substantiate the novel redox process postulated here.
lrrespective of this mechanistic query, the fact that glutathione and other biological thiols such as cysteine and
penicillamine (Table 1) quantitatively reduce dioxetanes to
their vicinal diols provides an effective means for the detoxification of such genotoxic agents in the cell. Indeed, in
this context it is important to mention that preliminary
studies have revealed that addition of glutathione diminishes the mutagenic activity of dioxetane 1 in the Ames
test. Furthermore, buthionine sulfoximine (an inhibitor of
glutathione synthesis) increases the cytotoxicity of dioxetane 1 in studies with HL60 (promyelocytic leukemia)
cells and S H E (Syrian hamster embryo) cells. Future studies plan using glutathione deficient bacterial strains and
cell lines to enhance the photogenotoxity of 1,2-dioxetanes.
generated and even isolated.“] Thus, bis(2,4,6-tri-tert-butylpheny1)diselane l a and apparently also bis(2,4,6-trimethylpheny1)diselane l b give dismutation equilibria with iodine:”] in solution the aryl(iodine) selenides 2a and 2b,
respectively, as well as the starting diselanes l a and l b
can be detected by ’H- and 77Se-NMR spectroscopy;[’]
crystalline iodine(2,4,6-tri-tert-butylphenyl) selenide 2a
consists of monomeric molecules with selenium-iodine
single bonding.’’]
R
la, R
R
,R
Za, R = t -C4H,
Zb, R = CH,
= t-C,H,
l b , R = CH,
Received: October 23, 1987 [Z 2487 IE]
German version: Angew. Chem. 100 (1988) 443
[I] a ) W. Adam, A. Beinhauer, B. Epe, R. Fuchs, A. Griesbeck, H. Hauer, P.
Miitzel, L. Nassi, D. Schiffmann, D. Wild in T. Friedberg, F. Oesch
(Eds.)’ Prrmarv Changes and Control Factors in Carcinogenesu,
Deutscher Fachschriften-Verlag, Wiesbaden 1986, pp. 64-67; b) J. W.
Lown, R. R. KOgdnty, K. R. Kopecky, Photobiochem. Photobiophys. I2
(1986) 295; c ) L Nassi, B. Epe, D. Schiffmann, W. Adam, A. Beinhauer,
A. Grieabeck, Carcrnogenesis (London) 8 (1987) 947; d) L. Nassi, D.
Schiffmann, A. Favre, W. Adam, R. Fuchs, Mutar. Res., in press.
121 W. Adam. G. Cilento, Angew. Chem. 95 (1983) 525; Angew. Chem. Int.
Ed Enyl 22 (1983) 529.
[3] H. Sies, Angew. Chem. 98 (1986) 1061; Angew. Chem. Int. Ed. Engl. 25
(1986) 1058.
[4] C. Berse, R. Boucher, L. Piche, Can. J. Chem. 37 (1959) 1733.
[5] H. H. Wasserman, E. Saito, J . Am. Chem. Sac. 97 (1975) 905.
161 B. S. Campbell, D. B. Denney, D. Z. Denney, L. S. Shih, J. Am. Chem.
Sac. 97 (1975) 3850.
17) a) f.L. Perrin, T. Arrhenius, J. Am. Chem Soc. 100 (1978) 5249; b) W.
Adam, J. W. Diehl, J . Chem. SOC.Chem. Commun. (1972) 797.
[XI Y. Watanabe, T. Numata, T. Iyanagi, S. Oae, Bull. Chem. SOC.Jpn. 54
(1981) 1163.
[9] G. B. Schuster, Acc. Chem. Res. 12 (1979) 366.
[lo] T. Wilson, D. Chia-Sen in M. J. Cormier, D. M. Hercules, J. Lee (Eds.):
Chemilrrmmescence and Bioluminescence. Plenum, New York 1973, pp.
265-283.
[ I 11 H. Lindley. Biochem. J. 82 (1962) 418.
[I21 a) R. L. Petersen, D. J. Nelsen, M. C. R. Symons, J. Chem Sac. Perkin
Trans 2 1978, 225; b) V. T. DSouza, R. Nanjudiah, J. Baeza, H. H.
Szmant, J . Org. Chem. 52 (1987) 1729.
[I31 G R. Buettner, A. G. Motten, R. D. Hall, C. F. Chignell, Photochem.
Photobiol. 44 (1986) 5.
[I41 C Bodea. 1. Silberg, Adu. Heterocycl. Chem. 9 (1968) 321.
The Reagent Diphenyldiselane/Iodine:
No Phenylselenenyl Iodide but a
Charge Transfer Complex with Cyclic Moieties**
By Silvia Kubiniok. Wov- Walther du Mont,*
Siegfried Pohl,* and Wolfgang Saak
The elements sulfur and selenium d o not react with elemental iodine to give stable molecular compounds containing chalcogen-iodine bonds. Only recently has it been
possible to demonstrate, in special cases, that-by cleavage of selenium-selenium bonds with iodine-uncharged
molecules with covalent selenium-iodine bonds can be
[‘I Prof. Dr W.-W. du Mont, Prof. Dr. S. Pohl,
DipLChem. S. Kubiniok, DipLChem. W. Saak
Fachbereich Chemie der Universitat
Carl~von-Ossietzky-Strasse9- I I, D-2900 Oldenburg (FRG)
[**I Properties of Chalcogen-Chalcogen Bonds, Part 9. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der
Chemiachen 1ndustrie.-Part 8: (11.
Angew Chem. Inr. Ed. Engl. 2711988J No. 3
The reagent diphenyldiselane I c / i ~ d i n e , ’ ~ which
’
Toshimitsu, Uemura, and Okano recommend for specific CC
coupling reactions, has been available commercially under
the name “phenyl selenenyl iodide”l4] [alternative to iodine(pheny1) selenide] for some time. Indeed chemical reactions with the “Toshimitsu reagent” suggest the formation
of a reactive species C6HSSeI 2c ;Is1
thus, e.g., it was possible to carry out the iodoselenation of I-hexyne with diphenyldiselane l c l i o d i n e in the same way as with the product
of the reaction of phenylselenenyl chloride with iodotrimethylsilane.[61
c.
lc
[“2c“]
[Zc]
+
H-E-R
-
HMseC6H
I
R
13C- and ”Se-NMR spectra of the solution obtained
upon reaction of l c with iodine, however, provided no reliable indication of the formation of molecular species
2c.I7] On addition of half an equivalent of iodine to l c in
chloroform, changes in the NMR shifts were observed but
no separate signals could be resolved for the reaction
product. When diselane l c is treated with an excess of iodine further shift changes occur compared to the I3C-NMR
quoted earlier for the Toshimitsu reagent.l7] These
findings leave it undecided whether a dismutation equilibrium between I d i o d i n e and 2c exists in solution which
is rapidly established on the NMR time scale, or whether a
(kinetically labile) charge transfer adduct is formed from
the diselane l c with iodine.
The product of the reaction of l c with iodine can be
prepared just as easily as the aryl(iodine) selenide 2a. Mixing of the reactants in petroleum ether 40/60 and crystallization at - 14°C afford reddish-black shiny crystals, which
after brief drying have the correct analytical composition.[’] If the product is left for a prolonged time (2-4 h)
under reduced pressure, it gradually loses some iodine:
this might explain why the compound was previously obtained only “almost pure” or “98%”.L4-61
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057U-0833/88/03U3-0431$ 02.50/0
43 1
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