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 Eschenmoseron the occasion of his 80th birthday

The fascination with B12-coenzymes, nature�s “most beauti-ful” cofactors,[1] is a reflection of the uniqueness and complex-ity of their structure and chemistry.[2, 3] B12 has also become aprominent testing ground for thoughts on the evolution of thecatalytic moieties of essential cofactors.[4] According toEschenmoser,[5] the corresponding corrin ligand may repre-sent a further evolved form of the hypothetical B12 progenitor,“protocobyrinic acid”. The attachment of methyl groups andthe characteristic appendage of a nucleotide loop may havearisen from (nonenzymatic) adaptation and self-constitution,respectively.[4] Herein we report on norvitamin B12 (1, Cob-cyano-5’’,6’’-dimethylbenzimidazolyl-176-norcobamide),[7] itsorganometallic analogue, methylnorcobalamin (2, Cob-methyl-5’’,6’’-dimethylbenzimidazolyl-176-norcobamide),and on two B12 derivatives that lack the methyl group of thecobamide ligand at C176. Our studies were induced by thediscovery of norpseudovitamin B12 (3, Scheme 1), the cyano-CoIII form of the cofactor of perchloroethylene reductasefrom Sulfurospirillum multivorans,[8] a natural “complete”B12 cofactor that lacks the methyl group at C176.

Norvitamin B12 (1) was prepared according to methodsdeveloped previously for the partial synthesis of vitamin B12

(4)[7,9, 10] and was obtained in 73% yield through the con-densation 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#utlerInstitute of Organic ChemistryInnrain 52aandCenter for Molecular BiosciencesLeopold-Franzens-Universit#t Innsbruck6020 Innsbruck (Austria)Fax: (+43)512-507-2892E-mail: bernhard.kraeutler@uibk.ac.at

A. Lyskowski, Prof. Dr. K. Gruber, Prof. Dr. C. KratkyInstitute of ChemistryKarl-Franzens-Universit#t GrazHeinrichstraße 28, 8010 Graz (Austria)

[**] We thank K.-H. Ongania for measuring FAB mass spectra. We aregrateful to Hoffmann-LaRoche for a gift of vitamin B12. The projectwas 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 werecollected at the EMBL beamline BW7b at DESY in Hamburg(Germany).

Supporting information for this article is available on the WWWunder http://www.angewandte.org or from the author.

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lized from aqueous acetone (see Scheme 2). The chromato-graphic behavior of 1 is similar to that of its homologue 4 andthe UV/Vis spectroscopic data is practically indistinguishable.Fast-atom-bombardment (FAB) mass spectra exhibited apseudomolecular ion at m/z 1341, that is, at 14 mass units lessthan that of 4. Similarly, the 1H and 13C NMR spectroscopicchemical-shift values[12] indicate the absence of the methylgroup at C176. Comparison of the NMR spectroscopic data of4[13, 14] with the spectra of 1 shows only the expected local-substituent effects of a methyl group on the chemical-shiftvalues (see the Experimental Section[15] and the SupportingInformation).

Crystals of 1 were grown from aqueous acetone and thestructure of 1 was determined at 0.85-? resolution by usingsynchrotron radiation (Figure 1).[15] The resultant structure isvery similar to that of 4.[16] A slightly longer Co�N bond fromthe cobalt ion to the lower axial base in 1 (2.047(5) ?compared with 2.011(10) ? in 4), a small increase in the basetilt angle (the difference of the angles Co�N3N�C9N and

Co�N3N�C2N) of 11.6(0.6)8 in 1 (compared with 9.7(1.1)8 in4), and a small decrease in the corrin fold angle from18.0(0.3)8 in 4 to 15.8(0.1)8 in 1 are all consistent with rathersimilar mechanochemical and steric effects of the nucleotidefunction in 1 and 4.[17] Minor differences between the twostructures are seen only in the region around the ethanol-amine linker and the phosphate and ribose moieties of thenucleotide loop. With the exception of the orientation of theamide group of side chain a, even the conformations of theamide side chains are virtually identical between 1 and 4.

Crystalline 2 was prepared from 4 in situ by methylationof electrochemically produced norcob(i)alamin (9)[18] withmethyltoluenesulfonate in 80% yield. The UV/Vis spectrumof 2 is practically indistinguishable from that of the homo-logue, methylcobalamin (5). The FAB mass spectra of 2exhibits a pseudomolecular ion atm/z 1330, that is, at 14 massunits less than that of 5. Likewise, the absence of the methylgroup at C176 is clearly indicated from comparison of thevalues of the 1H and 13C NMR spectroscopic chemical shifts[12]

of 2with those of 5[13, 19] (see the Experimental Section and theSupporting Information). Again, aside from the expectedlocal-substituent effects of the methyl group on the chemicalshift-values in the NMR spectra, there are no other significantdifferences.

The effect of the methyl group on the conformationalproperties of 4 and 5 could not be derived from the spectraldata or from the crystal structure of 1. Indeed, in the structureof 4 and in that of pseudovitamin B12 (6),

[8] the methyl groupat C176 occupies an uncrowded place, antiperiplanar to N174and anticlinal to the P atom of the phosphate linker (it alsooccupies the place of HproR(176) of 1) (Figure 2). The methylgroup at C176 thus does not specifically influence thenucleotide loop conformation of the known B12 coenzymesin their “base-on” forms, but is expected to destabilize thealternative staggered “base-off” conformations (see belowand Figures 2 and 3).

The methyl group at C176 is derived from threoninephosphate,[20] whereas for the ethanolamine linker of norcob-amides, serine has been considered as the biosyntheticprecursor.[8] The presence of the methyl group in 4 appearsto be a consequence neither of a restricted biosynthetic supply(threonine versus serine), nor of an inherent specific adapta-tion of the base-on form of the complete cobamides. How-ever, as discussed by Eschenmoser,[4,6] the methyl group andan R configuration at C176 is expected to allow for (rela-tively) less instable “precyclic” (base-off) conformations ofthe nucleotide appendage in favor of the formation of thecobalt-coordinated base-on form. On one hand, this couldassist the complete B12 structure to self-constitute,

[6] however,it could also influence the base-on/base-off equilibria ofcomplete cobamides. These cobamides are considered to bemolecular switches[21] that are in control of their organome-tallic redox chemistry. Such a property could also be criticalfor the binding and recognition of complete B12 derivatives(either in their base-on or their base-off forms) by biologicalmacromolecules, such as proteins[2, 3, 22,23] and oligonucleoti-des.[21, 24]

To assess the effect of the C176 methyl group on arepresentative B12 base-on/base-off equilibrium, the tendency

Scheme 1. Structural formulas of complete base-on corrinoids. Top:norvitamin B12 (1, R=H), vitamin B12 (4, R=CH3). Bottom: norpseu-dovitamin B12 (3, R=H), pseudovitamin B12 (6, R=CH3).

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of 2 to undergo acid-induced decoordination of its nucleotidebase was determined and compared with the correspondingprocess in 5.[25] In the base-on form of 5, the rather strongintramolecular coordination of the dimethylbenzimidazolebase is reflected by a low pKa(H-5+)= 2.90[25] (5 protonated atN3N). The acidity of the corresponding protonated base-offform of 2, that is, of H-2+, could be determined by UV/Visspectroscopy (see Figure 3), which provides a pKa(H-2+)=

3.24. As pKa(H-2+)�pKa(H-5+)= 0.34, 2 is therefore approx-imately twice as basic as 5 (with protonation at thedimethylbenzimidazole nitrogen N3N). From the two pKa

values, the nucleotide coordinated base-on state is deduced tobe disfavored at room temperature by a factor of about 2.1 in2, when compared with 5.

As was expected[4,5] in “complete” cobamides, such as 4 or5, the methyl group at C176 is influential. It helps constitutethe base-on form, as explained by qualitative conformationalanalysis (see Figure 2). Indeed, a remarkable long-distanceconstitutional effect of the methyl group at C176 is observedat the corrin-bound cobalt center. The methyl group assiststhe nucleotide base to find its coordination partner at adistance of 11 bonds away. Norcobamides, which are devoidof the methyl-group pivot, are predicted to have a highertendency to be base-off and to be, in general, reduced at morepositive potentials.[18, 26] This may be relevant in dehalogenat-ing anaerobes, one of which was shown to use the norcoba-mide norpseudo B12 as a cofactor.

[8]

The presence of the methyl group at C176 in cobamides isa feature of complete “B12 dinucleotides”: they provide amore stable base-on constitution and assist the (reversible)back binding, in a loop, of one nucleotide appendage to theother (cobalt–corrin). The propensity for the formation of

Scheme 2. Outline of the preparation of norvitamin B12 (1) and methylnorcobalamin (2) from vitamin B12 (4) via cobyric acid (7) (see theSupporting Information).[15]

Figure 1. Left: Crystal structure of norvitamin B12 (1), ball-and-stickmodel with C, N, O, P, Co atoms colored gray, blue, red, green, andpink, respectively. Right: Superposition of models of the 3D structuresof 1 (red) and of vitamin B12 (4)

[16] (yellow, C176 methyl labeled green).

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such a loop is inherent to the B12 structure and is conjecturedto be critical for the presumed role of B12 in primordial life.[4]

This nucleotide loop (as in B12) is unique in cofactors and ispreserved only in part in B12-dependent enzymes. Bothconstitutional types of B12 cofactors are found as eitherbase-on or base-off forms. In some organisms, the presenceof the methyl group at C176 of the “complete” cobamides

seems to offer no functional advantage, but may merely be aconsequence of the genetically programmed B12 biosynthesis.The methyl group at C176 thus appears to reflect the “fossilorigin” of the B12 structure

[5] and invites further investiga-tions, as are currently carried out in our labs.

Received: July 27, 2005Published online: December 22, 2005

.Keywords: bioorganometallic chemistry · conformationanalysis · molecular switch · structure elucidation · vitamin B12

[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.Int. Ed. Engl. 1988, 27, 5 – 40.

[5] A. Eschenmoser, Nova Acta Leopold. 1982, 55, No. 247.[6] A. Eschenmoser, F. Kreppelt, unpublished work; see F. Krep-

pelt, PhD thesis no. 9458, ETH-ZNrich 1991.[7] For a review of earlier work, see: W. Friedrich in Fermente,Hormone und Vitamine, Vol. III/2 (Eds.: R. Ammon, W.Dirscherl), Georg Thieme, Stuttgart, 1975, pp. 25.

[8] B. KrIutler, W. Fieber, S. Ostermann, M. Fasching, K.-H.Ongania, K. Gruber, C. Kratky, C. Mikl, A. Siebert, G. Diekert,Helv. Chim. Acta 2003, 86, 3698 – 3716.

[9] R. B.Woodward inVitamin B12, Proceedings of the 3rd EuropeanSymposium 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.Chem. Int. Ed. Engl. 1964, 3, 200 – 211.

[11] R. Bonnet in B12 (Ed.: D. Dolphin), Wiley, New York, 1982,pp. 201 – 243.

[12] R. Konrat, M. Tollinger, B. KrIutler in Vitamin B12 and B12-Proteins (Eds.: B. KrIutler, B. T. Golding, D. Arigoni), Wiley-VCH, 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= 5.59O 10�4m, 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= 4.51O 10�4m, 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 :1H NMR (300 MHz, D2O): d= 2.37 (3H, s; CH3), 2.39 (3H, s;CH3), 3.19 (2H, t; H2NCH2CH2O), 3.83 (1H, dd; J= 4.2 Hz, J=12.6 Hz; HaC5R), 3.95 (1H, dd, J= 2.7 Hz, J= 12.6 Hz, HbC5R),4.08 (2H, m; H2NCH2CH2O), 4.62 (1H, m; HC4R), 4.7–4.9(water signal overlaps with signals of HC2R, HC3R), 6.41 (1H,d, J= 4.5 Hz; HC1R), 7.45 (1H, s; aromatic CH), 7.53 (1H, s;aromatic CH), 8.34 ppm (1H, s; HC2N). UV/Vis (c= 9.97 O10�4m, 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. Struc-ture determination of 1: Crystals were grown from water/

Figure 2. Qualitative analysis of the effect of the C176 methyl group byusing idealized conformations around the (C175�C176) bond. Top:cobalamins (e.g. vitamin B12); bottom: norcobalamins (e.g. norvita-min B12). A torsion angle of approximately �608 is observed in thebase-on cobalamins and is required in the “precyclic” forms (base-offform in which the base is preoriented towards coordination at thecobalt center);[6] other staggered conformations (+608, 1808) are base-off and do not allow strain-free base-on forms. The methyl group atC176 of vitamin B12 is gauche and found to destabilize the 608 and1808 (base-off) conformations, but not the �608 (base-on) conforma-tion (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 ofmethylnorcobalamin (2) ([2]=0.45 mm ; at the indicated pH values,1.0m KCl, room temperature; see the Supporting Information).

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acetone. Diffraction data (space group P212121, a= 15.573 ?, b=22.846 ?, c= 24.583 ?,Rsym= 0.039) to a maximum resolution of0.85 ? were collected at 103 K by using synchrotron radiation(l= 0.8426 ?) on beam line BW7b at EMBL/DESY in Ham-burg. Refinement on F2 (7744 unique reflections, 1131 param-eters and 1693 restraints) converged at crystallographic residualsof R1= 0.0796 and wR2= 0.2137 for all reflections. Details aregiven in the supporting information. CCDC-278 482 (1) containsthe supplementary crystallographic data for this paper. Thesedata can be obtained free of charge from The CambridgeCrystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

[16] B. KrIutler, R. Konrat, E. Stupperich, G. FIrber, K. Gruber, C.Kratky, Inorg. Chem. 1994, 33, 4128 – 4139.

[17] C. Kratky, B. KrIutler in Chemistry and Biochemistry of B12(Ed.: R. Banerjee), Wiley, New York, 1999, pp. 9 – 41. The foldangle of the corrin ligand is defined (here) as the angle betweenthe best planes through atoms N1-C4-C5-C6-N2-C9-C10 (plane1) and C10-C11-N3-C14-C15-C16-N4 (plane 2); see the Sup-porting Information for atom numbering.

[18] B. KrIutler in Chemistry and Biochemistry of B12 (Ed.: R.Banerjee), Wiley, New York, 1999, pp. 315 – 339.

[19] M. Tollinger, T. DQrer, R. Konrat, B. KrIutler, J. Mol. Catal. A1997, 116, 147 – 155.

[20] D. Thibaut, F. Blanche, B. Cameron, J. Crouzet, L. Debussche, E.RQmy, M. Vuilhorgne in Vitamin B12 and B12-Proteins (Eds.: B.KrIutler, B. T. Golding, D. Arigoni), Wiley-VCH, Weinheim,1998, pp. 63 – 80.

[21] S. GschRsser, K. Gruber, C. Kratky, C. EichmNller, B. KrIutler,Angew. Chem. 2005, 117, 2324 – 2328; Angew. Chem. Int. Ed.2005, 44, 2284 – 2288.

[22] S. Fedosov, L. Berglund, N. U. Fedosova, E. Nexø, T. E.Petersen, J. Chem. Biol. 2002, 277, 9989 – 9996.

[23] C. L. Drennan, S. Huang, J. T. Drummond, R. G. Matthews,M. L. Ludwig, Science 1994, 266, 1669 – 1674.

[24] A. Nahvi, N. Sudarsan, M. S. Ebert, X. Zou, K. L. Brown, R. R.Breaker, Chem. Biol. 2002, 9, 1043 – 1049.

[25] K. L. Brown, S. Peck-Siler, Inorg. Chem. 1988, 27, 3548 – 3555.[26] D. Lexa, J. M. SavQant, Acc. Chem. Res. 1983, 16, 235 – 243.

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993Angew. Chem. Int. Ed. 2006, 45, 989 –993 � 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org