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Vitamin B12 A Methyl Group without a Job.

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Angewandte
Chemie
Molecular Switch
DOI: 10.1002/anie.200502638
Vitamin B12 : A Methyl Group without a Job?**
Philip Butler, Marc-Olivier Ebert, Andrzej Lyskowski,
Karl Gruber, Christoph Kratky, and Bernhard Kr"utler*
Dedicated to Professor Albert Eschenmoser
on the occasion of his 80th birthday
The fascination with B12-coenzymes, natures “most beautiful” cofactors,[1] is a reflection of the uniqueness and complexity of their structure and chemistry.[2, 3] B12 has also become a
prominent testing ground for thoughts on the evolution of the
catalytic moieties of essential cofactors.[4] According to
Eschenmoser,[5] the corresponding corrin ligand may represent a further evolved form of the hypothetical B12 progenitor,
“protocobyrinic acid”. The attachment of methyl groups and
the characteristic appendage of a nucleotide loop may have
arisen from (nonenzymatic) adaptation and self-constitution,
respectively.[4] Herein we report on norvitamin B12 (1, Cobcyano-5’’,6’’-dimethylbenzimidazolyl-176-norcobamide),[7] its
organometallic analogue, methylnorcobalamin (2, Cobmethyl-5’’,6’’-dimethylbenzimidazolyl-176-norcobamide),
and on two B12 derivatives that lack the methyl group of the
cobamide ligand at C176. Our studies were induced by the
discovery of norpseudovitamin B12 (3, Scheme 1), the cyanoCoIII form of the cofactor of perchloroethylene reductase
from Sulfurospirillum multivorans,[8] a natural “complete”
B12 cofactor that lacks the methyl group at C176.
Norvitamin B12 (1) was prepared according to methods
developed previously for the partial synthesis of vitamin B12
(4)[7, 9, 10] and was obtained in 73 % yield through the condensation of cobyric acid (7) (from hydrolysis of 4[7, 11]) with
(2-aminoethyl)-3’-(a-ribazolyl)diphosphate (8) and crystal[*] Dr. P. Butler, Dr. M.-O. Ebert, Prof. Dr. B. Kr#utler
Institute of Organic Chemistry
Innrain 52a
and
Center for Molecular Biosciences
Leopold-Franzens-Universit#t Innsbruck
6020 Innsbruck (Austria)
Fax: (+ 43) 512-507-2892
E-mail: bernhard.kraeutler@uibk.ac.at
A. Lyskowski, Prof. Dr. K. Gruber, Prof. Dr. C. Kratky
Institute of Chemistry
Karl-Franzens-Universit#t Graz
Heinrichstraße 28, 8010 Graz (Austria)
[**] We thank K.-H. Ongania for measuring FAB mass spectra. We are
grateful to Hoffmann-LaRoche for a gift of vitamin B12. The project
was supported by grants from the European Commission (Proj. No.
HPRN-CT-2002-00195), the Austrian National Science Foundation
(FWF, projects P-13595 and P-17132). X-ray diffraction data were
collected at the EMBL beamline BW7b at DESY in Hamburg
(Germany).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2006, 45, 989 –993
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
989
Communications
Scheme 1. Structural formulas of complete base-on corrinoids. Top:
norvitamin B12 (1, R = H), vitamin B12 (4, R = CH3). Bottom: norpseudovitamin B12 (3, R = H), pseudovitamin B12 (6, R = CH3).
lized from aqueous acetone (see Scheme 2). The chromatographic behavior of 1 is similar to that of its homologue 4 and
the UV/Vis spectroscopic data is practically indistinguishable.
Fast-atom-bombardment (FAB) mass spectra exhibited a
pseudomolecular ion at m/z 1341, that is, at 14 mass units less
than that of 4. Similarly, the 1H and 13C NMR spectroscopic
chemical-shift values[12] indicate the absence of the methyl
group at C176. Comparison of the NMR spectroscopic data of
4[13, 14] with the spectra of 1 shows only the expected localsubstituent effects of a methyl group on the chemical-shift
values (see the Experimental Section[15] and the Supporting
Information).
Crystals of 1 were grown from aqueous acetone and the
structure of 1 was determined at 0.85-? resolution by using
synchrotron radiation (Figure 1).[15] The resultant structure is
very similar to that of 4.[16] A slightly longer Co N bond from
the cobalt ion to the lower axial base in 1 (2.047(5) ?
compared with 2.011(10) ? in 4), a small increase in the base
tilt angle (the difference of the angles Co N3N C9N and
990
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Co N3N C2N) of 11.6(0.6)8 in 1 (compared with 9.7(1.1)8 in
4), and a small decrease in the corrin fold angle from
18.0(0.3)8 in 4 to 15.8(0.1)8 in 1 are all consistent with rather
similar mechanochemical and steric effects of the nucleotide
function in 1 and 4.[17] Minor differences between the two
structures are seen only in the region around the ethanolamine linker and the phosphate and ribose moieties of the
nucleotide loop. With the exception of the orientation of the
amide group of side chain a, even the conformations of the
amide side chains are virtually identical between 1 and 4.
Crystalline 2 was prepared from 4 in situ by methylation
of electrochemically produced norcob(i)alamin (9)[18] with
methyltoluenesulfonate in 80 % yield. The UV/Vis spectrum
of 2 is practically indistinguishable from that of the homologue, methylcobalamin (5). The FAB mass spectra of 2
exhibits a pseudomolecular ion at m/z 1330, that is, at 14 mass
units less than that of 5. Likewise, the absence of the methyl
group at C176 is clearly indicated from comparison of the
values of the 1H and 13C NMR spectroscopic chemical shifts[12]
of 2 with those of 5[13, 19] (see the Experimental Section and the
Supporting Information). Again, aside from the expected
local-substituent effects of the methyl group on the chemical
shift-values in the NMR spectra, there are no other significant
differences.
The effect of the methyl group on the conformational
properties of 4 and 5 could not be derived from the spectral
data or from the crystal structure of 1. Indeed, in the structure
of 4 and in that of pseudovitamin B12 (6),[8] the methyl group
at C176 occupies an uncrowded place, antiperiplanar to N174
and anticlinal to the P atom of the phosphate linker (it also
occupies the place of HproR(176) of 1) (Figure 2). The methyl
group at C176 thus does not specifically influence the
nucleotide loop conformation of the known B12 coenzymes
in their “base-on” forms, but is expected to destabilize the
alternative staggered “base-off” conformations (see below
and Figures 2 and 3).
The methyl group at C176 is derived from threonine
phosphate,[20] whereas for the ethanolamine linker of norcobamides, serine has been considered as the biosynthetic
precursor.[8] The presence of the methyl group in 4 appears
to be a consequence neither of a restricted biosynthetic supply
(threonine versus serine), nor of an inherent specific adaptation of the base-on form of the complete cobamides. However, as discussed by Eschenmoser,[4, 6] the methyl group and
an R configuration at C176 is expected to allow for (relatively) less instable “precyclic” (base-off) conformations of
the nucleotide appendage in favor of the formation of the
cobalt-coordinated base-on form. On one hand, this could
assist the complete B12 structure to self-constitute,[6] however,
it could also influence the base-on/base-off equilibria of
complete cobamides. These cobamides are considered to be
molecular switches[21] that are in control of their organometallic redox chemistry. Such a property could also be critical
for the binding and recognition of complete B12 derivatives
(either in their base-on or their base-off forms) by biological
macromolecules, such as proteins[2, 3, 22, 23] and oligonucleotides.[21, 24]
To assess the effect of the C176 methyl group on a
representative B12 base-on/base-off equilibrium, the tendency
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 989 –993
Angewandte
Chemie
Scheme 2. Outline of the preparation of norvitamin B12 (1) and methylnorcobalamin (2) from vitamin B12 (4) via cobyric acid (7) (see the
Supporting Information).[15]
Figure 1. Left: Crystal structure of norvitamin B12 (1), ball-and-stick
model with C, N, O, P, Co atoms colored gray, blue, red, green, and
pink, respectively. Right: Superposition of models of the 3D structures
of 1 (red) and of vitamin B12 (4)[16] (yellow, C176 methyl labeled green).
of 2 to undergo acid-induced decoordination of its nucleotide
base was determined and compared with the corresponding
process in 5.[25] In the base-on form of 5, the rather strong
intramolecular coordination of the dimethylbenzimidazole
base is reflected by a low pKa(H-5+) = 2.90[25] (5 protonated at
N3N). The acidity of the corresponding protonated base-off
form of 2, that is, of H-2+, could be determined by UV/Vis
spectroscopy (see Figure 3), which provides a pKa(H-2+) =
Angew. Chem. Int. Ed. 2006, 45, 989 –993
3.24. As pKa(H-2+) pKa(H-5+) = 0.34, 2 is therefore approximately twice as basic as 5 (with protonation at the
dimethylbenzimidazole nitrogen N3N). From the two pKa
values, the nucleotide coordinated base-on state is deduced to
be disfavored at room temperature by a factor of about 2.1 in
2, when compared with 5.
As was expected[4, 5] in “complete” cobamides, such as 4 or
5, the methyl group at C176 is influential. It helps constitute
the base-on form, as explained by qualitative conformational
analysis (see Figure 2). Indeed, a remarkable long-distance
constitutional effect of the methyl group at C176 is observed
at the corrin-bound cobalt center. The methyl group assists
the nucleotide base to find its coordination partner at a
distance of 11 bonds away. Norcobamides, which are devoid
of the methyl-group pivot, are predicted to have a higher
tendency to be base-off and to be, in general, reduced at more
positive potentials.[18, 26] This may be relevant in dehalogenating anaerobes, one of which was shown to use the norcobamide norpseudo B12 as a cofactor.[8]
The presence of the methyl group at C176 in cobamides is
a feature of complete “B12 dinucleotides”: they provide a
more stable base-on constitution and assist the (reversible)
back binding, in a loop, of one nucleotide appendage to the
other (cobalt–corrin). The propensity for the formation of
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
991
Communications
seems to offer no functional advantage, but may merely be a
consequence of the genetically programmed B12 biosynthesis.
The methyl group at C176 thus appears to reflect the “fossil
origin” of the B12 structure[5] and invites further investigations, as are currently carried out in our labs.
Received: July 27, 2005
Published online: December 22, 2005
.
Keywords: bioorganometallic chemistry · conformation
analysis · molecular switch · structure elucidation · vitamin B12
Figure 2. Qualitative analysis of the effect of the C176 methyl group by
using idealized conformations around the (C175 C176) bond. Top:
cobalamins (e.g. vitamin B12); bottom: norcobalamins (e.g. norvitamin B12). A torsion angle of approximately 608 is observed in the
base-on cobalamins and is required in the “precyclic” forms (base-off
form in which the base is preoriented towards coordination at the
cobalt center);[6] other staggered conformations (+ 608, 1808) are baseoff and do not allow strain-free base-on forms. The methyl group at
C176 of vitamin B12 is gauche and found to destabilize the 608 and
1808 (base-off) conformations, but not the 608 (base-on) conformation (ribz = a-ribazole = 5’,6’-dimethylbenzimidazolyl-a-nucleoside).
Figure 3. The B12 “molecular switch”, as represented by 2 and 2-H+:
pH dependence of UV/Vis spectra of buffered aqueous solutions of
methylnorcobalamin (2) ([2] = 0.45 mm; at the indicated pH values,
1.0 m KCl, room temperature; see the Supporting Information).
such a loop is inherent to the B12 structure and is conjectured
to be critical for the presumed role of B12 in primordial life.[4]
This nucleotide loop (as in B12) is unique in cofactors and is
preserved only in part in B12-dependent enzymes. Both
constitutional types of B12 cofactors are found as either
base-on or base-off forms. In some organisms, the presence
of the methyl group at C176 of the “complete” cobamides
992
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[1] J. Stubbe, Science 1994, 266, 1663 – 1664.
[2] Vitamin B12 and B12-Proteins (Eds.: B. KrIutler, B. T. Golding, D.
Arigoni), Wiley-VCH, Weinheim, 1998.
[3] Chemistry and Biochemistry of B12 (Ed.: R. Banerjee), Wiley,
New York, 1999.
[4] A. Eschenmoser, Angew. Chem. 1988, 100, 5 – 40; Angew. Chem.
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[5] A. Eschenmoser, Nova Acta Leopold. 1982, 55, No. 247.
[6] A. Eschenmoser, F. Kreppelt, unpublished work; see F. Kreppelt, PhD thesis no. 9458, ETH-ZNrich 1991.
[7] For a review of earlier work, see: W. Friedrich in Fermente,
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Dirscherl), Georg Thieme, Stuttgart, 1975, pp. 25.
[8] B. KrIutler, W. Fieber, S. Ostermann, M. Fasching, K.-H.
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Helv. Chim. Acta 2003, 86, 3698 – 3716.
[9] R. B. Woodward in Vitamin B12, Proceedings of the 3rd European
Symposium on Vitamin B12 and Intrinsic Factor (Eds.: B.
Zagalak, W. Friedrich), Walter de Gruyter, Berlin, 1979,
pp. 37 – 87.
[10] a) W. Friedrich in Vitamin B12 and Intrinsic Factor (Ed.: H. C.
Heinrich), Enke, Stuttgart, 1962, pp. 62 – 72; b) K. Bernhauer, O.
MNller, F. Wagner, Angew. Chem. 1963, 75, 1145 – 1188; Angew.
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[11] R. Bonnet in B12 (Ed.: D. Dolphin), Wiley, New York, 1982,
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[12] R. Konrat, M. Tollinger, B. KrIutler in Vitamin B12 and B12Proteins (Eds.: B. KrIutler, B. T. Golding, D. Arigoni), WileyVCH, Weinheim, 1998, pp. 349 – 368.
[13] K. Brown in Chemistry and Biochemistry of B12 (Ed.: R.
Banerjee), Wiley, New York, 1999, pp. 197 – 237.
[14] A. M. Calafat, L. G. Marzilli, J. Am. Chem. Soc. 1993, 115, 9182 –
9190.
[15] Selected spectroscopic data: 1: UV/Vis (c = 5.59 O 10 4 m, H2O):
548(3.85), 518.5(3.80), 407(3.47), 360(4.36), 321.5(3.81), 304.5(3.88), 277.5(4.10); FAB-MS: 1343.5 (13), 1342.5 (22), 1341.5 (24,
[M+H]+), 1317.6 (57), 1316.4 (100), 1315.5 (94, [M+H CN]+);
2: UV/Vis (c = 4.51 O 10 4 m, 0.1m phosphate buffer, pH 7.25):
518.5 (3.83), 373.5 (3.93), 339 (4.01), 314.5 (4.00), 279 (4.15), 265
(4.18); FAB-MS: 1332.6 (24), 1331.6 (43), 1330.6 (52, [M+H]+),
1317.6 (68), 1316.5 (100), 1315.6 (76, [M+H CH3]+); 8:
1
H NMR (300 MHz, D2O): d = 2.37 (3 H, s; CH3), 2.39 (3 H, s;
CH3), 3.19 (2 H, t; H2NCH2CH2O), 3.83 (1 H, dd; J = 4.2 Hz, J =
12.6 Hz; HaC5R), 3.95 (1 H, dd, J = 2.7 Hz, J = 12.6 Hz, HbC5R),
4.08 (2 H, m; H2NCH2CH2O), 4.62 (1 H, m; HC4R), 4.7–4.9
(water signal overlaps with signals of HC2R, HC3R), 6.41 (1 H,
d, J = 4.5 Hz; HC1R), 7.45 (1 H, s; aromatic CH), 7.53 (1 H, s;
aromatic CH), 8.34 ppm (1 H, s; HC2N). UV/Vis (c = 9.97 O
10 4 m, 0.1m phosphate buffer, pH 7.25): 286.5(4.05), 278.5(4.07),
248(4.21). ESI-MS: 439.97 (20, [M+K]+), 423.97 (5, [M+Na]+),
404.03 (5), 403.02 (20), 402.01 (100, [M+H]+), 146.97(10).
Further details are given in the supporting information. Structure determination of 1: Crystals were grown from water/
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 989 –993
Angewandte
Chemie
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
acetone. Diffraction data (space group P212121, a = 15.573 ?, b =
22.846 ?, c = 24.583 ?, Rsym = 0.039) to a maximum resolution of
0.85 ? were collected at 103 K by using synchrotron radiation
(l = 0.8426 ?) on beam line BW7b at EMBL/DESY in Hamburg. Refinement on F2 (7744 unique reflections, 1131 parameters and 1693 restraints) converged at crystallographic residuals
of R1 = 0.0796 and wR2 = 0.2137 for all reflections. Details are
given in the supporting information. CCDC-278 482 (1) contains
the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
B. KrIutler, R. Konrat, E. Stupperich, G. FIrber, K. Gruber, C.
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angle of the corrin ligand is defined (here) as the angle between
the best planes through atoms N1-C4-C5-C6-N2-C9-C10 (plane
1) and C10-C11-N3-C14-C15-C16-N4 (plane 2); see the Supporting Information for atom numbering.
B. KrIutler in Chemistry and Biochemistry of B12 (Ed.: R.
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S. GschRsser, K. Gruber, C. Kratky, C. EichmNller, B. KrIutler,
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Angew. Chem. Int. Ed. 2006, 45, 989 –993
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