Partial Synthesis of Coenzyme B12 from Cobyric Acid

Article abstract of DOI:10.1002/hlca.201700170

Here we report the direct chemical synthesis of coenzyme B₁₂ (AdoCbl) from Co_β-5′-deoxyadenosylcobyric acid (AdoCby) and the preparation of the latter from crystalline CN,H₂O-cobyric acid (CN,H₂OCby). AdoCby is a suggested common key intermediate in the biosynthesis of AdoCbl and of other cobamides in microorganisms. AdoCby was thoroughly characterized by spectroscopic means, including homo-nuclear and hetero-nuclear NMR, as such data are not available in published work. AdoCbl was prepared from AdoCby in one-step in over 85% yield, by covalent attachment in aqueous solution of the integral B₁₂-nucleotide moiety using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC·HCl) and N-hydroxybenzotriazole (HOBt) as coupling reagents. By the same procedure crystalline vitamin B₁₂ (CNCbl) was also prepared in 92% yield from CN,H₂OCby. Coordination of the B₁₂-nucleotide base at the Co_α-face of AdoCby or of CN,H₂OCby was indicated to assist in the efficient covalent coupling at the activated f-side chain function to furnish the complete corrinoids AdoCbl and CNCbl.

Full text of DOI:10.1002/hlca.201700170

1
1
Florian J. Widner , Fabian Gstrein and Bernhard Kräutler *
Institute of Organic Chemistry and Center of Molecular Bioscience Innsbruck (CMBI), University of
Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria, bernhard.kraeutler@uibk.ac.at
nd
Dedicated to Prof. Albert Eschenmoser on the occasion of his 92 birthday
Here we report the direct chemical synthesis of coenzyme B12 (AdoCbl) from Co
deoxyadenosylcobyric acid (AdoCby) and the preparation of the latter from crystalline CN,H
cobyric acid (CN,H OCby). AdoCby is a suggested common key intermediate in the biosynthesis of

-5’-
2
O-
2
AdoCbl and of other cobamides in microorganisms. AdoCby was thoroughly characterized by
spectroscopic means, including homo-nuclear and hetero-nuclear NMR, as such data are not
available in published work. AdoCbl was prepared from AdoCby in one-step in over 85% yield, by
covalent attachment in aqueous solution of the integral B12-nucleotide moiety using 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC*HCl) and N-hydroxybenzotriazole (HOBt) as coupling
reagents. By the same procedure crystalline vitamin B12 (CNCbl) was also prepared in 92 % yield from
CN,H
2
OCby. Coordination of the B12-nucleotide base at the Co

-face of AdoCby or of CN,H OCby was
2
indicated to assist in the efficient covalent coupling at the activated f-side chain function to furnish
the complete corrinoids AdoCbl and CNCbl.
Keywords: Amide bond • biomimetic synthesis • cobalamin • porphyrinoid • vitamin B12
Introduction
Nucleotide containing ‘complete’ B12-derivatives, like vitamin B12 (cyanocobalamin, CNCbl, 1),
coenzyme B12 (5’-deoxyadenosylcobalamin, AdoCbl, 2) and methylcobalamin (MeCbl) play
[
1-4]
indispensable roles in the metabolism of various microorganisms, animals and humans.
Keys to
their ubiquitous biological functions are their special structures, their organometallic chemistry,
[
5-7]
their redox chemistry and photochemistry.
However, only certain prokaryotes have developed
[
8, 9]
the capacity to biosynthesize natural corrinoids.
So far, two pathways of their biosynthesis have
been elucidated, one aerobic (oxygen dependent), and the other one anaerobic (oxygen
independent). For both of these routes the light sensitive organometallic 5’-deoxyadenosyl-cobyric
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acid (AdoCby, 3) is a suggested key intermediate, from which coenzyme B12 (AdoCbl, 2) is
[
8, 9]
‘
completed’ by formal biosynthetic attachment of the B12-nucleotide moiety of the cobalamins.
Figure 1. Structural formulas (left) of the cob(III)alamins vitamin B12 (CNCbl, L = CN, 1) and coenzyme B12 (AdoCbl, L = 5’-deoxy-5’-adenosyl, 2) and (right)
dicyano-Co(III)-forms of cobyrinic acid, of its activated hepta-cyanomethyl ester and of cobyric acid (CN
respectively).
2 2 2
Cbic, CN Cbic cyanomethyl ester and CN Cby,
The unique structures of cyano,aquo-cobyric acid (CN,H
2
O-Cby, 4) and vitamin B12 (CNCbl, 1) (see
Figure 1) are known from pioneering single crystal x-ray analysis in the laboratories of
[
10, 11]
[12, 13]
D.C.Hodgkin,
subsequently also prepared in about 40% yield by chemical attachment of the B12-nucleotide (R-2-
amino-1-methylethyl)-3’-(-ribazolyl)-diphosphate (5) at the f-acid function of CN,H O-Cby (4),
O-Cby (4) became the key target of the (formal)
following the isolation of CNCbl (1) from natural sources.
CNCbl (1) was
2
[
14]
activated with ethyl chloroformate. Hence, CN,H
2
total synthesis of vitamin B12 (1), and was the product of its total synthesis by Eschenmoser and
[
15, 16]
[17]
coworkers
and by Woodward and coworkers in the 1970es. The Woodward group then
reported the preparation of CNCbl (1) in 73% yield from synthetic CN,H
2
O-Cby (4) by condensation
with synthetic (R-2-amino-1-methylethyl)-3’-(-ribazolyl)-diphosphate (5), thus actually also
[
18]
completing the total synthesis of vitamin B12 (1).
In a fundamentally different approach, Bartels and Eschenmoser prepared crystalline vitamin B12 (1)
in 44% yield from undifferentiated, activated cobyrinic acid (cyano,acetato-cobyrinic acid
heptacyanomethyl ester, see Figure 1) via highly selective covalent attachment of the B12 nucleotide
portion (R-2-amino-1-methylethyl)-3’-(-ribazolyl)-diphosphate (5) at the ‘natural’ f-side chain,
[
19]
followed by ammonolysis of the six remaining activated ester groups. The amazing selectivity of
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this B12-reconstitution experiment was rationalized by pre-coordination of the nucleotide moiety to
the cobalt center of the ()-isomeric mixture of the CN,acetato-cobyrinic acid hepta-ester,
allowing for an outstandingly selective condensation of the pre-coordinated B12-nucleotide (5) at the
f-side chain. This type of a reconstitution of vitamin B12 (1) delineated a ‘primitive’ (non-enzymatic)
path from ‘incomplete’ and undifferentiated cobyrinic acid to the specific structure of ‘complete’
[
19, 20]
corrinoids, such as the cobalamins.
Figure 2. Outline of the partial syntheses of coenzyme B12 (AdoCbl, 2) and of vitamin B12 (CNCbl, 1) from cyano-aquo-cobyrinc acid (CN,H
2
OCby, 4) in aqueous
solution. i) HOBt, EDC*HCl, H
for further details).
2
O; ii) = NaBH
4
, 5’-iodo-5’-deoxyadenosine, MeOH/10 mM phosphate buffer pH=7 (1:1), argon; iii) HOBt, EDC*HCl, H O (see text
2
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Crystalline coenzyme B12 (AdoCbl, 2) was isolated in the early 1960es as corrinoid product of a
[
21]
‘
guided’ biosynthesis in Clostridium tetanomorphum. The genuine cofactor of this organism is the
[
22]
crystallization resistant pseudocoenzyme B12, as was also discovered in the Barker laboratories. X-
ray analysis of crystals of AdoCbl (2) revealed its enigmatic organometallic nature. AdoCbl (2) was
subsequently prepared by partial chemical synthesis, via reduction of aquocobalamin (H OCbl) or
[
23]
2
[
24, 25]
vitamin B12 (CNCbl, 1), followed by adenosylation at the cobalt center.
Herein, we report the partial synthesis of Co
aquo,cyano-cobyric acid (CN,H O-Cby, 4) and the direct preparation of AdoCbl (2) and CNCbl (1) in
aqueous solution via covalent attachment of the B12-nucleotide moiety (R-2-amino-1-methylethyl)-
’-(-ribazolyl)-diphosphate, 5) to AdoCby (3) or CN,H OCby (4), respectively (see Figure 2).
Furthermore, the cobyric acid derivatives AdoCby (3) and CN,H OCby (4) were tested qualitatively

-5’-deoxyadenosyl-cobyric acid (AdoCby, 3) from
2
3
2
2
for their capacity to coordinate N-methyl-imidazole in aqueous medium, in order to gain insights
into the coordination tendency of imidazoles to such Cbys. Along the lines of Eschenmoser’s
[
19]
pioneering experiments, such a pre-cordination of the benzimidazole base of the B12-nucleotide
[
26]
moiety would increase the effective molarity
of the latter for condensation reactions at the f-side
chain of cobyric acids. This would represent a relevant factor in accelerating the amide-forming
reaction via a properly pre-organized intramolecular path, and leading to an increase of the chemical
yield of the desired cobalamins.
Results and Discussion
Preparation of Co
β
-5’-deoxyadenosyl-cobyric acid (AdoCby, 3) from CN,H O-cobyric acid (4).
2
2
.00 mg of crystalline Co

CN,Co
 2
H O-Cby (4) were dissolved in MeOH/potassium phosphate buffer
(10mM, pH=7, see Supporting Information, Figure S1). The Co(III)-corrinoid was reduced under
anaerobic conditions using an excess of sodium borohydride and transformation into cob(I)yric acid
was verified by a UV/Vis-spectrum (see Supp. Info Figure S2). 1.2 equivalents of 5’-iodo-5’-
deoxyadenosine were added under protection from air and daylight, and an immediate color change
to orange was observed (and formation of AdoCby (3) – see Supporting Information, Figure S2).
From the reaction mixture, 1.85 mg of AdoCby (3, 74%) was isolated as orange powder.
The UV/Vis-spectrum of Co
absorption maxima at 457, 378 and 303 nm, similar to published spectra of AdoCby (3), and of
β
-5’-deoxyadenosyl-cobyric acid (AdoCby, 3, see Figure 3) showed
[
27]
[
28]
Co -5’-deoxyadenosylcobinamide (AdoCbi). The CD-spectrum of AdoCby (3) (see Supp. Info Figure
β
[
29]
S3) displayed the band structure of AdoCbi. A (positive ion) ESI-MS spectrum of AdoCby (3)
+
showed the pseudo-molecular ion [C55
H
77CoN15
O
11] as base-peak at m/z=1182.4 and its sodium
+
adduct [C55
H
76CoN15NaO11] at m/z=1204.4 (see Supporting Information, Figures S4 and S5).
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1
A 500 MHz H-NMR spectrum of AdoCby (3) in D
2
O showed resonances for all carbon-bound H
C5RL
atoms, including the characteristic doublet- and triplet-like signals at high field for Co-bound H
2
of the Ado ligand (see Figure 4). In the low field region, the protons HC2L and HC8L of the adenine
base were detected at 8.20 ppm and 8.01 ppm, respectively. The proton signals of the ribose moiety
1
1
could be assigned on the basis of a set of continuous H, H-COSY correlations from the C5RL protons
at high field (at 0.35 and 0.50 ppm) all the way to HC1RL at 5.59 ppm (see Supporting Information,
Figure S8). The two diastereotopic protons at C5RL were assigned to Hre at 0.35 ppm (H
.5 Hz, J = 8.8 Hz) and to Hsi at 0.50 ppm (H
A
C5RL, dd, J =
1
9
B
C5RL, d, J = 8.8 Hz), in analogy to assignments in H-NMR
1
1
spectra of coenzyme B12 (2). The H, H-COSY spectrum shows a large coupling for H
HC4RL (9.5 Hz) and a small unresolved coupling for H C5RL (Hsi) ↔ HC4RL, consistent with torsion
angles (-169° and 75°, respectively) observed in the crystal of coenzyme B12 (2)
A
C5RL (Hre) ↔
B
[
2]
.
Furthermore,
1
1
H
B
C5RL (Hsi) exhibits strong H, H-ROESY correlations with HC3RL, HC4RL and HC19 and a weak
1
1
H, H-ROESY correlation to H C81. H C5RL (H ), on the other hand, shows a strong correlation with
2
A
re
HC3RL but none to HC19. These data indicate the adenosyl group attached at the -face of the
1
1
cobalt-center of AdoCby (3) (see Supporting Information, Table S2). H, H NOE spectra, furthermore,
indicated a similar positioning of the 5′-deoxyadenosyl moiety above the rings C and D (see Supp.
[
28]
[30,31]
Info Figure S7), as observed in AdoCbi
HC8L↔H C12B, HC1RL↔H C17B, HC1RL↔H
C5RL↔HC19 found here in the spectra of AdoCby (3), provide evidence for very similar
and coenzyme B12 (2).
The major NOE cross peaks
3
3
3
3
C12B, HC4RL↔H C12B, HC4RL↔HC19 and
H
2
[
28]
conformations of its adenosyl moiety as deduced for AdoCbi , as well as in AdoCbl (2) “base on”
[
30]
[31]
and protonated “base off” forms.
Such NOE correlations were interpreted as a dynamic
equilibrium between two major conformations, one of them similar to that found in the crystal
[
31]
structure, and the other with the adenosyl group rotated 50° counterclockwise.
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1
3
With few exceptions, the chemical shifts of corresponding protons and C-atoms in the NMR spectra
of AdoCby (3) and AdoCbi are very similar (see Supp. Info Table S1). However, the H C172 protons
2
are shifted up-field by about 0.45 ppm in the AdoCby (3) spectrum. Likewise, among the carbon
atoms of AdoCby (3) and of AdoCbi, only C173 is shifted significantly downfield by 2.8 ppm in
AdoCby (3), reflecting the different carboxylic acid function in AdoCby (3) and in AdoCbi, a free acid
group vs. a carboxamide, respectively. Interestingly, in spite of their very similar environment, the
H C5RL protons of the adenosyl-moiety of AdoCby (3) are also shifted up-field (by 0.08 and 0.24
2
[
28]
ppm) compared to AdoCbi.
Partial synthesis of coenzyme B12 (2) from Co -5’-deoxyadenosyl-cobyric acid (AdoCby, 3).

Coenzyme B12 (AdoCbl, 2) was prepared in aqueous solution by activation of the carboxylic acid
*
function of AdoCby (3) with 1-ethyl-3-(-3-dimethylaminopropyl)-carbodiimide (EDC HCl) and N-
hydroxybenzotriazole (HOBt). Thus, AdoCby (3, 1.0 mg, 0.83 µmol), B12-nucleotide (5, 0.50 mg, 1.16
µmol) and HOBt (1.6 µmol) were dissolved in 750 µl water at 0°C. EDC*HCl (8.72 µmol, dissolved in
2
50 µl of water) was added. The reaction was monitored via HPLC (see Supporting Information,
Figure S9).The stirred reaction mixture was first kept for 2 h at 0°C, then for additional 22 h at RT, till
full conversion to coenzyme B12 (2). After workup and crystallization from water/acetone, 1.1 mg
(85% yield) of coenzyme B12 (AdoCbl, 2) were obtained.
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The spectra of here prepared AdoCbl (2) confirmed its identity. Hence, its UV/Vis spectrum in H O
2
[
21]
corresponded to the published spectrum, with absorption maxima at 523, 488 and 430 nm (see
Supp Info Figure S10). Likewise, a CD-spectrum of AdoCbl (2) in H O (see Supp. Info Figure S11)
2
[
32]
1
matched the reported CD-data of AdoCbl (2). A fully assigned H-NMR spectrum of AdoCbl (2) (see
Supp. Info Figure S12) was consistent with the known structure and showed chemical shifts very
[
30]
similar as published. An ESI-MS spectrum of the here prepared AdoCbl (2) (see Supp. Info Figure
+
S13) displayed the signals of a sodium adduct [C72
H
100CoN18NaO17P] at m/z= 1602.4 (as base-peak)
+
and of the proton adduct [C72H101CoN18
O17P] at m/z= 1579.5.
Partial synthesis of vitamin B12 (CNCbl, 1) from CN,H
2
O-cobyric acid (CN,H OCby, 4).
2
Crystalline CN,H
2
O-cobyric acid (4, 1.0 mg, 1.02 µmol), B12-nucleotide (5, 0.65 mg, 1.43 µmol) and
HOBt (0.30 mg, 2.24 µmol) were dissolved in 900 µl water at 0°C. EDC*HCl (13.86 µmol, dissolved in
1
00 µl of water) was added. Crystalline CN,H
2
OCby (4) represents the Co

-cyano form according to
[
11]
its x-ray crystal structure.
solution in almost a 1:1-mixture (see H-NMR analysis in D O, Supporting Information, Figure S1).
However, Co

-cyano and the Co -cyano forms are soon present in

1
2
Only the latter would allow for coordination of the B12-nucleotide (5) at the -face, proposed to be
helpful for the actual (intramolecular) coupling step. Direct monitoring of the reaction mixture by
HPLC (see Supporting Information, Figure S14) also indicated rapid equilibration of Co
the Co -cyano forms of CN,H O-Coby (4). In the event, the stirred reaction mixture was first kept for
h at 0°C, then for 5 h more at RT, till full conversion was analyzed, and nearly quantitative

-cyano and

2
2
formation of vitamin B12 (1). Vitamin B12 (1) was crystallized from water/acetone and was obtained
with 92% yield.
[
32, 33]
The UV/Vis- and CD-spectra of the here prepared CNCbl (1) matched the known spectra
(see
1
Supp. Info Figures S15 and S16). The 500 MHz H-NMR spectrum of the here prepared vitamin B12 (1)
(
see Supp. Info Figure S17) and the published spectrum matched with chemical shift differences less
[
34]
than 0.1 ppm. An ESI-MS spectrum of CNCbl (1) (see Supp. Info Figure S18) showed the sodium
adduct [C63
[
+
H
88CoN14NaO14P] at m/z 1377.7 as base-peak, as well as signals of the proton adduct
+
C
63
H
89CoN14
O
14P] at m/z 1355.5.
Investigations on the coordination properties of AdoCby (3) and CN,H OCby (4) towards N-methyl-
2
imidazole (MeIm): The synthetic work carried out here counted on a coordination of the DMB-
nitrogen of the B12-nucleotide loop (5) to the cobalt center, as setting the stage for an efficient
intramolecular amidation-step of the activated f-carboxylic side chain (see Figure 5). This strategy
followed the essential structural component of the mechanism laid out in the B12-reconstitution
[
19]
experiment, so beautifully designed by Eschenmoser and Bartels. In order to judge the relevance
of this process for our synthetic work, the tendency of DMB to undergo cobalt-coordination in
CN,H OCby (4) and AdoCby (3) was to be tested. In fact, N-methyl-imidazole (MeIm) was used here
2
as a surrogate of 5,6-dimethylbenzimidazole (DMB), as poor solubility of DMB in aqueous solution
inhibited its practical use in the spectral studies. Hence, the coordination behavior of N-methyl-
imidazole to the cobalt center of CN,H OCby (4) and AdoCby (3) was studied at room temperature.
2
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UV/Vis-spectra of AdoCby (3) and of CN,H
solution (pH=7) and MeIm was added to increasing concentrations. Due to the presence of both
coordination isomers of CN,H OCby (4) in aqueous solution, axial MeIm coordination is presumed to
occur with similar strengths at the α- or the β-side of the Co-center. Binding of MeIm to CN,H OCby
4) led to a shift of the absorbance maxima to longer wavelengths (see Supp. Info Figure S19) and to
a final spectrum resembling the one of CNCbl (1). Based on the absorbance change at 550 nm an
2
OCby (4) were recorded in buffered aqueous
2
2
(
4
-1
effective average binding constant of MeIm was derived as Kon (MeIm/CNCby) = 1.2∙10 M . The here
analyzed coordination of MeIm to CN,H OCby (4) could be compared to intramolecular coordination
of the DMB-base in CNCbl (1), which exhibits a concentration independent K’on = 2.9∙10 at 25°C.
2
5
[6,34]
Binding of MeIm to Co -5’-deoxyadenosylcobyric acid (AdoCby, 3) was studied similarly. It
β
was weaker and much higher concentrations of MeIm were needed, in order to see a significant
effect in UV/Vis-spectra (see Supporting Info Figure S20). Based on the absorbance changes a rough
-
1
binding constant Kon (MeIm / AdoCby) = 0.8 M was estimated at room temperature. This value, when
[
35]
compared with the concentration independent Kon in AdoCbl (2) of 14.3 (at 25°C), again indicated
a high effective molarity of the DMB-base of about 10M for intramolecular coordination to the
4
Co(III)-center. Furthermore, imidazole binding to AdoCby (3) was indicated to be about 1.5∙10 fold
weaker than to CN,H
adenosyl group.
2
OCby (4), reflecting the thermodynamic trans-effect of the CN-ligand vs. the
Clearly, the intramolecular coordination of the DMB-base in AdoCbl (2) and in CNCbl (1) is
remarkably more effective at low overall concentrations, than binding of (external) MeIm to the
Co(III)-centers of AdoCby (3) and CN,H
2
OCby, respectively. In previous studies in ethylene glycol,
MeIm was found to coordinate with methylcobinamide about 2.5 times better than 1,5,6-
[
36]
trimethylbenzimidazole. Likewise, taking into account the different bases (MeIm or DMB), the
direct comparison of these two values is very qualitative, but it points to a high effective molarity (of
the order of 10 M) of the DMB-base in CNCbl (1) and in AdoCbl (2). In the situation, where amide
formation was considered, the terminal amino group of the coordinated B12-nucleotide (5) would be
selected with, approximately, the same magnitude over the one in the free B12-nucleotide 5. This
argument would suggest the intramolecular path from CN,H
AdoCbl (2) to be faster by up to 10 times at the (sub)mM concentrations of the reaction partners in
2
O-Cby (4) or AdoCby (3) to CNCbl (1) or
4
our synthetic experiments.
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Figure 5. Coordination of the B12-nucleotide moiety 5 at the lower face of the corrin-bound Co(III)-center of AdoCby (3) or of CN,H
nucleotide moiety for efficient intramolecular amide bond formation to the f-side chain of the activated cobyric acid derivatives AdoCby (3) or CN,H
4).
2
OCby (4) prepares the B12
-
2
OCby
(
Conclusions
AdoCby (3) is a key common intermediate in the biosynthesis of adenosylcobamides, such as
[
8, 9]
[37]
coenzyme B12 (2),
synthesis of Co
as well as in diverse pathways of ‘B12-salvaging’. Here, a reliable partial
β
-5’-deoxyadenosylcobyric acid (AdoCby, 3) from CN,H
2
O-cobyric acid (CN,H OCby, 4)
2
is reported, as well as the first extensive spectroscopic characterization of AdoCby (3). These studies
have provided a spectroscopic data set consistent with the expected structural similarity of AdoCby
[
28]
(3) with the previously characterized analogue Co
β
-5’-deoxyadenosylcobinamide (AdoCbi),
and
likely to be useful in future biological studies.
The ‘complete corrinoids’ vitamin B12 (CNCbl, 1) and coenzyme B12 (AdoCbl, 2) could be obtained
in a single step, high yield partial synthesis by coupling the B12-nucleotide moiety (5) to the
corresponding cobyric acid derivatives, CN,H
2
OCby (4) or AdoCby (3), respectively, using EDC*HCl
and HOBt in aqueous solution. The same reagents were used in DMF in the recent synthesis of
[
38]
vitamin B12 mimics with a peptide backbone. In our experiment, crystalline CNCbl (1) was obtained
in a yield of 92%. The corresponding assembly of AdoCbl (2) took place with a yield of about 85%.
Pre-coordination of the B12-nucleotide moiety 5 to the ‘lower’ -face of the Co-center is a suggested
crucial element in obtaining a higher effective reaction rate in the amide-forming coupling step of
the partial synthesis of CNCbl (1) and AdoCbl (2) (and, thus, helpful for obtaining a good yield of
these cobalamins). This conclusion is congruent with qualitative coordination experiments, also
done, using 1-methylimidazole (Me-Im). From these we deduce pre-coordination of the DMB unit of
the B12-nucleotide moiety 5 to the cobalt-center of CN,H OCby (4) or AdoCby (3) to be relevant
2
under the conditions of our synthesis experiments, increasing the ‘effective’ concentration of its
terminal amino group and accelerating the amide-bond forming step.
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The strategy of the pre-coordination of the B12-nucleotide moiety 5, considered here, has
similarly been employed recently in the last step of the total synthesis of 5’-deoxyadenosyl-
rhodibalamin (AdoRhbl), the rhodium homologue of coenzyme B12 (AdoCbl, 2), from 5’-
[
39]
deoxyadenosyl-rhodibyric acid (AdoRhby, the Rh-analogue of AdoCby, 3). An analogous synthesis
strategy may also be helpful in the preparation of other transition metal analogues of the
[
40]
cobalamins, i.e. of ‘metbalamins’ carrying other metals than (the cobalamin’s) cobalt. Some of
[
39]
such structural similars of vitamin B12 (1) or of coenzyme B12 (2)
are in the focal point of current
[
41]
[41, 42]
B
12-research and may represent effective ‘antivitamins B12’.
The biosynthetic completion of AdoCbl (2) from AdoCby (3) depends upon several enzyme
[
8,9]
catalyzed steps
and does not involve direct attachment of the complete B12-nucleotide 5 to
AdoCby (3). However, the central biosynthetic relevance of the adenosylated cobalt-corrin AdoCby
3) as organometallic precursor of AdoCbl (2) is reflected by its apparently obligatory intermediacy in
(
the two studied branches of B12-biosynthesis. In this sense, the here reported partial synthesis of
AdoCbl (2) from AdoCby (3) may be classified as having a biomimetic element. Furthermore, as the
‘
intact’ B12-nucleotide (R-2-amino-1-methylethyl)-3’-(-ribazolyl)-diphosphate (5) has proven to be a
surprisingly effective structural complement of the cobyrinic acid moiety in Bartels’ and
[
19]
Eschenmoser’s successful studies of the self-assembly of vitamin B12 (CNCbl, 1), our experiments
suggest that a correspondingly selective formation of the organometallic cofactor coenzyme B12
(AdoCbl, 2) might represent an interesting alternative for the direct formation of this metabolically
active ‘complete’ cobalamin.
Experimental Section
Solvents:
Water (H
99.9%, HPLC gradient grade); acetone (Fluka, puriss p.a); acetonitrile (MeCN; BDH prolabo, >99.9%, HPLC gradient grade); diethyl ether
distilled); tert-butyl methyl ether; toluene (Fluka, >99.7%); D O (Euriso-top, 99.96%D).
2 2
O; purified over reversed Osmose); ddH O (Millipore MiliQ Academic, HPLC gradient grade); methanol (MeOH; BDH prolabo,
>
(
2
Reagents:
Acetic acid (AcOH; AnalaR Normpure, 100%), hydrochloric acid (12 mol/l; AnalaR Normpure, 35%);
benzoic acid crystalline (practical lab); sodium hydrogencarbonate (NaHCO ; practical lab); sodium
borohydride (NaBH ; Fluka, purum p.a.); potassium cyanide (KCN); dipotassium hydrogenphosphate
HPO ; Fluka, p.a); potassium dihydrogenphosphate (KH PO ; Roth, p.a.); iodine (Sigma Aldrich,
3
4
(K
2
4
2
4
puriss p.a.); triphenylphosphine (Sigma Aldrich, 99%); N-methylimidazole (MeIm) (Fluka p.a. >99%,
GC); N-hydroxybenzotriazole (HOBt) hydrate (12% water; Sigma Aldrich >97%); 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl; Fluka, purum p.a.); vitamin B12 (CNCbl,
crystalline; Hoffmann La Roche, 1); B12-nucleotide (R-2-amino-1-methylethyl)-3’-(-ribazolyl)-
[
19, 43]
diphosphate, 5) was obtained from partial hydrazinolysis of CNCbl (1),
hydrolysis of CNCbl (1);
dicyano-cobyric acid by
[
44, 45]
[46]
5’-iodo-5’-deoxyadenosine was prepared as reported
.
Spectroscopy:
UV/Vis spectra: Agilent Technologies Cary 60; max(log) in nm, sh signifies shoulder. CD-spectra:
-
1
-1
JASCO J715 spectrophotometer; max/

min and  in nm, Δ(molar circular dichroism) in [l mol m ],
0
This article is protected by copyright. All rights reserved.

1
13
1
sh signifies shoulder. H- and C-NMR spectra: Varian Unity Inova 500 spectrometer; H-NMR (500
MHz), coupling constant J in Hz, multiplicities abbreviated as s, d, t, q, m (singlet, doublet, triplet,
1
3
1
13
1
13
quartet, multiplet). C-NMR signal assignments from H, C-HSQC and H, C-HMBC spectra. For
[
47]
atom numbering, see Supp. Information Figure S6. ESI-MS: Finigan LCQ classic, positive ion mode;
data reported as: m/z (relative intensity in % in reference to the base peak); samples were desalted
and injected with MeOH.
HPLC:
Hitachi Pump, type L-2130; Diode Array Detector L2450 L-P19596-1/07 (detection at =520 nm);
injection: manual loading of the injection loop (~70 l); stationary phase: Phenomenex HyperClone
ODS (C18); column parameters: 250 x 4.60 (5 m; # 522787-11); mobile phase: potassium phosphate
buffer 10mM pH=7; solvent composition: Potassium phosphate buffer 10mM pH=7/MeOH: 0-25 min
(5%-90% MeOH), 25-33 min (90%-95% MeOH), 33-35 min (95% MeOH), 35 min-40min (95%-5%
MeOH).
2
Preparation of CN,H O-cobyric acid (4):
Crystalline cyano-aquo-cobyric acid (CN,H
2
OCby, 4) was prepared from dicyanocobyric acid following
[
44, 45]
1
a published procedure.
CN,H
2
OCby (4) was identified by its UV/Vis, ESI-MS and H-NMR spectra,
as well as by comparison by HPLC with a sample provided by Prof. Ray Bonett (Queen Mary College,
London). In D
2
O a roughly 1:1 mixture of the Co

-CN and Co

-CN isomers of (CN,H OCby, 4) was soon
2
1
present, as seen in H-NMR spectra (see Supp. Info Figure S1)
-
5
UV/VIS (c = 4.0∙10 M, H
2
O): max(log()) = 526 (3.82), 495 (3.85), 353 (4.36), 273 (3.98). ESI-MS:
+
+
9
80.4 (16, [M+Na] , C47
H67CoN11NaO
8
); 960.5 (16.9), 959.5 (66.0) 958.5 (100, [M+H] ,
+
C
47
H
68CoN11
O
8
).
Preparation of Coβ-5’deoxyadenosylcobyric acid (AdoCby, 3):
In a round bottom flask fitted with a UV/Vis cell, crystalline CN,H OCby (4, 2.00 mg, 2.1 µmol) was
2
dissolved in 500 µl MeOH and 500 µl 10 mM phosphate buffer pH=7. The solution was purged with
argon and degassed using freeze-pump-thaw cycling. NaBH (10 mg, 130 equiv.) was added under
argon and the apparatus was purged with argon again. After half an hour the reaction mixture had
turned brown, and 10 mg of NaBH were again added. After 1 h, benzoic acid (25.0 mg, 100 equiv.)
was added, followed by another portion of 10 mg NaBH . After 4 hours a UV/Vis spectrum indicated
4
4
4
full conversion to a Co(I)-species (see Supp. Info Figure S2). All further operations were carried out in
the dark. 5’-iodo-5’-deoxyadenosine (1.0 mg, 1.2 equiv.) was added under argon and the reaction
mixture was stirred for half an hour, when a UV/Vis-analysis indicated conversion to AdoCby (3).
After addition of 5 ml phosphate buffer pH=5 (50 mM) the reaction mixture was desalted over a Sep-
Pak column. The mixture was purified over a RP column using phosphate buffer pH=7 (10mM) with
increasing amounts of MeCN. At 2% MeCN unidentified cobyric acid derivatives were eluted as small
red bands. At 7% MeCN AdoCby (3) and CN,H O-Cby (4) were separated as two bands and were
2
eluted with 10% MeCN. Fractions containing AdoCby (3) were identified by HPLC, and were
combined. Solvents were removed on a rotary evaporator. The orange residue was dissolved in
1
00µl MeOH and precipitated by dropwise addition to diethyl ether. The precipitate was washed
with acetone and dried (HV, overnight), and 1.85 mg (74% yield) of Co -5’-deoxyadenosyl-cobyric
β
acid (AdoCby, 3) were obtained as an orange powder.
This article is protected by copyright. All rights reserved.

-
5
UV/Vis (c= 2.5∙10 M, H O): max(log(ε)) = 457 (3.85), 378 (3.80), 303 (4.24), 262 (4.44). CD (c=
2
-
4
1
(
.25∙10 M, H
2
O): max/min (Δ): 564 (1.1), 500 (-1.2), 432 (-1.8), 401 (0.4), 378 (-0.5), 330 (5.6), 302
1.3, sh), 282 (-1.1), 240 (-3.4).  : 525, 408, 391, 360, 291, 223. H-NMR (500 MHz, 298 K, D O,
1
0
2
-
3
c=7.0∙10 M):  = 0.35 (t, J = 9.5 Hz, 1H, H
.84 (s, 3H, H C12B), 1.12 (s, 3H, H C17B), 1.41 (s, 3H, H
C7A), 1.80 (m, 1H, H
J = 3.7 Hz, J = 15 Hz, 2H, H
.47 (m, 2H, H C181), 2.48 (m, 2H, H
ddd, J = 4.4 Hz, J = 2.2 Hz, J = 10.0 Hz, 1H, HC18), 3.39 (dd, J = 3.7Hz, J = 4.8 Hz, 1H, HC13), 3.71 (d,
A
C5RL), 0.50 (d, J = 8Hz, 1H, H
B
C5RL), 0.80 (s, 3H, H
3
C1A),
0
H
3
3
3
C2A), 1.58 (s, 3H, H
3
C12A), 1.78 (s, 3H,
C81), 2.09 (dd,
C171), 2.34 (m, 2H, H C132),
C32), 2.60 (d, J=13Hz, 2H, H C21), 2.77
3
2
C81), 1.93 (m, 1H, HC4RL), 2.05 (m, 1H, H
C172), 2.18 (m, 2H, H C131), 2.22 (m, 2H, H
C82), 2.54 (m, 2H, H
2
C31), 2.21 (m, 1H, H
2
2
2
2
2
2
2
2
2
2
(
1
4
8
H; HC3RL), 3.75 (m, 1H, HC8), 4.20 (d, J=8.8 Hz, 1H, HC3), 4.39 (dd, J = 4.4 Hz, J = 4.8 Hz, 1H, HC2RL),
.62 (d, J = 10.3 Hz, 1H, HC19), 5.59 (d, J = 4.4 Hz, 1H, HC1RL), 6.71 (s, 1H, HC10), 8.01 (s, 1H, HC8L),
+
+
.20 (s, 1H, HC2L). ESI-MS: m/z = 1204.5 (29, [M+Na] , C55
H
76CoN15NaO11 ); 1184.4 (24.6), 1183.4
+
+
+
+
(68.0), 1182.4 (100, [M+H] , C55
H
77CoN15
O
11 ); 932.6 (7, [M+H-(adenosyl)] , C45
H
65CoN10
8
O ),
β
Preparation of coenzyme B12 (2) from Co -adenosyl-cobyric acid (AdoCby, 3):
All operations were carried out in the dark. In a 10 ml round bottom flask, 1.0 mg of AdoCby (0.83
µmol, 3), B12-nucleotide (5, 0.50 mg, 1.4 equiv) and HOBt (0.25 mg, 1.6 µmol) were dissolved in 750
µl H
2
O and cooled in an ice bath. The round bottom flask was closed with a septum and the solvent
*
purged with argon. With a syringe EDC HCl (1.70 mg, 10.5 equiv.), dissolved in 250 µl ice-cold ddH
2
O,
was added. The reaction mixture was stirred at 0°C for 2 hours and subsequently at RT. After 8 h,
*
1
.70 mg (10.5 equiv.) of EDC HCl, dissolved in 50 µl ddH
2
O, were added. The reaction mixture was
stirred at RT overnight. The conversion was monitored via HPLC. After full conversion, the reaction
mixture was purified on a RP-column using phosphate buffer pH=7 10mM with increasing gradient of
MeCN. The red fractions of coenzyme B12 (2) were eluted with 10% MeCN, were combined and the
solvents were removed using a rotary evaporator. The red residue was dissolved in 200 µl of MeOH
and precipitated by the dropwise addition to a solution of 2ml tert-butyl methyl ether. To complete
the precipitation the solution was stored at 5 ꢀ3°C overnight. Then solvents were carefully removed
and the precipitate was washed 3 times with acetone. The red solid was dried under high vacuum
for 24 hours. 1.2 mg of coenzyme B12 (2, 91%) were obtained as a red powder. For crystallization, the
red solid was dissolved in 400 µl of MeOH, transferred in equal portions into two small tubes, where
solvents were removed and the residue was again dried under vacuum. The red residues were
dissolved in 20 µl H O and 10 µl of acetone. Then the tubes were closed with aluminum foil, into
2
which a small hole was made. The small tubes were put into vial, which contained about 4 ml
acetone. The crystallization batches were stored at 5 ꢀꢁ°C for 14 days and 10 µl portions of
acetone were added once every two days. When crystals had separated, the mother liquor was
removed and the crystals were washed with acetone and dried under high vacuum for 24 hours.
Some seed crystals were taken aside and the remaining AdoCbl (2) was dissolved in water,
transferred into a vial and evaporated again on the rotary evaporator. The residue was dissolved in
1
00 µl H O and acetone (300 µl) was added dropwise until the point of turbidity. In addition, the
2
separated seed crystals were added and the mixture was left at ꢂ ꢁ°C for 5 days. The red crystals
of coenzyme B12 (AdoCbl, 2) formed were separated from the mother liquor and dried under high
vacuum for 24 hours (yield: 1.10 mg, 85%).
This article is protected by copyright. All rights reserved.

-
5
UV/Vis (c = 2.96∙10 M, H O): max(log(ε)) = 523 (3.90), 488 (3.82), 430 (3.64), 375 (4.00), 339 (4.1),
2
5
-
3
3
2
H
H
1
H
H
16 (4.11). CD (c = 8.9∙10 M, H
33 (0.25), 323.5 (-0.1), 299 (1.3);  at 515, 449, 406, 375, 337, 328, 321, 246. H-NMR: (500 MHz,
2
O): max/min(Δ): 554 (-2.3), 481 (2.7), 429 (-0.7), 386 (1.3), 359 (-2.5),
1
0
-
3
98 K, D
2
O, c = 1.7∙10 M):  = 0.44 (s, 3H, H
C12A), 0.88 (m, 2H, H C82), 1.17 (d, J = 6.6 Hz, 3H, H
C17B), 1.34 (s, 3H, H C17B), 1.50 (d, J = 8.8 Hz, H C5RL), 1.69 (s, 3H, H
C82), 1.73 (m, 2H, H C81) , 1.77 (m, 2H, H C172), 1.97 (m, 1H, H
C31), 2.08 (m, 1H, H C171), 2.18 (s, 6H, H C11N, H C10N), 2.19 (m, 1H, H
C132), 2.38 (m, 2H, H C21), 2.45 (m, 1H, H C171), 2.5 (m, 2H, H C32), 2.54 (m, 2H, H
C181), 2.87 (d, J = 10.3 Hz, CH13), 3.12 (dd, J= 7.3 Hz, J= 10.7 Hz, 1H, CH18), 3.20 (m, 2H,
175), 3.27 (m, 1H, HC89), 3.55 (m, 2H, H C175), 3.68 (m, 1H, HC3RL), 3.72 (m, 1H, HC3R), 4.07 (m,
3
C1A), 0.55 (t, J = 8.8 Hz, 1H, H
C177), 1.30 (s, 3H, H
C7A), 1.71 (m, 1H, H
C31), 2.06 (m, 1H,
C71), 2.2 (m, 2H,
C132), 2.62
A
C5RL), 0.84 (s, 3H,
C12B), 1.33 (s, 3H,
C71),
3
2
3
3
3
3
B
3
2
.71 (m, 1H, H
2
2
2
2
2
2
3
3
2
2
2
2
2
2
(m, 2H, H
2
CH
2
2
1
1
H, HC4R), 4.10 (m, 1H, HC3), 4.2 (m, 1H, HC2R), 4.24 (d, J = 10.3 Hz, HC19), 4.33 (ddd, J = 2.2 Hz, J =
1Hz, 1H, HC176), 4.51 (dd, J = 3.7 Hz, J = 4.8 Hz, HC2RL), 5.54 (d, J = 3.7 Hz, HC1RL), 5.91 (s, 1H,
HC10), 6.21 (s, 1H, HC4N), 6.22 (d, J = 2.93 Hz, HC1R), 6.91 ( s, 1H, HC2N), 7.13 (s, 1H, HC7N), 7.98 (s,
1
H, HC8L), 8.19 (s, 1H, HC2L); signals according to MTBE: 3.25 (s, CH
3
-O), 1.19 (s, (CH
3
)
3
C-O). ESI-MS:
+
+
+
m/z = 1617.5 (9, [M+K] , C72
C
H
100CoKN18
O
17P ); 1604.4 (12.62), 1603.4 (46.61), 1602.4 (100, [M+Na] ,
+
+
+
72
H
100CoN18NaO17P ); 1581.5 (5.4), 1580.5 (10.0), 1579.5 (11, [M+H] , C72
H
101CoN18
O
17P ); 1351.6
+
+
+
(10, [M+Na-(adenosyl)] ,
C H88CoN13NaO14P ); 1239.3 (7, [M-(-ribazole phosphate)]
62
+
2+
2+
2+
C
C
58
H
H
84CoN16
88CoN13Na
O
11 ); 812.8 (30, [M+2Na] , C72
H
100CoN18Na O17P ); 687.9 (12.7, [M-(adenosyl)+2Na] ,
2
2
+
62
2
O14P ).
2
Preparation of vitamin B12 (CNCbl, 1) from CN,H O-cobyric acid (4):
In a 5 ml round bottom flask, crystalline CN,H
2
O-cobyric acid (4, 1.0 mg, 1.02 µmol), HOBt (0.30 mg,
2
.2 equiv.) and B12-nucleotide (5, 0.65 mg, 1.4 equiv.) were dissolved in 900 µl H
2
O. The flask was
*
closed with a septum and the solvent purged with argon. With a syringe a solution of EDC HCl (2.10
mg, 10.5 equiv.) in 100 µl ice-cold H O was slowly added. The reaction mixture was cooled and
2
stirred for 2 hours in an ice bath, which was then removed. The reaction progress was monitored via
HPLC and full conversion was observed after 7 hours (see Supp Info Figure S14). The reaction
mixture was purified via RP18-column chromatography using potassium phosphate buffer pH=7 10
mM with an increasing content of MeCN. At 10% MeCN vitamin B12 (1) eluted as red band. The red
CNCbl (1) fractions were collected, solvents removed on the rotary evaporator and desalted via RP18
Sep-Pak cartridge. A red methanolic eluate was obtained that was dried on the rotary evaporator.
The residue was dissolved in 100 µl H O, transferred into a vial for crystallization at room
2
temperature and acetone was added dropwise until turbidity (about 300 µl). After 4 hours the vial
with crystallization batch was put in a refrigerator at 5°C overnight. On the next day, the supernatant
was removed and the crystals were washed with acetone three times. The red crystals of vitamin B12
(CNCbl, 1) were dried under high vacuum for 24 hours (yield: 1.30 mg, 92%).
-
5
UV/Vis (c = 2.5 ∙10 M, H
3
2
O): max(log(ε)) = 549 (3.99), 521 (3.94), 408 (3.62), 360 (4.51), 322 (3.95),
-
4
05 (4.02), 277 (4.25). CD (c = 1.45∙10 M, H O): max/min (Δ) = 548 (-0.7), 527 (-0.4), 483 (-1.5), 433
2
(
2
1
4.8), 362 (-6.1), 333 (-1.0), 326 (-1.3), 316 (-0.4), 309 (-0.7), 277 (1.8), 251 (-3.8); 
0
at 463, 382, 302,
C1A), 0.99 (m, 1H, H C81),
C2A), 1.41
1
-3
61, 242. H-NMR (500 MHz, 298 K, D
2
O, 2.1∙10 M):  = 0.42 (s, 3H, H
3
2
.16 (s, 3H, H
3
C12A), 1.22 (d, J = 6.6 Hz, 3H, H
3
C177), 1.35 (s, 3H, H
3
C17B), 1.37 (s, 3H, H
3
This article is protected by copyright. All rights reserved.

(
1
H
3
2
8
1
s, 3H, H
3
C12B), 1.78 (m, 1H, H
.98 (m, 2H, H C131), 2.07 (m, 2H, H
C11N), 2.22 (s, 3H, H C10N), 2.37 (m, 2H, H
C151), 2.58-2.5 (m, 1H, H C32), 2.6 (m,1H, H
.65 (m, 1H, H C181), 2.71 (m, 1H, H C181), 2.72 (d, J = 12.5 Hz, 1H, HC18), 2.92 (dd, J = 14 Hz, J =
,0Hz, H C157), 3.3 (d, J = 10.3 Hz, 1H, HC13), 3.39 (dd, J = 5.14 Hz, J = 11.5 Hz, 1H, HC8), 3.57 (d, J =
4,7 Hz, 1H, H C157), 3.72 (dd, J = 4.4 Hz, J = 8.4 Hz, 2H, HC5R), 3.89 (m, 1H, HC5R), 4.03 (d, J = 8.8
2
C81), 1.80 (m, 1H, H
C81), 2.16 (m, 1H, H
C21), 2.50 (s, 3H, H
C71), 2.6 (m, 2H, H
2
C171), 1.83 (s, 3H, H
C172), 2.20 (m,1H, H
C51), 2.51 (m, 1H, H
C132), 2.65 (m, 1H, H
3
C7A), 1.95 (m, 2H, H
C71) 2.22 (s, 3H,
C171), 2.54 (s,
C172),
2
C31),
2
2
2
2
3
3
2
3
2
H, H
3
2
2
2
2
2
2
2
2
Hz, 1H, HC4R), 4.05 (d, J = 11.0 Hz, 1H, HC19), 4.15 (d, J = 8.8 Hz , 1H, HC3), 4.24-4.29 (m, 1H, HC2R),
4
1
.24-4.29 (m, 2H, H
2
C176), 6.05 (s, 1H, HC10), 6.31 (d, J = 2.9Hz, HC1R), 6.47 (s, 1H, HC4N), 7.05 (s,
+
+
H, HC2N), 7.24 (s, 1H, HC7N). ESI-MS: m/z = 1393.5 (13, [M+K] , C63
H
88CoKN14
O
14P ); 1377.7 (80,
+
+
+
+
[
M+Na] , C63
H88CoN14NaO14P ); 1357.5 (36.6), 1356.5 (74.0), 1355.5 (100, [M+H] , C63
H89CoN14O14P ).
Supplementary Material
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/MS-number.
Acknowledgements
We thank Christoph Kieninger for help with NMR measurements, and Prof. Ray Bonnett (London,
UK) for an authentic reference sample of crystalline CN,H O-cobyric acid. We are grateful for
2
financial support of our work by the Austrian Science Fund (FWF, P-28892).
Author Contribution Statement
1
These two authors contributed equally. The experiments were carried out by F. J. W. and F. G.
under the supervision of B.K., and the manuscript was written by F. J. W., F. G. and B.K.
References
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.