Structural Analysis of Biomolecules DOI: 10.1002/anie.200603901 Novel Approaches to the Experimental Charge Density of Vitamin B 12 ** Birger Dittrich, Tibor Koritsanszky, Anatoliy Volkov, Stefan Mebs, and Peter Luger* Knowledge of the steric, dynamic, and electronic contribu- tions to intermolecular interactions is of fundamental impor- tance to the modeling of biochemical processes. X-ray diffraction has become a widely applied experimental tool for gaining this knowledge, since the analysis of high- resolution diffraction data can lead not only to the solid- state structure, but also to the corresponding electron density (ED). [1] In spite of the potential applicability to larger systems of biological interest, [2–5] X-ray ED studies have been almost exclusively restricted to small molecules, [6] because the assumption of perfect periodicity becomes less realistic for large unit-cell structures. This issue is already a problem for systems of intermediate size, such as the alkylcobalamines (R  Cbl), whose crystal structures have been described as notoriously inaccurate, [7] owing to the disorder of side chains and solvent molecules. Alkylcobalamines are cofactors of mammalian enzymes that catalyze methyl-transfer, isomerization, and redox reac- tions. The first two reactions involve the breaking and re- forming of the Co  C R bond, presumably through a reversible heterolytic or homolytic cleavage. [8,9] Since the alkylcobala- min coenzymes are inactive, and their binding to the protein does not involve any change in the equatorial ligation of the cobalt atom by the corrin ring, the main issue in cobalt- enzyme research is to understand how the axial Co  C R bond is labilized by the apoenzyme. [10] A detailed analysis of the electronic configuration of the cobalt center and the covalent character of the Co  C R bond should provide some insight into the destabilization of the bond through intermolecular forces. Quantum-chemical studies on cobalt corrinoids have been limited to geometry-optimized models for the coenzymes (alkyl corrin complexes), the sizes of which were reduced through the truncation of side chains. [11] More recent studies have been based on density functional theory (DFT) calcu- lations and have utilized experimental structures determined from relatively high-resolution diffraction data. Calculations on CNCbl, [12] Me Cbl, [13] and Ado Cbl (Ado = 5’-deoxy-5’- adenosyl) [14] have shown that the polarity of the Co  C R bond increases in the order R = CN, Ado, Me, even though the equatorial ligation of the cobalt cation (that is, the Co  N eq bonds) remains unchanged upon variation of the axial R group. Modern X-ray ED studies on transition-metal com- plexes [6] have combined d-orbital population analyses [15] with density-based topological analyses, [16] allowing a quanti- tative characterization of the metal–ligand interactions. [17] Since, for these interactions, the ED at the bond critical point (BCP) is low, whereas the bond-parallel curvature (l 3 ) is very high, the effects of the radial basis functions [18] can be pronounced; the limitations of the standard multipole model [19] are becoming increasingly evident. The present study has the following novel components: a) We were able to crystallize a new solvate of vitamin B 12 with a low solvent content (twelve water and three propanol molecules per molecule of cyanocobalamin in Figure 1. SCHAKAL [33] representationofthemolecularstructureofthe solvateofvitaminB 12 (Cblue,Hwhite,Coorange,Ngreen,Ppurple, Ored)withtwelvewater(Oyellow)andthreepropanolmolecules (Cbronze),oneofwhichispartiallydisordered.Thedashedredlines representhydrogenbonds. [*] Dipl.-Chem.S.Mebs,Prof.Dr.P.Luger FreieUniversitätBerlin InstitutfürChemieundBiochemie/Kristallographie FreieUniversitätBerlin Fabeckstrasse36a,14195Berlin(Germany) Fax:(+ 49)30-838-53464 E-mail:luger@chemie.fu-berlin.de Dr.B.Dittrich ChemistryM313,SchoolofBiomedicalandChemicalSciences UniversityofWesternAustralia 35StirlingHighway,Crawley,WA6009(Australia) Prof.Dr.T.Koritsanszky DepartmentofChemistry MiddleTennesseeStateUniversity P.O.Box68,Murfreesboro,TN37132(USA) Dr.A.Volkov DepartmentofChemistry StateUniversityofNewYorkatBuffalo 747NaturalSciencesComplex,Buffalo,NY14260-3000(USA) [**] ThisworkwassupportedbytheDeutscheForschungsgemeinschaft (grantsSPP1178,Lu222/29-1,andKo1350/4-1)andbytheAus- tralianSynchrotronResearchProgram,whichisfundedbythe CommonwealthofAustraliaundertheMajorNationalResearch FacilitiesProgram.B.D.thankstheASRPforapostdoctoral fellowship. Angewandte Chemie 2935 Angew. Chem. Int. Ed. 2007, 46, 2935–2938 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim