A simple, convenient method to synthesize cobalamins: Synthesis of homocysteinylcobalamin, N-acetylcysteinylcobalamin, 2-N-acetylamino-2- carbomethoxyethanethiolatocobalamin, sulfitocobalamin and nitrocobalamin

Article abstract of DOI:10.1039/b610158e

Glutathionylcobalamin, nitrocobalamin and sulfitocobalamin are important cobalamin metabolites isolable from human tissues. Herein we demonstrate that a procedure used to synthesize and isolate γ-glutamylcysteinylcobalamin and glutathionylcobalamin in aqueous solution in high yield and purity can be used to synthesize other novel, biologically relevant thiolatocobalamins, including d,l-homocysteinylcobalamin, N-acetyl-l-cysteinylcobalamin (Na⁺ salt) and 2-N-acetylamino-2-carbomethoxy-l-ethanethiolatocobalamin, as well as other non-alkylcobalamins, such as sulfitocobalamin (Na⁺ salt) and nitrocobalamin. This uncomplicated, general procedure will assist researchers in identifying unknown cobalamin metabolites isolated from biological samples, and researchers interested in studying the uptake and intracellular cobalamin processing mechanisms utilizing non-alkylcobalamin derivatives that are not yet commercially available. The X-ray structure and XAS spectrum of N-acetyl-l-cysteinylcobalamin are also presented. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b610158e

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PAPER
www.rsc.org/dalton | Dalton Transactions
A simple, convenient method to synthesize cobalamins: synthesis of
homocysteinylcobalamin, N-acetylcysteinylcobalamin,
2
-N-acetylamino-2-carbomethoxyethanethiolatocobalamin, sulfitocobalamin
and nitrocobalamin†
a,b,c
a,b,c
d
b
Edward Suarez-Moreira,
Luciana Hannibal,
Clyde A. Smith, Roberto A. Chavez,
a,c
a,b
Donald W. Jacobsen and Nicola E. Brasch*
Received 17th July 2006, Accepted 31st August 2006
First published as an Advance Article on the web 21st September 2006
DOI: 10.1039/b610158e
Glutathionylcobalamin, nitrocobalamin and sulfitocobalamin are important cobalamin metabolites
isolable from human tissues. Herein we demonstrate that a procedure used to synthesize and isolate
c-glutamylcysteinylcobalamin and glutathionylcobalamin in aqueous solution in high yield and purity
can be used to synthesize other novel, biologically relevant thiolatocobalamins, including
+
D,L-homocysteinylcobalamin, N-acetyl-L-cysteinylcobalamin (Na salt) and 2-N-acetylamino-2-
carbomethoxy-L-ethanethiolatocobalamin, as well as other non-alkylcobalamins, such as
+
sulfitocobalamin (Na salt) and nitrocobalamin. This uncomplicated, general procedure will assist
researchers in identifying unknown cobalamin metabolites isolated from biological samples, and
researchers interested in studying the uptake and intracellular cobalamin processing mechanisms
utilizing non-alkylcobalamin derivatives that are not yet commercially available. The X-ray structure
and XAS spectrum of N-acetyl-L-cysteinylcobalamin are also presented.
Introduction
Corrinoid-dependent enzymes are widespread in nature and play
1
key roles in human, animal and microbial metabolism. Two
known B12-dependent enzymes exist in humans: methylcobalamin
(
(
MeCbl)-dependent methionine synthase and adenosylcobalamin
AdoCbl)-dependent methylmalonyl-coenzyme A mutase. The
2–4
clinical hallmarks of B12 deficiency are megaloblastic anemia
5
(
‘pernicious anemia’), and/or neuropathies. Fig. 1 gives the
structure of the two coenzyme forms of vitamin B12 and related
12 derivatives (cobalamins) found in humans. Upon reaching
B
cells, cobalamin derivatives are converted to MeCbl and AdoCbl
by currently ill-defined mechanisms. Many studies have been
carried out over the past several decades involving the extraction
and identification of cobalamins from mammalian cells, tissue
and blood, in addition to other biological samples such as
foods and seaweed. Adenosylcobalamin, methylcobalamin and
+
+
6
aquacobalamin (H
2
OCbl ↔ HOCbl + H ; pK
a
= 7.8) are the
7–10
major cobalamin metabolites isolated from biological samples.
ꢀ
−
Fig. 1 Structures of naturally occurring cobalamins: X = 5 -deoxy-
3
Sulfitocobalamin (SO
3
Cbl ) is also isolable from mammalian
and there are also reports of the isolation of
Cbl) from biological sources.
adenosyl, adenosylcobalamin (coenzyme B12); X = CH
, methylcobal-
9–19
cells and foods,
−
2−
amin; X = H
2
O/OH , aquacobalamin/hydroxycobalamin; X = SO
3
,
11,20
nitrocobalamin (NO
2
Whether or
−
−
sulfitocobalamin; X = NO , nitrocobalamin; X = CN , cyanocobalamin;
2
X = glutathione, glutathionylcobalamin. The positions of the protons that
1
a
resonate in the aromatic region in the H NMR spectrum of cobalamins
School of Biomedical Sciences, Kent State University, Kent, OH, 44240
b
Department of Chemistry, Kent State University, Kent, OH, 44240. E-mail:
nbrasch@kent.edu
(B2, B4, B7, R1 and C10) are also shown.
c
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic,
not cyanocobalamin is truly ‘naturally occurring’ is controversial;
some studies report small amounts of this derivative, especially in
Cleveland, OH, 44195
d
Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo
7,21
smokers.
Park, CA, 94025
Thiol derivatives of B12, thiolatocobalamins, were first iden-
tified in the 1960s, but have not attracted much attention until
1
†
Electronic supplementary information (ESI) available:
spectra of Na[SO Cbl] (Fig. S1) and NO
0.1039/b610158e
H NMR
3
2
Cbl (Fig. S2). See DOI:
18,19,22–44
1
recently.
Glutathionylcobalamin (GSCbl) is an important
This journal is © The Royal Society of Chemistry 2006
Dalton Trans., 2006, 5269–5277 | 5269

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31–33
cobalamin metabolite in mammals,
and is more active than
is formed in biological systems to any appreciable extent, NO
2
Cbl
other cobalamins in promoting methionine synthase activity in
rabbit spleen extracts. We proposed that GSCbl (or a closely related
thiolatocobalamin adduct) is a precursor of the two coenzyme
rather than NOCbl will be isolated after typical (aerobic) isolation
and purification procedures.
70,71
Finally, we note that there are numerous studies concerning the
isolation and identification of cobalamin metabolites from biolog-
41
forms of vitamin B12, AdoCbl and MeCbl. Formation of GSCbl
+
8
−1
72–77
from H
2
OCbl and glutathione is irreversible (Kobs ∼ 3 × 10 M
ical samples which report ‘unknown’ analogues of cobalamin.
◦
at pH 7.4, 25 C) and rapid (t1/2 ∼ 3 s for [GSH] = 5 mM,
To assist in their identification, simple procedures are required
to synthesize potential candidates with different b-axial ligands,
allowing spectroscopic and chromatographic comparison of un-
known cobalamin metabolites with known standards. In addition,
pure, well characterized cobalamin derivatives will assist re-
searchers investigating intracellular cobalamin processing. Simple
synthetic procedures are also useful when expensive, radioactive
cobalamin derivatives are desired for studies on the uptake and/or
conversion of cobalamin derivatives in humans, animals and cell
◦
45
pH 7.4, 37 C). An alternative role for GSCbl was also recently
proposed, in which the formation of GSCbl prevents B12 from
24
being scavenged by xenobiotics. Finally, McCaddon and co-
workers suggested that GSCbl and related thiolatocobalamins
might be more effective than currently available pharmaceutical
+
B
12 forms (CNCbl and H
2
OCbl ) in treating B12-related conditions
34,35
associated with oxidative stress such as Alzheimer’s disease.
However, the apparent clinical benefit of combining N-acetyl-L-
+
40,78–80
cysteine with H
2
OCbl in patients with Alzheimer’s disease or
models.
mild cognitive impairment requires formal clinical trials to prove
34,46
or refute such observations.
Experimental
In this paper we describe the synthesis of three novel
thiolatocobalamins: D,L-homocysteinylcobalamin (HcyCbl), the
sodium salt of N-acetyl-L-cysteinylcobalamin (Na[NACCbl]),
Hydroxycobalamin hydrochloride (HOCbl·HCl), 98% (stated pu-
rity by manufacturer) was purchased from Fluka. The percentage
and
2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobal-
of water in HOCbl·HCl (·nH
2
O) (batch-dependent, typically 10–
amin (NACMECbl). The thiol ligand structures are shown in
Fig. 2. HcyCbl is of interest, since it is potentially a naturally occur-
ring cobalamin metabolite. In the MeCbl-dependent methionine
synthase reaction, a methyl group is transferred from methylte-
trahydrofolate to homocysteine via MeCbl to generate methionine
and tetrahydrofolate. Impairment of this reaction leads to elevated
serum levels of homocysteine, which is associated with an in-
creased risk of cardiovascular, peripheral vascular and cerebrovas-
1
5%), was determined by converting HOCbl·HCl to dicyanocobal-
− −1
2
amin, (CN)
cm
Cbl (0.10 M KCN, pH 10.0, e368 nm = 30.4 mM
(99%), MES, KNO (99%) and D,L-homocysteine
−1 81
). NaN
3
3
were from ACROS Organics and glutathione (GSH, 98%; i.e., in
its reduced form) was purchased from Aldrich. NaOAc (99%),
NaNO
2
(>98%), NaSO (>98%) and N-acetyl-L-cysteine were
3
obtained from Sigma. Water was purified using a Barnstead
Nanopure Diamond water purification system and/or HPLC
grade water. All thiol solutions were prepared directly before use.
pH Measurements were made with a Corning Model 445 pH
meter in conjunction with a Mettler-Toledo Inlab 423 electrode
at room temperature. The electrode was filled with 3 M KCl–
saturated AgCl solution, pH 7.0. The electrodes were standardized
with standard BDH buffer solutions at pH 4.01 and 6.98. Solution
pH was adjusted using HCl or NaOH solutions as necessary.
47–52
cular diseases.
In addition, moderately raised serum homocys-
teine levels are more prevalent in patients suffering from neurolog-
5
3–56
ical diseases such as Alzheimer’s disease,
and appear to predict
57,58
cognitive decline.
benefits of co-administrating H
Na[NACCbl] is also of interest, given the
+
2
OCbl and N-acetyl-L-cysteine to
and the possibility that NACCbl , rather
34,46
−
Alzheimer’s patients,
than the individual components themselves, is responsible for the
beneficial effects of this approach. The X-ray structure and XAS
1
H NMR spectra were recorded on an Inova 500 MHz or a
spectrum of Na[NACCbl]·18H
2
O are also reported.
Bruker 400 MHz spectrometer equipped with a 5 mm probe at
◦
room temperature (22 ± 1 C). Solutions were prepared in D
2
O
or MES buffer (pD 5.50) and TSP (3-(trimethylsilyl)propionic-
,2,3,3-d acid, sodium salt) was used as an internal standard.
2
4
UV-Vis spectra were recorded on a Cary 5000 spectrophotometer
◦
equipped with a thermostatted cell changer (25.0 ± 0.1 C),
operating with WinUV Bio software (version 3.00). Electrospray
mass spectra were recorded using a BRUKER Esquire-LC mass
spectrometer. Mass spectra for all cobalamins were recorded in the
positive mode, except for sulfitocobalamin, which was recorded in
the negative mode.
For experiments conducted under anaerobic conditions, solu-
tions were degassed using three freeze–pump–thaw cycles and
argon using standard Schlenk techniques. Air-free manipulations
were carried out on a Schlenk line or in an MBRAUN Labmaster
Fig. 2 Structure of the thiol ligands.
We were also interested to see whether our procedure could
be used to synthesize other non-thiol cobalamin derivatives,
such as the sodium salt of sulfitocobalamin (Na[SO
130(1250/78) glovebox.
3
Cbl]) and
nitrocobalamin (NO Cbl). Although both are isolable from
2
Synthesis of N-acetyl-L-cysteine methyl ester
1
1–17,20
biological samples,
characterized,
NO
and are well studied and structurally
neither are commercially available.
23,29,38,43,59–69
N-Acetyl-L-cysteine methyl ester was prepared according to a
82
2
Cbl is also of interest, since if nitrosylcobalamin (NOCbl)
modified published procedure. Briefly, CH
3
OH (1.6 ml, 39 mmol)
5
270 | Dalton Trans., 2006, 5269–5277
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and concentrated HCl (180 ll) were added slowly to a solution
D,L-Homocysteinylcobalamin (HcyCbl)
of N-acetyl-L-cysteine (NAC, 640 mg, 3.9 mmol) in chloroform
◦
The synthesis of this derivative was carried out under strictly
anaerobic conditions. An anaerobic solution of D,L-Hcy (395 lml,
191 mM, 75.4 lmol, 1.2 mol equiv.) in MES buffer (0.1 M, pH
(
9 ml). The reaction mixture was refluxed for 3 h at 66 C, taken to
dryness by rotary evaporation and the product recrystallized from
1
ethyl ether. Yield: 405 mg (59%). H NMR: (d, ppm) 1.29 (t CH),
∼
6) was added drop wise to an anaerobic solution of HOCbl·HCl
2
.07 (s, MeCO), 2.97 (d CH ), 3.78 (s MeO).
2
(
98.7 mg, 62.8 lmol) in MES buffer (0.80 ml, 0.1 M, pH ∼6)
with stirring, and the reaction allowed to proceed for 5 min at
0
Determination of the thiol concentrations
◦
C. The product precipitated upon dripping into a degassed
◦
The total reduced thiol concentration was determined using the
chilled acetone solution (−20 C), and was filtered, washed with
◦
◦
83
Ellman’s procedure. One part of the thiol solution was diluted
chilled acetone (20 ml, −20 C). The product was dried at 50 C
−
1
−2
with five parts of Ellman’s reagent (32 lg ml dithionitrobenzoic
under vacuum (2 × 10 mbar) overnight. Yield: 64.4 mg (70%).
acid and 1.25 mM EDTA in 0.5 M TRIS, pH 8.5) and the
The purity assessed by conversion to dicyanocobalamin was 97%.
absorbance determined at 412 nm (e = 13.6 mM− cm ).
1
−1
1
2
H NMR (D
O, d, ppm): 6.10 (s C10), 6.28 (d R1), 6.38 (s B4),
1
6
.95 (s B2), 7.20 (s B7). The cobalamin purity assessed by H
NMR spectroscopy was ∼98%. ES-MS, m/z: 1464.8 (calcd for
Synthesis of thiolatocobalamins
+
[
HcyCbl + H] , C66
H
98CoN14
O
16PS = 1464.6); 1486.7 (calcd for
+
All syntheses were carried out under red-light-only conditions, due
[HcyCbl + Na] , C H CoN NaO PS = 1486.6); 732.5 (calcd
6
2+
6
97
14 16
43
to the potential light sensitivity of thiolatocobalamins. Unless
specifically stated otherwise, all syntheses were carried out under
aerobic conditions.
for [HcyCbl + 2 H] , C H CoN O PS = 732.8); 743.4 (calcd
6
6
99
14 16
2
+
for [HcyCbl + Na + H] , C H CoN NaO PS = 743.8).
6
6
98
14
16
Synthesis of non-thiol cobalamins
Sulfitocobalamin, sodium salt (Na[SO
N-Acetyl-L-cysteinylcobalamin, sodium salt (Na[NACCbl])
3
Cbl]). A Na
2
SO solu-
3
A solution of N-acetyl-L-cysteine (263 lml, 284 mM, 74.7 lmol,
tion (266 mM, 519 ll, 138.1 lmol, 1.3 mol equiv.) was added drop-
wise to a HOCbl·HCl solution (162.3 mg, 103.3 lmol) in MES
1
.1 mol equiv.) in MES buffer (0.1 M, pH ∼6) was added drop-
wise to a solution of HOCbl·HCl (107 mg, 67.9 lmol) in MES
◦
buffer (pH 6.0, 1.60 ml), and the mixture left to react at 0 C (ice
buffer (0.80 ml, 0.1 M, pH ∼6) with stirring, and the reaction
bath) for 2 h. The product precipitated upon dripping into acetone
◦
was allowed to react for 30 min at 0 C. The product precipitated
◦
(
20 ml, −20 C), and was washed with acetone (20 ml), diethyl
◦
upon dripping into a chilled acetone solution (−20 C), and was
◦
ether (−20 C) and dried overnight under vacuum. Yield: 145.7 mg
◦
filtered, washed with chilled acetone (20 ml, −20 C) and diethyl
81
(
98%). The purity assessed by conversion to dicyanocobalamin
◦
◦
ether (10 ml, −20 C). The product was dried at 50 C under
1
was 99 ± 2%. H NMR (D
2
O, d, ppm): 5.92 (s C10), 6.15 (d
−
2
vacuum (2 × 10 mbar) overnight. Yield: 90 mg (87%). The
64
R1), 6.33 (s B4), 6.84 (s B2), 7.07 (s B7). The cobalamin purity
81
purity assessed by conversion to dicyanocobalamin was 95 ±
1
assessed by H NMR spectroscopy was 98%. ES-MS, m/z: 1408.5
1
2
%. H NMR (D
2
O, d, ppm): 6.10 (s C10), 6.28 (d R1), 6.40 (s
−
−
(
calcd for [SO
3
Cbl] , C62
H
89CoN13
O17PS = 1409.5).
B4), 6.96 (s B2), 7.20 (s B7). The cobalamin purity assessed by
1
84
H NMR spectroscopy was ∼98%. ES-MS, m/z: 1492.3 (calcd
Nitrocobalamin (NO
nitrocobalamin was similar to that for Na[NACCbl], except that
a NaNO solution (61.1 mM, 100 lml, 61.1 lmol, 1.2 mol equiv.)
2
Cbl). The procedure for the synthesis of
+
for [NACCbl + 2 H] , C67
for [NACCbl + H + Na] , C67
(
(
H
98CoN14
O
17PS = 1492.6); 1513.9 (calcd
+
H
97CoN14NaO17PS = 1514.6); 746.5
2
2
+
calcd for [NACCbl + 3 H] , C67
calcd for [NACCbl + Na + 2 H] , C67
H
99CoN14
O
17PS = 746.8); 757.7
was added drop-wise to a HOCbl·HCl solution (80.0 mg,
2
+
H
98CoN14NaO17PS =
50.9 lmol) in MES buffer (pH 6.0, 0.90 ml), and the mixture left
◦
7
57.8).
to react at 0 C (ice bath) for 2 h. The product precipitated upon
dripping into acetone (5 ml), and was washed with acetone (20 ml)
◦
and dried overnight under vacuum at 60 C. Yield: 151 mg (92%).
2
-N-Acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin
81
The purity assessed by conversion to dicyanocobalamin test was
O, d, ppm) 6.20 (s C10), 6.28 (d R1), 6.42 (s
B4), 6.74 (s B2), 7.20 (s B7). The cobalamin purity assessed by H
(
NACMECbl)
1
9
9 ± 2%. H NMR (D
2
The procedure was similar to that for Na[NACCbl], except
that a solution of N-acetyl-L-cysteine methyl ester (NACME,
70
1
NMR spectroscopy was 98%. ES-MS analysis, m/z: 1375.7 (calcd
249 lml, 250 mM, 62.2 lmol, 1.2 mol equiv.) in MES buffer
+
for [NO
2
Cbl + H] , C62
H
90CoN14
O
16P = 1376.6); 1398.6 (calcd for
was added to a HOCbl·HCl solution (81.5 mg, 51.8 lmol)
+
[
[
[
NO
NO
NO
2
2
2
Cbl + Na] , C62H
89CoN14NaO16P = 1398.6); 689.0 (calcd for
in MES buffer, and the mixture left for 30 min to react at
2+
Cbl + 2 H] , C62
H
91CoN14
H
O
16P = 688.8); 699.9 (calcd for
◦
0
C. Yield: 66.3 mg (85%). The purity assessed by the di-
2+
Cbl + Na + H] , C62
90CoN14NaO16P = 699.8).
81
1
cyanocobalamin test was 96 ± 2%. H NMR (D
2
O, d, ppm):
6
.10 (s C10), 6.30 (d R1), 6.40 (s B4), 6.95 (s B2), 7.21 (s
X-Ray diffraction studies on N-acetyl-L-cysteinylcobalamin
sodium salt)
1
B7). The cobalamin purity assessed by H NMR spectroscopy
(
+
was ∼98%. ES-MS, m/z: 1506.6 (calcd for [NACMECbl + H] ,
C
Na] ,
68
H
100CoN14
O
17PS = 1506.6); 1527.9 (calcd for [NACMECbl +
An aliquot of a NAC solution (182 mM, 40 lL, in HPLC grade
O) was added to a solution of HOCbl·HCl (100 lL, 36 mM,
in H O), NaCl (1.4 mg) added, and the solution mixed until
+
C
68
H
99CoN14NaO17PS
=
1528.6); 753.7 (calcd for
H
2
2
+
([NACMECbl + 2 H] , C68
H
101CoN14
O17PS = 753.8); 765.0 (calcd
2
2
+
for [NACMECbl + Na + H] , C68
H
100CoN14NaO17PS = 764.8).
everything dissolved. The solution was kept at room temperature
This journal is © The Royal Society of Chemistry 2006
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Table 1 Crystal data and structure refinement parameters for Na[NACCbl]·18H
2
O
Parameter
Value
Empirical formula
FW/g mol
T/K
Wavelength/A˚
NaC67
1838
100
132 14
H N O35PCoS
−
1
0.817975
P2
Space group
1 1 1
2 2
a/A˚
16.153
21.076
25.449
8663.8(8)
4
b/A˚
c/A˚
V/ A˚ 3
Z
−
1
Absorption coefficient/mm
F(000)
0.31
3208
Limiting indices
Reflections collected/unique
−19 ≤ h ≤ 19, −25 ≤ k ≤ 25, −32 ≤ l ≤ 32
53901/17617
R
merge and Rsym
0.105, 0.119
Full-matrix least squares on F
17617/0/1143
0.986
2
Refinement method
Data/restrains/parameters
2
GOF on F
R factors (I > 4r(I))
R1 = 0.1030, wR2 = 0.2645
R factor (all data)
R1 = 0.1082
Largest difference peak and hole/e A˚
3
+1.3 and −1.1
in a closed glass vial protected from light. Small crystals were
16106 reflections, with F > 4rF. Additional data collection and
o
observed after 24 h. Suitable crystals for X-ray diffraction studies
refinement statistics are given in Table 1.
CCDC reference number 615032.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b610158e
◦
were obtained after leaving the sample in a fridge at 4
for 5 d.
C
A crystal (∼0.3 × 0.1 × 0.1 mm) was mounted under paraffin
oil in a nylon loop and flash frozen in liquid nitrogen. Diffraction
experiments were carried out on beamline BL11-1 at the Stanford
Synchrotron Radiation Laboratory (SSRL). Data were collected
on an ADSC Q-315 CCD detector using X-rays produced by a
Measurement of the Co(III) absorption spectrum
The cobalt absorption edges for the Na[NACCbl] sample were
measured on SSRL beamline BL9–2 from the frozen crystal used
for data collection (irradiated sample) and from a freshly mounted
frozen crystal which had not been exposed (unirradiated sample),
using X-rays produced by a 16 pole wiggler insertion device
through a flat Rh coated collimating mirror, a liquid nitrogen
cooled double Si(111) crystal monochromator and a toroidal
focussing mirror. The spectra were collected in fluorescence mode
between 7500 and 7900 eV using a Canberra/Eurisys Si drift
detector with a total acquisition time of 440 s. The spectra were
normalized by dividing the sample fluorescence at each point by
the fluorescence at the inflection point of the first EXAFS peak (at
7.784 eV). The first derivative of the spectra were calculated using
˚
2
6 pole wiggler insertion device, with a wavelength of 0.81798 A
(
15160 eV) from a side scattering bent asymmetric cut Si(111)
crystal monochromator. Two data sets were collected, both
◦
consisting of 90 1 images with a crystal to detector distance of
9
7 mm and covering the same range of phi angle. The first high-
resolution pass had an exposure time of 15 s and the second pass
had an exposure time of 1 s to record the strong low resolution
reflections discarded from the first pass due to overloading of the
CCD detector. The data were processed with the program XDS
85
and scaled together with the program XSCALE. Bijvoet pairs
were not merged and no absorption correction was applied. A
total of 53901 reflections were measured to a nominal resolution
87
˚
of 0.77 A, resulting in a final unique dataset of 17617 reflections
the program AUTOCHOOCH which is based on the Kramers–
Kronig transformation algorithm and the inflection point or
threshold energy taken as the minimum of this transformation.
with a merging R factor of 0.105.
The structure was solved by Patterson methods to locate the
cobalt, phosphorus and sulfur atoms, then the lighter atoms
located by difference Fourier synthesis, as implemented in the
86
Results and discussion
program SHELXS. The structure was refined by full matrix
least-squares methods using SHELXL. All non-hydrogen atoms
were refined with anisotropic thermal parameters and hydrogen
atoms were added in idealized positions and refined in riding
positions. A correction for the anomalous scattering from cobalt
at 15160 eV was applied during refinement. Additional difference
electron density peaks were modelled as water molecules. One of
the hydroxyl oxygen atoms of the ribose moiety appeared to have
two distinct conformations and these were modelled with 50%
occupancy. The final crystallographic R factor, R1, was 0.1023 for
Synthesis and characterization of D,L-homocysteinylcobalamin,
+
N-acetyl-L-cysteinylcobalamin (Na salt) and 2-N-acetylamino-
2
-carbomethoxy-L-ethanethiolatocobalamin
Previously one of us reported procedures for synthesizing and
isolating the thiolatocobalamins c-glutamylcysteinylcobalamin
and glutathionylcobalamin in aqueous solution in high yield and
purity by the addition of a small excess of thiol to a highly concen-
trated solution of aquacobalamin, followed by the addition of
5
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1
◦
Table 2 UV-Vis and H NMR spectroscopy data for cobalamins at pH/pD 5.5 (0.10 M MES buffer); electronic spectra were recorded at 25.0 C and
NMR spectra were collected at room temperature
1
UV-Vis spectroscopy data
H NMR spectroscopy data: chemical shift (ppm)
Cobalamin
k
max/nm
B7
B2
B4
R1
C10
+
H
GSCbl
2
OCbl
349
372
372
372
372
365
354
411
428
428
428
428
418
413
525
534
534
534
534
517
532
7.18
7.19
7.19
7.19
7.20
7.17
7.20
6.54
6.95
6.95
6.95
6.95
6.94
6.74
6.47
6.39
6.40
6.40
6.38
6.43
6.42
6.26
6.28
6.28
6.28
6.28
6.25
6.28
6.30
6.09
6.09
6.09
6.10
6.02
6.20
a
333
333
333
333
312
−
NACCbl
NACMECbl
HcyCbl
SO
NO
−
3
Cbl
2
Cbl
a 1
H NMR chemical shifts from ref. 84.
acetone to precipitate the product after completion of the
Similar spectra were observed for the other thiolatocobalamins.
Since we used a racemic mixture (D,L-Hcy) of the ligand,
two stereoisomers of HcyCbl are formed, making the peak at
6.10 ppm appear as a doublet when it actually corresponds to
27,43
reaction.
A similar procedure was also used to synthe-
size pentafluorophenylthiolatocobalamin and cyclohexylthiolato-
25,26
cobalamin in methanol.
Herein we report that this procedure
1
can be utilized to synthesize the novel and biologically rele-
vant thiolatocobalamin derivatives HcyCbl, Na[NACCbl], and
NACMECbl in high purity (>95%) and in good yield (>70%).
To obtain a pure product, it was necessary to synthesize HcyCbl
under anaerobic conditions. All other syntheses were carried out
under aerobic conditions. It is not entirely clear why the synthesis
of HcyCbl must be carried out under anaerobic conditions while
the other thiolatocobalamins are successfully synthesized in the
two separate species. H NMR chemical shifts in the aromatic
region and UV-Vis wavelength maxima for the new derivatives
are summarized in Table 2, together with previously reported
84
values for glutathionylcobalamin. From this table it can be seen
that there is excellent agreement between the chemical shifts and
wavelength maxima for all thiolatocobalamins.
The percentage of cobalamin impurities present in the products
1
can also be estimated from the aromatic region of the H NMR
presence of air. It has previously been reported that the close
spectrum, and was found to be ∼2% for HcyCbl, Na[NACCbl],
and NACMECbl. The percentage of non-corrinoid products
(salts) in the product can be determined by converting the
thiolatocobalamin to dicyanocobalamin after drying the product
at 50 C under vacuum (e367 nm = 30.4 mM cm ), and was found
to be ≤5%.
+
proximity of a ligand NH
3
group to the Co–S bond could be
responsible for the poor stability of the related cysteinylcobalamin
43
and cysteinylglycinylcobalamin derivatives. The products were
1
◦
−1
−1 81
characterized by ES-MS, H NMR spectroscopy, and UV-Vis
1
spectroscopy. Fig. 3 gives the H NMR spectrum of the aromatic
region for HcyCbl. The expected five signals attributable to the
B2, B4, B7 protons of the a-dimethylbenzimidazole nucleotide,
the C10 proton of the corrin ring and R1 proton of the ribose
X-Ray structure of Na[NACCbl]·18H
2
O
Crystals suitable for X-ray diffraction studies were obtained
for the sodium salt of N-acetyl-L-cysteinylcobalamin from a
concentrated solution of HOCbl·HCl, with 2 molar equivalents
of NAC and 1% NaCl in water. Crystal data and structure
84
are observed at 6.95, 6.38, 7.20, 6.10 and 6.28 (d), respectively.
refinement parameters are given in Table 1. Na[NACCbl]·18H
2
O
crystallizes in the orthorhombic space group P2 with one
1
2 2
1 1
molecule per asymmetric unit. Analysis of the crystal packing by
comparison of the ratios of the c : a and b : a unit cell dimensions
show that these crystals are typical of cluster I packing (c : a =
6
9
1
.575, b : a = 1.305). The crystal structure of the cobalamin
molecule has been described exhaustively in the literature and the
current Na[NACCbl]·18H O structure (Fig. 4A) does not deviate
2
markedly from the cobalamin structures known to date. The
individual cobalamin molecules are oriented in the crystal such
that the plane of the corrin ring is roughly parallel to the ab plane
of the unit cell. However, the neighbouring cobalamin molecules
are not perfectly parallel with each other, which gives rise to
layers of zig-zagged planes when viewed perpendicular to the bc
plane, separated by layers of solvent molecules. The axial base and
the N-acetyl-L-cysteinyl ligand extend into these solvent layers.
When viewed down the 2-fold screw along the c axis, long solvent
channels are clearly evident. This solvent structure has been
modelled in Na[NACCbl] as 18 water molecules. The majority
of the solvent molecules are directly hydrogen bonded to either
1
Fig. 3 H NMR spectrum of D,L-homocysteinylcobalamin in D
5
7
2
O, pD
.9 (0.1 M MES buffer), 22 ± 1 ◦C. Five major signals are observed at
.20, 6.95, 6.38, 6.28, and 6.10 ppm, corresponding to the resonances of
the B7, B2, B4, R1 and C10 protons (see Fig. 1).
This journal is © The Royal Society of Chemistry 2006
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Fig. 4 Thermal ellipsoid plot (30%) of Na[N-acetyl-L-cysteinylcobalamin]·18H
2
O. (A) View of the entire cobalamin complex. The N-acetyl-L-cysteine
is bound to the Co through the sulfur atom (Co–S bond distance = 2.250(3) A˚ ) and the carboxylate O of the NAC ligand is bound to a Na cation. Two
+
+
waters bound to the Na cation are also shown. Other solvent molecules are not shown for clarity. (B) Close-up view of the N-acetyl-L-cysteine ligand.
Table 3 Comparison of the Co coordination sphere in a number of Co–S containing cobalamins
a
b
c
b
Na[NACCbl]
Na[c-GluCysCbl]
[(NH
2
)
2
CSCbl]Cl
[NCS]Cbl
4 3
NH [SO Cbl]
Co–S
2.25
2.06
1.88
1.92
1.93
1.88
17.5
2.27
2.05
1.89
1.90
1.91
1.89
24.2
2.22
2.01
1.85
1.90
1.91
1.88
2.25
1.99
1.89
1.92
1.92
1.90
14.9
2.23
2.13
1.87
1.91
1.89
1.89
Co–N3B
Co–N21
Co–N22
Co–N23
Co–N24
Corrin fold/
◦
14.9
16.3
a
b
c
From ref. 43. From ref. 88. Also see ref. 59. From ref. 69.
an oxygen or a nitrogen atom, although one water molecule is
hydrogen bonded to other water molecules only. There is evidence
The N-acetyl-L-cysteine ligand is bound to the cobalamin
through the sulfur atom as expected (Fig. 4B), with a Co–S
˚
˚
for the presence of a sodium ion at a distance of 2.61 A from one of
bond distance of 2.250(3) A, similar to the bond lengths ob-
the oxygen atoms (O74) of the carboxylate group of the cysteinyl
ligand. Although it was initially modelled as a water molecule, it
served in other Co–S containing cobalamin structures determined
43,59,69,88
previously (Table 3).
Furthermore, the four in-plane Co–
˚
was found to be within 3 A of five other oxygen atoms (O74 and
N bonds and the axial base Co–N bond are all very similar to
˚
four water molecules, at an average distance of 2.87 A), hence it
those reported elsewhere. Comparison of the Na[NACCbl]·18H
2
O
was changed to a sodium ion in the final stages of the refinement.
This is consistent with the presence of a negative charge at the
cysteinyl carboxylate, and the observation of a sodium ion near
the glutamyl carboxylate in the c-GluCysCbl structure. There
are significant intermolecular contacts in the crystal lattice, and
although there are five direct nitrogen–oxygen hydrogen bonds
linking neighbouring cobalamin molecules, the majority of the
interactions involve water-mediated hydrogen bonds. Although
structure with that of c-GluCysCbl is rather interesting, in that
whereas the upward corrin fold in the latter was found to be
◦
the largest yet observed in a cobalamin structure (24.2 ), the
43
◦
corrin fold angle in Na[NACCbl]·18H
2
O of 17.5 is significantly
less, although still slightly larger than some of the other Co–S
containing cobalamins. This is rather intriguing given that the
Co–S and Co–N bond lengths show very little variation between
these two structures, although it was observed in the c-GluCysCbl
that there was a hydrogen bonding interaction between N40 and
43
the final R factor of 10.24% is typical of the cobalamin structures,
˚
this could possibly be explained by a combination of inherent
disorder in the crystal lattice due to the relative lack of direct
contacts versus water-mediated interactions, and the presence
of disorder in the solvent structure itself. Attempts were made
during the latter stages of the refinement to model the disorder
solvent but this gave negligible improvement to the overall R
factors.
the cysteinyl sulfur (N–S distance of 3.41 A), and it was suggested
that this could contribute to the larger upward fold of the corrin
4
3
ring. A similar interaction could exist in Na[NACCbl]·18H
2
O,
although the orientation of the N40 and S1 atoms are not entirely
optimal for hydrogen bonding, in that the C71–S1–N40 angle is
close to 90 and the N–S distance is 3.55 A. However, there could
still be a weak interaction and it is conceivable that the strength
◦
˚
5
274 | Dalton Trans., 2006, 5269–5277
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of this intramolecular hydrogen bond may have an impact on the
amount of pucker in the corrin ring.
molecule, including cleavage of disulfide bonds, decarboxylation
of acid residues, changes in crystal packing and photoreduction
of metal centers in metalloproteins.
90,91
A change in the threshold energy has been interpreted as
additional evidence for significant photoreduction by the X-ray
Co(III) X-ray absorption spectrum
89
The cobalt X-ray absorption spectroscopy (XAS) spectrum from
beam. The threshold energy (or inflection point) of the absorp-
tion edge, calculated from the first derivative of the edge spectra
(see inset) using the Kramers–Kronig algorithm implemented in
AUTOCHOOCH, is the same within experimental error (7721.3 ±
0.2 and 7721.4 ± 0.2, respectively) for the unirradiated and
irradiated Na[NACCbl] crystals. This result combined with the
reasonable Co–S and Co–N bond lengths suggest that radiation
damage of Na[NACCbl] probably has a minimal effect on the Co
oxidation state and the first coordination sphere ligands during X-
ray data collection in this particular case. The threshold frequency
has also previously been used as an indicator of the oxidation
state of the cobalt center, with the threshold frequency supposedly
7
650 to 7850 eV for the Na[NACCbl] crystal used in the X-
ray diffraction experiments (irradiated NACCbl) and a second
Na[NACCbl] crystal from the same crystal batch which had not
previously been exposed to X-rays (unirradiated NACCbl) are
given in Fig. 5. The overall shape of the XANES region of the
unirradiated Na[NACCbl] crystal is reminiscent of the XANES
spectra for unirradiated CNCbl and H OCbl . The XANES
spectrum of the crystal used for the X-ray diffraction data collec-
tion shows a significant drop in the intensity of the first maximum,
+
89
2
+
as was also previously observed for CNCbl and H
2
OCbl upon
89
prolonged exposure to X-rays. In this study, the samples were
exposed to X-rays during the collection of the XAS spectra and
for extended periods in between as a simulation of typical X-ray
exposure during X-ray structure data collection. In the case of
Na[NACCbl], the irradiated sample was produced during normal
synchrotron X-ray diffraction data collection (the total exposure
time was between 25–30 min, including initial test images), and
the XANES spectrum of the irradiated sample was measured
subsequently to determine whether any damage had occurred. To
our knowledge, the current results with Na[NACCbl] is the first
reported example showing that irradiation damage does indeed
occur to cobalamins during X-ray diffraction data collection. The
92
increasing with increasing oxidation state. However, the value
of 7721.3 eV obtained for unirradiated Na[NACCbl] is closer
to that reported for Cbl(I) (7721.0 ± 0.2) rather than Cbl(II)
(7722.0 ± 0.2), or the X-ray insensitive AdoCbl (7722.0 ± 0.2) and
89
MeCbl (7722.5 ± 0.2), suggesting that the threshold frequency
may not always be a good indicator of the oxidation state of
the cobalt center. Values of 7721.8 ± 0.25 and 7721.0 ± 0.2
have been previously reported for GSCbl and cysteinylcobalamin,
respectively.
29
XANES spectrum of GSCbl has also been previously reported,
Synthesis and characterization of sulfitocobalamin and
nitrocobalamin
and resembles the irradiated Na[NACCbl] XANES spectrum. In
this study, XAS and EXAFS spectra were recorded 6–9 times,
hence it is possible that photoreduction of the sample did occur.
It has been proposed that irradiation of cobalamins may lead to
Although equilibria and rate constants for the formation of a
wide range of non-alkylcobalamin derivatives in aqueous solution
89
61
mixed Co(III)/Co(II) oxidation states, which may ultimately be
responsible for the abnormally long Co–N bond lengths observed
for Cbl-bound methylmalonyl-CoA mutase and glutumate mutase
as a consequence of the formation of six-coordinate Cbl(II)
ligated via S, O or N atoms to the cobalt(III) have been reported,
very few studies report the actual isolation of these compounds in
a pure form. Excess ligand is used to ensure the reaction goes to
completion, requiring a subsequent column chromatography de-
salting step (typically Amberlite XAD-2 or XAD-4) to remove the
remaining unreacted ligand prior to isolation of the product. A
Sephadex C-25 column chromatography step may also be required
to separate the product from unwanted cobalamin side-products.
Our method employs very high, practically saturated, concentra-
tions of aquacobalamin in addition to high ligand concentrations,
so that the desired product is rapidly and completely formed using
essentially molar equivalents of ligand. The requirement of a lower
mole equivalent of ligand for complete formation of the product
at higher aquacobalamin concentrations is readily understood
from the definition of the formation constant for the complex.
Since the product solution is so concentrated, the product is
easily isolated in high yield by the addition of cold acetone. In
addition, since high concentrations of ligand are also used, the
percentage of ligand which is converted to other forms during
the reaction can be significantly reduced, so that excess ligand is
not required to compensate for side reactions involving the ligand.
89
species. It is well established in protein crystallography that
X-ray exposure during diffraction data collection generates free
radicals which cause extensive radiation damage to the protein
For example, the percentage of reduced thiol which undergoes B12
-
Fig. 5 X-Ray absorption spectrum of unirradiated (bold) and radiated
catalyzed aerial oxidation to its inactive disulfide form (disulfides
2
Na[NACCbl]·18H O between 7500 and 7900 eV. The inset shows the
44
do not react with aquacobalamin ) during the synthesis of
thiolatocobalamins from aquacobalamin is considerably less if
the synthesis is carried out at high concentrations, and can remove
first derivative of the spectrum, with the negative peak position (7721.3
and 7721.4 eV for the irradiated and unirradiated samples respectively)
corresponding to the position of the inflection point or threshold energy.
This journal is © The Royal Society of Chemistry 2006
Dalton Trans., 2006, 5269–5277 | 5275

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the need for a large excess of the reduced thiol and/or anaerobic
interested in synthesizing cobalamin derivatives to assist in the
identification of unknown cobalamins isolated from biological
samples, and those interested in testing the biological activities
of different cobalamin derivatives in studies designed to elucidate
uptake and intracellular B12 processing mechanisms.
conditions.
Sulfitocobalamin (SO
metabolite,
−
3
Cbl ) is a biologically important B12
11–17
and there are several reports concerning the
15,37,93
synthesis and isolation of this derivative.
These methods
require a large excess of sulfite to obtain complete formation of
−
SO
3
Cbl , requiring subsequent de-salting of the product using
Acknowledgements
column chromatography. B12-catalyzed oxidation of sulfite to
sulfate can also result in the product being contaminated by
the byproduct sulfatocobalamin. It was of interest to see if our
procedure could be applied to the synthesis and isolation of pure
This research was funded by Kent State University (NEB),
the National Heart Lung and Blood Institute of the National
Institutes of Health (HL71907, DWJ), the National Science
Foundation (REU; CHE-0353737) and the Ohio STEM Alliance
Initiative (RAC).
20
Na[SO Cbl] in high yield, and indeed this cobalamin was readily
3
synthesized in the presence of air using 1.2 mol equiv. sulfite
without the need for either anaerobic conditions or subsequent de-
1
salting by column chromatography to remove excess sulfite. The H
NMR spectrum of Na[SO
supplementary information (ESI)† and clearly demonstrates that
3
Cbl] is given in Fig. S1 in the electronic
References
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B. Krautler, Biochem. Soc. Trans., 2005, 33, 806–810.
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