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Chemistry and Biochemistry of -Lipoic Acid.

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Chemistry and Biochemistry of a-Lipoic Acid
BY PROF. DR. ULRICH SCHMIDT AND DR. P. GRAFEN
CHEMISCHES LABORATORIUM, UNIVERSITAT FREIBURG (GERMANY)
AND
PR1V.-DOZ. DR. H.W. GOEDDE
A3T. BIOCHEMISCHE GENETIK, INSTITUT
UNIVERSITAT FREIBURG (GERMANY)
FUR HUMANGENETIK UND ANTHROPOLOGIE,
The principal physiological function of Iipoic acid appears to 02 its action as coenzyme in
the oxidative decarboxylation of a-0x0 acids. In order to find out whether Iipoic acid is
essential to higher organisms, structurally modified lipoic acids were prepared as potential
antagonists. Only 4-oxalipoamide promotes the growth of S. faecalis 8043; the other
substances tested are inactive or else inhibit growth. In experiments to determine the
substrate specificity of lipoamide oxidoreductase and its inhibition by structurally modified
lipoic acids, no strong inhibitors were discovered. - Ring strain, spectra, and ring cleavage
of compounds containing the 1,2-dithiolane ring are discussed in relation to the biological
function and the reactivity of Iipoic acid.
A. Introduction
Lipoic acid (1) first became known as a growth factor
for many bacteria and protozoa under the name of
acetate-replacing factor or protogen-A [l -71. It occurs
widely in plants and animals [7al, and acts as a coenzyme
in many enzyme-catarysed reactions, particularIy in
oxidative decarboxylations. Several reviews on the
chemistry and biochemistry of lipoic acid have been
published [8-lo]. The present paper gives a detailed
survey of the chemical properties and enzymatic
function of lipoic acid. The still controversial effect of
lipoic acid on photosynthesis and pharniacological
reactions is only briefly mentioned.
Table 1 shows the enzymes involved in lipoic acid-dependent reactions. The cc-0x0-acid oxidases (or dehydrogenases) mentioned, e. g . pyruvate oxidase and
a-oxoglutarate dehydrogenase, are multienzyme complexes.
111 B. M. Guirard, E. E. Snell, and R. J. Williams, Arch. Biochem. Biophysics 9, 381 (1946).
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(31 L. G . Coho and V. Babb, J. biol. Chemistry 174, 405 (1948).
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17al For chromatographic detection, cf. 11421.
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Enzymes. Academic Press, New York 1960, p. 195.
846
Table I. Enzymes and enzyme complexes participating in lipoic
acid-dependent reactions.
Enzyme
Origin
Ref
Pyruvate dehydrogenase
complex
Pig heart muscle
pigeon breast muscle
plants
yeast
E. colt
S.faecalis
M. tuberculosis
Cl. sporogenes
Micrococcus pyogenes
var. aureus
Cl. saccharobufyricum
A.aerogenes
a-Oxoglutarate
dehydrogenase complex
[12,13,15,
21.52-621
[I3 161
[21,27,32,
33,361
[42,431
Pig heart muscle
pigeon breast muscle
E. coli
S. faecalis
Lipoic acid reductasetransacetylase or
-succinylase
S. faecalis
E. coli
Lipoamide
oxidoreductase
Pig heart muscle
1631
t26.B-3 1,
641
~
yeast
E. coli
153-58,
65-77]
I781
[26,28,30,
31,37,73,
79-811
Lipoamidase
Pigeon liver
S. faecalis or yeast
~~
Lipoic acid activating
system
B. coli
S. faecalis
[a] Pyruvate; Ib] a-Oxoglutarate;
acid; [d] a-Lipoic acid.
[c] (-)-6-S-Acetyldihydrolipoic
- -~- ..-
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Angew. Chem. internat. Edit./ Vol. 4 (1965) No. 10
B. The Structures of Enzymes Containing
Lipoic Acid
The most thoroughly investigated enzyme containing
lipoic acid is the pyruvate oxidase of E. coli, a multienzyme complex with a molecular weight of about
4 . 8 106
~ [25,26]. It consists of pyruvate decarboxylase
(2), lipoic acid reductase-transacetylase (3), and lipo[14] R.S.Schweet and K.Cheslock, J. biol. Chemistry 199,749(1952).
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U.S.A. 47, 753 (1961).
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(1961).
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PC 33 (1961).
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chem. SOC.82, 896 (1960).
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235, 1924 (1960).
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J. biol. Chemistry 193, 721 (1951).
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(London) 166, 439 (1950).
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1421 M . I. D o h and I. C. Gunsalus, Federat. Proc. 11,203 (1952).
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113 (1953)
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Maruzen, Tokio 1958, p. 71.
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Yol. 4 (1965)
I No.
10
amide oxidoreductase (4). The molecular weights of
these three enzymes are 1.83 x lO5,1.6x 106, and 1.12x 105
respectively ; lipoic acid reductase-transacetylase probably consists of sub-units with a molecular weight of
26000 each.
___
[55] R. L . Searls, J. M. Peters, and D. R. Sanadi, J . biol. Chemistry 236, 2317 (1961).
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45, 697 (1959).
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(1960).
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PC 32 (1960).
1591 D. R. Sanadi, J. W.Littlefield, and R. M . Bock, J. biol. Chemistry 197, 851 (1952).
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Metabolism.TheJohnsHopkinsPress,Baltimore 1951,Vol. 1,p.370.
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327 (1952).
[63] I. C. Gunsalus in W. D. McElroy and B. Glass: The Mechanism of Enzyme Action. The Johns Hopkins Press, Baltimore 1954.
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SOC.78, 1763 (1956).
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(1961).
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(1960).
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(1962).
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(1962).
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(1959).
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(1960).
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793 (1939).
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[78] E. Cutolo, Arch. Biochem. Biophysics 64, 242 (1956).
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[80]M . Koike and L. J. Reed, J . biol. Chemistry 235, 1931 (1960).
[8 1] M. Koike, P. C. Shah, and L. J. Reed, J. biol. Chemistry 235,
1939 (1960).
[82] G. R. Seaman and M. D. Dell Naschke, J. biol. Chemistry
213, 705 (1955).
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232, 123 (1958).
[84] L. J . Reed, M. Koike, M. E. Levitch, and F. R. Leach, J.bio1.
Chemistry 232, 143 (1958).
[84a] The following abbreviations will be used:
= adenosine diphosphate
ADP
= adenosine triphosphate
ATP
= coenzyme A
CoA
= 2,6-dichlorophenolindophenol
DCP
FAD
= flavinadenine dinucleotide
HETPP = 2-(a-hydroxyethy1)thiamine pyrophosphate
Lip(SH)z = dihydrolipoic acid
Lips2
= lipoic acid
NAD
= nicotinamide adenine dinucleotide
NADH = hydrogenated NAD
NADP = nicotinamide adenine dinucleotide phosphate
= thiamine pyrophosphate
TPP
[85] G. R. Seaman, J . biol. Chemistry 234, 161 (1959).
[86] K. Suzuki and L. J . Reed, J. biol. Chemistry 238,4021 (1963).
[87] L. J . Reed and B. G. De Busk, Federat. Proc. 13,723 (1954).
847
The whole complex appears to contain 16 molecules of decarboxylase, 64 sub-units of lipoic acid reductase-transacetylase, and 8 molecules of lipoamide oxidoreductase. This
opinion has been confirmed by Reed et al. [25,26]by electronmicroscopic studies. According to these authors the complex
has the shape of a disk about 350 8, in diameter and about
250 A in height. The diameters given for the enzyme components (2), (3), and (4) are 75, 40, and 64 A, respectively.
Two superimposed rings, each consisting of 8 molecules of
pyruvate decarboxylase (2) and 4 molecules of lipoamide
oxidoreductase (4 ) , surround a n aggregate of 64 lipoic acid
reductase-transacetylase sub-units (3) arranged in four piles.
A model is shown in Figure 1.
+
Pyruvate
NAOH +H‘
NAO’
i i
TPP
Acetyl-dihydrohpoic acid
Fig. 2. Reactions participating in the oxidation of pyruvate to acetylcoenzyme A by the pyruvate oxidase multienzyme complex.
[*I Although the three enzymes have actually been isolated, the revised
nomenclature of the Enzyme Commission groups together the decarboxylating and the lipoic acid-reducing and -acetylating activities
under the heading pyruvate: lipoate oxidoreductase (acceptor-acetylating), E.C. 1.2.4.1. The corresponding 2-oxoglutarate degrading enzyme
is 2-oxoglutarate: lipoate oxidoreductase (acceptor-acylating) E.C.
1.2.4.2.
Fig. I . Model of the structure of pyruvate dehydrogenase [25.261.
Large spheres: pyruvate dehydrogenase (2) ; Medium spheres: lipoamide oxidoreductase (41 ; Small spheres: subunits of the lipoic acid
reductase-transacetylase (3).
Reprinted from Science 145,930 (1964) with permission of the copyright
owner.
C. Coenzyme Action of Lipoic Acid in Oxidase and
Dehydrogenase Systems
The mode of reaction of these oxidases will be explained
here using the most thoroughly investigated substrate,
i.e. pyruvate, as an example. The coenzymes required
for the overall oxidative decarboxylation quat ti on (a)]
of pyruvate are thiamine pyrophosphate (TPP), Mg2+,
nicotinamide adenine dinucleotide (NAD), lipoic acid
(I), coenzyme A, and flavin adenine dinucleotide
(FAD) [84a].
CH3-CO-CO2H
TPP, Mg*+
+
+ NAD+ + COA-SH lipoic
acid, F A D
CH3-CO-SCoA + NADH + H + + COZ
In yeast cells there are two routes for the conversion of
pyruvate into acetyl-coenzyme A. Equation (b) shows a
Pyruvate
+ acetaldehyde
Pyruvate decarboxylase
NAD’ (hlADP)+
-+ acetate
Acetaldehyde dehydrogenase
acetyl-SCo A
ATP, Mg2+, CoA-SH
Thiokinase
+
(b)
[88] H . Holzer, Angew. Chem. 73, 721 (1961).
[89] H . W. Goedde, Int. 2. Vitaminforsch. 33, 18 (1963).
[90] H . W. Goedde, C. Stahlmann, B. Ulrich, and H . Holzcr,
Biochem. Z., in the press.
1911 H. Holzer and K . Beaucamp, Biochim. biophysica Acta 46,
225 (1961).
848
[***I
E.C.
1.6.4.3.
reaction sequence the intermediates of which were identified
by Holzer and Goedde [23]. Cutolo [78] discovered in 1956
that yeast cells contain lipoamide oxidoreductase. I n 1957,
the present authors [23] showed that in yeast, pyruvate can
also be oxidized directly since yeast contains a pyruvate
oxidase system which behaves like pyruvate oxidase from
animals and bacteria [Equation (a)]. Reaction (b) takes place
in the yeast cytoplasm, and Reaction (a) in the mitochondria.
I. Pyruvate Decarboxylase
The synthesis of 2-(~-hydroxyethyl)thiaminepyrophosphate (HETPP) is catalysed by pyruvate dehydrogenase:
(a)
The reaction is catalysed by the enzymes (2), (3), and
( 4 ) described in Section B. It starts with the formation
of “active acetaldehyde”, i. e. 2-(~-hydroxyethyl)thiamine pyrophosphate (HETPP) 188-911, via a thiamine
pyrophosphate (TPP)-pyruvatecompound [91]. Figure 2
shows the coniponent reactions.
TPP, Mgz+
I**] The transacetylating activity with respect to CoA is described by
the Enzyme Commission as acetyl-CoA: dihydrolipoate S-acetyltransferase, E.C. 2.3.1.12.
6%
HETPP
The subsequent reactions leading to acetyl-coenzyme A have
also been elucidated, except for a few details concerning the
action of lipoic acid. [“W]-HETPP obtained by both enzymatic [92] and chemical methods 193,941 has been converted to [14C]-acetyl-CoA [95].
1921 H. Holzer, H . W. Goedde, K. H. Goggel, and B. Ulrich, Biochem. biophysic. Res. Commun. 3, 599 (1960).
[93] H. W. Goedde, B. Ulrich, K. G . Blume, and H . Holzer, Angew. Chem. 73, 772 (1961).
[94] H . Holzer, H . W. Goedde, and B. Ulrich, Biochem.biophysic.
Res. Commun. 5, 447 (1961).
[95] H . W. Goedde, H . Inouye, H . Holzer, Biochim. biophysica
Acta 50, 41 (1961).
Angew. Chem. internat. Edit. / Vol. 4 (1965) / No.I0
11. Lipoic Acid Reductase-Transacetylase
and treating the product with dihydrolipoic acid [Eq. (h)].
This second step is catalysed by lipoic acid transsuccinylase.
This enzyme, originally known as thiol transacetylase,
was first detected in extracts of E. coli by Gunsalus
[40,63,64]. It catalyses the acetylation of lipoic acid ( I )
and the transfer of the acetyl group from acetyldihydrolipoic acid ( l a ) to coenzyme A.
ATP
(1)
-
+ Coenzyme A
~ ~ ( c H , ) , - c o ~ H
( d)
HS
S-COCH,
+ Acetyl-CoA
t TPP
+ Dehydrolipoic acid
(e)
Acetylthiamine pyrophosphate (acetyl-TPP) was postulated
as a n intermediate in Reaction (d). This highly unstable
compound [96-981 would be the first oxidation product of
active acetaldehyde (HETPP). Experiments by Dus et al. [27]
indicated the occurrence of acetyl-TPP in reactions with nonphysiological electron acceptors. Holzer and Goedde 1991
have demonstrated the formation of acetate from pyruvate
in reaction sequences involving TPP, yeast pyruvate decarboxylase, and 2,6-dichlorophenolindophenol. Further experiments [IOO] suggested the intermediate formation of
acetyl-TPP according to Reaction (f), since acetate was
detected and lipoic acid was not involved.
Pyruvate
+
Succinyl-SCoA
ADP
+ P + Succinyl-SCoA
(g)
+ Lip(SH)2
+
CoA-SH
+ Succinyl-S-Lip(SH)
(h)
The S-succinyldihydrolipoic acid is quantitatively determined
as succinylhydroxamic acid obtained by reaction with hydroxylamine (see also [lOS]).
(la)
(la)
+ Succinate + CoA-SH
+ HETPP + acetyl-TPP
--f
acetate
(f)
However, on repeating this experiment with various enzymes
[11,23,51]and acceptors, and starting with [14C]-HETPP
instead of pyruvate, we were unable to detect any intermediate
formation of [14C]-acetyl-TPP [loll.Nothing is known yet
about the occurrence of acetyl-TPP in the oxidation of
pyruvate or of a-oxoglutarate under physiological conditions.
In the reactions described in Fig. 2, (-)-dihydrolipoic
acid appears to be the biologically active form. Reed
[32,84] has shown that lipoic acid reductase-transacetylase can react with bound or free S-acetyldihydrolipoic acid, as does lipoamide oxidoreductase. In contrast, enzyme-bound lipoic acid is essential to Reaction
(a.
a) Removal of Enzyme-Bound Lipoic Acid Using
Lipoamidase, and Reactivation of the Apoenzyme
Lipoic acid reductase-transacetylaseis the only enzyme
of the pyruvate oxidase complex containing proteinbound lipoic acid. The latter can be removed using an
enzyme from pigeon liver [82,106]. According to Reed,
lipoic acid is joined by a peptide linkage to the &-amino
group of one of the lysine residues in the enzyme
protein [33]. Correspondingly, lipoamide and dihydrolipoamide are more effective than lipoic acid in model
reactions [34,107,108]. Reed [33,109] has shown that
N(z)-DL-lipcyI-L-lysine is reduced three times as fast as
DL-lipoamide by NADH and lipoamide oxidoreductase
from E. coli.
The isolation of acetyl and succinyl thioesters of dihydrolipoic acid by Gunsalus [63,102,103] proved that
lipoic acid acts as an acyl acceptor. S-AcetyldihydroIipoic acid Bq. (d)] is formed by a redox process, in
which the oxidation of the aldehyde to the carboxylic
acid is accompanied by reductive cleavage of the 1,Zdithiolane ring of lipoic acid. The dihydrolipoic acid is
acetylated exclusively at the C-6 sulfur atom [63,83].
Two enzymatic reactions are required to link lipoic acid to
the apoenzyme: (a) the ATP-dependent activation of lipoic
acid to form lipoyladenylate, and (b) the transfer of the
lipoyl residue to the apoenzyme. Lipoic acid reductasetransacetylase from which lipoic acid has been removed, can
be reactivated by incubating it with ATP plus lipoic acid (or
synthetic lipoyladenylate) and a lipoic acid-activating system
from S. fuecnlis or E . coli 183,1091.
Sunudi et al. [52,104] and Goldmunn [44] have prepared
lipoic acid reductase-transacetylase from pyruvic oxidase
obtained from M . tuberculosis. Huger et al. [28-301 have
purified the enzyme from E. coli mutants. According to Reed
[84],the enzyme reacts with both enantiomers of lipoamide.
Hager and Kornberg [30] developed a test for lipoic acid
transsuccinylase obtained from E. coli mutants using
succinate thiokinase to prepare succinyl-coenzyme A [Eq.(g)]
b) Type of Linkage and Amino-Acid Sequence at the Site
of the Lipoic Acid-Protein Link
1961 R. Breslow and E. McNeIis, J. Amer. chem. SOC. 82, 2394
(1960).
1971 C.P. Nash, C . W . Olsen, F. G. White, and L. L. Ingraham,
J. Amer. chem. SOC. 83,4106 (1961).
1981 K . Daigo and L. J. Reed, J. Amer. chem. SOC.84,659(1962).
I991 H . Holzer and H . W. Goedde, Biochem. Z . 329, 192 (1957).
[lo01 H. Hoizer and R. M . M . Crawford, Nature (London) 188,
410 (1960).
[loll H. W. Goedde and B. Ulrich, unpublished work.
[lo21 I. C. Gunsalus, I. Vitaminol. (Kyoto) 4 , 52 (1958).
[lo31 I. C. Gunsalus: Abstr. Amer. chem. SOC. 133rd Meeting
1958,3c.
[104,1051 D . R. Sanadi, M . Langley, and F. White, J. biol. Chemistry 234, 183 (1959).
Angew. Chem. internat. Edit. 1 Vol. 4 (1965) 1 No. 10
0x0-acid oxidases containing radioactively labelled
lipoic acid can be prepared in two ways:
(a) By culturing E . coli strains in the presence of [35S]-lipoic
acid.
(b) By allowing lipoamidase t o act o n 0x0-acid oxidases. The
apoenzyme formed is isolated by centrifugation at 144000 g
and is reactivated by incubation with ATP, [35S]-lipoic acid,
and the lipoic acid-activating system.
0x0-acid oxidases containing labelled lipoic acid have
been oxidized with performic acid and hydrolysed for
3 h at 100°C with 12 N HCI. It was possible to isolate
[lo61 G. R. Seaman, J. Amer. chem. SOC.76, 1712 (1954).
11071 V. Massey, Biochim. biophysica Acta 30, 205 (1958).
[lo81 H. W . Goedde, unpublished work.
[lo91 H. Nuwa, W. T. Brady, M. Kaike, and L. J. Reed, J. Amer.
chem. SOC.81,2908 (1959).
849
N(E)-lipoyl-L-lysine (4a) [109al, the hydrolysis of which
gave L-lysine and lipoic acid in the ratio of 1:1 [41,45,83].
This result has been confirmed by Goldman.
Reed treated highly purified enzyme preparations from E. coli
containing [35S]-lipoic acid with performic acid and partially
hydrolysed the product. The [3sS]-peptides of lipoic acid were
isolated and identified. From pyruvate dehydrogenase the
sequence glycine-asparagine-lipoyllysine-alanine was obtained, while in oxoglutarate dehydrogenase the sequence
threonine-asparagine-lipoyllysine-valine-(valine,
leucine)-glutamine [112] was present.
III. Lipoamide Oxidoreductase
This flavin-adenine dinucleotide enzyme catalyses the
reoxidation of dihydrolipoic acid in the presence of
NAD:
Dihydrolipoic acid f NAD'
+ lipoic acid + NADH + Hf
(i)
Lipoamide oxidoreductase has been studied since 1958
[35,53-58,65-73,79,84,104,107,113-1171.
It is identical
with the diaphorase [119-1211 isolated from heart muscle by
Straub 11181 and Savage [74], and can be estimated photoNADH
+ H+ f Acceptor + NAD+ + Acceptor-Hr
ti)
metrically by Reactions (i) or (j). In the latter, hexacyanoferrate(II1) or 2,6-dichlorophenolindophenol (DCP) can act
as hydrogen acceptors.
Unlike lipoic acid reductase-transacetylase, lipoamide
oxidoreductase is not specific for either (+)- or (-)lipoic acid. However, a lipoamide oxidoreductase
obtained by Basu and Burma [122] from Spinacia
oleracea reacts only with (-)-dihydrolipoic acid.
Flavin-adenine dinucleotide (FAD) can dissociate reversibly from the enzyme. The enzyme is decolorized by
[l09a] Biotin, which plays an important part in carboxylation
reactions, (e.g. in fatty acid synthesis) likewise occurs bound to
lysine. Biocytin [N(~)-biotinyl-~-lysine]has been isolated from
yeast autolysates [IlO, 1111.
11101 R. L . Peck, D. E. Wow, and K. Folkers, J. Amer. chem.
SOC.74, 1999 (1952).
11111 L. D. Wright, E. L. Cresson, H. R. Skeggs, T. R. Wood,
R. L. Peck, D . E. W o f , and K . Folkers, J. Amer. chem. SOC.74,
1996 (1952).
[I 121 K. Daigo and L . J. Reed, J. Amer. chem. SOC.84,666 (1962).
[I131 V. Massey, Q . H. Gibson, and C. Veeger, Biochem. J. 77,
341 (1960).
[I141 C . J. Lusty, J. biol. Chemistry 238, 3443 (1963).
11151 H . W . Goedde, P. Grafen, and U. Schmidt, Biochem. Z . 339,
23 (1963).
I1161 H. W. Goedde, C. Stahlmann, P. Grafen, and U. Schmidt,
Arch. Mikrobiol. 45, 359 (1963).
[117] H. W . Goedde and U. Schmidt, unpublished work.
11181 F. B. Straub, Biochem. J. 33, 787 (1939).
[I191 H . v. Euler and H. Hellstrdm, Hoppe-Seylers Z . physiol.
Chem. 252, 31 (1938).
[120] J. G. Dewan and D. E. Green, Nature (London) 140, 1097
(1937).
[I211 J. G. Dewan and D . E. Green, Biochem. J. 32, 626 (1938).
11221 D. K . Basu and D. P. Burma, J. biol. Chemistry 235, 509
(1960).
NADH or dihydrolipoic acid but the color is restored
on addition of lipoic acid. This is taken to indicate that
enzyme-bound dihydrolipoic acid is oxidized by FAD,
and not directly by NAD, as had previously been
assumed [63]. Thus the electron transfer sequence is
probably [37,80]
oxoglutarate
--f
lipoic acid
--f
FAD
+ NAD.
a) Properties of Lipoamide Oxidoreductase
Lipoamide oxidoreductase has been detected in the pyruvate
oxidase and oxoglutarate dehydrogenase complexes of many
organisms (see Table 1) [36,53,57,80,81,123]. Highly purified
preparations of lipoamide oxidoreductase are intensely
yellow, exhibit fluorescence, and have absorption maxima
a t 273, 359, and 456 m p with a shoulder at 480 m p [37].
The highly purified enzyme contains 1.16 % of FAD = 0.56 %
of riboflavin [74,76]. Its fluorescence and the absorption at
456 m p disappear in the presence of excess NADH. The
enzyme is inhibited by p-chloromercuribenzoate, and reactivated by glutathione [123].
p H optima of 5.6 to 5.9 and 6.5 to 7.0 are found for lipoic
acid and DL-lipoamide, respectively [107]. By reduction of
the flavoprotein in various NADH/NAD equilibria Sanadi
found a reduction potential of about -0.34 volt at p H 7.0 and
22OC for lipoamide oxidoreductase from pig heart oxoglutarate dehydrogenase [56,57].
Turnover number: Measurements using Reaction (j) show
that 8500 moles of substrate react per minute with 105 g of
enzyme protein [76] (for data on the reaction of lipoic acid,
see [71,115] and Table 2).
It was originally thought that lipoamide oxidoreductase
resulted from the breakdown of cytochrome reductase.
Massey [7 11 however disproved this by isolating both
enzymes separately from pig heart.
b) The Mode of Action of Lipoamide Oxidoredrrctase
Lipoic acid transacetylase-reductase containing bound lipoic
acid is inhibited by arsenite even in concentrations as low as
10-3-10-5
M [40,52,59,66,125-1281. (For inhibition experiments using other arsenic compounds or heavy metal
cations, see [104,105,128,129]). The inhibition can be
reversed by the addition of dithiols, e . g . 2,3-dimercaptopropanol.
Highly purified lipoamide oxidoreductase, which does
not contain bound lipoic acid, is also inhibited by
11231 Note added in proof: According to Lusty and Singer,
J. biol. Chemistry 239, 3733 (1964), 23% of the lipoamide oxidoreductase contained in heart mitochondria is present in a-oxoglutarate dehydrogenase; 52% are found in pyruvate oxidase,
and the remaining 25% are present either as free enzyme or
linked to an as yet unknown enzyme complex. In liver mitochondria the figures are 6%, 66%, and 28%, respectively. The
fraction denoted "free lipoamide oxidoreductase" is identical in
electrophoretic mobility, substrate specificity, and arsenite inhibition with lipoamide oxidoreductase isolated from pig heart or
beef liver by the usual procedures.
11241 M . M. Weber and N. 0 . Kaplan, Science (Washington) 123,
844 (1956).
11251 D. R. Sanadi, M . Langley, and F. White, Biochim. biophysics Acta 29, 218 (1958).
11261 R. A . Peters, Symposia SOC.exp. Biol. 3, 36 (1949).
[127] R. A. Peters, H. M . Sinclair, and R. H. S. Thompson, Biochem. J. 40, 516 (1946).
[I281 0. K. Reiss, J. biol. Chemistry 233, 789 (1958).
11291 0. K . Reiss and L. HelIermann, J. biol. Chemistry 231, 557
(1958).
Angew. Chem. internat. Edit. / Vol. 4 (1965) / No. 10
arsenite (or Cd2“) but only if it is reduced by simultaneous incubation with NADH [55,72,66,75]. After reduction, 2 moles of SH groups per mole of enzyme can
be detected [55]. Apparently a disulfide bridge between
two cysteine residues is cleaved by NADH. By ultracentrifugation experiments in 6.5 M urea, Mussey [65,
1301 was able to show that on reduction of the enzyme
its molecular weight decreases to about half. This was
taken to indicate that the disulfide bridge cleaved by
NADH links two peptide chains in the native enzyme.
When lipoamide oxidoreductase is inhibited by arsenite,
only Reaction (i) is affected, i.e. the enzyme loses the
ability to catalyse the oxidation of dihydrolipoic acid
(or the reduction of lipoic acid). However, it still catalyses the transfer of hydrogen from NADH to hexacyanoferrate(II1) or 2,6-dichlorophenolindophenol,i.e.
Reaction (j) is not affected by arsenite [55,72]. From
this it follows that NADH first reduces the FAD component of the enzyme (Scheme 1). The FADH formed
then transfers hydrogen to the disulfide bridge of the
enzyme, thus forming two SH groups. Under physiological conditions, the disulfide bridge is reduced by dihydrolipoic acid. FAD accepts the hydrogen from the
SH groups (with reformation of the disulfide bridge)
and transfers it to NAD. Non-physiological hydrogen
acceptors (hexacyanoferrate(II1) or 2,6-dichlorophenolindophenol) accept hydrogen directly from FADH, i. e.
without SH groups being involved. Therefore, catalysis
of Reaction (j) is independent of arsenite.
Lip(SH)2
NADH
NAD
x
IFDH
free radicals in the reduced enzyme. The intramolecular
electron transfer from enzyme dithiol to flavin proceeds via
an intermediate with an absorption peak at about 530 mp.
An intermediate denoted as a “charge-transfer complex” or
as the “red form” was studied by Sanadi et al. and also by
Massey et al. [30,52,55,58,65,66,68,69,72,113]. Its structure
remains unidentified.
D. Studies on Modified Lipoic Acids 11331
No avitaminoses or metabolic alterations associated
with lipoic acid deficiency have so far been recorded.
Whether lipoic acid is an essential factor for higher
organisms can be decided only by the use of lipoic acid
antagonists. A lipoic acid-free diet can hardly be
maintained, since the substance is widely distributed in
foodstuffs and presumably is formed by the intestinal
flora.
Several laboratories [134-1 371 have synthesized lipoic
acid derivatives in which the carboxy group was altered
and the number of ring members was varied. Only one
of the diastereomers of 8-methyllipoic acid exhibited a
detectable inhibitory effect on the growth of bacteria.
Lipsz
We have synthesized compounds resembling lipoic acid
[138,139a, 139b], to investigate whether the distance of
four methyl groups between the carboxy group and the
1,Zdithiolane ring or the length of the (eight-membered)
carbon chain is necessary for the action of lipoic acid.
Accordingly lipoic acids with the CH2 groups replaced
by heteroatoms (S, 0)and, in addition, an acid in which
the ring was displaced toward the carboxy group were
synthesized. Further, hydroxy groups and carbonyl
oxygen were introduced into the ring or side-chain, since
these substituents often give rise to antagonistic effects
in biologically active substances. Table 2 gives a list
of these compounds.
Intermediate
absorbing
[at 5 3 0 m p
The growth of the following microorganisms depends o n
lipoic acid: Tetrahymena pyriformis, Corynebacterium bovis,
E. coli mutants and lactic acid bacilli. For the growth of the
first two organisms lipoic acid is an absolute requirement. In
other bacteria (Lactobacillus casei, L. arabinosis, S. lacris,
S. faecalis R., S. faecalis 8043) lipoic acid reverses the
inhibition of growth caused by substances such as propionate.
In our studies we used S. faecalis 8043 the growth of which
is inhibited by 1-3 mg/ml propionate but which starts to
grow again after the addition of lipoic acid or some of its
analogues (cf. Fig. 3). The rate of growth was determined by
measuring the turbidity of a diluted sample of a culture
with a Zeiss PMQ I1 spectrophotometer at 660 mp [116].
]
1
Hydrogen
acceptor
kD
Scheme 1. The mode of action of lipoamide oxidoreductase 155,721.
[1331 For the chromatographicdetection of lipoic acid, see 11421.
[134] M. W. Bullock, J. J. Hand, and E. L . R. Stokstad, J. Amer.
chem. SOC.79, 1975 (1957).
11351 R . C. Thomas and L. J. Reed, J. Amer. chem. SOC.78, 6150
(1956).
Savage [74] showed that the addition of NADH to lipoamide
oxidoreductase alters the spectrum. The compound formed
is stable and it appears to be analogous to flavin semiquinone
[131J (see also [57,113]). The absorption maximum at 530 mp
of the reduced enzyme disappears on addition of arsenite or
cadmium ions. ESR-spectrometric investigations showed no
[I301 V . Massey, T . Hofmann,and G. Palmer, J. biol. Chemistry
237, 3820 (1962).
[131] H.Beinert, J. Amer. chem. SOC.78, 5323 (1956).
Angew. Chem. internat. Edit.
Vol. 4 (1965)
I No. 10
[I361 E. L . R. Stokstad, Federat. Proc. 13, 712 (1954).
[137] E. L . Patterson, J. V. Pierce, E. L . R. Stokstad, C. E. Hofman, J. A. Brockman j r . , F. P. Day, M. E. Macchi, and T . H.
Jukes, J. Amer. chem. SOC.76, 1823 (1954).
11381 U. Schmidt, H. Alpes, J. C. Loewenguth, P. Crafen, and
H. W.Goedde, Liebigs Ann. Chem. 666, 201 (1963).
[139a] U . Schmidt, P. Crafen, and H . W. Goedde, Liebigs Ann.
Chem. 670, 157 (1963).
1139bl U. Schmidt, J . Malone, and H. W. Goedde, unpublished
work.
85 1
Table 2. Michaelis constants KM and turnover numbers for the reaction of modified lipoic acids with lipoamide oxidoreductase.
__
103 K~
[mole/l]
Modified lipoic acid [a]
H-Z-(CH2)4-COOH
H-Z--(CH~)~-CONHZ
(1)
(5)
2.3
2.8
Turnover
nurnberx 10-3
1831
Ref.
3.15
14.0
HobcHzoH
H- Z-(CHz)1-CO-NH-
Hz
(6)
1.54
-
H3C ‘N
-
-
3.7
6.25
4.0
11.0
4.65
2.4
13.1
2.54
-
-
-
H-Z-CHz-O-(CH2)2-COOH
H-Z-CCH~-O-(CHZ)Z-CONH~
H-Z-CH2-CH(CH3)-(CH2)z-COOH
H-Z-(CHr)z-S-CHz-COOH
H-Z-CO-(CHZ)J-COOH
H-Z-(CHz),-CHF--COOH
CHa-(CH2),-Z--COOH
z=
[a1
(10)
(11)
(12)
(13)
(14)
5.0
(151
3.2
(161
-
H~C-FH-H&.s’S
I. Growth and Inhibition Studies on S . faecalis 8043with
Modified Lipoic Acids [116,138,139a, 139bl
The results of the growth test are shown in Fig. 3 [116].
Replacement of a C H 2 group in the chain between the
carboxy group and the ring by a r S-atom [3-thialipoic
acid (13)] or an 0-atom [4-oxalipoic acid ( l o ) ] leads
to compounds which are either inactive or have a very
weak activating effect on the growth of S. .faecalis 8043.
02 -
t
0 -02-
w
rl
5-OL -
-061
r
10’
Fig. 3. Rate of growth of S. fuccutis 8043 in the presence of modified
lipoic acids 11161.
Abscissa: Concentration of modified lipoic acid in 10-9 mole/l.
Ordinate: Natural logarithm of the change in extinction AE at 660 m s
in a given time relative to a standard solution containg no bacteria.
Thesmoothed curveswere normalized to InE=O at c = 5 x 10-9/6molc/l.
Compounds (7), (12). ( S ) , and (9) inhibit growth, (161. (14)>and (13)
have no effect, while (11) like lipoic acid ( I ) and 4-oxalipoic acid ( l o ) ,
stimulates growth.
Introduction of a methyl group [4-methyllipoic acid
(12)] at the CH2-chain produces an inhibitory effect
[ 139~1D . Sorg, Doctorate Dissertation, Universitat Freiburg
1961; A . Liittringhaus and D . Sorg, Liebigs Ann. Chem., in the
press.
11401 German Pat. Appl. M 57881 IVd/fZp (Aug. 20th, 1963),
E. Merck. inventor: U. Schmidt.
852
0.96
9.87
-
whereas introduction of a keto group [S-oxolipoic acid
(14)] does not. Substitution on the ring carbons leads to
compounds with inhibitory action, e.g. 7-methyllipoic
acid (7), 7-hydroxylipoicacid (8), 7-oxolipoic acid (9),
and also the 8-methyllipoic acid synthesized by Stockstad. Alterations in ring size or in the position of the
disulfide bridge [2,4-lipoic acid (Is)]lead to loss of
activity.
11. Inhibition and Substrate Specificity of Lipoamide
Oxidoreductase [115,138,139a,139bl
Since lipoamide oxidoreductase containing modified
lipoic acids still reacts with free lipoic acid according
to Equation (i), this enzyme is particularly suitable for
the investigation of inhibition and substrate specificity.
Michaelis constants, turnover numbers, and inhibition
constants were determined by the method of Massey
[53,66,71,107] (see Table 2). 7-Methyllipoic acid (7)
and 3-thialipoic acid (13) are the only acids which give
rise to competitive inhibition of the reduction of lipoic
acid. The inhibition constant €or 7-methyllipoic acid
was 2 . 8 ~ 1 0 - 3 ,and that for 3-thialipoic acid 8 . 0 ~ 1 0 - 4 .
The following conclusions may be drawn from the
results :
2,4-Lipoic acid (16) is inert, i.e. it is unable to form an
enzyme-substrate complex. Replacement of the carbon atoms
3 or 4 in Iipoic acid by a hetero-atom leads to complete loss
of activity in 3-thialipoic acid (13) and to reduced affinity in
4-oxalipoic acid (10). The amide (11) of this latter acid is
the only derivative of lipoic acid which has almost the same
activity as lipoamide. 4-Methyl- (12) and 5-oxolipoic acid
(14) have properties similar to those of 4-oxalipoic acid, i. e.
both the reaction rate and enzyme affinity are reduced.
Substitution on C-7 produces varying effects. 7-Methytlipoic
acid (7) is not transformed by lipoamide oxidoreductase but
has a n inhibitory effect, whiIe 7-hydroxylipoic acid (8) is
entirely inactive. 7-Oxolipoic acid (9) is transformed more
rapidly than the physiological substrate, which indicates an
activating effect of the carbonyl group o n the S-S bridge. The
amides of lipoic acid and 4-oxalipoic acids show high turnover
numbers and relatively high Michaelis constants as does
lipoylpyridoxamine ( 6 ) (see also [71,107]). 2-Fluorolipoic
acid (15) [117] exhibits no inhibitory action.
Angew. Chem. internat. Edit. 1 Vol. 4 (1965) I No. 10
E. Further Reactions of Lipoic Acid
The main physiological function of lipoic acid appears
to be its role as coenzyme during the oxidative decarboxylation of tc-0x0 acids. Oxidative splitting of diacetyl
by S. faecalis requires the presence of lipoic acid in
addition to that of TPP and Mg*+ 11411. Acetyldihydrolipoic acid is formed intermediately.
Calvin et al. [143-1501 have discussed the participation
of Iipoic acid in photosynthesis. Studies on green algae
with [35S]-lipoic acid led to the characterization of
several biologically active forms of the acid indicating
its participation in the Hill reaction. However, other
investigations produced conflicting results [151]. Some
evidence for the participation of dihydrolipoic acid in
the Hill reaction is provided by the arsenite sensitivity
of the latter reaction, which is catalysed by chloroplast
preparations from spinach leaves [l52].
F. The Pharmacology of Lipoic Acid
The therapeutic use of lipoic acid in liver diseases has
not so far given concordant results. The findings of a
response of hepatic coma to lipoic acid have not been
confirmed, but a beneficial effect on liver afflictions has
been reported from numerous sources [153-1591. Many
publications have described the protective and curative
effect of lipoic acids in heavy-metal poisoning (As, Pb,
Hg, Se, Ni(CO)4). The effect produced is frequently
superior to that of BAL [159a]. A similar beneficial
.___-
[I411 M . Brenner, A . Niederwieser, and C . Pataki, Experientia
17, 145 (1961).
11421 L . Reio, J. Chromatogr. 4 , 458 (1960).
[I431 M . Culcin, H . Grisrbach, and R . C.Fuller, J. Amer. chem.
SOC.77, 2659 (1955).
[I441 M . Calvin and J. A. Barltrop, J. Amer. chem. SOC.74, 6153
(1952).
[145]D. F. Bradey and M . Calvin, Arch. Biochem. Biophysics
53, 99 (1954).
11461 M . Calvin, Angew. Chem. 68, 263 (1956).
11471 J. A . Barltrop, P. M . Hayes, and M . Calvin, J. Amer. chem.
SOC.76, 4348 (1954).
[I481 M . Calvin, Federat. Proc. 13, 697 (1954).
I1491 D . F. Bradley and M . Calvin, Proc. nat. Acad. Sci. U.S.A.
41, 563 (1955).
11501 H . Grisebach, R . C . Fuller, and M. Calvin, Biochim. biophysics Acta 23, 34 (1957).
[I511 R. Lumry, .
I
.
D . Spikes, and H . Eyring, Annu. Rev. Plant
Physiol. 5, 271 (1954).
[I521 D. I. Arnon, M . B. Allen, and F. R. Whatley, Biochim. biophysics Acta 20,449 (1956).
11531 Pharm. Symp. on Thioctic Acid, Naples 1955;Chem. Abstr. 51, 8153 (1957).
11541 Shutari Nakai, Naika HBkan 6, 1189 (1959); Chem. Abstr. 54, 1 1 274 (1960).
11551 S. Tusaka: Second Japanese Symp. on Thioctic Acid.
Tokyo 1959.
[I561 T. Yoshida: Third Japanese Symp. on Thioctic Acid.
Osaka 1960.
[I571 H. Redetzki, H . Bloedron, and H . W . Buns, Klin. Wschr.
34, 845 (1956).
11581 W. Eger, Arzneimittel-Forsch. 7, 601 (1957).
11591 W . Eger, Arztl. Forsch. 11, 251 (1957).
[ 159al 2,3-Dimercaptopropanol.
Angew. Chem. internaf. Edit.
Vol. 4 (1965) No. I 0
effect has been reported in liver damage induced experimentally by CCl4, ally1 alcohol, and narcotics. The
clinical testing of easily resorbed lipoic acid derivatives,
e.g. /6) [140], which are readily incorporated into
lipoic acid-containing enzymes, appears to be promising
[140] (Lipoic acid: D L ~ O(mouse) 160-275 mg/kg intraperitoneally).
G. Reactivity, Ring Cleavage, and
Ultraviolet Absorption of 1,2-Dithiolanes
The biological activity of lipoic acid depends on the
great reactivity of the disulfide bond of the dithiolane
ring. Earlier attempts to interpret this reactivity of (fivemembered) 1,2-dithiolanes over the (six-membered) 1,2dithianes and open-chain disulfides by the higher strain
in the five-membered ring [160], and a calculation of the
strain from the shift of the ultraviolet absorption towards
longer wavelengths [161] were unsuccessful. The strain
in the dithiolane ring is only 4-6 kcal/mole [162,163].
Also, it is not permissible to use thermodynamic data,
such as the energy content as expressed by the ring
strain, to interpret kinetic factors (reactivity).
Such a relationship is found only in very simple cases,
e.g. in unsubstituted 1,Zdithiolane and 1,Zdithiane.
Here no mesomeric groups are present to influence the
S-S bond and no deformation of the ring through
steric interactions between substituents is possible, so
that bathochromic shift and ring strain are predominantly determined by the interplanar angle. Even so, no
quantitative relationship exists among reactivity, ring
strain, and ultraviolet absorption.
I. Ring Strain in 1,2-Dithiolanes
The angle 9 (Fig. 4) which the two R-S bonds form
in strain-free open-chain disulfides is about 90 [164].
Under these conditions the repulsion between the lone
p,-electron pairs of the sulfur atoms is at a minimum,
and an interchange of these electrons with the free d-
Fig. 4. Interplanar angles rp in aliphatic disulfides.
[160] J. G . Affleck and G. Dougherty, J. org. Chemistry 15, 865
(1950).
[161]J. A. Baltrop, P. M . Hayes, and M . Calvin, J. Amer. chem.
SOC.76, 4348 (1954); M . Calvin, Federat. Proc. I S , 697 (1954).
[I621 S.Sunner, Nature (London) 176, 217 (1955).
11631 A . Fava, A . Iliceto, and E. Camera, J. Amer. chem. SOC.79,
833 (1957).
11641 0.Foss in N. Kharasch: Organic Sulfur Compounds. Pergamon Press, London 1961,Vol. I, p. 75.
853
levels of the neighboring sulfur atoms is possible to
give the S - S bond a partial double bond character
(p,-d,
bonding) [165-1671. The energy required for
rotation about the S-S bond, i. e. for a reduction of the
interplanar angle from 90 " to 0 ",was found to be 3-14
kcal/mole [168-1701. Measurements by FehPr on disulfane seem to confirm the lower value of 3 kcal/mole
[168].
I t has not been resolved whether the bond between "normal"
divalent sulfur atoms is formed by a n overlap between pure
p- or sp-hybridized orbitals. The bond length gives no
indication of the nature of the bond since the "normal"
length of the single S-S bond is unknown. In the case of
disulfides with potentially mesomeric groups in the conjugated position with respect to the disulfide bridge ( e . g . in
aromatic disulfides), the electrons of the suffur atoms are
included in the mesomeric system. The homolysis of the S-S
bond in aromatic disulfides depends on the polarity of the
p-substituents on the benzene ring [171]. An increase in the
electron donating character of the substituents leads to a
more ready splitting of the S-S bond in diary1 sulfides by a
radical mechanism.
Incorporation of the disulfide group into a carbocyclic
ring without deformation of the interplanar angle of
about 90 " is possible only in large rings. With decreasing
number of ring members (7 6 5) 'p becomes smaller
and hence the strain increases. For the 1,Zdithiolane
ring 'p is only about 27 ". The double bond character
of the S-S bond (p,-d,
bonding) disappears and
almost maximum repulsion of the p,-electron occurs.
In addition, the deformation and alteration of the bond
angles at the carbon and sulfur atoms have an effect on
the strain of the dithiolane ring.
---f
--f
From a determination of bond lengths and angles by X-ray
analysis [I731 of 1,2-dithiolane-4-carboxylic acid [172] a
value of 16-26 kcal/mole was calculated for the strain energy
[171a]. More recent thermochemical measurements have
indicated a value of only 4 kcal/mole for unsubstituted
dithiolane and a value of 3.5 kcal/mole for lipoic acid [162].
between the ultraviolet absorption of cyclic disulfides
and their interplanar angle 'p. The interplanar angle
diminishes with decreasing size of the ring and the ultraviolet absorption is displaced toward longer wavelengths
with a simultaneous fall in the molar extinction (sixmembered ring: 'p = 60" [172], h,,, = 286 mp: fivemembered ring: 'p = 27" [172], Amax = 330 mp). By
iricorporation into polycyclic systems [1724 the dithiolane ring can be flattened with a further shift of the
absorption peak [1741 towards longer wavelengths. Tetramethylation at C-3 and C-5 has a bathochromic
effect [175,175a].
Polar and potentially mesomeric substituents have a n
appreciable effect o n the UV absorption of the dithiolane
ring. Electron attracting groups are weakly hypsochrornic
[176]. Carbonyl groups in the neighborhood of the sulfur
atoms displace the absorption peaks towards shorter wavelengths. According t o Bergson [177], however, it is not the
absorption of the S-S chromophore which is observed here,
but the absorption of the carbonyl group which is disturbed
by the S atoms (Table 3).
Table 3. Ultraviolet absorption of organic disulfides.
I
Disulfide
Dimethyl disulfide
Di-t-butyl disulfide
1,Z-Dithiolane
3-Aminomethyl-l,2-dithiolane
hydrochloride
3,3,5.5-Tetramethyldithiolane
1,2-Dithiolane-3-carboxylicacid
1,2-Dithiolane-3,5-dicarboxylicacid
3-Acetyl- 1,2-dithiolane
Lipoic acid (I)
7-Methyllipoic acid (7)
7-Hydroxylipoic acid (8)
5-Oxolipoic acid (14)
Amax
[mu]
255
ca. 230
330
322
358
280
250
307
333
350
340
312
In his study of the electronic spectra of aliphatic disulfides,
Bergson [178] considers the S-S chromophore as a n isolated
x-electron system and its excitation as a transition from a
nonbonding x(3px) level to a nonbonding a(a3p) level (Fig.5).
II. Ultraviolet Absorption of 1,2-Dithiolanes
In comparison to the open-chain disulfides, the ultraviolet absorption of 1,Zdithiolanes is strongly displaced
toward longer wavelengths [161]. If there are no substituent effects, a simple relationship can be recognized
[I651 H . Krebs, Z . Naturforsch. 126, 795 (1957).
11661 0. Foss and 0. Tjomsland, Acta chem. scand. 44, 1799
(1958).
[167] G. Cilento, Chem. Reviews 60, 147 (1960).
[I681 F. F e h t and R. Schulze-Rettmer, Z . anorg. allg. Chem. 295,
262 (1958).
11691 D. W. Scott et al., J. Amer. chem. SOC.72, 2424 (1950); 74,
2478 (1952); 76, 1488 (1954); 80, 3547 (1958).
[I701 L. Pauling: The Nature of the Chemical Bond. 2nd edit.
Cornell University Press, Ithaca 1948.
[I711 U. Schmidt and A . Miiller, Liebigs Ann. Chem. 672, 90
(1964).
[171a] In this calculation the rotational barrier for S-S bonds
was assumed to be 12-14 kcal/mole. However, this value appears
much too high.
[I721 0. Foss and L. Schotte, Acta chem. scand. 11, 1424 (1957);
0. Foss and T. Reistad, ibid. I I , 1427 (1957); 0. Foss and
0.Tjomsland, ibid. 12, 1810 (1958).
[172a] G . Bergson, Acta chem. scand. 14, 222 (1960).
[173] G. BergsonandL. Schotte, Actachem.scand. 12,367(1958);
Ark. Kemi 13, 43 (1958).
854
I
w
0
9--
90"
Fig. 5. Dependence of excitation energy AE on the interplanar angle q
in the electronic spectra of aliphatic disulfides [178].
[I741 L. Schotte, Ark. Kemi 9, 309 (1956).
[I751 G. Claeson, Svensk kem. Tidskr. 69, 395 (1957).
[175a] It is not clear whether this effect is produced by the inductive effect of the methyl groups on the 5-S bond or whether it is
a consequence of the flattening of the ring brought about by
steric hindrance between the methyl groups. Disubstitution of
adjacent carbon atoms by alkyl or hydroxy groups can likewise
produce displacement of the absorption peak when the substituents produce flattening of the ring due to steric hindrance.
(7-hydroxylipoic acid: hmax = 340.5 rnp; 7-methyllipoic acid:
Amax = 350 mp).
[I761 G. Bergson, Acta Univ. Upsaliensis 13, 24 (1962).
[I771 G. Bergson and A. L. Delin, Ark. Kemi 18, 489 (1962).
[I781 G. Bergson, Ark. Kemi 12, 233 (1958); 18, 409 (1962).
Angew. Chem. internaf.Edit. / Yol. 4 (1965) 1 No, I 0
Because of the different symmetries of the TC- and a-levels
with respect to the S-S bond axis, the energy of the groundstate but not that of the excited state must depend on the
interplanar angle 9.The result is the angle-dependence of the
excitation energy AE (Fig. 5 ) .
transition state (17) of the nucleophilic opening of a disulfide ring by a thiolate the three sulfur atoms lie in a
straight line, and the two sulfur-sulfur distances are
equal; the valence angle a t the attacked sulfur atom is decreased t o 90 O and the bond to be opened is lengthened.
111. The Reactivity of 1,2-Dithiolanes
The fact that 1,Zdithiolanes are more reactive at the
S-S bond than open-chain disulfides becomes apparent
in radical, electrophilic, and nucleophilic reactions :
Polymerization to linear disulfides is characteristic of
1,2-dithiolanes. Therefore, solutions of unsubstituted
1,Zdithiolane can be kept only for short periods. The
low activation energy (8.6 kcal/mole) [161] and the low
collision rate for the thermal polymerization of 1,2dithiolane in iso-octane point to a free-radical chain
mechanism. Opening of the S-S bond by cyanopropyl
radicals is much faster in dithiolanes than in dithianes,
but the latter still react more rapidly than open-chain
disulfides [171].
Electrophilic persulfate oxidation to thiosulfinates does
not succeed in the case of open-chain disulfides and
(seven-membered) dithiepanes. Dithianes react slowly
and dithiolanes most rapidly [161]. Hence the electrophilic attack of the oxidizing agent on a pair of pzelectrons of one of the S-atoms of the disulfide group
proceeds all the more rapidly (the activation energy
decreases) the less the electrons participate in the lielectron system of the S-S bond. This participation of
the p,-electrons diminishes as the interplanar angle 'p
decreases (i.e. falls in the order open-chain > sevenmembered ring > six-membered ring > five-membered
ring disulfides). In addition a bimolecular reaction on a
relatively rigid ring has lower entropy of activation than
a reaction on a flexible chain. The increase in the rate
of electrophilic attack on the disulfide bond on passing
from open-chain disulfides to the dithiolane ring via
dithiepane and dithiane is thus caused by a decrease in
activation energy a n d activation entropy. Hence the
interplanar angle (ring strain) and the reactivity of the
disulfide group towards electrophilic and radical reagents
alter analogously.
Nucleophilic reactions at the sulfur atoms in cyclic
disulfides have received little attention in the past.
Activation energies for the reaction of 1,Zdithiolane
[13 kcal/mole; Equation (m)] and of dibutyl disulfide
[14.45 kcal/mole; Equation (n)] with butanethiolate
differ only slightly [163] and the difference found may
be within the experimental error (the Arrhenius curve
of Reaction (m) is not linear).
R-S-S-R
+
R-SQ
R-~-s-R
+
R-SO
(n)
R = C,H,
The rate of Reaction (m) is almost lo4 times greater than
that of Reaction (n). This must be caused mainly by a much
lower activation entropy in the case of Reaction (m). In the
Angew. Chem. internat. Edit. 1 Vol. 4 (1965)
No:lO
The ground state of 1,Zdithiolane (valence angle at the S
atom: 92'; S-S bond length: 2.1 A) is therefore much more
similar geometrically to the transition state than is the
ground state of a n open-chain disulfide (valence angle at S:
107 O ; S-S distance: 2.05 A). The transition state is therefore
reached with greater ease from the 1,Zdithiolane ground
state than from that of the open-chain disulfide. The somewhat greater S-S distance in the cyclic disulfide possibly
explains the somewhat lower activation energy of Reaction
(m) [178a].
Molecular models show that the formation of a n analogous
transition state in dithianes is much more difficult. It is
accompanied by a n appreciable deformation of the C-C
valence angles and the elimination of the staggered groundstate arrangement of the CH2 groups. Not surprisingly,
sensitive dithianes are therefore less sensitive towards
nucleophilic cyanide splitting than are dithiolanes and openchain disulfides [179].
H. Recent Lipoic Acid Syntheses [IS01
Numerous routes lead to octanoic acids carrying
halogen, hydroxy, or ether groups on the carbon atoms
6 and 8. Treatment of these with HI/thiourea [18l],with
sodium disulfide [182a, 182b], thiocyanate [182b], thiosulfate [182b], or thioacetate [182b] leads to the formation of lipoic or dihydrolipoic acid. The lengthening
of the carbon chain from heptanoic acids by means of
the Prins reaction [183] or by acylation of ketones and
enamines, e.g. by reaction of cyclopentenylpyrrolidine
[178a] R. E. Davis, J. Amer. chem. SOC.85, 3050 (1963), has
pointed out the general relationship between the S - S bond
length (which varies between 1.97 and 2.2 A) and the activation
energy of the nucleophilic S - S bond opening. For many compounds the activation energy for the cyanide splitting is given
by Ea = 99.9/r3 [kcal/mole] and for the sulfite splitting by
Ea = 1 lO/r3 [kcal/mole].
[179] A . Schoberl and H. Gruifie, Liebigs Ann. Chem. 614, 66
(1958).
11801 Synthesis of optically active lipoic acid: K. Folkers et al.,
J. Amer. chem. SOC.77, 5144 (1955); [182a]. Synthesis of lipoamide, lipoyl chloride, lipol: A . F. Wagner, E. Walton, G. E.
Boxer, M . P. Pruss, F. W . Holly, and K. Folkers, J. Amer. chem.
SOC.78, 5079 (1956); T. Kishi, J. pharmac. SOC.Japan (Yakugakuzasshi) 81, 787 (1961); Chem. Abstr. 55, 247183. (1961). Acyldihydrolipoic acids: J. Nakano, J. pharmac. SOC.Japan (Yakugakuzasshi) 76, 943, 1207 (1956)l.
11811 See 11851; U.Schmidt and P . Grafen, Chem. Ber. 92, 1177
(1959); A . Segre, R . Viterbo, and G. Parisi, J. Amer. chem. SOC.
79, 3503 (1957).
[182a] D . S. Acker and W . J . Wayne, J. Amer. chem. SOC.79,
6483 (1957).
[182b] S. Yurugi, T. Fushini, and M . Murata, J. pharmac. SOC.
Japan (Yakugakuzasshi) 80, 1686 (1960); Chem. Abstr. 55, 12287
(1961).
11831 E. A . Braude, R. P . Linstead, and K . R. H. Woolridge,^
J. chem. SOC.(London) 1956, 3074; K. Hagele, Doctorate Dissertation, Universitat Freiburg 1956; German Published Pat. Appl.
1046016 (Oct. 13th, 1956), Merck, inventors: A . Liittriizghaus and
K . Hagele.
855
with P-butoxypropionyl chloride [184], cannot compete
on a technical scale with the synthesis by addition of
ethyl adipate chloride to ethylene to form the S-chloro6-oxooctanoate [185]. After elimination of HC1 to
form 6-oxo-7-octenoicacid,dihydrolipoic acid is obtained
directly by treatment with HzS/H2 in the presence of
MoS, under vigorous conditions [186]. However, it
appears to be difficult to perform this reaction on a
large scale. Reduction of the 8-chloro-6-0x0-octanoate
with NaBH4 to form the 6-hydroxy compound proceeds
particularly readily. The latter is converted into lipoic
acid via 6,8-dichloro-octanoic acid [187]. Addition of
alcohol or carboxylic acids to 6-0x0-7-octenoic acid
and catalytic hydrogenation to form ethers or esters
of 6,8-dihydroxyoctanoic acid, which can be readily
transformed into lipoic acid, have been described [188].
Advantages for the large-scalemanufacture are provided
by the addition of ethyl adipate chloride to acetylene
[I841 S. Yurugi, T. Fushini, and M . Murata, J. pharmac. SOC.
Japan (Yakugakuzasshi) 80, 1165 (1960); Chem. Abstr. 55, 4503
(1961).
[1851 M. W. BuIIock, J. A. Brockman, E. L. Patterson, J. V.
Pierce, and E. L. R. Stockstad, J . Amer. chem. SOC.74, 3455
(1952); M . W. Bullock, J . A. Brockman, E. L. Patterson, J . V .
Pierce, M. H . v. Saltza, F. Sanders, and E. L. R. Stockstad, ihid.
76, 1828 (1954).
[I861 M. W. Bullock, J. H. Hand, and E. L. R. Stockstud,
J. Amer. chem. SOC.79, 1978 (1957).
[187] cf. [183]; L. J. Reed and Ching-J Niu, J. Amer. chem.
SOC.77, 416 (1955).
[188] S. Yurugi, M. Murata, and T. Fushini, J. pharmac. SOC.
Japan (Yakugakuzasshi) 81, 299 (1961); 80, 1317, 1686 (1960);
Chem. Abstr. 55, 14302, 5335, 12287 (1961).
and treatment of the reaction product with alcohol to
form 8,8-dialkoxy-6-oxo-octanoic
acid. This compound
is then hydrogenated to form 8-alkoxy-6-hydroxyoctanoic acid, whose esters or lactones readily yield
dihydrolipoic acid on treatment with HI/thiourea [189].
- S-S -CH2-CH2-yH-S-
S-
n2won
HOS - C HZ-CHZ-FH-SH
R
(18)
*
R
H&-qH-F
H2C,S/S
(1)
Almost all these syntheses proceed via dihydrolipoic
acid. This compound can be oxidized to form lipoic
acid with 12 or FeC13, or better catalytically with air/
FeC13 [185]. Varying amounts of polymeric disulfides
are obtained as by-products, and, provided the molecular weight is not too high, can be thermally depolymerized in vacuo. Suprisingty, aqueous solutions of the
alkali metal salts of polymeric lipoic acids are depolymerized by small quantities of alkali [190]. Possibly
this reaction proceeds via a mercaptosulfenic acid (18)
to lipoic acid.
Received: November 27th, 1964 [A 4491243 IEI
German version: Angew. Chem. 77,900 (1965)
Translated by Express Translation Service, London
11891 U. Schmidt and P. Grafen, Chem. Ber. 92, 1177 (1959);
German Patent 1134370 (April 4th, 1958), E. Merck, inventor:
U.Schmidt; Y. Deguchi, J . pharmac. SOC.Japan (Yakugakuzasshi) 80,933 (1960); D. S. Acker, J. org. Chemistry 28,2533 (1963).
[190] R. C. Thomas and L. J. Reed, J. Amer. chem. SOC.78,6148
(1956).
The Use of Electron Paramagnetic Resonance in Organic Chemistry
BY DR. F. SCHNEIDER, D1PL.-PHYS. K. MOBIUS, AND DIPL.-PHYS. M. PLAT0
AEG-FORSCHUNGSINSTITUT, BERLIN-REINICKENDORF
The more recent applications of’electron paramagnetic resonance (EPR) to organic chemistry include the investigation of triplet states, donor-acceptor complexes, short-lived freeradical intermediates, and reaction kinetics. The principles of the theoretical calculations
required to obtain the maximum information from EPR spectra are outlined for free
radicah containing x-electron systems. EPR intensities, line widths, hyperfine splitting,
and g factors permit the determination of free-radical yields, ionic charges, rotafion jrequencies in rotamers, electron density distributions, electronegativities, intramolecular
torsion angles, solvent efects (resulting from hydrogen bonding), and x-orbital energies. The production of free radicals with the aid of initiation reactions in flow systems, by
irradiation, and by electrolysis seems particularly promising.
I. Introduction
Absorption spectroscopy has always enjoyed an imPortant Position among the Physical methods used in
analytical chemistry. In addition to the classical ultraviolet and infrared absorption spectroscopy, electron
paramagnetic
(EPR) and nuclear magnetic
resonance (NMR) spectroscopy have also become im-
8 56
portant in recent years. However, electron paramagnetic
resonance (EPK) spectroscopy [*I has not yet become SO
widely used as infrared and nuclear magnetic resonance
spectroscopy. There are two reasons for this: infrared
and nuclear magnetic resonance spectroscopy are more
applicable, and the spectra can be identified by
[+I The frequently used abbreviation ESR (electron-spin resonance) is less general than EPR, since the former does not
take into account orbital magnetism.
Angew. Chem. internut. Edit.
1 Vol. 4 (1965)
No. 10
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