Coα-(1H-Imidazolyl)-Coβ-methylcob(III)amide: Model for protein-bound corrinoid cofactors

Article abstract of DOI:10.1002/1522-2675(20000906)83:9<2295::AID-HLCA2295>3.0.CO;2-T

Coα-(1H-Imidazol-1-yl)-Coβ-methylcob(III)amide (4) was synthesized by methylation with methyl iodide of (1H-imidazol-1-yl)cob(I)amide, obtained by electrochemical reduction of Coα-(1H-imidazol-1-yl)-Coβ-cyanocob(III)amide (5). The spectroscopic data and a single-crystal X-ray structure analysis indicated 4 to exhibit a base-on constitution in solution and in the crystal. The crucial lengths of the axial Co-N and Co-CH₃ bonds also emerged from the crystallographic data and were found to be smaller by 0.1 and 0.02 A, respectively, than those in methylcob(III)alamin (2). The data of 4 support the view, that the 'long' axial Co-N bonds as determined by X-ray crystallography for the B₁₂-dependent methionine synthase, for methylmalonyl-CoA mutase, and for glutamate mutase represent stretched Co-N bonds. The thermodynamic effect (the 'trans influence') of the 1H-imidazole base in 4 on the organometallic reactivity of this model for protein-bound organometallic B₁₂ cofactors was examined by studying Me-group-transfer equilibria in aqueous solution and using (5',6'-dimethyl-1H-benzimidazol-1-yl)cobamides (cobalamins) as reaction partners (Schemes 2-5, Table). In comparison with methylcob(III)alamin (2), 4 was found to be destabilized for an abstraction of the Co-bound Me group by a Co(III) electrophile. In contrast, the abstraction of the Co-bound Me group by a radical(oid) Co(II) species was not significantly influenced thermodynamically by the exchange of the nucleotide base. Likewise, exploratory Me-group-transfer experiments with Me-Co(III) and nucleophilic Co(I) corrinoids at pH 6.8 provided an apparent equilibrium constant near unity. However, this finding also was consistent with partial protonation of the imidazolylcob(I)amide at pH 6.8, suggesting an interesting pH dependence of the Me-group-transfer equilibrium near neutral pH. Therefore, the replacement of the 5',6'-dimethyl-1H-benzimidazole base by an 1H-imidazole moiety, as observed in methyl transferases and in C-skeleton mutases, does not by itself strongly alter the inherent reactivity of the B₁₂ cofactors in the crucial homolytic and nucleophilic-heterolytic reactions involving the organometallic bond, but may help to enhance the control of the organometallic reactivity by protonation/deprotonation of the axial base.

Full text of DOI:10.1002/1522-2675(20000906)83:9<2295::AID-HLCA2295>3.0.CO;2-T

Helvetica Chimica Acta ± Vol. 83 (2000)
2295
Coa-(1H-Imidazolyl)-Cob-methylcob(III)amide:
Model for Protein-Bound Corrinoid Cofactors
by Mario Fasching, Wolfgang Schmidt, and Bernhard Kräutler*
Institut für Organische Chemie, Leopold-Franzens-Universität Innsbruck, Innrain 52a, A-6020 Innsbruck
and Erhard Stupperich
Angewandte Mikrobiologie, Universität Ulm, Oberer Eselsberg M23, D-89079 Ulm
and Andrea Schmidt and Christoph Kratky*
Institut für Chemie, Strukturbiologie, Karl-Franzens-Universität, Heinrichstrasse 28, A-8010 Graz
Dedicated to Prof. Albert Eschenmoser on the occasion of his 75th birthday
Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)amide (4) was synthesized by methylation with methyl iodide
of (1H-imidazol-1-yl)cob(I)amide, obtained by electrochemical reduction of Coa-(1H-imidazol-1-yl)-Cob-
cyanocob(III)amide (5). The spectroscopic data and a single-crystal X-ray structure analysis indicated 4 to
exhibit a base-on constitution in solution and in the crystal. The crucial lengths of the axial CoÀN and CoÀCH3
bonds also emerged from the crystallographic data and were found to be smaller by 0.1 and 0.02 Š, respectively,
than those in methylcob(III)alamin (2). The data of 4 support the view, that the ꢀlongꢁ axial CoÀN bonds as
determined by X-ray crystallography for the B12-dependent methionine synthase, for methylmalonyl-CoA
mutase, and for glutamate mutase represent stretched CoÀN bonds. The thermodynamic effect (the ꢀtrans
influenceꢁ) of the 1H-imidazole base in 4 on the organometallic reactivity of this model for protein-bound
organometallic B12 cofactors was examined by studying Me-group-transfer equilibria in aqueous solution and
using (5',6'-dimethyl-1H-benzimidazol-1-yl)cobamides (cobalamins) as reaction partners (Schemes 2 ± 5,
Table). In comparison with methylcob(III)alamin (2), 4 was found to be destabilized for an abstraction of
the Co-bound Me group by a CoIII electrophile. In contrast, the abstraction of the Co-bound Me group by a
radical(oid) CoII species was not significantly influenced thermodynamically by the exchange of the nucleotide
base. Likewise, exploratory Me-group-transfer experiments with MeÀCoIII and nucleophilic CoI corrinoids at
pH 6.8 provided an apparent equilibrium constant near unity. However, this finding also was consistent with
partial protonation of the imidazolylcob(I)amide at pH 6.8, suggesting an interesting pH dependence of the Me-
group-transfer equilibrium near neutral pH. Therefore, the replacement of the 5',6'-dimethyl-1H-benzimidazole
base by an 1H-imidazole moiety, as observed in methyl transferases and in C-skeleton mutases, does not by itself
strongly alter the inherent reactivity of the B12 cofactors in the crucial homolytic and nucleophilic-heterolytic
reactions involving the organometallic bond, but may help to enhance the control of the organometallic
reactivity by protonation/deprotonation of the axial base.
1. Introduction. ± The B12 cofactors, such as coenzyme B12 (ˆ(5'-deoxy-5'-
adenosyl)cob(III)alamin; 1) and methylcob(III)alamin (2), are highly complex,
naturally occurring organometallic compounds [1][2] (see Fig. 1). They are coenzymes
for a series of enzymes that catalyze very unusual biological reactions [2 ± 5]. At
present, coenzyme B12 (1) is known to be the cofactor of eleven enzymes (of B12-
dependent ribonucleotide reductase [6] and of ten other enzymes that catalyze
rearrangement reactions via radical intermediates [4][5]). Methylcob(III)alamin (2) is
the cofactor in a class of enzymes that catalyze the intermolecular transfer of Me groups

2296
Helvetica Chimica Acta ± Vol. 83 (2000)
[5][7 ± 9]. In all these enzymes, critical steps of their catalytic cycles depend upon the
rapid homolytic or heterolytic cleavage (and homolytic and heterolytic formation) of
the organometallic bond of the protein-bound B12 cofactors [2][4][5].
Since the early (nineteen)-sixties, isolated B12 cofactors have been the subject of
numerous structural investigations, both in the crystalline state [1][10 ± 12] and in
Fig. 1. Structural formulae of vitamin-B12 derivatives in the ꢀbase-onꢁ constitution: a) Cobalamins: (5'-deoxy-5'-
adenosyl)cob(III)alamin (ˆcoenzyme B12; R ˆ 5'-deoxy-5'-adenosyl; 1); methylcob(III)alamin (R ˆ Me; 2);
‡
cyanocob(III)alamin (ˆvitamin B12; R ˆ CN; 3), aquacob(III)alamin cation (R ˆ H2O; 8 ); andcob(II)alamin
(R ˆ unoccupied; 10). b) Imidazolylcobamides: Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (R ˆ Me;
4), Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide (R ˆ CN; 5), Coa-(1H-imidazol-1-yl)-Cob-aqua-
‡
cob(III)amide cation (R ˆ H2O; 7 ), and(1 H-imidazol-1-yl)cob(II)amide (R ˆ unoccupied, 9).

Helvetica Chimica Acta ± Vol. 83 (2000)
2297
solution [4][5][13][14]. Indeed, vitamin B12 (3) is prominent for being the first case of
a natural compound where the essential features of its structure were deduced by X-ray
analysis [10 ± 12][15]. The complex structure of the B12 cofactors implies a correspond-
ingly complicated biosynthesis [16 ± 19], laboratory synthesis [20][21], and chemistry of
B12 coenzymes [2 ± 5][14][22][23].
A well-studied feature of the isolated, intact ꢀcompleteꢁ B12 cofactors is the
intramolecular (ꢀbase-onꢁ) cobalt coordination of their unique nucleotide function (see
Fig. 1), which represents their most stable constitution in solution and in the solid state
[1][2][4][5][10 ± 14]. The first crystal structure of a B12-binding protein, of the B12-
binding domain of methionine synthase (MetH) from Escherichia coli, revealed a
surprisingly different situation for the protein-bound corrinoid cofactor methyl-
cob(III)alamin (2): here, the cofactor was observed in a ꢀbase-off/His-onꢁ constitution,
i.e., instead of the dimethylbenzimidazole base, the imidazole side chain of the protein
residue His759 was found to coordinate at the corrin-bound CoIII center of 2 (see Fig. 2)
[24 ± 26]. In two recent structure analyses of coenzyme-B12-dependent mutases,
methylmalonyl-CoA mutase (MCM) from Propionibacterium shermanii [26][27] and
in glutamate mutase (Glm) from Clostridium cochlearium [28], the corresponding
ꢀbase-off/His-onꢁ constitution (previously predicted from protein-sequence alignment
data [29]) of the bound corrinoid was also observed. In all three protein structures, the
Co-coordinating histidine and a H-bonded aspartate represent a conserved ꢀB12-
bindingꢁ motif, and they are part of a regulatory ꢀligand triadꢁ of (typically) three
amino-acid residues [25][26]. The role of the unique (a-configurated) ribazole function
of the B12 coenzymes in their ꢀbase-off/His-onꢁ constitution, therefore, does not concern
the control of the organometallic reactivity at the Co-atom, but primarily the anchoring
of the corrinoid ꢀdinucleotidesꢁ coenzyme B12 and methylcobalamin to the apoenzyme.
Contrasting these crystallographic findings on the Me-group transferase MetH [24],
and the two C-skeleton mutases, MCM and Glm [27][28], EPR-spectroscopic studies
on the mode of binding of coenzyme B12 to diol dehydratase (DD) and to B12-
dependent ribonucleotide reductase (RNR) indicated base-on binding of the corrinoid
cofactor [30 ± 32]. The finding on DD was confirmed by the crystal structure of the
inactive vitamin-B12 complex of this enzyme, which provided for the first time struc-
Fig. 2. Sketch of the coordination features of methylcob(III)amin (2) bound
ꢀbase-off/His-onꢁ in methionine synthase from E. coli [24 ± 26]

2298
Helvetica Chimica Acta ± Vol. 83 (2000)
tural clues concerning the binding of a corrinoid cofactor in its ꢀconventionalꢁ base-on
form [33].
Studies concerning the axial coordination properties of corrinatocobalts are of
interest, as their homolytic and heterolytic organometallic reactivity are central to their
biological role [2 ± 5][34]. Such studies have gained further relevance from the ongoing
crystallographic workon B 12-dependent enzymes. In the B12-binding domain of MetH,
the Co-coordinating imidazole moiety forms a H-bond to the carboxylate of an
aspartate, constituting the crucial part of the ꢀregulatory ligand triadeꢁ [24 ± 26]. The
latter has been suggested to undergo protonation/deprotonation as part of the catalytic
cycle, which involves shuttling between hexacoordinate CoIII and tetracoordinate CoI
states of the corrinoid. In this way, the Co-coordinated histidine apparently helps to
control the organometallic reactivity of the protein-bound corrinoid cofactor and to
mediate the influence of the proteinic environment [24 ± 26][35]. On the other hand,
the bonding pattern around the Co-center in the crystallographically investigated
coenzyme-B12-dependent enzymes are remarkable, as all of them show an unusually
ꢀlongꢁ axial CoÀN bond [26 ± 28][33]: the observed lengths of the CoÀN bonds in
MCM (2.53 Š), in Glm (2.27 ± 2.35 Š), and in DD (2.50 Š) are considerably longer
than those observed in the isolated corrinoid cofactors [1][10 ± 12][36]. The crystallo-
graphically observed ꢀlongꢁ axial CoÀN bond has been suggested to be relevant for the
activation of the protein-bound corrinoid cofactor towards homolysis of its CoÀC bond
[26][27][33]. However, the significance of these crystallographic data must not be
overestimated since their crystallographic resolution was inherently low (often > 2 Š),
and neither the oxidation state of the corrin-bound Co-centers nor the nature of their
upper axial coordination partners were always clear. In the two mutase structures
(MCM, Glm), presumably a mixture of corrinoid cofactors with different oxidation
states was present [26]. The length of this bond was also studied by X-ray absorption
spectroscopy, which yielded an indication for bond elongation for MCM [37] and a
more or less ꢀnormalꢁ bond length for Glm [38]. However, recently we were able to
show that under the conditions of a high-brilliance X-ray beam, photoreduction of the
cofactor may occur, which would then suggest that at least some of the crystallo-
graphically observed bond elongation is in fact an artifact of the experimental
technique [39]. However, at this stage, the ꢀlong bondꢁ still poses a puzzling
observation. The point has been made elsewhere [2] that comparison of the lengths
of the axial CoÀN bond in organo-cob(III)alamins and cob(II)alamin (10) [10 ± 12]
would not suggest a simple stretch of the axial CoÀN bond to lead to ꢀactivationꢁ of the
protein-bound corrinoid cofactor towards homolysis.
Clearly, further chemical consequences of the axial coordination of a nitrogenous
base to the Co-center of corrins are of interest [2][12][14]. Its effect on the reactivity of
organometallic groups at the other axial (trans) position has been discussed in the
context of the enigmatic mode of activation of protein-bound coenzyme B12 [2][40 ±
43]. Similar questions on the effect of the intramolecular coordination of the nucleotide
base on the reactivity of methylcobalamin were also posed and examined experimen-
tally [44]. So far, all such studies had to rely on a comparison between the reactivity of
cobalamins and corrinoids lacking a nucleotide function all-together. With the two
exceptions of the structures of the cyano-corrinoids Factor A [45] and Coa-(1H-
imidazol-1-yl)-Cob-cyanocob(III)amide [46], also all high-resolution crystal structures

Helvetica Chimica Acta ± Vol. 83 (2000)
2299
of ꢀcompleteꢁ B12 derivatives have involved cobalamins ((5',6'-dimethyl-1H-benz-
imidazol-1-yl)cob(III)amides) [1][10 ± 15]. Questions [24 ± 28][35] concerning the
consequences of the replacement of the 5',6'-dimethyl-1H-benzimidazole (Dmb)
moiety of cobalamins (such as of methylcob(III)alamin (2)) by an 1H-imidazole (Im)
moiety, such as in Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4), on the
structure and organometallic reactivity have so far not been addressed. We describe
here the synthesis and spectroscopic characterization in solution of Coa-(1H-imidazol-
1-yl)-Cob-methylcob(III)amide (4) and the crystal structure of this model for protein-
bound organocorrinoid cofactors, and report on experiments concerning its organo-
metallic reactivity.
2. Results and Discussion. ± 2.1. Preparation andSpectroscopic Characterization of
Coa-(1H-Imidazol-1-yl)-Cob-Methylcob(III)amide (4). The nonnatural complete
corrinoid Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide (5) was available from
guided biosynthesis [46]. The electrosynthesis of the organocobamide 4 from 5 and
MeI followed an established procedure [47]: Electrochemical reduction of 5 at a
potential of À 1.1 V vs. 0.1n calomel electrode by ca. 2 F/mol gave the highly O2-
sensitive imidazolylcob(I)amide 6À (see below, Scheme 4), characterized by the UV/
VIS spectrum. Treatment of the solution containing 6À with an excess of MeI was
accompanied by a color change (from green to red) of the reaction solution. The light-
sensitive Coa-imidazolyl-Cob-methylcobamide 4 was isolated in ca. 90% yield as bright
red crystals. This ꢀcompleteꢁ imidazolylcobamide 4 exhibited UV/VIS- and CD-
spectroscopic data typical for ꢀbase-onꢁ methylcobamides (see Fig. 3 for the UV/VIS
spectrum). In the FAB-MS, the base peakat m/z 1266.8 and fragments at m/z 1251.7
‡
indicated relatively stable pseudo-ions of intact 4 ([M ‡ H] ) and of the demethylated
‡
corrin fragment ([M ‡ H À CH3] ). Detailed NMR studies of 4 allowed the complete
assignment of the 1H-NMR spectrum, fully consistent with the ꢀbase-onꢁ constitution of
4 in neutral aqueous solution (see Fig. 4).
Fig. 3. UV/VIS Spectrum of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4) in aqueous solution (c ˆ
0.8 mm, 0.1m phosphate, pH 6.74)

2300
Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 4. 500-MHz 1H-NMR Spectrum of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4) in aqueous
solution (D2O, pH 7)
The 1H-NMR spectrum of 4 has similar features as that of methylcob(III)alamin (2) [48], i.e. high-field s at
À 0.21 and 0.49 ppm for the Co-bound Me group and MeÀC(1), respectively. The latter signal experiences a
related upfield shift by the Co-coordinated imidazole moiety in the spectrum of 4, as by the Co-coordinated
benzimidazole moiety in the spectrum of 2. The signal of MeÀCo of 4 is shifted upfield slightly in both the 1H-
and 13C-NMR spectra, compared to the corresponding data for 2. All the available spectroscopic information is
consistent with the expected (ꢀconventionalꢁ) base-on constitution of 4.
2.2. Preparation andSpectroscopic Identification of Coa-(1H-Imidazol-1-yl)-Cob-
‡
aquacobamide (7 ). The Coa-imidazolyl-Cob-methylcobamide 4 is rapidly decom-
posed by light, similar to other MeÀCoIII corrinoids, such as 2. As described for the
‡
preparation of Cob-aquacob(III)alamin (8 ) from 2 [49], the Cob-aquacob(III)amide
‡
7 was prepared by photolysis of the corresponding MeÀCoIII corrinoid 4 in aerated
‡
aqueous solution. The UV/VIS spectrum of an aqueous solution of 7 (at pH 6.3)
exhibits the typical broad band with two absorbance maxima near 520 nm and a sharp
1
‡
band near 360 nm (see Fig. 5). The H-NMR spectrum of an aqueous solution of 7
supports the assignment of its ꢀbase-onꢁ constitution.
2.3. Acid/Base Properties of Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)amide (4)
in Solution. The UV/VIS absorbance spectra of aqueous solutions of 4 are strongly pH-
dependent, indicating protonation of 4 at low pH with concominant decoordination of
the nucleotide base (see Fig. 6 and Scheme 1). A spectrophotometric analysis of
buffered aqueous solutions of 4 in the pH range between pH 0.3 and 7.8 allowed the
‡
determination of the pKa of protonated (ꢀbase-offꢁ) 4 ´ H as 4.3 Æ 0.1 at room
‡
temperature. The value of this pKa is higher by ca. 1.4 units than that of 2 ´ H , the
‡
corresponding pKa of the protonated, ꢀbase-offꢁ form 2 ´ H of methylcob(III)alamin
‡
(2) [50]. Comparison of the pKa of 2 ´ H (ˆ2.9) with that of protonated 5,6-dimethyl-
1-(a-d-ribofuranosyl)-1H-benzimidazole (ˆa-ribazole; 5.56 at 258) indicated the
equilibrium constant for the formation of ꢀbase-onꢁ methylcobalamin 2 to amount to
ca. 460 (at 258) [50]. The corresponding pKa of protonated 1-(a-d-ribofuranosyl)-1H-
imidazole is not available at this stage, but may be estimated to be close to 7, based on
the values for Me-group-transfer equilibria, determined here (see below).

Helvetica Chimica Acta ± Vol. 83 (2000)
2301
‡
Fig. 5. UV/VIS Spectrum of Coa-(1H-imidazol-1-yl)-Cob-aquacob(III)amide (7 ) in aqueous solution (0.02m
phosphate, pH 6.3)
Fig. 6. pH Dependence of UV/VIS Spectra of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4) in buffered
aqueous solutions (see Exper. Part for conditions)
Scheme 1

2302
Helvetica Chimica Acta ± Vol. 83 (2000)
2.4. Methyl-Group-Transfer Equilibria in Solution Characterizing the Organo-
metallic Reactivity of Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)amide (4). 2.4.1.
Preamble. Early observations have suggested the occurrence of Me-group transfer
reactions between various Co-complexes [23]. Methyl-group transfer from methyl-
cob(III)alamin (2) to diaquacob(III)inamide was reported by Hogenkamp and co-
workers [51]. Systematic studies on the equilibria in aqueous solution between
MeÀCoIII, CoII, and CoI forms involving the cob(III)alamin 2, cob(II)alamin (10), and
cob(I)alamin (11À; see below, Scheme 4), as well as the corresponding cobinamides
(methylcob(III)-, cob(II)-, and cob(I)inamide), were carried out and informed on the
(thermodynamic) effect of the Dmb base on the three CoÀC bond dissociation
energies in methylcorrinoids [2][44][52]. They indicated a small effect of the
coordinated Dmb base on the homolytic bond strengths of the investigated
methylcorrinoids, as well as on the related adenosylcorrinoids [44]. In contrast, these
experiments revealed significant stabilization of (ꢀbase-onꢁ) methylcob(III)alamin (2)
by the intramolecular coordination of the base against abstraction of the Co-bound Me
group by a nucleophile [44]. Exploratory experiments comparing cobalamins and (5'-
hydroxy-1H-benzimidazol-1-yl)cobamides (found in some methanogens [53]) also
indicated small effects of the differing bases on the heterolytic mode of nucleophilic
cleavage of the CoÀC bond, but negligible effects on the homolytic mode of cleavage
[52]. The experiments described here were carried out to compare the thermodynamic
(trans) effects of Im and of Dmb as nucleotide bases on the three elementary modes of
CoÀC bond cleavage of the methylcob(III)amides 2 and 4.
2.4.2. Equilibration Experiments with Methylcob(III)amides and Aquacob(III)a-
mides. The following experiments were carried out to investigate the Me-group-transfer
equilibrium indicated in Scheme 2, which allowed the determination of KIII/III of Eqn. 1.
‡
‡
KIII/III ˆ ([8 ] ´ [4])/([2] ´ [7 ])
(1)
Scheme 2
A buffered aqueous solution of about equimolar amounts of methylcob(III)alamin
‡
(2) and of Coa-(1H-imidazol-1-yl)-Cob-aquacob(III)amide (7 ) was stored in an
NMR tube at room temperature for 30 days with protection from light (reaction A). In
a parallel experiment (reaction B), a corresponding buffered solution of Coa-(1H-

Helvetica Chimica Acta ± Vol. 83 (2000)
2303
imidazol-1-yl)-Cob-methylcob(III)amide (4) and of aquacob(III)alamin chloride (8 ´
Cl) was also stored in an NMR tube at room temperature for the same period of time
with protection from light. The changes in the two solutions (A and B) were observed
1H-NMR-spectroscopically. The reaction mixture A only underwent minor, 1H-NMR-
spectroscopically manifest changes, but small signals due to traces of 4, of
‡
aquocobalamin cation 8 , and of an intermediate 12 (see below) appeared over the
course of the measurements. In contrast, the observed changes in reaction mixture B
were more apparent. Within the first 2 h, the signals of the intermediate 12 (no formula
‡
shown) had grown to almost their maximal intensity, while those of 2 and 7 appeared
more slowly, to reach by the end of the experiment (30 days) essentially the same
solution composition as in reaction A. The position of two signals of the intermediate
12, a singlet at À 0.36 ppm (a high-field position typical of signals due to Co-bound Me
groups) and a singlet at 0.95 ppm, would make it rather likely that 12 is a Coa-
methylcorrinoid in a ꢀbase-offꢁ form [54]. Further experiments are planned to reveal
the identity of 12.
For further analysis of both experiments, the two reaction mixtures were treated
with a slight excess of KCN in H2O (with protection from light) and were analyzed once
1
more by H-NMR spectroscopy. Accordingly, the cyanide treatment of both reaction
mixtures converted the aquacorrinoids in their cyano analogues and induced the
disappearance of the small remaining signals of 12. In the reaction mixture from
experiment A, the corrinoids 2 ± 5 were present in a ratio of 4.8 :1.2 :0.8 :2.5,
corresponding to a product ratio of 0.08 Æ 0.04 (see Table). Likewise, in the reaction
mixture from experiment B, the corrinoids 2 ± 5 were present in a ratio of
1.8 :1.1:0.3 :1.4, corresponding to an equilibrium value of 0.12 Æ 0.02. Excluding
significant equilibration after the cyanide quenching, the contents of the equilibrating
‡
‡
reaction mixtures (2, 8 , 4, and 7 ) would be reflected by the ratios of the four
corrinoids 2, 3, 4, and 5, respectively, and the equilibrium constant for the equation
given in Scheme 2 (see Eqn. 1) could be estimated as KIII/III ˆ 0.10 Æ 0.02 (see Table).
These experiments indicate that the thermodynamic enhancement of the electrophilic
demethylation of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide 4 by the coordi-
nated Im base is significantly larger than that by the Dmb base in methylcob(III)alamin
(2). This suggests a particularly strong stabilization of Coa-(1H-imidazol-1-yl)-Cob-
‡
aquacob(III)amide (7 ) by the intramolecular coordination of the Im base. In view of
Table. Concentration Ratios of Cyanocorrinoids 3 and 5 andof Methylcorrinoids 2 and 4, from Methyl-Group-
Transfer Experiments at Room Temperature andpH 6.8 andCalculated (apparent) Equilibrium Constants for
Methyl-Group-Transfer Equilibria
Starting materials
Products (rel. amount, Æ 10%)
Calc. ratio
(3 ´ 4)/(5 ´ 2)
Calc. (apparent)
equilibrium constants
2
3
4
5
‡
2, 7
4, 8
2, 9
4.77
1.83
0.61
1.80
1.22
1.31
1.24
1.09
1.01
1.04
1.04
1.05
0.76
0.30
0.90
3.62
1.01
1.70
2.52
1.43
0.72
1.55
1.08
1.30
0.08
0.12
2.06
1.34
0.80
1.05.
KIII/III ˆ 0.1 Æ 0.02
KII/III ˆ 1.7 Æ 0.4
‡
4, 10
2, 6À
4, 11À
KI/III(app) ˆ 0.9 Æ 0.2

2304
Helvetica Chimica Acta ± Vol. 83 (2000)
the observed strong upwards folding in aquacobalamin, ascribed to steric strain
imposed by the strongly coordinating, bulky Dmb base [55], an increased stabilization
‡
in 7 by the intramolecular coordination of the less bulky Im base is in agreement with
basic expectations on the coordination behavior of the corrin-bound CoIII-center and
on the higher nucleophilicity of Im (compared to Dmb).
2.4.3. Equilibration Experiments with Methylcob(III)amides and Cob(II)amides.
The following two experiments were carried out to investigate the Me-group-transfer
equilibrium given in Scheme 3, which led to the determination of KII/III of Eqn. 2.
KII/III ˆ ([10] ´ [4])/([2] ´ [9])
(2)
Scheme 3
A buffered aqueous solution of (1H-imidazol-1-yl)cob(II)amide (9) was prepared
by electrochemical reduction of Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide 5.
The extent of the reduction was monitored coulometrically and UV/VIS-spectroscopi-
cally (see below) [56]. The solution of 9 was then mixed (with protection from light and
air) with a buffered aqueous solution of an equimolar amount of methylcob(III)alamin
(2). The mixture was left to stand at room temperature (with protection from light and
air). After an equilibration time of 2 h, the mixture was taken out of the glove box, and
O2-saturated 1% HCN in MeOH was injected. The reaction mixture contained
significant amounts of the four corrinoids 2 ± 5 (according to TLC) and was further
1
analyzed by recording a 500-MHz H-NMR spectrum (see Table). Accordingly, the
corrinoids 2 ± 5 were present in a ratio of 0.61:1.0 :0.90 :0.72 (see Fig. 7), correspond-
ing to a ratio of products of 2.06 Æ 0.4.
A buffered aqueous solution of cob(II)alamin (10) was prepared likewise by
electrochemical one-electron reduction of vitamin B12 (3) (see below) [54]. An aliquot
of the solution of 10 was then mixed (with protection from light and air) with a buffered
aqueous solution of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4) and left to
1
stand at room temperature. After workup as described above, the H-NMR analysis
(500 MHz) revealed the presence of the corrinoids 2 ± 5 in a ratio of 1.8 :1.04 :3.6 :1.55,
corresponding to a ratio of products of 1.34 Æ 0.4 (see Table). Assuming insignificant
equilibration after the oxidative quenching, justified by the experiences with
aquacorrinoids [44], the contents of the equilibrating reaction mixtures (2, 10, 4, and
9) would be reflected by the ratios of the four corrinoids 2, 3, 4, and 5, respectively, and
the equilibrium constant for the equation given in Scheme 3 (see Eqn. 2) could be

Helvetica Chimica Acta ± Vol. 83 (2000)
2305
Fig. 7. Comparison of the low-fieldsection of the 1H-NMR spectra of Coa-(1H-imidazol-1-yl)-Cob-methyl-
cob(III)amide (4) andof the reaction mixture from methyl-group transfer starting with methylcob(III)alamin (2)
and(1 H-imidazol-1-yl)cob(II)amide (9). *: signals of 2, &: signals of 3, .: signals of 4, and &: signals of 5.
estimated as KII/III ˆ 1.7 Æ 0.4. This value indicates a slight stabilization of the
imidazolyl(methyl)cobamide 4 upon homolytic methylation, compared to methylco-
balamin 2. The (thermodynamic) effect of the coordination of the Im base of 4 on the
homolytic CoÀC bond strength thus is small, when compared to that of the Dmb base
of 2, but tends to stabilize the imidazolyl(methyl)cob(III)amide 4. A similar result is
likely to characterize also the related homolytic CoÀC bond strengths of adenosylco-
bamides, such as coenzyme B12 (1) and its imidazolyl analogue. Accordingly, the
substitution of the Dmb base by a histidine residue, as observed in the adenosylco-
bamide-dependent mutases MCM and Glm, is not likely to result in an inherently
increased reactivity towards homolysis of the CoÀC bond.
2.4.4. Exploratory Equilibration Experiments with Methylcob(III)amides and
Cob(I)amides. With the intention of investigating the Me-group transfer equilibrium
given in Scheme 4 and of determining the equilibration constant KI/III of Eqn. 3, the
following two exploratory experiments were carried out.
KI/III ˆ ([11À] ´ [4])/([2] ´ [6À])
(3)
In one experiment, crystalline methylcob(III)alamin (2) was dissolved in a buffered
aqueous solution (pH 6.8) containing a nearly equimolar amount of (1H-imidazol-1-
yl)cob(I)amide (6À), prepared electrochemically and identified by a UV/VIS spectrum
of the electrolysis product (see Fig. 12 in the Exper. Part). The mixture was left to stand
at room temperature for 15 min (with protection from light and air), after which time a

2306
Helvetica Chimica Acta ± Vol. 83 (2000)
Scheme 4
sample was analyzed by UV/VIS spectroscopy. The UV/VIS spectrum showed
absorbance characteristics of a mixture of cob(I)amides and of alkylcob(III)amides.
Thus, the flaskcontaining the reaction mixture was tightly stoppered and taken out of
the glove box. Injection of 0.5 ml of O2-saturated 1% HCN in MeOH into the flask
rapidly oxidized the mixture, which then consisted of significant amounts of all of the
four corrinoids 2 ± 5 (according to TLC). The 1H-NMR analysis (500 MHz) established
the presence of the corrinoids 2 ± 5 in a ratio of 1.2 :1.04 :1.0 :1.1, corresponding to a
ratio of products of 0.80 Æ 0.2 (see Table).
Likewise, a buffered aqueous solution of cob(I)alamin (11À), prepared electro-
chemically, was added to a buffered aqueous solution (pH 6.8) of an equimolar amount
of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4). The mixture was left to
stand at room temperature for 30 min (with protection from light and air), and then
analyzed and worked up as described in the preceding paragraph. The reaction mixture
again contained significant amounts of all of the four corrinoids 2 ± 5 (according to
1
TLC), and the H-NMR spectrum (500 MHz) indicated the corrinoids 2 ± 5 to be
present in a ratio of 1.3 :1.05 :1.7:1.3, corresponding to a ratio of the products of 1.05 Æ
0.2 (see Table). Based on earlier experiences with the analysis of Me-transfer mixtures
[44], significant equilibration during the oxidative quenching process can be assumed
to be unlikely. Accordingly, the contents of the equilibrating MeÀCoIII and CoI
corrinoids would be reflected by the ratios of the four cobamides 2 ± 5, and the
apparent equilibrium constant for the equation given in Scheme 4 could be estimated as
KI/III(app) ˆ 0.9 Æ 0.1. These results appear to suggest an unexpectedly similar
stabilization of methylcob(III)alamin (2) and of Coa-(1H-imidazol-1-yl)-Cob-meth-
ylcob(III)amide (4) by the intramolecular coordination of their nucleotide bases Dmb
or Im, respectively.
‡
However, from an earlier comparison of the pKa of 2 ´ H (ˆ2.9) with the pKa of
protonated a-ribazole (5.56 at 258), the equilibrium constant for the formation of ꢀbase-
onꢁ methylcob(III)alamin (2) was deduced to amount to ca. 460 (at 258) [50]. Indeed,
the size of KI/III(app) similarly suggests the equilibrium constant for the formation of
base-on Coa-(1H-imidazol-1-yl)-Cob-methylcobamide 4 to be roughly 500 too (at
room temperature). Accordingly, the pKa of protonated 1-(a-d-ribofuranosyl)-1H-
‡
imidazole would be indicated to exceed the pKa of 4 ´ H similarly and to amount to ca.

Helvetica Chimica Acta ± Vol. 83 (2000)
2307
7.0, at least. Indeed, such acid/base characteristics of 1-(a-d-ribofuranosyl)-1H-
imidazole or of the imidazolylcob(I)amide 6À would be consistent with the higher
acidity of protonated Dmb (pKa ˆ 6.0 [57]) than of protonated 1H-imidazole (pKa ˆ
7.0 [58]). However, assuming the acidity of the protonated Im base of the base-off
imidazolyl cobamides to be similar to that of the protonated 1-(a-d-ribofuranosyl)-1H-
imidazole, a significant fraction of the imidazolylcob(I)amide 6À would be expected to
be protonated at the solution pH of 6.8, and the protonated form of the
imidazolylcob(I)amide, i.e., 6 ´ H, would also have to be taken into consideration
(see Scheme 5).
Scheme 5
Indeed, the product ratios of the four cobamides 2 ± 5, therefore, provide the
apparent equilibrium constant KI/III(app), as given in Eqn. 4.
The product ratios represent, in fact, a measure of the extent of Me-group
equilibration between (all of) the cobalamins and the imidazolylcobamides present in
solution at a given pH (near neutral). Accordingly, near neutral pH KI/III(app) can be
correlated with KI/III by taking into account the pH-dependent presence of 6 ´ H (and of
11 ´ H). With pKa (11 ´ H) < pKa (6 ´ H) (pKa (11 ´ H) ca. 5.6 [47]), an approximate
correlation between KI/III(app) and KI/III for the case of equilibria at neutral pH or
slightly basic solutions (where [11 ´ H] is neglected) is given by Eqn. 5.
KI/III(app) ˆ ({[11À] ‡ [11 ´ H]}[4])/([2]{[6À] ‡ [6 ´ H]})
(4)
(5)
KI/III ˆ KI/III(app) ´ {1 ‡ 10[pK (6´H)ÀpH]
}
a

2308
Helvetica Chimica Acta ± Vol. 83 (2000)
Eqn. 5 shows that KI/III exceeds KI/III(app), but KI/III 92 KI/III(app) at pH values
close or slightly above the pKa of 6 ´ H. However, Eqn. 5 also indicates KI/III to exceed
KI/III(app) more significantly at pH values in the range between pKa (11 ´ H) and pKa
(6 ´ H). Accordingly, protonation of the axial base of the cob(I)amide 6À near neutral
pH is due to assisting (thermodynamically) the demethylation of the Coa-imidazolyl-
Cob-methylcob(III)amide 4 by nucleophiles. To explore the effects from (partial)
protonation of the imidazolylcob(I)amide 6À under the conditions of the Me-group-
transfer equilibria, further experiments at various (higher) solution pH values are,
therefore, needed and are currently in progress.
Our Me-group-transfer experiments indicated the Co-bound Me group to be
similarly deactivated against nucleophilic abstraction by the intramolecular coordina-
tion of the Dmb base of 2, as by the Im base of 4. Accordingly, the substitution of the
Dmb base by a histidine residue, as observed in MetH and as indicated in other Me-
group transferases [24 ± 26][59], is not likely to result in an inherently altered reactivity
towards nucleophilic heterolysis of the CoÀC-bond. However, the estimated pKa value
for protonated 1-(a-d-ribofuranosyl)-1H-imidazole is close to 7 and is significantly
more positive than those of protonated a-ribazole. The higher pKa values of protonated
imidazole bases, when compared to Dmb and other natural nucleotide bases, are likely
to be very significant from the enzymological point of view: protonation/deprotonation
steps involving the His-Asp pair of the regulatory ꢀligand triadꢁ were observed to
accompany both the enzymatic heterolytic demethylation/methylation reactions of
MetH near neutral pH, as well as the one-electron redox reactions involving protein-
bound cob(II)alamin (10) and cob(I)alamin (11À) [26]. Protonation/deprotonation
steps of protein-bound methylcorrinoids in their ꢀbase-off/His-onꢁ constitution thus
control and tune the thermodynamics of the Me- and electron-transfer reactions of
MetH and of other methyl transferases. Clearly, in methyl transferases, the substitution
of the Dmb base (and of other nucleotide bases, such as adenines) of the naturally
occurring complete corrinoids by the more basic imidazole base (of His in the ꢀligand
triadꢁ) thus contributes significantly to the accessibility of the crucial protonation/
deprotonation steps at physiological pH. On the other hand, it is far from being
understood, to what extent the more basic character of Im (compared to Dmb) and the
associated protonation/deprotonation steps would contribute to the function of the
protein-bound adenosyl-cobamides in the coenzyme-B12-dependent mutases.
2.5. Crystal-Structure Analysis of Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)-
amide (4). Crystals of 4 were grown from an aqueous acetone solution at 58, and X-
ray diffraction data were collected at cryotemperature (100 K) at one of the EMBL
beamlines at the DESY synchrotron in Hamburg, Germany. Structure refinement
against diffraction data extending to a crystallographic resolution of 1.0 Š converged at
a residual of R ˆ 0.0871 für all 9011 symmetry-independent reflections. Projections of
the B12 moiety of the crystal structure of 4 are shown in Fig. 8.
A superposition of the crystal structure of 4 with the B12 cofactors observed in the
ꢀbase-off/His-onꢁ proteins [24][27][28] reveals the imidazole ring of 4 to occur in a very
similar conformation (ꢀnorth-southꢁ orientation of the plane of the imidazole ring) with
respect to the corrin ring as the protein-derived imidazole of the protein-bound B12
cofactors (Figs. 9 and 10). The distance between the Co-center and the axially
coordinated 1H-imidazole N-atom of 4 is 2.09 Š, which is 0.10 Š shorter than the

Helvetica Chimica Acta ± Vol. 83 (2000)
2309
Fig. 8. Two projections of the crystal structure of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4)
Fig. 9. Structure superposition of the B12 cofactors obtainedfrom the crystal structures of methylmalonyl-CoA
mutase (MCM), glutamate mutase (Glm), andof the B 12-binding domain of methionine synthase (MetH;
green), with that of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4; black)

2310
Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 10. Structure superposition of the corrin ligands of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4;
black) with those of the B12 cofactors obtainedfrom the crystal structures of methylmalonyl-CoA mutase (MCM),
glutamate mutase (Glm), andof the B 12-binding domain of methionine synthase (MetH; green)
corresponding distance to the Dmb N-atom in the crystal structure of methylcob-
(III)alamin (2) [36]. This ꢀshorteningꢁ of the axial CoÀN bond as a consequence of the
replacement of Dmb by Im underpins the similar observation in the crystal structure of
5 [46]. The shorter bond in 4 (and in 5) correlates with the smaller size and the higher
‡
nucleophilicity of 1H-imidazole as compared to Dmb (pKa(Im ´ H ) ˆ 7.0 [ 57 ] ,
‡
‡
pKa(Dmb ´ H ) ˆ 6.0 [58]), and pKa(a-ribazole ´ H ) ˆ 5.6 [60]).
Rossi et al. [36]
present work
Ludwig et al. [25][26]
Lenhert and Hodgkin [1][10]
Shibata et al. [33]
Evans et al. [26][27]
Reitzer et al. [28]
Fig. 11. a) Comparison of axial bondlengths of crystalline methyl-corrinoids andof methylcob(III)alamin bound
ꢀbase-off/His-onꢁ in methionine synthase from E. coli [24 ± 26]. b) Comparison of axial bondlengths of crystalline
coenzyme B12 andof corrinoidcofactors boundin methylmalonyl-CoA mutase from P. shermanii [26][27], in
glutamate mutase from C. cochlearium [28], and in diol dehydratase [33].

Helvetica Chimica Acta ± Vol. 83 (2000)
2311
The length of the CoÀNax bond in the crystal structure of 4 (2.09 Š) is thus shorter
than the corresponding bond to the protein-derived 1H-imidazole moiety in proteins
with a methylcob(III)alamin (2) [24] or (5'-deoxy-5'-adenosyl)cob(III)alamin (1)
cofactor (see Fig. 11) [27][28][37]. These data appear to suggest that protein-bound
methylcob(III)alamin (2) in the ꢀbase-off/His-onꢁ constitution of methionine synthase
has a strained CoÀNax bond, since the crystallographically observed bond length was
reported to be 2.2 Š. However, the experimental observation has to be taken with
caution since the crystal structure of MetH is of relatively low resolution (2.4 ± 8.0 Š)
[24][26], and it is conceivable that, during the crystallographic refinement, the bond
length was constrained to the value (2.2 Š) observed in the structure of methylco-
b(III)alamin (2). Nevertheless, the presence of such a stressed CoÀNax bond would
favor the cleavage of both axial bonds to the CoIII center. This may be necessary during
the catalytic cycle of this enzyme (and of other methyl transferases), which involves
heterolytic release of the Cob-methyl group to be transferred to a nucleophile. Such a
heterolytic CoÀC bond cleavage results in a (tetracoordinated) CoI metal center.
An important and frequently cited structural parameter concerns the conformation
of the corrin ligand, whose most pronounced deviation from planarity involves a
ꢀfoldingꢁ of the corrin ring about an axis running ꢀeast-to-westꢁ, i.e., through the meso-
atom C(10) and intersecting the C(1)ÀC(19) bond [61]. This deformation was termed
ꢀupward foldingꢁ, and it was suggested to be involved in enzyme-assisted CoÀC
homolysis (for a discussion, see, e.g., [55]). The deformation is measured as the
dihedral angle between best planes through rings A ‡ B and C ‡ D, respectively.
Observation of a negative correlation of this parameter with the length of the CoÀNax
bond in crystal structures of cobalamins led to the conclusion that large upward folding
is primarily the result of steric repulsion between the Dmb base and the lower face of
the corrin ring [11]. This view was initiated by the observation of a small fold angle
(10.88) in the crystal structure of Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide (5)
[46]. In the crystal structure of Coa-(1H-imidazol-1-yl)-Cob-methylcob(III)amide (4),
the observed upward folding is 12.58, ca. 28 smaller than in the structure of
methylcob(III)alamin (2) (14.68) [36]. Lackof resolution of presently available
crystallographic evidence of proteins with a B12 cofactor [24][27][28][33] prevents a
detailed appraisal of the relevance of the cofactorꢁs ꢀupward foldingꢁ for the catalysis of
the corresponding enzymes.
The observed elongation of the CoÀNax bond (ca. 2.5 Š) of the protein-bound
coenzyme B12 (1) in the crystal structure of methylmalonyl-CoA mutase, on the other
hand, is experimentally highly significant. Therefore, this bond was considered as
strained [27]. Likewise, the value observed for the length of this bond in the crystal
structure of glutamate mutase (2.27 ± 2.35 Š) is significantly longer than the value
observed for crystalline 5. In view of recent XAS evidence [38][62], it remains to be
shown whether and how such a strained CoÀNax bond can be linked to activation
towards CoÀC bond homolysis in this class of coenzyme-B12-dependent mutases.
3. Conclusions. ± In the present work, we studied some main patterns of the
organometallic reactivity of (1H-imidazol-1-yl)cobamides and the details of the crystal
structure of Coa-(1H-imidazol-1-yl)-Cob-methylcobamide (4), considered to be a
model for protein-bound (organometallic) corrin(III)cofactors. This workwas

2312
Helvetica Chimica Acta ± Vol. 83 (2000)
intended to provide insights into relevant effects of the substitution of the Dmb base of
the cobalamins by an imidazole moiety, as observed (in the ꢀligand triadꢁ) in several B12-
dependent proteins. First of all, these studies supported the view that the observed
ꢀlongꢁ axial CoÀN bonds of the Co-coordinated histidine in MetH [26], in the C-
skeleton mutases MCM [26][27] and Glm [28] would represent stretched CoÀN bonds,
provided that these observations do not represent experimental artifacts [39]. The long
axial CoÀN bond has been suggested to contribute in a crucial way to the activation of
the corrin-bound Co-center of the B12 cofactors, relevant for the initiation of the radical
reactions [26][27]. One of us has raised the alternative that the ꢀstretched CoÀN bondꢁ,
if crystallographically significant, may not mainly play a role in the activation of the
bound (5'-deoxy-5'-adenosyl)cobamides towards homolysis of their CoÀC bond, but
may be relevant for a later stage of the enzymatic radical rearrangement reactions [2].
The studies presented here also provide further experimental insights into the
importance of the inherent basicity of the imidazole moieties of the His-Asp part of the
ꢀligand triadꢁ, of particular relevance in enzymatic Me- and electron-transfer reactions
that are accompanied by the coordination/decoordination of a N-ligand at the corrin-
bound Co-center.
Experimental Part
1. General. Materials. Methylcob(III)alamin (2), prepared as described in [48]. Crystalline cyanocobalamin
3, supplied by Hoffmann-La Roche & Co. Ltd.; aquacobalamin chloride 8 ´ Cl, Roussel Uclaf, water (purified
using Epure, BarnsteadCo. ); acetone, Hg, (Bu4N)PF6, MeI, AcOH, KCl, KCN, KH2PO4, K2HPO4, NaOAc,
aq. HCl soln., all Fluka puriss. p.a., or Fluka MicroSelect; Me3CN, ClCH2COOH, HCOOH, benzoic acid, Fluka
purum; H3PO4 crystallized puriss.; MeOH, CH2Cl2 redistilled; 1% HCN in MeOH [63]. O2-Sensitive
manipulations were done in a glove box (Mecaplex GB-80, < 15 ppm O2). The electrochemical syntheses were
carried out in an electrolysis cell with two compartments, separated by a medium porosity glass frit; Hg-pool
working electrode, Pt-wire counter electrode, 0.1n calomel electrode (0.1n CE) as reference electrode;
potentiostat Amel 550. pH values were determined with a WTW SenTix 41 electrode connected to a WTW
inoLab digital pH meter. Reaction mixtures were desalted with Waters Sep-Pak Classic RP-18 cartridges. TLC:
RP-18 F 254s TLC plates 0.25 mm (Merck No. 115389). UV/VIS Spectra: Hitachi-U3000; lmax (log e) in nm. CD
Spectra: Jasco J715; lmax or lmin (De) in nm. 1H-NMR Spectra: Varian Unity 500plus; d(H) in ppm referenced to
HOD (ˆ4.66). FAB-MS: Finnigan MAT 95S, positive-ion mode; 3-nitrobenzyl alcohol matrix; Cs gun.
2. Syntheses. Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)amide (4). In a glove box (<10 ppm O2), 18 mg
(14.1 mmol) of Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide (5) and 2.2 mg (18.3 mmol) of benzoic acid
were dissolved in 4 ml of 0.1m Bu4N(PF6)/MeOH. The red soln. was placed into the cathode chamber of a two-
compartment electrolysis cell and was stirred magnetically. It was reduced at a Hg electrode at a potential of
À 1.1 V vs. a 0.1n CE reference (at r.t.) for 4.5 h, when 2.44 Coulombs were consumed. To the resulting green
soln. of the (1H-imidazol-1-yl)cob(I)amide, 6.9 ml (140.9 mmol) of MeI were added under protection from light,
while the cathode was kept at the same potential. After 140 min, the mixture was transferred into a separatory
funnel (with protection from light), that contained 5 ml of H2O. The darkred mixture was extracted with CH 2Cl2
(3 Â 5 ml), the aq. phase evaporated, and the dried residue (18.8 mg) taken up in 0.1 ml of H2O. Acetone was
added dropwise, until a slight turbidity appeared. After storage in a refrigerator overnight, 16 mg (89.7%) of
crystalline 4 was obtained. TLC: uniform: UV/VIS (c ˆ 8.69 ´ 10À4m, H2O): 268(4.16), 314(4.07), 341(4.11),
376(4.07), 528(3.91); Fig. 3. CD (c ˆ 8.69 ´ 10À4m, H2O): 219 (37143), 258 (À45322), 284 (4314), 314 (À27490),
337 (À2511), 358 (À18218), 398 (13483), 417 (6401), 477 (37432), 564 (À11228). 1H-NMR (pH 5, H2O
(600 ml)/D2O (100 ml), 500 MHz; Fig. 4): À0.21 (s, b-MeÀCo); 0.49 (s, MeÀC(1)); 0.82 (s, 3 H); 1.06 (d, J ˆ 6.6,
3 H); 1.17 (s, 3 H); 1.28 (s, 3 H); 1.29 (s, 3 H); 1.36 (m, 1 H); 1.59 (s, 3 H); 1.64 (m, 1 H); 1.71 (m, 2 H); 1.79 (m, 1
H); 1.89 (m, 2 H); 1.93 (m, 2 H); 1.97 (m, 1 H); 2.04 (m, 1 H); 2.16 (m, 1 H); 2.18 (s, 3 H); 2.24 (s, 3 H); 2.30 (m,
1 H); 2.33 (m, 2 H); 2.35, 2.37 (m, 4 H); 2.55 (m, 2 H); 2.91 (t, J ˆ 5.9, 1 H); 3.04 (dt, J ˆ 14.4, 7.3, 1 H); 3.31 (dd,
J ˆ 10.3, 4.6, 1 H); 3.40 (dd, J ˆ 14.4, 1.9, 1 H); 3.56 (dd, J ˆ 12.7, 4.2, 1 H); 3.71 (dd, J ˆ 12.7, 2.5, 1 H); 3.74 (d,

Helvetica Chimica Acta ± Vol. 83 (2000)
2313
J ˆ 9.4, 1 H); 3.81 (d, J ˆ 8.8, 1 H); 3.88 (dt, J ˆ 8.1, 2.9, 1 H); 4.12 (dd, J ˆ 4.0, 1 H); 4.19 (dq, J ˆ 6.8, 1.9, 1 H);
4.47 (dt, J ˆ 7.9, 4.6, 1 H); 5.76 (s, 1 H); 7.78 (d, J ˆ 3.55, 1 H); 5.80 (s, 1 H); 6.61 (br. s, 1 H); 6.68 (s, 1 H); 6.71 (s,
1 H); 6.76 (br. s, 2 H); 6.79 (br. s, 1 H); 6.92 (br. s, 1 H); 6.93 (br. s, 1 H); 7.06 (br. s, 1 H); 7.46 (br. s, 1 H); 7.49
(br. s, 1 H); 7.52 (br. s, 1 H); 7.62 (br. s, 1 H); 7.76 (br. s, 1 H); 8.03 (t, J ˆ 6.1, 1 H). FAB-MS: 1268.8 (32), 1267.8
‡
‡
(61), 1266.8 (100, [M ‡ H] ); 1253.7 (26), 1252.7 (55), 1251.7 (82, [M ‡ H À CH3] ).
‡
Coa-(1H-Imidazol-1-yl)-Cob-aquacob(III)amide (7 ). A soln of 4 (6.0 mg, 4.5 mmol) in H2O (4 ml) was
saturated with O2 and irradiated with light (500 W) for 50 min. The H2O was evaporated, the residue taken up in
a minimal amount of H2O, and the product precipitated by diffusion of acetone vapor into the aq. soln. at r.t. The
precipitate was washed with acetone (3Â): 5.1 mg (85%) of pure Coa-(1H-imidazole-1-yl) Cob-hydroxocob-
(III)amide. TLC (RP18, MeCN/H2O 3 :7): Rf 0.05). UV/VIS (0.02m phosphate buffer pH 6.3; rel. e; Fig. 5): 273
(0.61), 355 (1.00), 410 (0.16), 532 (0.35). 1H-NMR (9 mm aq. phosphate, pH 6.8): 0.66 (s, MeÀC(1)); 1.12 (s, 3
H); 1.13 (s, 3 H); 1.20 (s, 3 H); 1.26 (s, 3 H); 1.42 (s, 6 H); 1.71 (s, 3 H); 2.37 (s, 3 H) superimposed by 1.1 ± 3.0
(various m, in total ca. 86 H), 3.2 ± 4.2 (m, ca. 13 H); 4.43 (m, HÀC(3')(Rib)); 5.44 (s, 1 H); 5.71 (s, 1 H); 6.16
(2s, 2 H); 6.69 (s, HÀC(5) (Im)).
‡
3. Determination of the pKa 4 ´ H . Aq. solns. of 4 (8 ´ 10À4m), 0.1m buffer (HCl, chloroacetate, acetate,
formate, or phosphate) and KCl (total ionic strength 1.0m) were prepared (with pH values 7.76, 6.50, 5.65, 4.48,
3.84, 3.24, 2.93, 2.10, and 0.30). Absorbance spectra of these solns. obtained at r.t. showed isosbestic points at
498, 388, and 334 nm (see Fig. 6). The spectral changes at 538, 455, and 304 nm were analyzed according to
Eqn. 6 by the method of least squares, where Ax is the absorbance at a given wavelength of the solution at pHx,
‡
and AAH and AAÀ are the corresponding absorbances of 4 ´ H and 4, determined (in duplicate) at the titration
end points. The intercepts resulting from these fits of the data determined at 538, 455, and 304 nm, resp., were
‡
4.22, 4.33, and 4.35, yielding a value of 4.3 Æ 0.1 for pKa (4 ´ H ).
pHx ˆ pKa ‡ log(jAx À AAH j / j AAÀ À Ax j )
(6)
4. Methyl-Transfer Experiments. 4.1 Equilibration Experiments with Methylcob(III)amides and Cob(III)-
amides (Scheme 2). The reactions were carried out in stoppered NMR tubes in 9 mm aq. phosphate buffers (10%
D2O), pH 6.8, and monitored directly by 1H-NMR. Reaction mixture A was produced by dissolving Coa-(1H-
imidazol-1-yl)-Cob-hydroxocob(III)amide (4.5 mg, 3.55 mmol) and methylcob(III)alamin (4.4 mg, 3.27 mmol;
2) in aq. phosphate (0.7 ml). Reaction mixture B was prepared likewise by dissolving 4 (3.5 mg, 2.77 mmol) and
aquacob(III)alamin chloride (5.3 mg, 3.83 mmol; 8 ´ Cl). Two hours after the preparation, the 500-MHz 1H-NMR
revealed significant changes in mixture B and only small changes in mixture A. Further NMR spectra were
recorded after 2, 8, 22, and 30 days. Before the final analysis, the equilibrated mixtures were treated each with
aq. KCN (0.5 mg, ca. 8 mmol) soln. yielding mixtures of Cob-methyl- and Cob-cyanocob(III)amides. The solvent
1
was evaporated at r.t., the residue of both mixtures dissolved in D2O, and the H-NMR recorded (see Table).
4.2. Equilibration Experiments with Methylcob(III)amides and Cob(II)amides (Scheme 3). By electro-
chemical one-electron reduction of Coa-(1H-imidazol-1-yl)-Cob-cyanocob(III)amide (3.6 mg, 2.8 mmol; 5) in
6 ml of a buffered O2-free aq. soln. (0.01m phosphate buffer, pH 6.8; 0.1m KCl) at À 1.0 V vs. 0.1n CE, a buffered
aq. soln. of 2.8 mmol of (1H-imidazol-1-yl)cob(II)amide (9) was prepared (UV/VIS (9 in H2O; rel. e): 264
(0.75), 312 (1.00), 473 (0.38)). The extent of the reduction was monitored coulometrically, and the UV/VIS of
the reduced reaction mixture exhibited the absorbance characteristics of a base-on cob(II)amide [58]. The soln.
of 9 was then mixed (with protection from light and air) with 0.5 ml of an aq. soln. of 2 (3.7 mg, 2.8 mmol),
buffered similarly (0.01m phosphate, pH 6.8; 0.1m KCl), and left to stand at r.t. (with protection from light and
air). After an equilibration time of 2 h, the stoppered reaction flaskwas taken out of the glove box and O 2-sat.
1% HCN/MeOH (0.5 ml) was injected (with protection from light). For further analysis, the mixture was first
desalted by filtration through an RP-18 cartridge and then examined by TLC (4 spots of 2 ± 5) and by 1H-NMR
(500 MHz) (see Table).
Likewise, by electrochemical one-electron reduction of vitamin B12 (3; 14 mg, 10.1 mmol) in 4 ml of a
buffered O2-free aq. soln. (0.01m phosphate buffer, pH 6.8; 0.1m KCl), a buffered aq. soln. of cob(II)alamin (10)
was prepared (coulometrical monitoring; UV/VIS base-on cob(II)alamin [58]; see above). An aliquot of 1 ml
of the soln. (containing 2.8 mmol) of 10 was then mixed (with protection from light and air) with 0.5 ml of an aq.
soln. of 4 (3.5 mg, 2.8 mmol), buffered similarly (0.01m phosphate, pH 6.8; 0.1m KCl). The mixture was left to
stand for 2 h at r.t. (with protection from light and air). The stoppered flaskwas taken out of the glove box,
treated with O2-sat. 1% HCN/MeOH, desalted, and analyzed by TLC (4 spots of 2 ± 5) and 1H-NMR as
described above (see Table).

2314
Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 12. UV/VIS Spectrum of electrochemically produced (1H-imidazol-1-yl)cob(I)amide (6À) in aqueous
solution
4.3. Equilibration Experiments with Methylcob(III)amides and Cob(I)amides (Scheme 4). By electro-
chemical reduction at À 1.1 V vs. 0.1n CE of 5 (3.5 mg, 2.7 mmol) in 5 ml of a buffered O2-free aq. soln., a
buffered aq. soln. of 2.7 mmol of (1H-imidazol-1-yl)cob(I)amide (6À) was prepared. (UV/VIS (6Àin H2O, rel. e):
280 (0.95), 296 (1.00), 386 (0.97), 453 (sh, 0.21), 546 (0.16); see Fig. 12). The extent of the reduction was
monitored amperometrically and UV/VIS-spectroscopically [50]. To the soln. of 6À, crystalline 2 (3.4 mg,
2.5 mmol) was added (glove box, protection from light). The mixture was left to stand at r.t. for 15 min (with
protection from light and air). UV/VIS Analysis indicated the presence of a mixture of cob(I)amides and
alkylcob(III)amides. The mixture was then taken out of the glove box, treated with O2-sat. 1% HCN/MeOH,
desalted, and analyzed by TLC (significant amounts of all 2 ± 5 and 1H-NMR as described in Exper. 4.2 (see
Table).
Likewise, by electrochemical reduction at À 1.1 V vs. 0.1n CE of vitamin B12 (14 mg, 10.3 mmol; 3) in 4 ml
of a buffered O2-free aq. soln., a buffered aq. soln. of 10.3 mmol of cob(I)alamin (11À) was prepared. The extent
of the reduction was monitored amperometrically and UV/VIS-spectroscopically [56]. One fourth of the soln. of
11À (ca. 2.7 mmol) were added (glove box, protection from light) to a soln. of crystalline 4 (3.5 mg, 2.8 mmol) in
0.5 ml of 10 mm phosphate buffer (pH 6.8). The mixture was left to stand at r.t. for 30 min (with protection from
light and air). UV/VIS Analysis indicated the presence of a mixture of cob(I)amides and alkylcob(III)amides.
The mixture was then taken out of the glove box, treated with O2-sat. 1% HCN/MeOH, desalted, and analyzed
1
by TLC (significant amounts of all 2 ± 5) and H-NMR (see Table).
5. Crystal-Structure Analysis of Coa-(1H-Imidazol-1-yl)-Cob-methylcob(III)amide (4). Crystals were
grown from H2O/acetone under exclusion of light. A crystal of approximate dimensions 0.2 Â 0.1 Â 0.1 mm was
removed from its mother liquor and immersed in a drop of hydrocarbon oil. After removing adhering mother
liquor from the surface of the crystal with a strip of filter paper, the crystal was picked up with a glass fiber and
shock-cooled by dumping into liq. N2 [46]. Low-temp. (92 K) intensity data (l 0.9076 Š) were collected at the
EMBL beamline X11 at DESY in Hamburg, Germany, using a MAR345 image-plate goniometer. The images
were integrated with the program DENZO [64], subsequent scaling steps were performed with the CCP4 [65]
program suite, yielding 17820 reflections (2qmax ˆ 548, resolution limit 1.0 Š), of which 9011 symmetry-
independent (Rint ˆ 0.0307). Space group P212121, a ˆ 15.95(1), b ˆ 22.10(1), c ˆ 25.92(1) Š. The structure was
solved with direct methods and refined against F2 quantities by full-matrix least squares. C57H83N13O14 plus 16
partly disordered solvent molecules; m ˆ 0.28 mmÀ1; dx ˆ 1.111 g cmÀ3; R ˆ 0.0857 for 8713 Fo > 4s(Fo) and
0.0871 for all 9011 reflexions (155 restraints); wR2 ˆ 0.2542. The following computer programs were used:
SHELXTL Version 5 [66]; SHELXL-93 [67]. Crystallographic data (excluding structure factors) have been
deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 144874.

Helvetica Chimica Acta ± Vol. 83 (2000)
2315
Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: ‡ 441223336033, e-mail: deposit@ccdc.cam.ac.uk).
We would like to thank Wolfgang Fieber, Christian Eichmüller, and Robert Konrat for NMR spectra,
Alexander Rieder and Karl-Hans Ongania for recording FAB mass spectra, and F. Hoffmann-LaRoche & Co.,
Basel, for a generous sample of vitamin B12. Crystallographic diffraction data for Coa-(1H-imidazol-1-yl)-Cob-
methylcob(III)amide were collected at the EMBL-beamline X11 at DESY in Hamburg, Germany. We
acknowledge financial support from the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung
through projects 11599 (CK) and 12639 (BK), and from the European Commission (TMR Project No. ERB
4061 PL 95-0307).
REFERENCES
[1] P. G. Lenhert, D. C. Hodgkin, Nature (London) 1961, 192, 937.
[2] B. Kräutler, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and B. T. Golding, Wiley-VCH,
Weinheim, 1998, p. 3.
[3] B. T. Golding, W. Buckel, in ꢀComprehensive Biological Catalysisꢁ, Vol. 3 Ed. M. Sinnott, Academic Press,
London, 1998, p. 239.
[4] R. Banerjee, ꢀChemistry and Biochemistry of B12ꢁ, John Wiley & Sons, New York, 1999.
[5] B. Kräutler, D. Arigoni, B. T. Golding, ꢀVitamin B12 and B12-Proteinsꢁ, Wiley-VCH, Weinheim, 1998.
[6] J.-A. Stubbe, S. Licht, G. Gerfen, D. Silva, S. Booker, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D.
Arigoni, and B. T. Golding, Wiley-VCH, Weinheim, 1998, p. 321.
[7] R. G. Matthews, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons, New York,
1999, p. 681.
[8] K. Sauer, R. K. Thauer, in ꢀChemistry and Biochemistry of B12ꢁ Ed. R. Banerjee, John Wiley & Sons, New
York, 1999, p. 655.
[9] S. W. Ragsdale, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons, New York,
1999, p. 633.
[10] J. P. Glusker, in ꢀB12ꢁ, Ed. D. Dolphin, Wiley-Interscience, New York, 1982, Vol. 1, p. 23.
[11] K. Gruber, G. Jogl, G. Klintschar, C. Kratky, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni,
and B. T. Golding, Wiley-VCH, Weinheim, 1998, p. 335.
[12] C. Kratky, B. Kräutler, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons, New
York, Vol. 1, 1999, p. 9.
[13] R. Konrat, M. Tollinger, B. Kräutler, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and
B. T. Golding, WILEY-VCH, Weinheim, 1998, p. 349.
[14] J. M. Pratt, ꢀInorganic Chemistry of Vitamin B12ꢁ, Academic Press, London, 1972.
[15] D. C. Hodgkin, J. Kamper, M. Mackay, J. Pickworth, K. N. Trueblood, J. G. White, Nature (London) 1956,
178, 64.
[16] A. R. Battersby, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and B. T. Golding, Wiley-
VCH, Weinheim, 1998, p. 47.
[17] A. I. Scott, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and B. T. Golding, Wiley-VCH,
Weinheim, 1998, p. 81.
[18] P. Renz, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons, New York, 1999,
pp. 557.
[19] J. C. Escalante-Semerena, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons,
New York, 1999, p. 577.
[20] A. Eschenmoser, Angew. Chem. 1988, 100, 5.
[21] R. B. Woodward, in ꢀVitamin B12ꢁ, Eds. B. Zagalakand W. Friedrich, W. deGruyter, Berlin, 1979, p. 37.
[22] D. Dolphin, ꢀB12ꢁ, Wiley-Interscience, New York, 1982, Vol. 1.
[23] W. Friedrich, ꢀVitamin B12 und verwandte Corrinoideꢁ, Thieme Verlag, Stuttgart, 1975, Vol. III/2.
[24] C. L. Drennan, S. Huang, J. T. Drummond, R. G. Matthews, M. L. Ludwig, Science (Washington, DC) 1994,
266, 1669.
[25] M. L. Ludwig, C. L. Drennan, R. G. Matthews, Structure 1996, 4, 505.
[26] M. L. Ludwig, P. R. Evans, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley and Sons,
New York, 1999, Vol. 1, p. 595.
[27] F. Mancia, N. H. Keep, A. Nakagawa, P. F. Leadlay, S. McSweeney, B. Rasmussen, P. Bosecke, O. Diat, P. R.
Evans, Structure 1996, 4, 339.

2316
Helvetica Chimica Acta ± Vol. 83 (2000)
[28] R. Reitzer, K. Gruber, G. Jogl, U. G. Wagner, H. Bothe, W. Buckel, C. Kratky, Structure 1999, 7, 891.
[29] E. N. G. Marsh, D. E. Holloway, FEBS Lett. 1992, 310, 167.
[30] A. Abend, R. Nitsche, V. Bandarian, E. Stupperich, J. Retey, Angew Chem. Int. Ed. 1998, 37, 625.
[31] M. Yamanishi, S. Yamada, H. Muguruma, Y. Murakami, T. Tobimatsu, A. Ishida, J. Yamauchi, T. Toraya,
Biochemistry 1998, 37, 4799.
[32] C. C. Lawrence, G. J. Gerfen, V. Samano, R. Nitsche, M. J. Robins, J. Retey, J. Stubbe, J. Biol. Chem. 1999,
274, 7039.
[33] N. Shibata, J. Masuda, T. Tobimatsu, T. Toraya, K. Suto, Y. Morimoto, N. Yasuoka, Structure 1999, 7, 997.
[34] J. Halpern, Science (Washington, DC) 1985, 227, 869.
[35] B. Kräutler, C. Kratky, Angew. Chem. Int. Ed. Engl. 1996, 35, 167.
[36] M. Rossi, M. F. Summers, L. Randaccio, P. J. Toscano, J. P. Glusker, L. G. Marzilli, J. Am. Chem. Soc. 1985,
107, 1729.
[37] E. Scheuring, R. Padmakumar, R. Banerjee, M. R. Chance, J. Am. Chem. Soc. 1997, 119, 12192.
[38] F. Champloy, G. Jogl, R. Reitzer, W. Buckel, H. Bothe, A. Michalowicz, W. Meyer-Klaucke, C. Kratky, J.
Am. Chem. Soc. 1999, 121, 11780.
[39] F. Champloy, K. Gruber, G. Jogl, C. Kratky, J. Synchrotron Rad. 2000, in press.
[40] R. G. Finke, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and B. T. Golding, Wiley-VCH,
Weinheim, 1998, p. 383.
[41] B. Kräutler, C. Kratky, W. Keller, J. Am. Chem. Soc. 1989, 111, 8936.
[42] J. H. Grate, G. N. Schrauzer, J. Am. Chem. Soc. 1979, 101, 4601.
[43] S. M. Chemaly, J. M. Pratt, J. Chem. Soc., Dalton Trans. 1980, 2259.
[44] B. Kräutler, Helv. Chim. Acta 1987, 70, 1268.
[45] J. Kopf, R. Bieganowski, W. Friedrich, K. Vondeuten, Z. Naturforsch. 1981, 36, 506.
[46] B. Kräutler, R. Konrat, E. Stupperich, G. Färber, K. Gruber, C. Kratky, Inorg. Chem. 1994, 33, 4128.
[47] B. Kräutler, in ꢀChemistry and Biochemistry of B12ꢁ, Ed. R. Banerjee, John Wiley & Sons, New York, 1999,
p. 315.
Â
[48] M. Tollinger, T. Derer, R. Konrat, B. Kräutler, J. Mol. Catal. 1997, 116, 147.
[49] D. Dolphin, in ꢀInorganic Synthesisꢁ, Ed. D. H. Busch, John Wiley & Sons, New York, 1980, Vol. 20 p. 136.
[50] K. L. Brown, J. M. Hakimi, D. M. Nuss, Y. D. Monteyano, D. W. Jacobsen, Inorg. Chem. 1984, 23, 1463.
[51] Y.-T. Fanchiang, G. T. Bratt, H. P. C. Hogenkamp, Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2698.
[52] B. Kräutler, in ꢀThe Biological Alkylation of Heavy Elementsꢁ, Eds. P. J. Craig, and F. Glockling, Royal
Society of Chemistry, London, 1988, p. 31.
[53] E. Stupperich, B. Kräutler, Arch. Microbiol. 1988, 149, 268.
[54] B. Kräutler, C. Caderas, Helv. Chim. Acta 1984, 67, 1891.
[55] C. Kratky, G. Färber, K. Gruber, K. Wilson, Z. Dauter, H.-F. Nolting, R. Konrat, B. Kräutler, J. Am. Chem.
Soc. 1995, 117, 4654.
Â
[56] D. Lexa, J. M. Saveant, Acc. Chem. Res. 1983, 16, 235.
[57] S. P. Datta, A. K. Grzybowski, J. Chem. Soc. B 1966, 136.
[58] M. T. Davies, P. Mamalis, V. Petrow, B. Sturgeon, J. Pharm. Pharmacol. 1951, 3, 420.
[59] U. Harms, R. K. Thauer, in ꢀVitamin B12 and B12-Proteinsꢁ, Eds. B. Kräutler, D. Arigoni, and B. T. Golding,
Wiley-VCH, Weinheim, 1998, p. 157.
[60] K. L. Brown, J. M. Hakimi, J. Am. Chem. Soc. 1986, 108, 496.
[61] V. B. Pett, M. N. Liebman, P. Murray-Rust, K. Prasad, J. P. Glusker, J. Am. Chem. Soc. 1987, 109, 3207.
[62] F. Champloy, M. Giorgi, M. A. M. Pierrot, J. Synchrotron Res. 1997, 4, 36.
[63] C. Caderas, B. Kräutler, in C. Caderas, ꢀAufbau von binuklearen und überbrückten Cobyrinsäureder-
ivatenꢁ, Ph.D. Thesis, ETH No. 7822, ADAG Administration & DruckAG Zürich, 1987.
[64] Z. Otwinowsky, in ꢀData Collection and Processingꢁ, Eds. L. Sawyer, N. Isaacs, and S. Bailey, Daresbury
Laboratory, Warrington WA44AD, England, 1993, p. 55.
[65] CCP4, Acta Crystallogr., Sect. D 1994, 50, 760.
[66] G. M. Sheldrick, ꢀSHELXTL Version 5ꢁ, Siemens Industrial Automation, Inc., Madison, WI., USA, 1990.
[67] G. M. Sheldrick, ꢀSHELXL-93, a Program for the Refinement of Crystal structures from Diffraction Data,
University of Göttingen, Göttingen, Germany, 1993.
ReceivedMay 26, 2000