the Mechanism of the Oxidation of Cob(I)alamins

Abstract

Spectroelectrochemical investigations of the reoxidation sequence of the reduced cob(I)alamin to the oxidized cob(III)alamin show that two different cob(II)alamin intermediates are formed during the processes which appear to correlate to base-on and base-off cob(II)-alamin species.     

Axial solvent coordination in "base-fff" cob(II)alamin and related co(II)-corrinates revealed by 2D-EPR

Detailed information on the structure of cobalt(II) corrinates is of interest in the context of studies on the coenzyme B(12) catalyzed enzymatic reactions, where cob(II)alamin has been identified as a reaction intermediate. Cob(II)ester (heptamethyl cobyrinate perchlorate) is found to be soluble in both polar and nonpolar solvents and is therefore very suitable to study solvent effects on Co(II) corrinates. In the literature, Co(II) corrinates in solution are often addressed as four-coordinated Co(II) corrins. However, using a combination of continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) and pulse ENDOR (electron nuclear double resonance) at different microwave frequencies we clearly prove axial ligation for Cob(II)ester and the base-off form of cob(II)alamin (B(12r)) in different solvents. This goal is achieved by the analysis of the g values, and the hyperfine couplings of cobalt, some corrin nitrogens and hydrogens, and solvent protons. These parameters are shown to be very sensitive to changes in the solvent ligation. Density functional computations (DFT) facilitate largely the interpretation of the EPR data. In the CW-EPR spectrum of Cob(II)ester in methanol, a second component appears below 100 K. Different cooling experiments suggest that this observation is related to the phase transition of methanol from the alpha-phase to the glassy state. A detailed analysis of the EPR parameters indicates that this transition induces a change from a five-coordinated (above 100 K) to a six-coordinated (below 100 K) Co(II) corrin. In a CH(3)OH:H(2)O mixture the phase-transition properties alter and only the five-coordinated form is detected for Cob(II)ester and for base-off B(12r) at all temperatures. Our study thus shows that the characteristics of the solvent can have a large influence on the structure of Co(II) corrinates and that comparison with the protein-embedded cofactor requires some caution. Finally, the spectral similarities between Cob(II)ester and base-off B(12r) prove the analogies in their electronic structure.     

Thermodynamic trans-Effects of the Nucleotide Base in the B12 Coenzymes

The thermodynamic effects of the nucleotide coordination on the Co-C bond strengths in the B ₁₂ coenzymes were analyzed. Methyl group transfer reactions from methylcob( III )inamides to cob( II )inamides and cob( I )inamides in neutral aqueous solution were used in equilibration experiments to determine the effect fo the intramolecular coordination of the nucleotide function on the Co-C bond dissociation energies of methylcob( III )alamin ( 4 ). In the equilibrium between 4 , cob( I )inamide ( 11 ), cob( I )alamin ( 10 ) and methylcob( III )inamide 6 (Scheme 2), 4 and 11 were found to predominate ( 4 + 11 ? 10 + 6 , equilibrium constant K_I/III≈0.004), while the equilibrium between 4 , cob( II )inamide 9 , cob( II )alamin ( 5 ), and 6 (Scheme 1) proved to be well balanced ( 4 + 9 ? 5 + 6 , equilibrium constant K_II/III=0.60). These equilibrium values indicate the nucleotide coordination to stabilize the Co–C bond in 4 both against homolysis (slight effect) and against nucleophilic heterolysis (considerable effect). They reflect a stabilization of the complete corrins 4 and 5 by the nucleotide coordination, which is also indicated for 4 and 5 by their (nucleotide) basicity. The latter information, where available for other organocobalamins, allows the analysis of the thermodynamicnucleotide trans effect there as well: e.g. in coenzyme B ₁₂ ( 1 ), the nucleotide coordination is found this way to weaken the Co–C bond towards homolysis by ca. 0.7 kcal/mol.     

Characterization of chlorovinylcobalamin,a putative intermediate in reductive degradation of chlorinated ethylenes

The first X-ray structure of a vinylcobalamin is reported. Chlorovinylcobalamin is formed in the reaction of cob(I)alamin with chloroacetylene. Subsequently, cob(I)alamin catalyzes the reduction of chlorovinylcobalamin to vinylcobalamin in the presence of excess titanium(III)citrate. Introduction of a chlorine onto the vinyl group of vinylcobalamin greatly changes its reduction potential. These results are discussed with respect to vitamin B12-catalyzed dechlorination of perchloroethylene, a pollutant on the priority list of the EPA.     

Mechanistic investigation of a novel vitamin B(12)-catalyzed carbon [bond] carbon bond forming reaction, the reductive dimerization of arylalkenes

In the presence of catalytic vitamin B(12) and a reducing agent such as Ti(III)citrate or Zn, arylalkenes are dimerized with unusual regioselectivity forming a carbon [bond] carbon bond between the benzylic carbons of each coupling partner. Dimerization products were obtained in good to excellent yields for mono- and 1,1-disubstituted alkenes. Dienes containing one aryl alkene underwent intramolecular cyclization in good yields. However, 1,2-disubstituted and trisubstituted alkenes were unreactive. Mechanistic investigations using radical traps suggest the involvement of benzylic radicals, and the lack of diastereoselectivity in the product distribution is consistent with dimerization of two such reactive intermediates. A strong reducing agent is required for the reaction and fulfills two roles. It returns the Co(II) form of the catalyst generated after the reaction to the active Co(I) state, and by removing Co(II) it also prevents the nonproductive recombination of alkyl radicals with cob(II)alamin. The mechanism of the formation of benzylic radicals from arylalkenes and cob(I)alamin poses an interesting problem. The results with a one-electron transfer probe indicate that radical generation is not likely to involve an electron transfer. Several alternative mechanisms are discussed.     

Evidence for the formation of a cis-dichlorovinyl anion upon reduction of cis-1,2-dichlorovinyl(pyridine)cobaloxime

The reduction of cis-1,2-dichlorovinyl(pyridine)cobaloxime, a model complex for the organometallic intermediate proposed in the dechlorination of trichloroethylene by cobalamin, was studied. Two mechanisms were considered for the Co-C bond cleavage following reduction. In the first, the Co-C bond cleaves to produce Co(I) and a chlorovinyl radical, while the second pathway results in the formation of Co(II) and a chlorovinyl anion. Four reducing agents, cobaltocene, decamethylcobaltocene, cob(I)alamin, and chromium(II), were used in the presence of H atom and proton donor species to identify the presence of chlorovinyl radical or chlorovinyl anion intermediates. Mechanistic conclusions were based on comparisons of the final product ratios of cis-dichloroethylene (cDCE) and chloroacetylene, which were found to have a direct relationship to the amount of proton donor available, with increased proton donor leading to increased cDCE production. The results support the intermediacy of a cis-1,2-dichlorovinyl anion.     

Dichloroacetylene is not the precursor to dichlorinated vinylcobaloxime and vinylcobalamin in cobalt catalyzed dechlorination of perchloro- and trichloroethylene

Vitamin B(12) catalyzes the reductive dechlorination of perchloroethylene (PCE), a process for which vinylcobalamins have been proposed as intermediates. Previous model studies have shown that PCE and trichloroethlylene (TCE) react with cob(I)aloxime to form cis-1,2-dichlorovinylcobaloxime (1). This compound could be formed by nucleophilic vinylic substitution of cob(I)aloxime on TCE or its syn-addition to dichloroacetylene. To evaluate the latter possibility, dichloroacetylene was reacted in this study with cob(I)aloxime. The major product was not complex 1 but a novel cobalt complex, indicating that dichloroacetylene is not involved in the reductive dechorination of PCE catalyzed by cob(I)aloxime. An X-ray structure of the major product was obtained showing an unexpected tricyclic structure in which one of the carbons of dichloroacetylene is a ligand to the metal and the second carbon has formed a C-C bond to one of the oxime carbons. This arrangement connects the axial and equatorial ligands. The cathodic peak potential of this complex is significantly more negative than that of previously characterized chlorinated vinylcobaloximes. Cob(I)alamin also reacts with chloroacetylene to provide cis-chlorovinylcobalamin in analogy to cob(I)aloxime, but it does not provide dichlorinated vinylcobalamins in the reaction with dichloroacetylene. Hence, dichlorinated vinylcobalt complexes detected in the reductive dechlorination of PCE catalyzed by cobaloximes or vitamin B(12) are not derived from a dichloroacetylene intermediate.     

Spectroscopic and computational studies of Co1+cobalamin: spectral and electronic properties of the "superreduced" B12 cofactor

The 4-coordinate, low-spin cob(I)alamin (Co1+Cbl) species, which can be obtained by heterolytic cleavage of the Co-C bond in methylcobalamin or the two-electron reduction of vitamin B12, is one of the most powerful nucleophiles known to date. The supernucleophilicity of Co1+Cbl has been harnessed by a number of cobalamin-dependent enzymes, such as the B12-dependent methionine synthase, and by enzymes involved in the biosynthesis of B12, including the human adenosyltransferase. The nontoxic nature of the Co1+Cbl supernucleophile also makes it an attractive target for the in situ bioremediation of halogenated waste. To gain insight into the geometric, electronic, and vibrational properties of this highly reactive species, electronic absorption, circular dichroism (CD), magnetic CD, and resonance Raman (rR) spectroscopies have been employed in conjunction with density functional theory (DFT), time-dependent DFT, and combined quantum mechanics/molecular mechanics computations. Collectively, our results indicate that the supernucleophilicity of Co1+Cbl can be attributed to the large destabilization of the Co 3dz2-based HOMO and its favorable orientation with respect to the corrin macrocycle, which minimizes steric repulsion during nucleophilic attack. An intense feature in the CD spectrum and a prominent peak in the rR spectra of Co1+Cbl have been identified that may serve as excellent probes of the nucleophilic character, and thus the reactivity, of Co1+Cbl in altered environments, including enzyme active sites. The implications of our results with respect to the enzymatic formation and reactivity of Co1+Cbl are discussed, and spectroscopic trends along the series from Co3+Cbls to Co2+Cbl and Co1+Cbl are explored.     

Ultrafast excited-state dynamics in vitamin B12 and related cob(III)alamins

Femtosecond transient IR and visible absorption spectroscopies have been employed to investigate the excited-state photophysics of vitamin B12 (cyanocobalamin, CNCbl) and the related cob(III)alamins, azidocobalamin (N3Cbl), and aquocobalamin (H2OCbl). Excitation of CNCbl, H2OCbl, or N3Cbl results in rapid formation of a short-lived excited state followed by ground-state recovery on time scales ranging from a few picoseconds to a few tens of picoseconds. The lifetime of the intermediate state is influenced by the sigma-donating ability of the axial ligand, decreasing in the order CNCbl > N3Cbl > H2OCbl, and by the polarity of the solvent, decreasing with increasing solvent polarity. The peak of the excited-state visible absorption spectrum is shifted to ca. 490 nm, and the shape of the spectrum is characteristic of weak axial ligands, similar to those observed for cob(II)alamin, base-off cobalamins, or cobinamides. Transient IR spectra of the upper CN and N3 ligands are red-shifted 20-30 cm(-1) from the ground-state frequencies, consistent with a weakened Co-upper ligand bond. These results suggest that the transient intermediate state can be attributed to a corrin ring pi to Co 3d(z2) ligand to metal charge transfer (LMCT) state. In this state bonds between the cobalt and the axial ligands are weakened and lengthened with respect to the corresponding ground states.     

Cob(I)alamin als Katalysator. 6. Mitteilung [1]. Bildung und Fragmentierung von Alkylcobalaminen,ein Gleichgewichtsprozess zwischen nukleophiler Addition und reduktiver Fragmentierung

Cob(I)alamin as Catalyst. 6. Communication [1]. Formation and Fragmentation of Alkylcobalamins: the Nucleophilic Addition – Reductive Fragmentation Equilibrium Isolated olefines can be saturated using catalytic amounts of cob(I)alamin in aqueous acetic acid; as electron source an excess of zinc dust is added to the solution containing the homogeneous catalyst. During this overall hydrogenation of isolated double bonds intermediate alkylcobalamins are formed (compare e.g. Schemes 2, 4, 5, 7 and 12). Clear evidence is presented that the nucleophilic attack on the isolated double bond is carried out by cob(I)alamin and not by cob(II)alamin also present in the system (see Scheme 3b and 3c). As this catalytic saturation of olefins depends on the pH of the solution, characterized by a slow reaction at pH = 7.0 compared to the same reduction in aqueous acetic acid (see Scheme 2, 2 → 4 , and Scheme 3a), it is reasonable to accept the participation of an electrophilic attack by a proton during the generation of alkylcobalamins. – We use the term nucleophilic addition to describe the formation of alkylcobalamins from a proton, an olefin and cob(I)alamin (compare Schemes 4–7 and 12). A special sequence of experiments showed the nucleophilic addition to be regioselective. Preferentially the higher substituted alkylcobalamin revealed to be produced. Therefore, the nucleophilic addition of cob(I)alamin follows the Markownikoff rule (compare chap. 4: formation and fragmentation of β-hydroxyalkylcobalamins). Under the reaction conditions applied the intermediate alkylcobalamins can be present in base-on and base-off forms. They are known to exist as octahedral complexes and might also be stable to some extent as tetragonal-pyramidal species. In addition the base-off forms can partially be protonated at the dimethylbenzimidazole moiety in aqueous acetic acid (compare Scheme 12). From this equilibrium of intermediate alkylcobalamins three modes of decay disclosed to be possible: (i) The reductive fragmentation leading to an olefin, a proton, and cob(I)alamin is the formal retro-reaction of the nucleophilic addition (see Schemes 2, 4 and 6–12). This equilibrium of an associated alkylcobalamin and the corresponding dissociation products revealed to be a fast process compared to the reductive cleavage of the Co, C-bond cited below (s. (iii)). (ii) As the second reaction pattern an oxidative fragmentation producing an olefin, a hydroxy anion (or water, respectively) and cob (III)alamin has been observed (see Schemes 7, 8, 10 and 12). (iii) The slow reductive cleavage of the Co, C-bond, initiated by addition of electrons (see [1a] [24]), was the third reaction path observed (see Schemes 2, 4–8 and 10–12). – The stereochemistry of the three transformations originating from the intermediate alkylcobalamins is unknown up to now. The antiperiplanar pattern of the fragmentation reactions presented in the Schemes has been chosen arbitrarily (see e.g. Scheme 12).     

ECE reaction pathways in the electrochemical reduction of dicyanocobalamin: Kinetics of ligand substitution in vitamin B12r cyanocob(II)alamin

The reduction of dicyanocob(III)alamin leads in a first stage to monocyanocob(II)alamin which can be partially converted into the base-off and base-on Co(II) complexes (B12r). The latter species are easier to reduce than the starting Co(III) complex leading to a single two-electron wave at low cyanide concentrations and/or low diffusion rates. Upon raising one of these two parameters two successive one-electron waves tend to be obtained corresponding to the Co(III)/Co(II) and Co(II)/Co(I) conversion respectively. The kinetics of the reduction process is investigated using potential-dependent potentiostatic chronoamperometry which allows a simpler analysis than cyclic voltammetry for systems involving a slow initial charge-transfer step. It is seen that the second electron, at the level of the first wave, comes from the electrode and not from the cyano-Co(II) complex in the solution. The reduction thus follows an ECE rather than a DISP-type mechanism in conditions where they can be distinguished by the usual electrochemical kinetic techniques. This contrasts with that which occurs in organic electrochemistry where the electron transfers are generally fast, while in the present case they are slow. The analysis of the reduction kinetics as a function of cyanide concentration gives some insight into the mechanism of the ligand substitution reaction at the Co(II). The kinetic data are discussed in terms of SN1-, SN2- and SNAr-like mechanisms.     

Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.     

Influence of environment on the electronic structure of Cob(III)alamins: time-resolved absorption studies of the S(1) state spectrum and dynamics

Transient absorption spectroscopy has been used to elucidate the nature of the S1 intermediate state populated following excitation of cob(III)alamin (Cbl(III)) compounds. This state is sensitive both to axial ligation and to solvent polarity. The excited-state lifetime as a function of temperature and solvent environment is used to separate the dynamic and electrostatic influence of the solvent. Two distinct types of excited states are identified, both assigned to pi3d configurations. The spectra of both types of excited states are characterized by a red absorption band (ca. 600 nm) assigned to Co 3d --> 3d or Co 3d --> corrin pi* transitions and by visible absorption bands similar to the corrin pi-->pi* transitions observed for ground state Cbl(III) compounds. The excited state observed following excitation of nonalkyl Cbl(III) compounds has an excited-state spectrum characteristic of Cbl(III) molecules with a weakened bond to the axial ligand (Type I). A similar excited-state spectrum is observed for adenosylcobalamin (AdoCbl) in water and ethylene glycol. The excited-state spectrum of methyl, ethyl, and n-propylcobalamin is characteristic of a Cbl(III) species with a sigma-donating alkyl anion ligand (Type II). This Type II excited-state spectrum is also observed for AdoCbl bound to glutamate mutase. The results are discussed in the context of theoretical calculations of Cbl(III) species reported in the literature and highlight the need for additional calculations exploring the influence of the alkyl ligand on the electronic structure of cobalamins.     

Structure-energy relations in methylcobalamin with and without bound axial base

The properties of the Co-C bond in methylcobalamin (MeCbl) are analyzed by means of first-principles molecular dynamics. The optimized structure is in very good agreement with experiments, reproducing the bent-up deformation of the corrin ring as well as the metal-ligand bond distances. The analysis of the binding energies, bond orders, and vibrational stretching frequencies shows that the axial base slightly weakens the Co-C bond (by 4%), while the alkyl ligand substantially reinforces the Co-axial base bond (by 90%). These findings support several experiments and provide insight into the conversion between the base-on and base-off forms of the MeCbl cofactor.     

Determination of hydroxyalkyl derivatives of cobalamin (vitamin B12) using reversed phase high performance liquid chromatography with electrospray tandem mass spectrometry and ultraviolet diode array detection.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) and ultraviolet diode array detection (UV-DAD), coupled on-line to reversed phase high performance liquid chromatography (HPLC), was used for the characterization of hydroxyalkyl derivatives of cob(I)alamin. The reduced form of vitamin B12, cob(I)alamin, denoted a supernucleophile due to its high nucleophilic strength, has shown promise as an analytical tool in studies of electrophilically reactive compounds in vitro and in vivo. A method for analysis of DNA-phosphate adducts was developed earlier utilizing the supernucleophilicity of cob(I)alamin to transfer alkyl groups from the phosphotriester configuration in DNA, with the formation of a Co-substituted alkyl-cobalamin (alkyl-Cbl) complex. For the purpose of identification and quantification of alkyl-Cbls at high sensitivity, an MS/MS method has been developed with application to a number of 2-hydroxyalkyl-cobalamins (OHalkyl-Cbls). The precursor oxiranes were reacted with cob(I)alamin, followed by clean-up and mass spectrometric analysis of the resulting OHalkyl-Cbls. It was found that ionization was highly dependent on solvent composition. By using acetonitrile/water/trifluoroacetic acid (TFA) (eluent I), the base peak was the doubly protonated molecule [M + 2H](2+), whereas acetonitrile/water/1-methylpiperidine (eluent II) yielded the singly protonated molecule [M + H](+) as the base peak. Excellent separation was obtained with eluent II, with good separation between stereoisomers, thus enabling the characterization of these by means of UV spectra. Limits of quantitation for 2-hydroxypropyl-cobalamin (OHPr-Cbl) were 0.2 and 2 pg/microL (or 0.1 and 1 fmol/microL) using selected ion recording (SIR) with eluent I and II, respectively. The obtained detection level should be sufficient for analysis of alkyl-Cbls from a wide range of toxicological applications.     

Reductive cleavage mechanism of methylcobalamin: elementary steps of Co-C bond breaking

Density functional theory has been applied to the investigation of the reductive cleavage mechanism of methylcobalamin (MeCbl). In the reductive cleavage of MeCbl, the Co-C bond is cleaved homolytically, and formation of the anion radical ([MeCbl]*-) reduces the dissociation energy by approximately 50%. Such dissociation energy lowering in [MeCbl]*- arises from the involvement of two electronic states: the initial state, which is formed upon electron addition, has dominant pi*corrin character, but when the Co-C bond is stretched the unpaired electron moves to the sigma*Co-C state, and the final cleavage involves the three-electron (sigma)2(sigma*)1 bond. The pi*corrin-sigma*Co-C states crossing does not take place at the equilibrium geometry of [MeCbl]*- but only when the Co-C bond is stretched to 2.3 A. In contrast to the neutral cofactor, the most energetically efficient cleavage of the Co-C bond is from the base-off form. The analysis of thermodynamic and kinetic data provides a rationale as to why Co-C cleavage in reduced form requires prior departure of the axial base. Finally, the possible connection of present work to B12 enzymatic catalysis and the involvement of anion-radical-like [MeCbl]*- species in relevant methyl transfer reactions is discussed.     

Interaction between super-reduced cobalamin and selenite

The kinetics of the reaction between the two-electron reduced form of cobalamin (super-reduced cobalamin, cob(I)alamin, or Cbl(I)) and sodium selenite in an alkaline medium is studied spectrophotometrically. It is shown that the selenite rapidly oxidizes Cbl(I) to cob(II)alamin (Cbl(II)). It is established that the active form of the oxidant is the protonated selenite anion (HSeO₃^-), which receives six electrons during the reaction and transforms into HSe^–. The reactions of cob(I)alamin oxidation by selenite and sulfite are compared.     

NO binding to cobalamin: influence of the metal oxidation state

Density functional and molecular orbital theory calculations on models for cobalamin suggest that NO binds similarly to the Co(II) and Co(III) oxidation states. However, Co(III) can bind water far more strongly than Co(II) as a sixth ligand, so that the competition between water and NO complexation strongly favors water for Co(III) in the gas phase. Although the Co(II) oxidation state is found to bind water slightly more strongly than NO in the gas phase, the inclusion of solvation effects via the polarizeable continuum model makes NO binding more favorable. Thus, the experimentally observed ability of cob(II)alamin to bind NO in aqueous solution is the result of its weak complexation with water and the relatively poor solvation of NO. Calculated vibrational frequencies support the interpretation of the cob(II)alamin-NO complex as being cob(III)alamin-NO-, although the DFT calculations underestimate the degree of charge transfer in comparison to Hartree-Fock calculations.     

Reduction‐Labile Organo‐cob(III)alamins via Cob(II)alamin: Efficient Synthesis and Solution and Crystal Structures of [(Methoxycarbonyl)methyl]cob(III)alamin

An efficient synthesis of Coβ‐[(methoxycarbonyl)methyl]cob(III)alamin ( 6 ) is reported as an example of a new method for the preparation of some easily reducible organo‐cob(III)alamins via the alkylation of cob(II)alamin. The procedure represents a considerable improvement compared to earlier methods that were based on an alkylation of cob(I)alamin. Thus, aquacob(III)alamin chloride ( 5 ⁺?Cl) was reduced to cob(II)alamin ( 4 ), either by controlled potential electrolytic reduction or with an excess of sodium formate as reducing agent. The solution of 4 was then treated with an excess of methyl bromoacetate while being reductively poised potentiostatically or kept reduced by the formate, to give crystalline 6 in a yield of up to 91%. The structure of 6 in aqueous solution was mainly established by the completely assigned ¹H‐ and ¹³CNMR spectra (Table 1). The NOE data (Table 2) were best rationalized by the presence of a single main conformation of the (methoxycarbonyl)methyl ligand. Single crystals of 6 were obtained by crystallization from an aqueous solution, and the crystal structure was determined by X‐ray analysis at cryotemperatures. The NMR and crystallographic data of 6 indicated similar structures in aqueous solution and in the crystal with the (methoxycarbonyl)methyl ligand preferring a ‘southern' orientation in each case.