Trans and cis effects of axial fluoroalkyl ligands in vitamin B12 analogues: relationship between alkyl- and fluoroalkyl-cobalamins.

CF2HCbl, CF3Cbl , and CF3CH2Cbl have been synthesized and characterized in solution by (1)H NMR and UV-vis spectroscopy, and their X-ray crystal structures have been determined using synchrotron radiation. The structure of CF3CH2Cbl is reported for the first time, whereas those of CF2HCbl and CF3Cbl are re-examined to obtain more precise structural data. Comparison of the structural data obtained with the alkylcobalamin analogues, MeCbl and EtCbl, indicates that the Co-C and Co-NB3 bond lengths are shorter in the fluoroalkylcobalamins. The structural data of the fluoroalkylcobalamins previously reported in the literature had been conflicting in this regard. Thus, a much less dramatic shortening of the two axial bonds was found for CF3Cbl, whereas in the case of CF2HCbl, the Co-NB3 bond length is shorter than in MeCbl. Direct comparison of the structures of CF3CH2Cbl and EtCbl indicates a large distortion of the axial fragment in the former case that can be attributed to steric effects. A number of previously reported correlations of the effect of the ß-ligand on the structure and properties of cobalamins are re-examined in light of the present results. Particular emphasis is placed on the axial fragment. This analysis substantially confirms and, with the new data reported here, adjusts and expands the data set for correlations between trans and cis influences of the ß-ligand of cobalamins and their structure (Co-X and Co-NB3 distances and corrin fold angle) and properties (UV-vis spectra, NMR spectra, and pK(base-off)).

Trans and cis influences and effects in cobalamins and in their simple models.

The interligand interactions in coordination compounds have been principally interpreted in terms of cis and trans influences and effects, which can be defined as the ability of a ligand X to affect the bond of another ligand, cis or trans to X, to the metal. This review analyzes these effects/influences in cobalamins (XCbl) and their simple models cobaloximes, LCo(chel)X. Important properties of these complexes, such as geometry, stability, and reactivity, can be rationalized in terms of steric and electronic factors of the ligands. Experimental evidence of normal and inverse trans influence is described in alkylcobaloximes for the first time. The study of simple B(12) models has complemented that on the more complex cobalamins, with particular emphasis on the properties of the axial L-Co-X moiety. Some of the conclusions reached for the axial fragment of simple models have also been qualitatively detected in cobalamins and have furnished new insight into the as yet unestablished mechanism for the homolytic cleavage of the Co - C bond in the AdoCbl-based enzymes.

Vitamin B12: unique metalorganic compounds and the most complex vitamins.

The chemistry and biochemistry of the vitamin B(12) compounds (cobalamins, XCbl) are described, with particular emphasis on their structural aspects and their relationships with properties and function. A brief history of B(12), reveals how much the effort of chemists, biochemists and crystallographers have contributed in the past to understand the basic properties of this very complex vitamin. The properties of the two cobalamins, the two important B(12) cofactors Ado- and MeCbl are described, with particular emphasis on how the Co-C bond cleavage is involved in the enzymatic mechanisms. The main structural features of cobalamins are described, with particular reference to the axial fragment. The structure/property relationships in cobalamins are summarized. The recent studies on base-off/base-on equilibrium are emphasized for their relevance to the mode of binding of the cofactor to the protein scaffold. The absorption, transport and cellular uptake of cobalamins and the structure of the B(12) transport proteins, IF and TC, in mammals are reviewed. The B(12) transport in bacteria and the structure of the so far determined proteins are briefly described. The currently accepted mechanisms for the catalytic cycles of the AdoCbl and MeCbl enzymes are reported. The structure and function of B(12) enzymes, particularly the important mammalian enzymes methyltransferase (MetH) and methyl-malonyl-coenzyme A mutase (MMCM), are described and briefly discussed. Since fast proliferating cells require higher amount of vitamin B(12) than that required by normal cells, the study of B(12 )conjugates as targeting agents has recently gained importance. Bioconjugates have been studied as potential agents for delivering radioisotopes and NMR probes or as various cytotoxic agents towards cancer cells in humans and the most recent studies are described. Specifically, functionalized bioconjugates are used as "Trojan horses" to carry into the cell the appropriate antitumour or diagnostic label. Possible future developments of B(12) work are summarized.

Carbamylation of N-terminal proline.

Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the α-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.

Axial bonding in alkylcobalamins: DFT analysis of the inverse versus normal trans influence.

Density functional theory has been applied to study the origin of the inverse and normal trans influence in alkylcobalamins. In order to cover the X-ray structural data available for alkylcobalamins with a variety of axial substituents, geometries of 28 related corrin-containing models have been optimized and analyzed. The BP86/6-31G(d) level of theory was applied which showed good reliability in reproducing the axial bond lengths. Comparison of experimental and calculated data allowed to conclude that the inverse trans influence is not a general feature of cobalamins, as it appeared from the experimental data analysis alone. Inverse trans influence is observed for the series of R groups with increasing bulk and electron donating ability. For the series of R groups having similar medium bulk, but differing significantly in the electron donating ability, normal trans influence was found. Finally, it was determined, that the axial bond lengths correlate well but differently in the two series of R groups with the orbital energies of the six molecular orbitals essential in axial interligand bonding.

Release of toxic Gd3+ ions to tumour cells by vitamin B12 bioconjugates.

Two probes consisting of vitamin B(12) (CNCbl) conjugated to Gd chelates by esterification of the ribose 5'-OH moiety, Gd-DTPA-CNCbl (1; DTPA = diethylenetriamine-N,N,N',N'',N''-pentaacetic acid) and Gd-TTHA-CNCbl (2; TTHA = triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), have been synthesised and characterised. The crystal structure of a dimeric form of 1, obtained by crystallisation with an excess of GdCl(3), has been determined. The kinetics of binding to and dissociation from transcobalamin II show that 1 and 2 maintain high-affinity binding to the vitamin B(12) transport protein. Complex 2 is very stable with respect to Gd(3+) release owing to the saturated co-ordination of the Gd(3+) ion by four amino and five carboxylate groups. Hydrolysis of the ester functionality occurs on the time scale of several hours. The lack of saturation and the possible involvement of the ester functionality in co-ordination result in lower stability of 1 towards hydrolysis and in a considerable release of Gd(3+) in vitro. Gd(3+) ions released from 1 are avidly taken up by the K562 tumour cells to an extent corresponding to approximately 10(10) Gd(3+) per cell. The internalisation of toxic Gd(3+) ions causes a marked decrease in cell viability as assessed by Trypan blue and WST-1 tests. On the contrary, the experiments with the more stable 2 did not show any significant cell internalisation of Gd(3+) ions and any influence on cell viability. The results point to new avenues of in situ generation of cytotoxic pathways based on the release of toxic Gd(3+) ions by vitamin B(12) bioconjugates.

Mechanistic behaviour of alkylcobaloximes and imino-oxime complexes related to vitamin B(12).

The ligand substitution reactions of complexes of the type trans-[(R)Co(Chel)S](+/0) with L, where chel = (DO)(DOH)pn = 2,2'-(1,3-diaminopropanebis(2-methyl-3-butanone)oxime), R = CH(3), L = imidazole, pyrazole, 1,2,4-triazole and 1-methylimidazole, and S = water and MeOH, and chel = (Hdmg)(2) = bis(dimethylglyoximate), R = CH(2)Cl, CH(2)Br, and CH(2)I, L = thiourea and pyridine, and S = water, were studied in detail as a function of temperature and pressure. The reported activation parameters (DeltaH, DeltaS and DeltaV) support the operation of a dissociative interchange (I(d)) mechanism. Complexes of the type trans-[RCo(Hdmg)(2)L] (R = CH(2)Cl, CH(2)Br, and CH(2)I; L = H(2)O and Py) were fully optimized at the B3LYP/LANL2DZp level, and the structural data support the mechanistic assignment based on the reported activation parameters. For the reaction of trans-[(CH(3))(2)Co((DO)(DOH)pn)] with acid, the activation parameters (DeltaH, DeltaS and DeltaV) were found to be 37 +/- 1 kJ mol(-1), -86 +/- 3 J mol(-1) K(-1) and -18.9 +/- 0.7 cm(3) mol(-1), respectively, and support a protonation mechanism.

Nanoporous crystals of calixarene/porphyrin supramolecular complex functionalized by diffusion and coordination of metal ions.

A highly nanoporous material has been obtained by self-assembly of calixarene and porphyrin building blocks. This supramolecular zeolite-like structure was successively functionalized by diffusion and coordination of metal ions to form a new bifunctionalized nanoporous material containing a porphyrinic pigment together with a metal center.

Time-dependent density functional theory study of cobalt corrinoids: Electronically excited states of methylcobalamin.

Time-dependent density functional theory (TDDFT) has been applied to the analysis of the electronic spectra of methylcobalamin (MeCbl) and its derivative in which the trans axial base was replaced by a water molecule (MeCbi[Single Bond]H(2)O). The latter corresponds to the situation encountered in strongly acidic solutions. The study primarily focuses on the accuracy of two functionals, the hybrid B3LYP and the gradient corrected BP86, in dealing with the electronic excitations. The high resolution crystal structure of MeCbl was the source of the initial coordinates. To generate the initial structures, the full MeCbl was simplified by replacing the corrin side chains by H atoms. The vertical excitation energies, together with the corresponding oscillator strengths, were calculated at the optimized BP86 and B3LYP structures of the ground electronic state of the complexes. The NBO analysis shows that the B3LYP functional gives a bonding description of the ground state as a more polarized covalent bond compared to that given by BP86. The latter functional has more covalent bonding and is thus more appropriate for modeling the axial bonding properties. To validate the accuracy of the present TDDFT analysis, the computed excitations were directly compared to the absorption spectra of MeCbl. In order to obtain a reliable agreement between experiment and theory, the two-parameter scaling technique was introduced, which compensates differently the low-energy and high-energy excitations. Electronic excitations strongly depend on the choice of the functional. Transitions involving corrin pi-->pi(*) excitations are better described by the B3LYP functional while transitions associated with metal-to-ligand (dpi-->pi(*)d) excitations are better described by BP86. These differences can be associated with the different bonding descriptions obtained by B3LYP and BP86.

Vitamin B12 transport proteins: crystallographic analysis of beta-axial ligand substitutions in cobalamin bound to transcobalamin.

Cobalamin (Cbl, vitamin B12) is an essential micronutrient that is synthesized only by bacteria. Mammals have developed a complex system for internalization of this vitamin from the diet. Three binding proteins (haptocorrin, intrinsic factor, transcobalamin (TC)) and several specific cell surface receptors are involved in the process of intestinal absorption, plasma transport and cellular uptake. The recent literature on the binding proteins is briefly reviewed. A structural study is presented addressing a unique feature of TC among the three proteins, i.e., the displacement of the weak Co(III)-ligand H2O at the upper (or beta) axial side of H2O-Cbl by a histidine side chain. We have investigated crystallographically the beta-ligand exchange on Cbl bound to TC by crystallization of bovine holo-TC in the presence of either cyanide or sulfite. The resulting electron density maps show that the histidine side chain has been displaced by an exogenous ligand CN(-) or SO(3)(-2)to a lower extent than expected based on their higher affinity for Co and excess concentration with respect to histidine. This may reflect either reduced affinities of CN(-) and SO(3)(-2)or the advantageous binding of the protein-integrated His-residue when competing for the beta-site of Cbl bound to TC. The loop hosting the histidine residue appears more flexible after disruption of the coordination bond His-Cbl but no other differences are observed in the overall structure of holo-TC. These structural results are discussed in relation to a possible physiological role of histidine substitution for H2O and regarding the role of beta-conjugated Cbl-analogues recently proposed for targeted delivery of imaging agents.

Stereoselective iodocyclization of (S)-allylalanine derivatives: gamma-lactone vs cyclic carbamate formation.

An efficient procedure for highly chemo- and stereoselective cyclization of (S)-allylalanine derivatives is reported (diastereomeric ratios up to 96:4) where the reaction course can be completely controlled by switching from gamma-lactones to cyclic carbamates simply with the proper choice of the amino acid protecting groups. Both processes are stereoconvergent and afford the (S,S)-products in high yields, short reaction times, and mild reaction conditions.

Structure of a 4:1:4 supramolecular assembly of neutral TiiiiPO cavitands and tetrakis(N-methylpyridinium)porphyrin iodide.

Four tetraphosphorylated TiiiiPO cavitands 1 encapsulate the pyridinium heads of a tetrakis(N-methylpyridinium)porphyrin iodide to form a 4:1:4 (host)4/guest4+/4I- complex. The single-crystal X-ray diffraction analysis shows the arrangement of the four cavities bound to the CH3N+ groups of the porphyrin moiety and the four iodide anions nested between the phenethyl substituents of the hosts. 1H NMR investigations show that the structure is preserved in chloroform solution and underscore the effect of the counteranions.

Structural study on ligand specificity of human vitamin B12 transporters.

Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the beta-hairpin motif beta3-turn-beta4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the beta-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.

Solvent polarity controls the helical conformation of short peptides rich in Calpha-tetrasubstituted amino acids.

The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.

Metal coordination by sterically hindered heterocyclic ligands, including 2-vinylpyridine, assessed by investigation of cobaloximes.

Structural and 1H NMR data have been obtained for cobaloximes with the bulkiest substituted pyridines reported so far. We have isolated in noncoordinating solvents the complexes CH3Co(DH)2L (methylcobaloxime, where DH = the monoanion of dimethylglyoxime) with L = sterically hindered N-donor ligands: quinoline, 4-CH3quinoline, 2,4-(CH3)2pyridine, and 2-R-pyridine (R = CH3, OCH3, CH2CH3, CH=CH2). We have found that the Co-N(ax) bond is very long in the structurally characterized complexes. In particular, CH3Co(DH)2(4-CH3quinoline) has a longer Co-N(ax) bond (2.193(3) A) than any reported for methylcobaloximes. The main cause of the long bonds is unambiguously identified as the steric bulk of L by the fairly linear relationship found for Co-N(ax) distance vs CCA (calculated cone angle, CCA, a computed measure of bulk) over an extensive series of methylcobaloximes. The linear relationship improves if L basicity (quantified by pKa) is taken into account. In anhydrous CDCl3 at 25 degrees C, all complexes except the 2-aminopyridine adduct exhibit 1H NMR spectra consistent with partial dissociation of L to form the methylcobaloxime dimer. 1H NMR experiments at -20 degrees C allowed us to assess qualitatively the relative binding ability of L as follows: 2,4-(CH3)2pyridine > 4-CH3quinoline approximately = quinoline approximately = 2-CH3pyridine > 2-CH3Opyridine > 2-CH3CH2pyridine > 2-CH2=CHpyridine. The broadness of the 1H NMR signals at 25 degrees C suggests a similar order for the ligand exchange rate. The lack of dissociation by 2-aminopyridine is attributed to an intramolecular hydrogen bond between the NH2 group and an oxime O atom. The weaker than expected binding of 2-vinylpyridine relative to the Co-N(ax) bond length is attributed to rotation of the 2-vinyl group required for this bulky ligand to bind to the metal center, a conclusion supported by pronounced changes in 2-vinylpyridine signals upon coordination.

Structural basis for mammalian vitamin B12 transport by transcobalamin.

Cobalamin (Cbl, vitamin B(12)) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal alpha(6)-alpha(6) barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H(2)O at the upper axial side of added H(2)O-Cbl is displaced by a histidine residue of the alpha(6)-alpha(6) barrel. Analysis of amino acid conservation on TC's surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the alpha(6)-alpha(6) barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.

Noncovalent synthesis in aqueous solution and spectroscopic characterization of multi-porphyrin complexes.

The interactions of the tetracationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (H(2)TMPyP) and its metallo derivatives (MTMPyP) (where M=copper(II), zinc(II), and gold(III) with the octa-anionic form (at neutral pH) of 5,11,17,23-tetrasulfonato-25,26,27,28-tetrakis(hydroxycarbonylmethoxy)calix[4]arene (C(4)TsTc) lead to a series of complex species whose stoichiometry and porphyrin sequence can be easily tuned. Crystallographic, spectroscopic, and diffusion NMR studies converge towards a common picture in which a central 1:4 porphyrin/calixarene unit serves as a template for the formation of more complex species. These species arise by successive, stepwise addition of single porphyrin molecules above and below the plane of the 1:4 central core to ultimately give a 7:4 complex. Noticeably, the stoichiometry of the various complex species corresponds to the actual concentration ratio of porphyrins and calixarenes in solution allowing the stoichiometry of these species to be easily tuned. This behavior and the remarkable stability of these species allow homo-porphyrin and hetero-(metallo)porphyrin species to be formed with control of not only the stoichiometry but also the sequence of the porphyrin array. The flexibility and ease of this approach permit, in principle, the design and synthesis of porphyrin arrays for predetermined purposes. For example, we have shown that it is very easy to design and obtain mixed porphyrin species in which a foreseen photoinduced electron-transfer is indeed observed.

Response of a designed metalloprotein to changes in metal ion coordination, exogenous ligands, and active site volume determined by X-ray crystallography.

The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability.

Electronic structure and bonding in hydroxocobalamin.

The electronic structure of hydroxocobalamin (OHCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbitals method (OLCAO). The X-ray crystal structure has been determined from synchrotron X-ray diffraction data and the geometry determined was used in the calculations. Comparison with the recently reported electronic structures of cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) shows that Mulliken charges (Q*) and bond orders (BO) vary only on the axial fragment.