[205] Preparation of the Reduced Forms of Vitamin B12 and of Some

[205] Preparation of the Reduced Forms of Vitamin B12
and of Some Analogs of the Vitamin B12 Coenzyme
Containing a Cobalt-Carbon Bond
By
D. DOLPHIN
Reduction of the cobalamins, which contain trivalent cobalt, proceeds
in two distinct steps. The first one-electron reduction product is vitamin
Bur which undergoes a further one-electron reduction to B 128 which may
be considered as either containing monovalent cobalt or being a trivalent
cobalt hydride.!
HT
HCo(l) ;::= Co(III)
is the most nucleophilic species known to exist in aqueous solution, 2
and this high nucleophilicity results in the ease of preparation of the B12
coenzyme and its analogs, since B l2s undergoes both rapid substitution
and addition reactions with a variety of electrophiles.3
B128
Preparation of the Reduced Forms of Vitamin
B12
A. Catalytic Reduction
Adams catalyst (5 mg) and a solution of hydroxocobalamin (20 mg)
(cyanocobalamin can be used in this preparation, but the reduction requires
longer to go to completion4) in water (10 ml) are placed in a 25-ml roundD. Dolphin, A. W. Johnson, and R. Rodrigo, Ann. N.Y. Acad. Sci. 112, 590 (1964).
G. N. Schrauzer, E. Deutsch, and R. J. Windgassen, J. Am. Chem. Soc. 90, 2441 (1968).
3 A. W. Johnson, L. Mervyn, N. Shaw, and E. L. Smith, J. Chem. Soc. p. 4146 (1963).
4 During the catalytic reduction of B 12, the cyanide ligand is reduced to methylamine [J. L. Ellingboe; J. I. Morrison, and H. Diehl, Iowa State Coli. J. Sci. 30, 263
(1955)], and this provides a method for the production of hydroxocobalamin from
vitamin B 12•
I
2
[205]
FORMS OF BI2 ANu ANALOGS CONTAINING Co-C BOND
35
bottomed flask which is attached to a standard hydrogenation apparatus.
The system is evacuated and refilled with hydrogen to 1 atmosphere; the
solution is magnetically stirred until the uptake of hydrogen has ceased.
This requires about 30 minutes, by which time the solution is dark brown
and has the spectrum shown in Fig. 1. The system is again evacuated, then
refilled with argon 6 ; the B 12r solution is removed from the catalyst by
filtration through a plug of cotton which has been washed with deoxygenated water.
In the absence of oxygen, solutions of Bur are indefinitely stable, and
providing that pipettes and receptacles have been flushed with an inert
gas, such solutions can be readily transferred without any oxidation
occurring.
There are no methods available for the catalytic reduction of vitamin
BIt to Bu•.
B. Controlled Potential Reduction
Reactions occurring at the surface of an electrode are dependent on the
potential of the electrode, and by controlling this potential specific oxida1.0
''I
'I
I
I
08
I \
I
I
I
.,0.6
I
I
u
c:
c
I
.J:l
~
I
I
\
\
I
\
\
"'
<lOA
\
J:>
\
\
\
\
\
'., ........
\, .. .
0.2
'300
400
500
700
Wavelength ( nm )
FIG. 1. A 2.88 X 10-& M solution, 1.O-cm cell. Hydroxocobalamin ( - - ) , B12r
(-----), Bu. (---).
I
Either nitrogen or argon are suitable for the deoxygenation, but argon is less ree.dily
displaced by air, because of its higher density, than nitrogenj it is, therefore, more
useful.
36
COBALAMINS AND COBAMIDES
[205]
tions or reductions can be carried out. The advantages of such electrolytic
reductions in the vitamin B12 series are their specificity, which enable one
to prepare B12r in the absence of Bus, and to carry out the reductions over
a range of pH values in a variety of buffers. However, the most important
advantage is that, unlike chemical reductions, no excess of reducing agent
is present.
Apparatus. Numerous electrolysis cells are described in the literature,6
and when modified so that the solutions can be deoxygenated they may be
used for the reduction of B 12 . Two cells (Figs. 2 and 3) are shown. The
first cell is convenient when dealing with volumes in the order of 50 ml
and the latter for volumes of 10 ml and less. The principal difference between the two cells is that in the former the standard electrode is in the
working half of the cell, and the potential of the mercury cathode can be
accurately measured, while in the latter the reference electrode is in the
auxiliary half of the cell since the introduction of a reference electrode
into the small cathode compartment is difficult. Both larger and smaller
cells than those described here can readily be constructed, bearing in
mind that the rate of reduction depends upon the surface area of the
cathode; and the more efficient the stirring the faster the rate of reduction,
since the cobalamin is reduced as rapidly as it arrives at the electrode
surface. It is necessary to use a diaphragm to prevent the cobalamin, which
is reduced at the cathode, from being reoxidized at the anode and to prevent
mixing of the anode electrolyte with the cobalamin solution. The diaphragm
consists of a sintered glass disk of fine porosity and an agar salt bridge.
Preparation of the Agar Salt Bridge. A stock gel for the salt bridge is
prepared as follows: agar (3 g) in distilled water (SO ml) is heated on the
water bath until the agar has dispersed, potassium chloride (31.3 g) is
added, and the volume is made up to 100 ml with distilled water. Heating
is continued, and the solution is deoxygenated with argon' for 10 minutes.
Argon is then passed over the surface of the solution, and heating is continued until a clear, bubble-free solution is obtained. A portion of this
solution is poured into the body of the electrolysis cell, which has previously been heated to about 100°, and the salt bridge is allowed to cool to
room temperature (rapid cooling of the salt bridge is not advisable, since
this may cause the gel to shrink from the walls of the cell). Shrinking will
also occur if the salt bridge is allowed to dry out; when the cell is not in
use, both compartments should be filled with a deoxygenated solution of
saturated potassium chloride.
The Standard Electrode. In the smaller of the two electrolysis cells,
II. M. Kolthoff and J. J. Lingane, "Polarography," Wiley (Interscience), New York,
1952.
[205]
FORMS OF B12 AND ANALOGS CONTAINING Co-C BOND
37
Ag electrode
Anode
FIG. 2. A cell for controlled potential reductions with the reference electrode in the
cathode compartment.
the standard electrode consists of a silver wire dipping into the anode
electrolyte, for the larger cell a separate standard electrode must be constructed. The body of this electrode should be of such a length that the
sintered disk is only 1 or 2 mm above the surface of the mercury cathode.
The salt bridge is prepared by pouring a portion of the hot deoxygenated
agar-Kel solution into the body of the electrode, which has been heated
to about 100°. When the salt bridge has cooled to room temperature, it is
covered with a deoxygenated solution of saturated potassium chloride to
which has been added one drop of a N silver nitrate solution. A silver
wire is held in the electrolyte by means of a serum cap (the size of the wire
is not critical, but a gauge of about 16 is convenient). When not in use, this
reference electrode should be stored in a deoxygenated saturated potassium
chloride solution.
38
[205]
COBALAMINS AND COBAMIDE8
Gas
inlet
Reference
electrode
Pt wire
FIG. 3. A cell for controlled potential reductions with the reference electrode in the
anode compartment.
The Anode. In both cells (Figs. 2 and 3) the anode electrolyte is a deoxygenated, saturated potassium chloride solution to which has been added
one drop of 1 N silver nitrate solution. The anode consists of a piece of
16-gauge silver wire.
Assembling the Apparatus. A potential (supplied by either a filtered
direct-eurrent power supply, or a lead storage battery and rheostat 7) is
applied to the. electrolysis cell through the mercury cathode, which is
connected to the negative terminal, and the silver anode. A Inilliammeter
(currents greater than 5 rnA are not encountered) is placed in this circuit
to measure the electrolysis current.
The mercury cathode and the reference electrode are connected to a
7
J. J. Lingane, "Electroanalytical Chemistry." Wiley (Interscience), New York, 1958.
[205]
FORMS OF B12 AND ANALOGS CONTAINING Co-C BOND
39
potentiometer (most pH meters can be used as potentiometers), and in
this manner the potential of the working mercury cathode can be monitored during the reduction. Since in the cell shown in Fig. 3 the reference
electrode is in the anode compartment, a correction must be made to allow
for the LR. drop between the electrode and the mercury surface. This
correction is made by determining the resistance between these two electrodes and multiplying this resistance by the current flowing through the
cell. The observed potential will be the true potential at the mercury
surface, which controls the course of the reduction, plus the computed
I.R. drop through the solution.
Preparation of Bur by Controlled Potential Reduction. A solution of
hydroxocobalamin (10 mg) (cyanocobalamin cannot be used in this preparationS) in 0.2 M phosphate buffer, pH 7.0 (50 ml), is placed in the cathode
compartment of the electrolysis cell shown in Fig. 2. The stirred cobalaInin
solution and saturated potassium chloride solution, in the anode compartment, are deoxygenated with a gentle stream of argon. 6 After 15 minutes
the flow of gas through the cathode compartment is stopped and both the
inlet and outlet tubes closed. The potential across the cell is then increased
until the potential of the mercury cathode, measured against the standard
silver-silver chloride reference electrode, is 0.7 V. The rate of stirring is
adjusted to maximize the current, an initial value of 1-2 rnA being observed, and the electrolysis is continued at 0.7 V until, after about 90
minutes, the current reaches a steady low value of about 0.05 mAo At this
time the reduction to B l2r is complete, and the resulting brown solution
has the spectrum shown in Fig. 1.
SiInilar preparations of B 12r in a wide range of acidic and basic buffers
can be carried out.
Preparation of Bu. by Controlled Potential Reduction. In the absence
of oxygen, B 12• in aqueous solution is oxidized to B l2r with the liberation
of hydrogen from water. The rate of oxidation is dependent upon the
hydrogen ion concentration, and the half-life of B l2• as a function of pH
is shown in Table.!.
When Bl2 is cheInically reduced under acidic conditions (e.g., zinc
and acetic acid) the large excess of reducing agent compensates for any
decomposition of B 12• that might occur. However, in controlled potential
reductions, where there is no excess of reducing agent and where the reduction may require up to 2 hours to go to completion, the reduction must be
carried out at a pH where the rate of reduction to Bu. is sufficiently faster
than its reoxidation. Thus, when the reduction is carried out in a phosphate
8
At pH 7, vitamin Bn displays only one well-defined reduction wave, which corresponds to the production of Bu•.
40
[205J
COBALAMINS AND COBAMIDES
TABLE 19
IlALF-LlFE OF B u, AS A FUNCTION OF
9
pH
Half-life
(min)
6.98
8.01
9.01
9.93
22
67
140
>240
pH
These data have been extrapolated from the paper by S. L. Tackett, J. W. Collat, and
J. C. Abbott, Biochemistry 1. 919 (1963).
buffer at pH 7, BUr and hydrogen are formed, but the concentration of
Bu. is low; while using a borate or Tris buffer at pH 9, Bus is produced
essentially quantitatively. A higher potential (1.3 V) is required to produce
B12s than that required for B 12r (0.7 V). At this higher potential, care must
be taken to ensure that the buffer is not electroactive. Electrolysis of buffer
alone at 1.3 V will show whether the buffer is electroactive, since at this
potential no current «0.05 rnA) should flow through the cell. Buffers
based upon phosphate, Tris, EDTA, and borate can be used, whereas
those based upon bicarbonate cannot. Since a standard silver electrode is
used, buffers containing ammonium ions must be avoided. At potentials
above 1.4 V vs. the silver-silver chloride electrode a considerable increase
in current is observed. This is not an indication that Bu. is being produced
at a faster rate but is due to the reduction of hydrogen ion, and for the
production of B 12• there is no advantage in using potentials greater than
1.35 V.
Procedure. A solution of hydroxo- or cyanocobalamin1o (3 mg) in borate
buffer, pH 9.2 (5 ml), was placed in the cathode compartment of the cell
shown in Fig. 3. A stream of argon, led in through a hypodermic needle in a
serum cap on the mercury side arm and out through a second needle in
the serum cap on the gas outlet tube, is passed through the stirred cobalamin
solution, and at the same time the electrolyte in the anode compartment
is also deoxygenated. Mter 15 minutes, the flow of argon through the
cathode is stopped and the hypodermic needles are removed. The applied
potential across the cell is increased until the potential across the silversilver chloride reference electrode and the mercury cathode is 1.35 V plus
the computed correction. The electrolysis is continued, the applied voltage
being adjusted so that the true potential at the cathode is maintained at
1.35 V, until the current has fallen to a steady low value of about 0.1 rnA.
III
When Bus is prepared from cyanocobalamin, cyanide ion is present at the end of the
reduction.
[205]
FORMS OF B12 AND ANALOGS CONTAINING Co--c BOND
41
At this stage reduction is complete, and the green solutionll has the spectrum shown in Fig. 1.
C. Chemical Reduction
Preparation of B 12r. A wide variety of reducing agents will reduce
vitamin B u , or hydroxocobalamin, to B12r.12 However, in the majority of
cases, varying amounts of Bu. are also present. Thus the reduction of
hydroxocobalamin with thiols13 gives brown species having electronic
absorption spectra identical to Bur; but while Bur is unreactive towards
alkylating agents, the "Bur" product by reduction with thiols is alkylated
to give the corresponding alkylcobalamin. We recommend in cases where
Bur is required in the absence of Bu. that chemical reducing agents are
not used.
Preparation of Bu•. The most convenient reduction is that brought
about by sodium borohydride. Although such reductions are usually complete within a few minutes, they occasionally stop at the brown Bur stage,
and the further reduction to Bu. then requires both a large excess of sodium
borohydride and an extended reaction time which results in some irreversible reduction of the corrin chromophore. It has been reported14 that addition of copper catalyzes the reduction, and we have found this to be so, but
this reducing system also reduces the alkylcobalamins which are normally
stable toward sodium borohydride by itself. Thus, the copper-catalyzed
reduction is unsuitable for the preparation of Bu. as a precursor of the
alkylcobalamins.
If, however, cobaltous ions are added to a cobalamin borohydride
solution, reduction is catalyzed without further reduction of alkylcobalamins. Thus, this reduction is suitable for the syntheses of both the
vitamin Bu coenzyme and its analogs.
Procedure. A solution16 of cyano- or hydroxocobalamin (50 mg) and
cobalt nitrate16 (1 mg) in water (10 ml) is deoxygenated with a stream of
argon. After 10 min a deoxygenated solution of sodium borohydride (15
mg) in water (1 ml) is added to the red cobalamin solution, which immediately turns brown and then blue-green, signifying that the reduction is
Solutions of Bu. when viewed in daylight are blue-green, but when viewed in artificial
light such solutions often look purple.
II J. A. Hill, J. M. Pratt, and R. J. P. Williams, J. Theoret. Bioi. 3,423 (1962).
13 D. Dolphin and A. W. Johnson, J. Chem. Soc. p. 2174 (1965).
14 G. N. Schrauzer, private communication, 1964.
16 Since solutions of BI2a are very susceptible to oxidation by air, they can be transferred
only with great difficulty, and it is advisable to prepare such solutions in a vessel
suitable for any subsequent reactions.
18 Any water-soluble cobaltous salt can be used.
11
42
[205]
COBALAMINS AND COBAMIDES
complete. A wide variety of reducing agents are available for the reduction
of vitamin Bu to Bus, and further examples are given in the section on the
preparation of the alkylcobalamins.
Preparation of Alkyl and Acyl Analogs of the Vitamin
B12
Coenzyme
General Considerations. The preparation of such analogs is based upon
the reaction of B 12s with suitable electrophiles, and the principal reactions
are summarized in Fig. 4.
Limitations. The limiting factors in the synthesis of Bl2 coenzyme
analogs by nucleophilic displacement reactions are primarily steric, and
they may either limit the rate of the reaction or effect the stability of the
coenzyme analog once it has been formed. Thus, no reaction is observed
between B 12s and neopentyl chloride, while the reaction between secondary
halides such as a-chloropropionie acid and 2-bromobutane is fast, but the
resulting coenzyme analogs are not sufficiently stable to survive the purification procedures.
Although steric considerations are of primary importance in determining stability, certain coenzyme analogs that are sterically feasible
require considerable care during their isolation. Thus, benzylcobalamin
is rapidly decomposed by oxygen, due to the ready homolytic cleavage
of the cobalt-carbon bond, and can be isolated only when oxygen is vigorously excluded during all stages of its preparation. Similarly, the analog
derived from methyl acrylate is base labile, due to the acidity of the tl-hydrogen atoms, and care must be taken to maintain neutrality during its
preparation.
The addition of Bl2s to alkenes and alkynes also results in the formation
of coenzyme analogs, and as well as steric considerations concerning the
stability of the products, the alkenes which are less susceptible to nucleophilic attack than the corresponding alkynes must be activated by being in
FIG. 4. ReactioIIII of B u ,.
[205]
FORMS OF B12 AND ANALOGS CONTAINING Co-C BOND
43
conjugation with an electron withdrawing group. Thus, whereas acetylene
reacts with B 12• to give vinylcobalamin, no reaction is observed with
ethylene, although acrylonitrile and methyl acrylate react rapidly to give
the corresponding alkylcobalamin.
The low solubility of the cobalamins in most solvents, coupled with
the need for solvents that are stable toward reducing agents, restricts the
choice for the preparation of coenzyme analogs to water, methanol, ethanol,
and aqueous acetic acid.
Both the vitamin BI2 coenzyme and its analogs are light sensitive, especially in solution, and the manipulations given below should be carried out
using the minimum intensity17 of light that one can conveniently work in as
well as shielding reaction flasks, chromatographic columns, etc., with
aluminum foil.
Purification by Extraction through Phenol. The procedure of extraction
through phenol removes water-soluble salts from cobalamins. A number of
modifications of the original procedure18 have been made, but in our hands
many such procedures often result in emulsions. We have found, however,
that the following technique is both reproducible and free from the problem
of emulsions.
The aqueous cobalamin. soluti()n is extracted with one-fifth of its volume
of a solution of phenol in methylene chloride. A stock solution may be
prepared by dissolving phenol (100 g) in methylene chloride (100 ml). The
organic layer is separated, and the aqueous layer is reextracted with successive aliquots of phenol-methylene chloride until no further color is
extracted. The combined organic extracts are washed with distilled water
(2 X 1/5 the volume of the organic layer), and the organic layer is diluted
with methylene chloride to 10 times its original volume. The cobalaInin
is reextracted from the organic layer with aliquots of distilled water (1/20
the volume of the organic layer) until no color remains in the organic
layer. The combined aqueous extracts are then washed with methylene
chloride (3 times the volume of the combined aqueous layer) to remove
traces of pheno1. The methylene chloride remaining in the aqueous phase
is removed when the solution is reduced in volume, or may be removed by
passing a stream of nitrogen through the solution.
Chromatography. Thin-layer chromatography on cellulose 19 or silica
ge11 9,20 is useful for the rapid analysis of cobalaInins, but the close similarity
in R, values of many cobalaInins makes paper chromatography more
Reactions involving such light-sensitive materials can be carried out in a dark room
using a photographic "safe light."
18 P. Laland and A. Klem, Acta Moo. Scand. 88, 620 (1936).
18 M. Brenner and A. Niederwieser, Vol. XI, p. 39.
10 Pre coated TLC plates which are suitable for the chromatography of coba.la.mins are
available from both the Eastman Kodak Company a.nd Brinkmann Instruments, Inc.
11
44
[205]
COBALAMINS AND COBAMIDES
reliable, and it is advisable to use three solvent systems when determining
the composition or purity of cobalamins.
Ascending chromatography was carried out on Whatman No.1 paper
using the following three solvent systems (ratios given by volume, the top
layer being used for systems I and II) :
Solvent I: Butan-2-ol-water-25% ammonium hydroxide (50: 36: 14)
Solvent II: n-Butanol-ethanol-water (50:15:35)
Solvent III: n-Butanol-propan-2-ol-water (37: 26: 37)
Mobilities are quoted relative to cyanocobalamin (RCN)'
PROCEDURES
The following specific procedures are presented as representing typical
syntheses of coenzyme analogs, and although a number of different reducing
agents are employed to show the scope of the reactions, we recommend
1.0
I
I
\
I
I
08
I
I
I
I
I
.,u
0.6
\
c
0
.0
(;
«'"
.0
0.4
\
0.2
/
V
./
300
400
500
600
Wavelength ( nm )
FIG. 5. Methylcobalamin, 2.60 X 10-6 M; 1.0-cm cell, water ( - - ) , 0.05 N Hel
(---).
[205]
45
FORMS OF B12 AND ANALOGS CONTAINING Ca-C BOND
using either the cobalt-catalyzed sodium borohydride reaction or controlled
potential reduction whenever possible.
Methylcobalamin. 21 •22 A solution of cyano- or hydroxocobalamin (100
mg) and cobalt nitrate (1 mg) in water (10 ml) is placed in a 25-ml Erlenmeyer flask, stoppered with a serum cap, and deoxygenated with a gentle
stream of argon. After 10 minutes, a deoxygenated solution of sodium
borohydride (20 mg) in water (0.5 ml) is added (foaming may occur at
this stage and the rate of flow of argon may need to be adjusted). The
solution immediately turns brown and then blue-green. After a further
5 minutes, methyl iodide (200 mg) is added whereupon the solution turns
yellow-orange. The cobalamin in solution is purified by extraction through
phenol (see above), and the resulting aqueous solution is reduced to 5 ml
on a rotary evaporator, the heating bath being kept below 50°. This solution
is placed on a column of carboxymethyl cellulose (30 X 2 cm) which has
1.0
\
\
\
\
0..8
\\
\
"
I
~
u
c
"
.c
v
.,o
I~
I \
0..6
Qj
I
\
'1\_
\,/ \
\
.c
<!
0..4
\
\
0.2
30.0.
400
Wavelength ( nm
600
I
FiG. 6. Acetylcoba1amin, 2.75 X 10-' M; 1.O-cm cell, wa.ter ( - - ) , 0.05 N HCl
(---).
11 O. Muller and G. Muller, Biochem. Z. 336, 299 (1962).
2tE. L. Smith, L. Mervyn, P. W. Muggleton, A. W. Johnson, and N. Shaw, Ann. N.Y.
Acad. Sci. 112,565 (1964).
46
[205]
COBALAMINS AND COBAMIDES
1.0
I
\
\
0..8
I
I
\
\
\
'"
0.6
I.)
c
C
Ll
;:;
Vl
Ll
«
0..4
0.2
-------~--,
30.0.
40.0.
500.
60.0.
Wavelength (nm )
FIG. 7. Carboxymethylcobalamin, 2.20 X 10-6 M; l.O-cm cell, water ( - - ) ,
0.05 N HC} (- - -).
been washed with 0.1 N hydrochloric acid (20 ml) and then water until the
washings are neutral, and eluted from the column with water. The eluate
is reduced in volume to 1 ml and treated with acetone until the solution
shows a faint turbidity. On standing overnight, bright red crystals of
methylcobalamin are deposited. These are collected by filtration, washed
with acetone, and air dried. Yield: 91 mg.
Light absorption: Fig. 5.
RCN values: solvent I, 1.9; solvent II, 2.7; solvent III, 1.3.
Acetylcobalamin.21 •22 Cyano- or hydroxocobalamin (50 mg) in 10%
acetic acid (10 ml) is magnetically stirred and deoxygenated with a stream
of argon. After 10 minutes, zinc dust (1 g) is added. The solution rapidly
turns brown and then after a further 10 minutes, blue green. Acetyl chloride,
or acetic anhydride (150 mg), is added and the resulting yellow-brown
solution is filtered immediately to. remove unreacted zinc. The pro.duct in
the filtrate is purified by extraction through phenol, and the resulting
aqueous layer is treated in the manner described above for methylcobalamin. The product crystallizes from aqueous acetone as large red needles.
Yield: 37 mg.
[205]
47
FORMS OF B12 AND ANALOGS CONTAINING CerC BOND
Light absorption: Fig. 6.
RoN values: solvent I, 0.44 (decomposes to B 12b); solvent 11,1.8; solvent
III, 1.3.
Carboxymeth'!,ilcobalamin. 21 •22 A magnetically stirred solution of cyanoor hydroxocobalamin (100 mg) in 15% ammonium chloride (15 ml) is
deoxygenated with a stream of argon; after 10 minutes, zinc dust (1 g)
is added. The solution turns brown and then blue green. Monochloroacetic
acid (200 mg) is added to the stirred solution which immediately turns
orange-brown. This solution is filtered, and the filtrate is purified by extraction through phenol. The resulting aqueous solution is treated in the manner described for methylcobalamin, and the product is crystallized from
aqueous acetone as red needles. Yield: 71 mg.
Light absorption: Fig. 7.
RoN values: solvent I, 0.81; solvent II, 1.6; solvent III, 1.0.
fJ-Hydroxyethylcobalamin. 22 Cyano- or hydroxocobalamin (5 mg) in
phosphate buffer, pH 9 (5 ml), is reduced at a potential of 1.3 V vs. a
silver-silver chloride reference electrode until the current decreases to a
1.0
0.8
I
\
I
I
\
\
\
\
'"
0.6
u
\
\
c
\
15
\
0
.0
01)
.0
«
0.4
\
\
\
,
,f\.
\
\
I 'If'
IJ/\
'\
\..'
\
\
\",
"-
0.2
'--"-
300
~-
400
500
--...-
600
Wavelength ( nm )
FIG. 8. p-HydroxyethylcobaIamin, 2.19 X la-1 M; 1.O-em cell, water ( - - ) ,
O.05N He} (---).
48
[205]
COBALAMINS AND COBAMIDES
steady low value of about 0.1 mAo Ethylene oxide is then passed into the
solution23 for 2 minutes; by this time the color has changed from green to
orange red. The cobalamin in solution is purified by extraction through
phenol, and the resulting aqueous layer is treated in the manner described
for methylcobalamin. The product crystallizes from aqueous acetone as
large red needles. Yield: 4.7 mg.
Light absorption: Fig. 8.
RCN values: solvent I, 1.2; solvent II, 1.5; solvent III, 0.95.
Vinylcobalamin. 22 To a solution of cyano- or hydroxocobalamin (50
mg) in methanol (20 ml) is added cobalt nitrate (1 mg) in water (0.2 ml).
The resulting solution is deoxygenated with a stream of argon, and after
10 minutes a deoxygenated solution of sodium borohydride (10 mg) in
water (0.5 ml) is added. The solution rapidly turns blue green after passing
through a brown stage. Acetylene is passed through this solution13 for 2
minutes; by this time the solution has turned orange red. The flow of
acetylene is then stopped, but the argon is continued for 10 minutes longer.
1.0
0.8
'"c
0.6
<.)
o
.Q
(;
f'
,"-
'"
.Q
<!
OA
/
"
\
~
'--0.2
300
400
500
600
Wavelength ( nm )
FIG. 9. Vinylcobalamin, 1.82 X 10-6 M; 1.0-cm cell, water ( - - ) , 0.1 N Hel
(---).
23
It is important that there be no air in the tube connecting the gas tank to the reaction
vessel.
[205]
FORMS OF B12 AND ANALOGS CONTAINING Co-C BOND
49
The resulting solution is taken to dryness, and the residue is dissolved in
water (5 ml). This product is purified from aqueous solution by extraction
with phenol, and treated in the manner described for methylcobalamin.
The product crystallizes from aqueous acetone as very small red needles.
Yield: 41 mg.
Light absorption: Fig. 9.
RCN values: solvent 1,2.0; solvent II, 2.4; solvent III, 1.5.
Ethynylcobalamin. 22 A solution of cyano- or hydroxocobalamin (50
mg) in 0.2 M EDTA (10 ml) is deoxygenated with argon for 10 minutes.
Chromous acetate 24 (20 mg) is added, and when the solution had turned
purple green, bromoacetylene26 (100 mg) is swept into the reaction mixture
with a stream of argon. This is continued for a further 10 minutes after
the bromo acetylene has been passed into the reaction vessel. The red
solution is purified by extraction through phenol, and the resulting aqueous
10
08
0.6
QJ
U
c;
a
.0
~
.0
<l:
04
v-
I
\
0.2
300
400
500
600
Wavelength ( nm)
FIG. 10. Ethynylcobalamin, 1.90 X 10-5 M; 1.0-cm cell, water ( - - ) , 5.0 N Hel
(---).
M. R. Hatfield, in "Inorganic Syntheses," Vol. III, p. 148. McGraw-Hill, New York,
1950.
25 When bromoacetylene is prepared and purified by the method of L. A. Bashford,
H. J. Emeleus, and H. V. A. Briscoe [J. Chem. Soc. p. 1358 (1938)], no bromovinyl
cobalamin is produced.
24
50
[205]
COBALAMINS AND COBAMIDES
solution is treated in the manner described for methylcobalamin. The
product crystallizes from aqueous acetone as deep red needles. Yield: 38 mg.
Light absorption: Fig. 10.
ReN values: solvent I, 1.45; solvent II, 2.2; solvent III, 1.4.
Electronic Absorption Spectra
The electronic absorption spectra of the cobalamins have not yet been
given detailed theoretical interpretations. However, general trends exist
within the system, and the spectra are useful for both qualitative and
quantitative analysis.
When the cyanide ligand of vitamin B12 is replaced by other ligands
such as H 20, OH-, NH a, or pyridine, only small changes in the spectra
occur (Table II).
TABLE II
ELECTRONIC ABSORPTION SPECTRA OF COBALAMINS VB. LIGAND
Principal absorption bands (nm)
(E X 10-4 in parentheses)
Ligand
CN-
361
351
356
357
359
H 20
NHa
OHPyridine
(2.75)
(2.26)
(2.30)
(1.75)
(2.30)
518
505
513
513
513
(0.80)
(0.71)
(0.72)
(0.72)
(0.72)
548
530
538
538
538
(0.85)
(0.75)
(0.76)
(0.77)
(0.76)
However, when the cyanide ligand of vitamin B12 is replaced by a ligand
such as methyl, there is a considerable change in the spectrum, and the
"coenzyme type" of spectrum is observed. Since the extent of the conjugated chromophone in the corrin ring and the valency of the cobalt are
the same in both vitamin B12 and the B12 coenzyme and its analogs, the
differences in the observed spectra are due to the influence of the sixth
ligand, ligands such as vinyl and ethynyl give spectra which are half way
between those of cyano- or hydroxycobalamin and methylcobalamin
(Figs. 1, Q, 9, and 10 and Table III).
TABLE
III
ELECTRONIC ABSORPTION SPECTRA OF COBALAMINS VB. LIGANDS
Ligand
-CHa
-CH=CH 2
-CsaCH
-C==N
Principal absorption bands (nm)
(e X 10-4 in parentheses)
340
340
340
322
(1.27)
(1.31)
(1.01)
(0.74)
377 (1.05)
375 (1.21)
369 (1.52)
361 (2.75)
528
528
538
518
(0.79)
(0.30)
(0.68) 552 (0.73)
(0.80) 548 (0.85)
TABLE IV
PRINCIPAL ABSORPTION BANDS OF THE PRoTONATI!lD
Ligand
5'-Deoxyadenosyl
Methyl
Vinyl
Ethynyl
SPECIES
Bands (nm) (e X 10-4 in parentheses)
264
(3.81)
264
(2.48)
260
(2.18)
266
(2.02)
275
(2.73)
278
(1.91)
278
(2.10)
277
(1.78)
286
(2.20)
286
(2.04)
286
(2.08)
287
(1. 73)
305
(2.06)
304
(2.24)
325
(1.45)
320
(1.40)
315
(1.88)
317
(1.85)
350
(1.07)
350
(1.00)
327
(1.63)
333
(1.27)
460
(0.79)
457
(0.75)
347
(1.04)
378
(0.87)
382
(0.79)
384
(0.80)
462
(0.83)
461
(0.89)
52
COBALAMINS AND COBAMIDES
[206]
Thus from cyano- through ethynyl- to methylcobalamin there is a continuous change in the electronic absorption spectra, and this change can
be correlated with the electron withdrawal of the ligands, i.e., -C=N >
-C=CH > -CH=CH 2 > -CHao
In dilute mineral acid, the vitamin B12 coenzyme and its alkyl and acyl
analogs are protonated and change color from red to yellow. The principal
absorption bands for some typical protonated species are given in Figs.
5-10 and Table IV. Examination of the spectra of the protonated and
nonprotonated alkylcobalamins in the region of 280 nm shows26 that in
the protonated species the 5,6-dimethylbenzimidazole is protonated and
no longer coordinated to the cobalt. Electrophoresis of such protonated
species has shown that only one proton is involved, indicating that water
is coordinated in place of the benzimidazole. This results in an increase in
electron density on the cobalt and a general shift of the absorption bands
to shorter wavelengths.
26
G. H. Beaven, E. R. Holiday, E. A. Johnson, B. Ellis, and V. Petrow, J. Pharm. 2,
944 (1950).