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New Developments in the Field of Vitamin B12 Enzymatic Reactions Dependent upon Corrins and Coenzyme B12.

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New Developments in the Field of Vitamin B I Z :Enzymatic
Reactions Dependent upon Corrins and Coenzyme B12
By G. N. Schrauzer[*]
Simple corrins such as vitamin B I Z and vitamin B12 coenzyme catalyze a variety of unusual
enzymatic reactions of which some are still without analogy in organic or organometallic
chemistry. The mechanisms of these reactions are currently the subject of lively discussion.
The present review focuses attention on new ideas about the mode of action of vitamin
B l 2 coenzymes in enzymatic reactions.
1. Introduction
1.1. Outline
The first article of the present series described some of
the more important reactions of the cobalt atom in corrins
and in vitamin Blz model compounds[’]. In this article we
shall discuss corrin-dependent enzymatic reactions with particular emphasis on questions concerning the mechanism of
action ofcorrinoid coenzymes. Details dealing with the biologi-
The known corrin-dependent enzymes may be placed in
two major groups. The first group comprises enzymes in which
simple corrins, e.g. vitamin Blz itself, are utilized as cofactors.
These are shown in Table 1. Enzymes which require vitamin
B12 coenzymes such as 5’-deoxyadenosylcobalamin as cofactors fall into the second group (see Table 2). It is therefore
Table 1. Enzymes dependent on simple corrins [4].
Enzyme
Source(s)
Reaction catalyzed [a]
Methane synthetase
e. g. Merhanosarcina barker!,
Mrrf~‘riii)hucrrrirrmM . o. H .
c,
Methylarsane synthetase
Methanobacterium M . 0.H .
C,
Acetate synthetase
Clostridium rhermoaceticum
C, + C 0 2
Methionine synthetase
Escherichia coli
-
Cofactors
Reductant (H2)
(ATP, H2, “coenzyme M”)
CII,
+
A<)*
ATP, H2,“coenzyme M” (?)
AsH,..(CHd.
Reductant
CH,COP
-
Adenosylmethionine
(reducing conditions)
5-CH3-THFA + H o r n o c y s r e i n e
T H F A + Alethmnine
[a]
_C
., denotes one-carbon precursors of the methyl
. group,
. also including compounds which already possess a reactive methyl group, r.g. 5-CH3-THFA
[ = 5-methyltetrahydrofolic acid, see eq. (k) for formula].
cal sources, properties, and isolation of the respective enzymes
will not be given, nor will we mention purely medical aspects
such as vitamin B I Zdeficiency syndromes, physiological transport mechanisms and the like, as these topics are adequately
treated elsewhere”. ’I. Moreover, the complexity of the corrindependent enzymatic reactions necessitated further limitations
of coverage, as it turned out to be impossible to discuss
all enzymatic reactions in full detail and to include historical
developments within the available space. The view can be
taken that proposed mechanisms of enzymatic reactions can
be considered as valid only after they have been simulated,
at least in principle, in suitable model systems under nonenzymatic conditions. Following the discovery of vitamin Blz
model compounds in our laboratory, we as well as other
workers have attempted to elucidate the mechanisms of corrindependent enzymatic reactions along these lines. In spite of
considerable effort, there is still much controversy and uncertainty concerning the mode of action of corrinoid coenzymes.
I believe that this is in part a consequence of insufficient
communication between the workers in this highly interdisciplinary field. Moreover, new results of enzymological studies
continue to appear in the biochemical literature, which in
part necessitate a modification of current mechanistic ideas.
It is therefore important to summarize the present status
of this intriguing area of research.
reasonable to treat the reactions of the enzymes of the two
groups separately.
1.2. Nomenclature
We shall use the same abbreviated nomenclature as outlined
in Ref. [‘I. 5’-Deoxyadenosylcobalamin (1) (Fig. 1) is simply
CH, C H, C ON Hz
Me
CH,CH,CONH,
Adenine
0
FIC
HOCH,
I\
[*] Prof. Dr. G. N. Schrauzer
University of California at San Diego, Revelle College
La Jolla, Calif. 92093 (USA)
Angew. Chem. Int. Ed. Engl. 16, 233-244 ( 1 9 7 7 )
HO
PH
0
(1)
Fig. 1. Structureofcoenzyme B I Z( 1 ) [43,44]. In place of the 5‘-deoxyadenosyl
group, vitamin B12 bears a cyano group at the cobalt atom and vitamin
BI za a hydroxyl group.
233
Table 2. Coenzyme BI2-dependentenzymes [4]
Enzyme
Source(s)
Reaction catalyzed
Dioldehydrase
Aerobacter
aerogenes
Lactobacillus sp.
Clostridium sp.
CH,CH(OH)CH,OH
GIyceroldehydrase
Ethanolamine
deaminase
Ribonucleotide
reductase
MethylmalonylCoA mutase
e. g. Lactobacillus
leickmannii
Propionibacterium skermanii
Sheep liver
-
--
CH,(OH)CH(OH)CH,OH
CH,(NHz)CH,OH
R(SH)z + GTP
-
+ H,O
CH,CH,CHO
CH,(OH)CH,CHO + H,O
CH,CHO
+
NH,
R(S,) + d G T P
+
Mol. Wt.
Cofactors
?
K”(NH?)
188000
520000
Km(NH?)
Km(NH:, Rbm)
70000
H20
Effector(e.g.dGTP)
56 000
HO2CCH(CH3)COCoA 4 IiO,CCH,CH,CO%oA
165000
Glutamate
mutase
u-Methylene
glutarate
mutase
e. g. Clostridium
tetranomorpkum
Ciostridium
barkeri
L-P-lysine
aminomutase
Clostridium
sticklandii
(I)
(11)
ca. 60000 [a] Km(Rbm),
Mg2m(Mn2e).
160000
ATP,pyruvate
D-u-lysine
aminomutase
Clostfidium
sricklandii
(I)
(11)
ca. 60000
160000
Km(Rb”),MgZm(Mn2”).
ATP, pyridoxal phosphate
Ornithine
aminomutase
Clostridium
sticklandii
(I)
ca. 60000
160000
none(?)
17000
ca. 170000
(11)
[a] Twocomponents; protein [I binds coenzyme BI2,
denoted as “coenzyme BIZ”.All other abbreviations are either
readily understood or explained in the text. The typical vitamin
B I Z model compounds are called cobaloximes.
2. Enzymatic Reactions Dependent on Simple Corrins
The enzymes listed in Table 1 occur in the presence of
simple corrins such as e. g. vitamin Blza (hydroxocobalamin).
Certain methane-producing bacteria utilize corrin derivatives
in which bases other than 5,6-dimethylbenzimidazoleare present in the molecule, e.g. “Factor III”,which contains 5-hydroxybenzimidazole, or corrins in which no axial base is present,
e. g. “Factor B” (cobinamide)r5’.The formation of methylarsanes presumably does not require an independent enzyme
and may be considered as a by-product of methanogenesis
in the presence of added arsenic.
2.1. Methane Biosynthesis
A number of bacterial species produce methane as the terminal product of the metabolism of cellulose, sugars, fatty
acids, alcohols, formaldehyde, serine,but also from COz.These
organisms are as a rule difficult to cultivate. Among the bestknown are Methanosarcina barkeri and Methanobacterium M .
0. H . Other methane-producing bacteria that may be mentioned in this context are those of the genus Methanococcus
as well as Methanobacterium ruminantium, Methanospirillium,
and Methanobacterium formicium. To illustrate the difficulties
of isolation and cultivation of some of these organisms we
quote the example of Methanococcus vanneillii, which was
isolated from San Francisco Bay sludge but subsequently
became lost. A deep-frozen tube of cells, stored for over 12
years was still available, however, from which Stadtman was
able to obtain viable cells for renewed c~ltivation[~?
Considerable amounts of methane are produced from sewage sludge, a source of combustible gas that could in future
234
become cohmercially interesting. Prior to World-War 11,
2 x lo6 cubic meters of methane were collected per year in
the disposal plant of Stuttgart, Germany. This amount was
sufficient to provide fuel for 110 municipal motor vehicles[6!
Methane is also formed in the rumen of cows and other
animals. A fully grown cow may generate as much as 200
liters of methane per day[51.This is considered to be an undesirable side-reaction during the digestive process as it lowers
the feed-efficiency. Methane is produced in the human intestine
too, in amounts which depend on the type and quantity of
food ingested.
2.2. Methane-Biosynthesis in Viituo
Methane bacteria can be cultivated in media containing
ammonia as the source of nitrogen; B-vitamins as well as
rumen fluid are added to promote growth. COz, methanol,
ethanol, formate, or acetate can be employed as sources of
C1,while hydrogen is commonly used as the reducing agent.
The pure methane synthetases have not yet been isolated;
they appear to be extremely sensitive, and most of the available
information on methanogenesis in uitro was obtained from
experiments with intact cells or cell-free extracts[51.A variety
of compounds contain reactive methyl groups or precursors
of the methyl group for methane production. These include:
serine, pyruvate, 5-methyltetrahydrofolicacid (5-CH3-THFA),
5,1O-methylene-tetrahydrofolicacid, methylcobalamin, other
co-methylated corrins and even methyl-derivatives of vitamin
B I Z model corn pound^^'^. Extracts from Methanobacterium
M . 0.H . require catalytic amounts of ATP for methane production. Additional cofactors are presumably also necessary; most
interestingly, 2-rnercaptoethylsulfonate, HS-CH2CH2SO;,
has been found to be one such factor. This compound was
shown by Wore et a[.[’]to possess the properties of a coenzyme
and was for this reason named “coenzyme M”. In the absence
of ATP, this compound is S-methylated by methylcobalamin;
the product is reductively demethylated by a different enzyme
Angew. Ckem. Int. Ed. Engl. 16, 233-244 ( 1 9 7 7 )
to yield methane. The reductive demethylation, which regenerates coenzyme M, is inhibited by tripolyphosphate[8]. It is
not yet known if methane is formed exclusively via methylated
coenzyme M or whether the S-methylation-demethylationis
not a side-reaction. On the other hand, there can be no doubt
that methylated corrins are formed in the terminal stages
of methanogenesis. A corrin-containing protein present in
cell-extracts of Methanobacterium ornelianskii, which is actually
FH3
(yo)
fl
7H3
RSH
7
($0)
R'
YH3
Ha
(CH3')+CH4
($0)
s
so
+@
H
'
(a)
sR
(C0)
R'
A mechanism of enzymatic methane production was formulated along these lines and is schematically represented in
Scheme 2[14]. At the time of this proposal, little was known
about coenzyme M. Since coenzyme M is a thiol, we expected
3;;>
[$o"']- e n z y m e
Biogenic methyl
donor
-CH:
Biogenic reductant
[CH,O]
2e0
Her
CH,
I c o '1 - e n z y m e
Scheme 2. Proposed mechanism of methane biosynthesis.
a symbiotic association of Methanobacterium M . 0. H . with
a hydrogen-producing organismrg1,can be converted into a
propylated derivative on reaction with propyl iodide. This
causes inhibition of methane formation, but exposure of the
protein to light restores activity. It thus may be assumed that
the enzyme-bound corrin is converted into the n-propyl-derivative and that the Co-C bond in the latter is cleaved on
irradiation with light. Methanogenesis is also inhibited by
a variety of halogenated hydrocarbons, including freon and
DDT" O- *I. Chloromethylcobalamins act as competitive inhibitors of methane formation. How methane is produced from
the methylated corrin cofactor does not follow clearly from
any of these experiments. It was assumed for some time that
the reaction occurs via free methyl radicals['31, but such a
mechanism must now be considered as unlikely.
it to react with methylcobalamin according to eq. (a). This
has since been confirmed['51, but we cannot say, of course,
whether coenzyme M reacts in this manner under enzymatic
conditions as well.
L
J
The S-methylation of coenzyme M, which has been observed
under enzymatic conditions, can also be formulated, as methylcobalt complexes react with thiolate ions according to eq.
(b)"].
23. Mechanism of Methane Synthesis
Scheme 1 surveys the most important reactions which occur
in cell extracts of methane-producing bacteria. A fully satisfying mechanism of methanogenesis cannot be formulated on
the basis of the available enzymological evidence. We considered the possibility that methane is formed from the methylated corrin through the reductive cleavage of the Co-C
Substrates
I
C,- R e d u c t i o n
I
Active
methyl
I*
'1
,
Eq. (b) could thus be considered as an acceptable model
of the enzymatic demethylation of methylcobalamin by coenzyme M.
Methanogenesis
1
c o e n z y m e n/I e t c .
Cell extract
R e d u c t a n t : H,
Scheme 1. Enzymatic methane synthesis
bond" 41. This reaction occurs readily under nonenzymatic
conditions; thiols and dithiols are some of the reductants
that can be used for this purpose (see [ll). The reaction occurs
as shown in eq. (a), it thus involves an axial attack of cobalt
by the thiol:
Anyew. Chem. Int. Ed. Engl. 16, 233-244 ( 1 9 7 7 )
The fact that methylcobaloximes are demethylated to yield
methane in functional cell-extracts of Methanobacterium M .
o. H . is of considerable interest. Methane is produced under
these conditions only if catalytic amounts of a corrin and
ATP are added"'. This suggested a mechanism in which the
235
cobaloxime-bound methyl group is first transferred to the
corrin cofactor, followed by the actual enzymatic demethylation process. A reversible exchange of methyl groups in terms
of eq. (c) occurs readily under nonenzymatic conditions[I6].
However, this interpretation leads to mechanistic difficulties.
Thus, if a transfer of the methyl group were the main
reaction, the rates of methane evolution from different methylcobaloxime substrates should either all be the same or should
at least be related to the relative tendency of methylcobaloximes to undergo transfer of the methyl group according to
eq. (c). However, this is not the case. It was observed instead
that the relative rates of methane production depend on the
nature of the axial bases. Other authors suggested that this
was mainly due to differences in the solubilities of the different
cobaloximes studied" '1, but a more detailed comparison of
the solubilities of the model compounds in polar solvents did
not reveal any signijkant correlation with the rates of methane
observed with intact cells of this organism under conditions
of methanogenesis. The reaction is inhibited by added selenite.
Methylcobalamin acts as the donor of the methyl group["],
and obviously has little if anything in common with the methylation of arsenic by certain fungi, e.g. Penicillium glaucum.
The corrin-dependent formation of methylarsanes must be
closely related with methane biosynthesis, as methane is always
formed as well. Methylcobalamin and methylcobaloximes
methylate arsenic under the conditions of reductive Co-C
bond cleavage['61. The reaction is inhibited by selenite, but
the latter is of course reduced to selenide and ultimately
forms methyl selenide. Ethylcobalamin, on the other hand,
does not give rise to ethylarsane, under neither enzymatic['']
nor nonenzymatic conditions[l61.
With ethylcobaloximes, only traces of ethylarsane are
formed[l6].There thus exist plausible model reactions for
methylarsane biosynthesis as represented in Scheme 3.
4
,"$
B i o g e n i c methyl
donor
-CH:
A@'
[CH3'1
-%CH,As2"
li
I
Scheme 3. Postulated mechanism of enzymatic methylarsane synthesis.
production["]. A statistically significant direct association
( P = 0.05) between the relative rates of enzymatic methane
production and the nonenzymatic reaction according to eq.
(a) was observed using experimental data for 13 different
compounds. The linear correlation coefficient increases to
r = 0.87 ( P< O.Ol), if the calculations are restricted to methylcobalamin and methylcobaloximes (7 data points)['']. These
results suggest that methane may well arise from methylcobalamin after the manner of Scheme 2 and that methylcobaloximes are reductively demethylated under the enzymatic conditions by way of a reaction which occurs with a displacement of
the axial base by a biogenic reductant, possibly a thiol o r
dithiol.
Thus methane biosynthesis is seen to be a relatively complicated process. It at least seems clear why corrins are utilized
for such a comparatively simple biochemical reaction: It is
now well established that the CO(I)supernucleophiles of vitamin B12 (vitamin B12s)are perhaps the most effective scavengers of activated methyl groups in nature. O n the other
hand, corrin-independent mechanisms of methanogenesis may
exist as well, as is suggested by the reductive demethylation
of methylated coenzyme M[19], but these processes are not
yet understood. Further details of methane biosynthesis will
not be discussed, particularly those concerning the reduction
of C 0 2 to active methyl groups. Such reactions are part
of the metabolism of C1-compounds and are corrin-independent processes.
The inhibitory effect of selenide on arsenic methylation
is attributed to a competition between the methylation of
arsenic and a nucleophilic displacement of the cobalt-bound
methyl group in terms of eq. (d).
L
J
The conversion of arsenic to methylarsanes under conditions
of methanogenesis may be regarded as a bacterial detoxification process. The observed methylation of mercuric ion should
also bementioned in this context. Hg2+ ion reacts with methylcobalamin rapidly to produce Hg(CH3)+['l -231, a process
which may have some toxicological ~ignificance[*~!
2 5 . Acetate Biosynthesk
Acetate (or acetic acid) is formed as terminal product of
the fermentation of glucose, cellulose, etc. by Clostridium ther51. The net reactions which occur are summarized
rno~ceticum[~
in eqs. (e) to (g)[261.
C&1@6
8 H
+
+ 2 HzO
2 COz
---j
2 CH,COOH + 2 C O z + 8 H
(e)
CH,COOH + 2 H,O
(f)
2.4. Methylarsane Biosynthesk
The addition of arsenate to cell extracts of Methanobacterium
M . 0. H . yields derivatives of methylarsanes, i. e. A s H ( C H ~ ) ~
and A s H ~ ( C H ~ )The
[ ~ ~ methylation
~.
of arsenic was also
236
Cell-free extracts of Cl. thermoaceticum convert the methyl
group of added methylcobalamin to acetate as a whole, i.e.
the reaction occurs without loss or exchange of hydrogen
atoms of the methyl
This reaction thus represents
Angew. Chem. Int. Ed. Enql. 16, 233-244 (1977)
a rare example of a bacterial COz assimilation. It occurs
under reducing conditions and is inhibited by SH-blocking
reagents. The conversion of methylcobalamin to acetate was
first assumed to occur as shown in eq. (h)[281.
yH2
+
I3
Co'lQ-enzyme
CH,S-CH,CH~CH-COO~)
E n z y m e - bound
v i t a m i n BIZ,
However, a reaction of this type is unlikely as it assumes
the removal of a proton from the cobalt-bound methyl group,
in contrast to the observations mentioned above. A methylation of COz by methylcobalamin in analogy to known reactions of Grignard reagent also does not occur, since methylcobalamin or related organocobalt complexes are insufficiently
reactive. However, the formation of acetate from methylcobalt
complexes was demonstrated under conditions of reductive
Co-C bond cleavage. This led to the proposal of acetate
biosynthesis shown in Scheme 4[163291.
Biogenic r e d u c t a n t
2 ea&[Co
ll-enzyme
Alkaline conditions are required for the nonenzymatic synthesis of methionine from methylcobaloxime and homocysteine, since a nucleophilic displacement according to eq. 0)
occurs only in the presence of the homocysteinyl dianion.
In neutral solution, homocysteine causes reductive cleavage
of the Co-C bond [see eqs. (a) and (b)].
On the other hand, attempts to demonstrate the formation
of methylcobalamin from 5-CH3-THFA and vitamin B l z r
or Blzs have thus far not been successful under nonenzymatic
conditions [eq. (k)].
It must be assumed that reaction according to eq. (k) can
only occur after a specific activation of the 5-CH3 group,
1
id
Scheme 4. Postulated mechanism of acetate biosynthesis from methylcobalamin.
Hence, acetate biosynthesis, the enzymatic methylation of
arsenic, and methanogenesis appear to be closely related reactions. The reaction of CO2 with CH; must occur in an essentially proton-free enzymic environment, for otherwise methane
would be formed preferentially. It would be of interest to
examine if Cl. therrnoaceticum methylates arsenic or produces
methane under certain conditions.
2.6. Methionine Synthesis by Eschevichia coli
E. coli contains an enzyme which transfers the methyl group
from 5-methyltetrahydrofolic acid (5-CH3-THFA) to homocysteine according to eq. (i)[30-361.
5 - C H3-T H F A
+
I;TH2
HS-C H, CHzCH-COOH
Cobalamln- enzymc
P
(+ Adenosylmethtommne)
y
THFA
+
(i)
2
CH,S-CH,CH,CH-COOH
Corrin-dependent methionine synthesis also occurs in animal tissues but not in plants. S-Adenosylmethionine (AMe)
also appears to be a cofactor and was shown to methylate
enzyme-bound vitamin BIzsmore rapidly than 5-CH3-THFA.
The terminal step of methionine biosynthesis is assumed to
occur according to eq. (i), a reaction which has also been
simulated under nonenzymatic conditions using methylcobaloxime as the donor of the methyl group.
Angew. Chem. l n t .
Ed. Engl. 16, 233-244 (1977)
THFA
R = -NH-CH-CHzCHZ-COOH
I
COOH
or that the methylation of the enzyme-bound corrin by 5-CH3THFA is an indirect reaction. The question how reaction
(k)occurs is one of the unresolved problems in all corrin-dependent methyl group transfer reactions. The role of adenosylmethionine in methionine biosynthesis has also not been fully
elucidated, even though reaction (1) occurs nonenzymatically
with no difficulty.
0
R-S-CH,
+
I
Adenosyl
00~1~
-----)
R-S
I
(1)
Adenosyl
R = CHzCHzCH-COOH
I
NH2
237
It should be mentioned in passing that thiols are reductively
methylated on reaction with formaldehyde in the presence
of catalytic amounts of vitamin Blz, [eq. (m)]137*3s1.
€ I 0 OH
(I,
(n)
+ I,"
(A~I.~cIu~~L
Adenine
c"ndll!"n\)
+
CHz=o
+
R-S'
icJp
b
S-R
'
YH2
HO OH
(7)
(4)
+ 2 @, + Hm
fioii" c!Y''o
(m)
CH3-S-R
rise to S'deoxyadenosine (7) (see Scheme 5)" ? The same compound (7) can also be formed from ( 4 ) in solvents containing
labile hydrogen atoms, e.g. in alcohols [eq. (n)].
Coenzyme B12 is also decomposed by alkaline cyanide.
Most Co-C bond cleavage reactions of ( I ) are irreversible,
except the recombination of vitamin Blzr with ( 4 ) and, possibly, the base-induced elimination to give vitamin Blzs and
(3)[40?
-
It would be of interest to demonstrate a corrin-dependent
enzymatic methylation of homocysteine in analogy to eq.
(m), utilizing substrates such as 5,1O-methylene-tetrahydrofolic
acid, which contains the C1-unit at the oxidation level of
formaldehyde.
3. Coenzyme B1*-Dependent Enzymatic Reactions
3.2. Coenzyme B12 Biosynthesis
3.1. Properties and Reactions of Coenzyme B12
Coenzyme BIZ ( I ) is synthesized by a variety of microorganisms and was first isolated by Barker et al. from extracts
of Cl. t e t a n o r n ~ r p h u m421.
[ ~ ~In
, the terminal step of the biosynthesis of ( I ) , the adenosyl residue from ATP is transferred
to the cobalt atom under reducing conditions with attendant
formation of tripolyphosphate [eq. ( 0 ) ] [ ~ ~ , ~ ~ 1 .
At least nine enzymes are presently known in which coenzyme Blz is a required cofactor (see Table 2). The structure
of the coenzyme (see Fig. 1) does not provide any clue as
to how it functions in enzymatic reactions. It is commonly
assumed that a cleavage of the Co-C bond occurs if the
coenzyme is bound to the apoenzymes, but how this activation
takes place is a matter of controversy. Coenzyme B I Z or
5'-deoxyadenosylcobalamin (1) is stable in the solid state
and in neutral aqueous solution. An irreversible decomposition
into hydroxocobalamin (vitamin B1za), adenine, and D-erythro2,3-dihydroxypent-4-enal(2) occurs on reaction with acids[391.
In alkaline solution, the Co-C bond is cleaved by way of
an elimination reaction, giving rise to the formation of vitamin
B l Z sand of 4',5'-anhydroadenosine (3)[401. O n irradiation,
vitamin B1Zr and the 5'-deoxyadenosyl radical ( 4 ) are formed,
indicating that the Co-C bond is cleaved homolytically. The
radical ( 4 ) terminates under anaerobic conditions to yield
the cyclic adenosinoid (5)1411. In the presence of air, ( 4 )
is oxidized primarily to (6)[42]. A reductive cleavage of the
Co-C bond on reaction of (1 ) with thiols or dithiols, gives
P H YH
Adenine
ATP
+
V i t a m i n BlZs
-
C o e n z y m e B,,
+ Tripolyphosphate
0)
Reaction according to eq. (0) could not yet be verified
under nonenzymatic conditions. The enzymatic reaction presumably involves a specific activation of the C-0-P
bond
of ATP. In laboratory syntheses of ( I ), vitamin Blzs is reacted
with 2',3'-protected derivatives of adenosine, usually the tosylates and the protecting groups are subsequently removed
under mildly acidic conditions.
3.3. Dioldehydrases
In cell extracts of Aerobacter aerogenes an enzyme was
']
catalyzes the conversion
discovered by Abeles et ~ l . [ ~which
OH
+ CH,=CHCH-CHCH=O + [&on'] + Xo
Lr
(2)
V i t a m i n BIZ,
T
NHz
HX (Cleavage by acids)
Adenine
Anaerobic
conditions
HO OH
Reductive cleavage,
*
7I
+
e.g with RSH
I
V i t a m i n B12,
V i t a m i n B,,,
Goll'l
Adenine
(4)
HO OH
(31
Scheme 5. Reactions of coenzyme B I Z ( 1 ) (see also Fig. 1).
238
Angew. Chem. I n t . Ed. Engl. 16, 233-244 (1977)
(0)
of D , L - ~,2-propanediol and of ethylene glycol to propionaldehyde and acetaldehyde, respectively [eq. (p)].
Coenzyme-enzyme complex.
R-CH-OH
K*
* R-CH,
I
I
CHz-OH
C H=O
+ HzO
aldehyde exchanges protons with the enzyme-bound (3) as
shown in Scheme 6. According to this mechanism, the coenzyme-substrate hydrogen exchange is thus considered to be
(P)
A
R = CH, o r H
A Lactobacillus contains a related enzyme which causes
the conversion of glycerol into hydroxypropionaldehyde[48!
This reaction may occur during alcohol fermentation and
is undesirable as it gives rise to the formation of acrolein
on subsequent distillation. Dioldehydrase from A . nerogenes
has since been isolated in relatively pure form. The apoenzyme
is knownJo contain thiol groups, some of which are presumably located in the vicinity of the coenzyme binding site. It
was observed only a short time ago, however, that the enzyme
also converts 1,2-butanediol into butyraldehyde and 2,3butanediol into 2-butanone, for example[491;these substrates
were previously considered to be inhibitors. The functional
holoenzyme is inactivated by oxygen. The relative oxygensensitivity depends on the pretreatment of the apoenzyme.
If solutions of the apoenzyme are prepared and stored under
careful exclusion of oxygen, more highly oxygen-sensitive
holoenzyme samples are obtained than when the apoenzyme
is dissolved and maintained aerobically. We assume[5o1that
the apoenzyme can exist in at least two active modifications,
which differ from each other in their content of free SH
groups. Exposure of the apoenzyme to air seems to cause
an oxidation of free SH groups to disulfide, giving rise to
the formation of a the less oxygen-sensitive holoenzyme. This
view is supported by the fact that the oxidized modification
may be in part converted back into the reduced form on
reaction with mild reducing
3.3.1. Mechanism of Enzymatic Dehydration of Diols
1,2-Diols can be converted into aldehydes or ketones quite
easily on reaction with acids. Thus, phenylglycol is converted
to phenylacetaldehyde simply by steam-distillation of a solution of the diol in 1 2 % aqueous HZSO4. But what is the
mechanism of the coenzyme B12-catalyzed dehydration of
glycol to acetaldehyde? P-Hydroxyethylcobaloximes were
found to decompose into acetaldehyde and cobaloxime(1) in
alkaline solution. This led us 10 years ago to propose that
an analogous reaction occurs in the enzymatic dehydration
of diols [see eq. (q)][511.
Since glycols as such d o not react with vitamin BIzs or
cobaloximes(i), the assumption was made that the diol substrates are specifically activated on interaction with the
enzyme[291.We furthermore suggested that enzyme-bound vitamin Blzs is generated from coenzyme Bl2 by way of a baseinduced elimination. The 4',5'-anhydroadenosine (3) formed
during this activation of the coenzyme was assumed to remain
in the vicinity of the active site. To explain the demonstrated
exchange of substrate protons with the hydrogen atoms at
the 5'-position of the coenzyme, we proposed that the product
Angew. Chrm. Inr. Ed. Engl. 16, 233-244 ( 1 9 7 7 )
f
pjo'lo
X;zrFt,",
Reaction with
Reversible activation
of coenzyme B,, ( I )
4
A
O=CHCH,CH3
L
+
Intermediate o r
transition state
A
(-)
+
""<O
HO
CH2
&oil']
CH,CH=CH-O~
+
CH-a
HCH
I
I
CH3
Product
(1)
Regeneration of coenzyme B,,
and removal of product
from active site
Scheme 6. Suggested mechanism for enzymatic conversion of D,L-I ,l?-propanediol into propionaldehyde [29].
independent of the actual catalytic conversion of diols to
aldehydes and is instead assumed to occur between the 4'-protons of (3) and the labile P-protons of the product aldehyde
generated at the active site. Such ionic proton-exchange reactions can be readily simulated in model systems[291.In Section
3.3.2 we shall discuss an alternative mechanistic proposal
in which the same processes are formulated via free radicals.
The mechanism in Scheme 6 is supported by the fact that
the oxygen-sensitive modification of dioldehydrase-holoenzyme is inactivated by NzO almost as efficiently as by Oz[501.
In the first part of this series, we already pointed out that
N 2 0is a specific oxidant of vitamin B12 s . Thus, the inactivation
of the holoenzyme by NzO demonstrates the presence of
enzyme-bound vitamin B1zs. We recently also obtained
evidence for the presence of (3) in the functional holoenzyme[5z1. In spite of these observations, the proposed
mechanism in Scheme 6 has thus far not been accepted by
other workers in the field. It has been argued, that our
mechanism does not fully explain the observed hydrogen-isotope exchange effects[53],but these and other objections will
not be discussed here in view of more extensive counterarguments presented in Ref. [541. We still consider the mechanism
in Scheme 6 to be correct in principle, but since an alternative
mechanism has been proposed which is based on different
assumptions, we shall discuss this mechanism as well.
239
3.3.2. Alternative Mechanism
Abeles and other workers believe that the diol dehydration
occurs by way of free radicals155! The assumption is made
that coenzyme B12 ( I ) is converted into the active form
by homolysis of the Co-C bond. The resulting 5'-deoxyadenosyl radical ( 4 ) in turn abstracts a hydrogen atom from the
glycol substrate and is converted into 5'-deoxyadenosine (7).
The glycol radical (8) is subsequently assumed to react with
vitamin BIzr to yield 1,2-di-hydroxyethylcobaiamin(91, an
intermediate which is postulated to rearrange into the hydrate
of formylmethylcobalamin ( I 0) o r formylmethylcobalamin
( I 1 ), respectively [eq. (r). The enzyme has been omitted from
the formulas].
B
U
In the terminal step of the reaction, formylmethylcobalamin
(I I ) was suggested to yield acetaldehyde by a reaction involving homolysis of the Co-C bond and a hydrogen abstraction
reaction between the resulting acetaldehyde radical and a
5'-hydrogen atom of 5'-deoxyadenosine (7). This hypothetical
reaction sequence is outlined in eq. (s).
.CHz-R
f4)
+
C H 3 C H = 0 T- ( 1 ) + C H 3 C H = 0
U
This radical mechanism was assumed in order to explain
the observed appearance of 3H-labeled acetaldehyde in the
reaction of unlabeled glycol with the holoenzyme containing
3H in the 5'-position of the coenzyme1561.If the view is taken,
that this exchange of hydrogen involves the coenzyme and
the substrate glycol, it would indeed be necessary to consider
radical reactions of the type shown in eq. (r) or (s). However,
there is in fact evidence that 3H-exchange reactions also occur
between enzyme-bound coenzyme and product aldehyde. This
would mean that the hydrogen-exchange reactions are reversible even though the glycol dehydration is irreversible.
Attempts to verify the hypothetical reactions (r) and (s) met
with little success. Model experiments indicated that compounds of type ( 9 ) are either extremely unstable or incapable
of existence; the postulated rearrangement of ( 9 ) into (10)
thus could not be demonstrated. Compound ( 9 ) could be
240
regarded as a "corrinohydrin" of glycolaldehyde. However,
we have previously shown that the equilibrium in reaction
(t) is far on the left side, over a wide p H rangers7"].
PH F:
R-CH-C-R
+
-[Co']O
L.3
+ H'
t
( -1
?H ?H
R-CH-C-R
I
(t)
R = H o r CH,
Glycol radicals generated by a radiolysis reaction of glycols
were in addition shown to have no tendency to form ( 9 )
on reaction with vitamin B12,[57b1. Another argument against
the free-radical mechanism of glycol dehydration was obtained
in attempts to verify reaction eq. (s). Formylmethylcobalamin
( I I ) decomposes thermally as well as on irradiation in solution
to yield acetaldehyde and hydroxocobalamin wirhout the intermediate appearance of uitamin B12r[581[see eq. (4) in the first
part of this series]. Thus it is not possible to support this
alternative mechanism of enzymatic diol dehydration by means
of direct experimental studies. This is not to say that free-radical rearrangements of glycol radicals to acetaldehyde radicals
cannot occur. Acetaldehyde is formed, for example, in the
reaction of glycol with Fenton's reagent. It has also been
reported that traces of acetaldehyde are generated if methylcobaloxime is photolyzed in the presence of glycol, provided
that the reaction is performed in acidic solution[591.We studied
the same reaction in neutral media. Under these conditions,
the glycol radicals generated seem to dimerize to yield erythrito1 and related compounds157a1.
Since a homolysis of the CoC bond of coenzyme BIZ was considered to represent the
mechanism of its enzymatic activation, it should also be
pointed out that such a process is improbable for energetic
reasons. Traces of (7) have been detected after the work-up
of enzyme solutions incubated with radioactively labeled ( I ).
This was interpreted to suggest that (7) participates in the
catalytic process of glycol dehydration. However, since the
same compound is formed as the product of a reductive cleavage of the Co-C bond of (I) and dioldehydrase contains
thiol groups, (7) may also be the by-product of a side-reaction
rather than a catalytic intermediate [see eq. (n) and Scheme
51.
ESR measurements, finally, have been performed on functional dioldehydrase. Under certain experimental conditions,
paramagnetic species were observed, but the signals are not
very characteristic and could not be identified unambiguously.
3.4. Ethanolamine-Deaminase (Ethanolamine-Ammonia Lyase)
An enzyme which catalyzes the conversion of ethanolamine
into acetaldehyde and ammonia [eq. (u)] was discovered in
Clostridium S P . [ ~ ' ] and shown to require ( 1 ) for activity. The
Coenzymeenzyme complex
YH,-OH
CH2-NHz
+
+ H20
CH,CH=O
+
N H ~ O H (u)
~
enzyme was studied in detail by Babiorl6']. The apoprotein
has a MW of 520000 and on treatment with 5 M guanidinium
hydrochloride is cleaved into inactive subunits of MW 51 OOO.
The oxygen atom of ethanolamine becomes the oxygen atom
of product acetaldehyde and remains associated with the same
carbon atom. The enzyme appears to be related to some
Angew. Chem. I n t . Ed. Engl. 16, 233-244 ( 1 9 7 7 )
with respect to the nucleotide base component. However,
minor modifications of the ribose moiety have a pronounced
effect on the ability of the substrates to be reduced. The
added effector is assumed to be bound by the apoenzyme
but probably not in the vicinity of the active site. It is believed
to induce a conformational change of the protein which in
turn enhances substrate binding and/or reduction. Incubation
of the enzyme with 5'-3H-labeled coenzyme B I Z causes the
appearance of tritium in the water solvent during substrate
reduction[671.At low enzyme concentrations the amount of
tritium released is proportional to the yields of reduced product. If the reaction is carried out in 3 H 2 0 , tritium is found
both in the enzyme-bound (1) as well as in the product
deoxyribonucleotide. The displacement of the OH group by
H in the C'-2 position of the ribose moiety occurs with net
retention of configuration. The apoprotein appears to contain
thiol groups in the vicinity of the coenzyme binding site.
Vitamin Blz, does not exhibit coenzyme activity but is reduced
to vitamin Blz, upon the addition of a thiol and an effector.
The resulting solution shows ESR signals of vitamin Bizr
with highly resolved 14N-superhyperfine splitting[681,indicating that the 5,6-dimethylbenzimidazoleremains attached to
cobalt under these conditions. In functional ribonucleotide
reductase holoenzyme ESR-acetive species have been observed
as
The signals show a certain degree of similarity
to those observed in other coenzyme B1z-dependent enzymes
but could not yet be identified.
extent with dioldehydrase, especially since glycol can function
as a stoichiometric substrate. If [3H]-2-aminoethanol is
employed as the substrate, 3H enters into the 5'-position of
the coenzyme. For reaction (u), both ionic and free-radical
mechanisms were therefore postulated as will be outlined
below.
3.4.1. Mechanism of Enzymatic Deamination of Aminoethanol
that ethanolamine-ammonia
We found in our
lyase is inactivated by oxygen as well as by NzO, and hence
concluded that the active enzyme contains a bound Co(1)-corrin. Using l4C-labeled coenzyme B I Z we were also able to
detect traces of labeled (3) on careful work-up of preparations
of the functional holoenzyme, while (7) could not be seen.
Accordingly, a mechanism was postulated for the enzymatic
reaction which is shown in Scheme 7[621.We see no possibility
to support a free-radical mechanism on the basis of model
experiments.
I
Enz yni e
NH3
CH,CH=O
pi--
enzyme
3.5.1. Mechanism of Enzymatic Reduction of Ribonucleotides
Exchange of acetaldehyde protons
with enzyme-bound (3J
Regeneration of coenzyme and
CH,CH=O
+ NH,O
Scheme 7. Proposed ionic mechanism of enzymatic deamination of ethanolamine by ethanolamine deaminase (abbreviated version)l6''.
3.5. Ribonucleotide Reductase
An enzyme in Lactobacillus leichmannii catalyzes the reduction of ribonucleotides to deoxyribon~cleotides[~~~
641. 5'-
For the mechanism of ribonucleotide reduction, schemes
have been put forward which involve free radicals [e.y. ( 4 ) ]
and in which 5'-deoxyadenosine ( 7 ) is assumed to be an
intermediate carrier of hydrogen. It has been suggested, for
example, as a working hypothesis, that ( 4 ) abstracts a hydrogen atom from the dithiol r e d ~ c t a n t c ~but
~ l , since thiols are
known to be efficient scavengers of radicals it would seem
to be unlikely that such mechanisms can be part of the catalytic
process. An ionic mechanism was proposed on the basis of
our own experiments[l6], however, which appears to be in
accord with most of the available experimental evidence. This
mechanism was deduced from observations made in the study
of the reductive cleavage of alkylcobalt complexes by thiols
and dithiols[' 61.These reactions may be regarded as prototypes
of corrin-catalyzed reductions of substrates [eq. (w)].
X
-C H-
Deoxyadenosylcobalamin is the coenzyme, a thioredoxinsystem the reductant, but for in vitro experiments, the latter
may be replaced by dithiols such as dihydrolipoic acid (12)
or dithioerythritol (13) [eq. (v)].
X T P + R(SH),
Coenzymesnzyme complex
(f
* d X T P + R/ S I + H,O
Effector)
Certain deoxyribonucleotides accelerate the reduction of
the substrates and are for this reason considered to act as
effectors. A similar enzyme was detected in Lactobacillus acidophilus and Rhizobium melioti; it is possibly also present in
a variety of other organisms[65,6 6 1 .The enzyme is non-specific
Angew. Chrm. Int. Ed. Engl. 16, 233-244 (1977)
(v)
'SH
' S
Along these lines, the mechanism given in Scheme 8 was
proposed. It has not been possible as yet to demonstrate
the presence of enzyme-bound vitamin BlZsin the functional
h o l ~ e n z y m e [ 'using
~ ~ NzO, since the enzymatic reduction of
ribonucleotides is also not greatly inhibited by oxygen. However, a surprising observation was made in a subsequent study
241
anything in common becomes plausible if it is assumed that
the same processes are rate-determining in both systems. Presumably, the axial coordination of the thiols is a concentrationand buffer-dependent step and responsible for the observed
results. We again assume that the activation occurs by way
of a p-elimination to yield enzyme-bound vitamin B I z s and
( 3 ) , respectively, although direct evidence is not yet available.
on the effects of buffers in reductive Co-C bond cleavage
reactions. Jacobsen and Huennekens reported in 1969 that
the rate of enzymatic ribonucleotide reduction is dependent
on the type and concentrations of the buffers
The authors interpreted their results by assuming chaotropic
effects of the buffer anions on the enzyme protein. We in
turn investigated the effects of buffer-type and -concentration
on the rates of methane production from methylcobaloxime
in the presence of dithioerythritol (13) and dihydrolipoate
( I 2). A surprising similarity between the nonenzymatic rates
of methane production and the enzymatic rates ofribonucleotide
reduction was obserued[l6],the results are shown in Figure
CH, 4 - T P
HOJ"Q
3.6. Coenzyme B1z-Dependent Mutase Reactiom
C Hz-0-T P
HO+&
+Enzyme,
+ Coenzymeenzyme complex
(- O H 9
HO
H
o
Base
Enzyme
25
-@
pB a s e
Base
3'
HOPBase
[Co]- e n z y m e
P
-Coenzyrne-enzyme complex
[yo]-enzyme
The presently known coenzyme B1*-dependent mut a s e ~ ~ ' ~ -are
' ' ~shown in Table 2. They exhibit high substratespecificity, the reactions are reversible and take place without
proton-exchange with the solvent. However, an exchange between protons of the substrate and hydrogen at the 5'-position
of the coenzyme has been demonstrated. It has also been shown
that hydrogen migrates between two neighboring carbon
atoms, and all reactions may beformally considered to involve
the reversible removal and attachment of a substituent R
to positions 3 and 2 of propionic acid [eq. (x)].
CHz-CHz-COOH
A
+ =++
CHS-CH-COOH
I
R
H-S'
R = -CO-CoA,
CHz-O-TP
H
I
Base
(Y)
Scheme 8. Postulated mechanism of enzymatic ribonucleotide reduction [16].
T P = triphosphate.
2. The observed relative rates of the reductions occurring
in both systems are directly and significantly correlated
[ r = 0.71,P< 0.01][' '1. This remarkable similarity between two
reactions which cannot be immediately recognized as having
dl
CJ
etc
We considered a mechanism in which an organocobalt
intermediate undergoes isomerization according to eq. (y)["]
as a part of the overall rearrangement process.
CH,-O-TP
Base
-CH(NH,)COOH, CH,=C-COOH
Reactions of this type have been observed in model systems
[see eq. (h) in the first article]. However, it would have to
be assumed that C-C bonds of the substrates are reversibly
broken or at least labilized to yield complexes of the type
assumed in eq. (y), and no good examples are known to
date for such reactions. As early as 1964 Ingraham considered
that the rearrangement of methylmalonyl-CoA occurs via
organocobalt intermediates of type (14)[821.We studied complexes of type (15) as possible models for the reverse conversion of succinyl-CoA into methylmalonyl-CoA but were unable
to observe a reaction of this type [eq. (z)]['~, 83a1
!
1
i.05 0 1 0.2
0.4 1005 0.1
0.2 0.4 1.05 01 0.2 0 1 1.05 01 0.2 OL
c Imol/ll-
Fig. 2. Relative rate profiles of enzymatic reduction of ribonucleotide (----)
and of methane formation from methyl(aquo)cobaloxide (-)
in tris and
phosphate buffer with dithioerythritol ( 1 3 ) and dihydrolipoic acid ( 1 2 )
as reductant [16]. cTel,relative reaction rate; c, concentration of buffer. a)
Tris buffer + ( 1 3 ) : b) phosphate buffer + ( 1 3 ) ; c ) tris buffer
112);
d) phosphate buffer + ( 1 2 ) . Kind of ribonucleotide: GTP; amount of thiols
+
0.17M.
242
Lowe and Ingraham succeeded in 1971 in performing at
least one reaction which could be considered a possible model
for the rearrangement of (14)to (15)['"]; however, even this
reaction cannot be fully compared with enzymatic process
(see, e. g.t41). It is particularly difficult to formulate a mechanism
for the reverse reactions, i. e. the conversion of succinyl-CoA
into methylmalonyl-CoA. In spite of more recent encouraging
reports from other w o r k e r ~ [ ~ it~ is
, ~necessary
~],
to treat all
attempts to explain the mechanisms of these intriguing reacAngew. Chem. I n t .
Ed. Engl. 16, 233-244 ( 1 9 7 7 )
tions with caution, until more evidence becomes available
and the role of the enzyme in the reactions is better known.
3.7. Aminomutases
Extracts of CI. sticklandii catalyze the reversible conversion
of a-lysine into P-lysine. Analogous isomerizations were
observed with arginine and ornithine. In all reactions an amino
group and a hydrogen atom appear to migrate simultaneously
between two carbon atoms. All known aminomutases consist
of two protein components (see Table 2). In some cases,
enhanced activity is observed upon the addition of bivalent
metal cations, e . g . Mg2+ or Mn”, or by catalytic amounts
of ATP. At least one aminomutase also requires pyridoxal
phosphate as cofactor. In view of the complexity of these
enzymes, possible mechanisms will not be discussed.
B12 coenzymes in enzymatic reactions. It also indicates, however, that serious problems remain to be solved even in the
simpler systems. Although model studies seem to lead in
the right direction, it cannot be denied that there are still
wide gaps between the current biochemical and organometallic
interpretations of the available experimental data. The most
important difference of opinion concerns the function of coenzyme B1 itself: The biochemical studies led to the conclusion
that the coenzyme promotes free-radical reactions of the substrates and that its adenosyl moiety acts as an intermediate
carrier of substrate hydrogen. Model work, as performed by
the author, has produced mechanistic proposals in which
free radicals are avoided and the hydrogen-exchange phenomena are formulated as ionic processes which are not directly
associated with the actual conversion of the substrates to
the products. A decision between the two divergent views is
left to the reader.
3.8. Coenzyme B12 Analogs
It is obvious that our understanding of the mechanism
of action of coenzyme B I Z( 1 ) is closely linked to the question
of how the coenzyme is activated by the enzyme. Only two
alternatives remain to be considered: Does the activation
occur via a homolytic or heterolytic cleavage of the cobalt-carbon bond? To obtain additional experimental information
that might prove useful toward the solution of this problem
various analogs of coenzyme B1 have been synthesized[87]
and tested for coenzyme activity. It was soon found that
even minor modifications of the structure of ( 1 ) cause a
loss or diminution of activity. The behavior of only one coenzyme B l z analog, ( I 6 ) , will be described here. The substitution
of the ribose oxygen by a CH2 group causes a significant
diminution of coenzyme activity in the dioldehydrase syswhich has been attributed to the greater stability
of the Co-C bond in ( I 6 ) as compared to ( I ). However,
( 1 6 ) also undergoes Co-C bond cleavage on reaction with
strong basesrs8].
We have recently observed that the activity of ( 1 6 ) in
dioldehydrase can be increased by exposing the pseudoholoenzyme to
The effect was attributed to a lightinduced heterolytic cleavage of the Co-C bond of enzymebound (16). In alkaline aqueous solution, ( 1 6 ) was found
to yield vitamin B12S[8glon irradiation with strict exclusion
of air. We suggested that the Co-C bond of the coenzyme
in dioldehydrase is not necessarily closed after each substrate
turnover[881,in accord with the general features of the mechanism in Scheme 6.
4. Concluding Remarks
This review shows that we may eventually come to understand all the details of the mechanism of action of vitamin
Angew. Chem. I n t . Ed. Engl. 16, 233-244 (1977)
This review was prepared with support by National Science
Foundation Grant C H E 76-10890.
Received: August 9, 1976 [A 155 IE]
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[89] In addition t o vitamin B I ~ . ,vitamin B I z 1is formed in the photolysis of
( 1 6 ) ; the photolysis must be performed in specially vacuum-degassed
cuvettes to eliminate traces of oxygen. If this is done carefully, the
major photolysis product is vitamin BL2*The photolysis of dioldehydrase incubated with ( I ) does not produce any stimulation, but
instead a slight diminution of enzymatic activity. The irradiation experiments with the enzyme must also be conducted as strictly anaerobically as possible [SS].
C 0M MU N I CAT10N S
Synthesis of Thiol and Selenol Esters from Carboxylic
Acids and Thiols or Selenols, r e s p e c t i v e l y C * * ]
By Hans-Joachim GaisC*]
Dedicated to Professor Robert B. Woodward on the occasion
of his 60th birthday
In contrast to their 0-analogs, thiol esters (I 1 and selenol
esters (2) frequently exhibit toward nucleophiles a higher
and more selective reactivity, which is enhanced even further
by activation with heavy metal ions or oxidizing agents. These
properties make (I) and (2) valuable acyl transfer agents
and permit selective transformations in complex molecules.
[*I
Dr. H.-J. Gais
lnstitut fur Organische Chemie der Technischen Hochschule
Petersenstrasse 22, D-6100 Darmstadt (Germany)
[**I Support of this work by Professor K. Hafner is gratefully acknowledged.
244
Angew. Chem. lnt. Ed. Engl. 16 ( 1 9 7 7 ) N o . 4
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