Solution, Crystal and in Silico Structures of the Organometallic Vitamin B 12

The natural vitamin B 12 -derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring-contracted corrin ligand. Here, we describe the synthesis of the Rh(III)-corrin acetylrhodibalamin ( AcRhbl ) from biotechnologically produced metal-free hydrogenobyric acid and analyze the effect of the replacement of the cobalt-center of the organometallic vitamin B 12 -derivative acetylcobalamin ( AcCbl ) with its group-IX homologue rhodium, to give AcRhbl . The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide-appended corrin-ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium-corrin AcRhbl joins the small group of B 12 -mimics classified as ‘antivitamins B 12 ’, isostructural metal analogues of the natural cobalt-corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes.


Introduction
The natural B 12 -derivatives are important cobalt complexes that encase the metal within a ring-contracted corrin ligand. [1 -3]The particular mutual interplay of cobalt and corrin in the B 12 -cofactors is essential for their fundamental biological functions, which are indispensable in all kingdoms of life. [4]The corrin ligand incarcerates cobalt-ions very tightly, [5,6] and provides a ring-contracted and skewed helical coordination environment for the metal-ion, acting as a unique entatic state module that activates the B 12cofactors for enzyme catalysis. [7]However, the exceptional chemical basis of cobalt for its role in B 12cofactors remains intriguing. [2,8]n line with studies on metal-analogues of the cobalamins (Cbls) [9 -12] we have become particularly interested in gaining access to well characterized rhodium analogues of the natural B 12 -derivatives, in order to study the effect of the replacement of the natural cobalt-centers by the homologous transition metal rhodium. [13,14]s discovered in the course of the comparison of the structures of coenzyme B 12 (5'-deoxy-5'-adenosylcobalamin, AdoCbl) and of 5'-deoxy-5'-adenosylrhodibalamin (AdoRhbl), the structural features of the latter suggested the corrin ligand to provide a slightly better fit for the Rh(III)-center, rather than for the natural Co(III)-ion. [9]A similar conclusion resulted from a comparison of the crystal structures of chlorocobalamin (ClCbl) and chlororhodibalamin (ClRhbl). [12]Most importantly, these previous investigations established largely common coordination-chemical features of the Rh(III)-and Co(III)-ions in Rhbls and Cbls, respectively, and indicated a comparable metal-center mediated (structural and kinetic) trans-influence of the axial ligands. [12]erein, we describe acetylrhodibalamin (AcRhbl), a previously unknown organometallic Rhbl, prepared by a combination of biological and chemical synthesis steps, and which has been structurally characterized in aqueous solution as well as in a single crystal.[17][18] In both of these organometallic acyl-corrins we observe the acetyl group in an approximate East-West orientation with respect to the metallocorrin moiety.This unprecedented conformational type of a B 12 -derivative with an unsaturated cobalt-bound carbon center [19,20] stimulated a more fundamental analysis of the organometallic bonding in AcRhbl and AcCbl by computational means, details of which are also reported herein.

Results and Discussion
Acetylrhodibalamin (AcRhbl) was prepared by attachment of the complete B 12 -nucleotide [2,21] to the 'incomplete' acetyl-rhodibyrate (AcRhby), newly synthesized in two steps from hydrogenobyric acid (Hby) [7] (see Scheme 1).The terminal condensation reaction of AcRhby with the B 12 -nucleotide was carried out in aqueous solution following methodology developed for the final step of the partial syntheses of vitamin B 12 and coenzyme B 12 , originally, [22] and later also adapted for the total synthesis of the organometallic rhodibalamin (Rhbl) AdoRhbl. [9]This methodology exploits the pre-coordination of the B 12 -nucleotide to the metal-center and the amide-formation at the f-propionate side chain in a particularly efficient intramolecular process assembling the nearly strainless 19-membered ring of the 'nucleotide loop', a concept pioneered by Eschenmoser in studies on the self-constitution of the Cbls [2] (for details, see [21]).In the present work, this synthesis step furnished crystalline AcRhbl in a yield of 58 %.The 'incomplete' acetyl-Rh(III)-corrin AcRhby was synthesized in high yield by sodium borohydride (NaBH 4 ) reduction of raw chlororhodibyrate in aqueous solution, furnishing a tentatively identified Rh(I)rhodibyrate intermediate, followed by subsequent acetylation at the presumed highly nucleophilic Rh(I)center by acetic anhydride (AcOAc).Hence, the typical organometallic and redox chemistry of natural B 12derivatives, [23] exploited in the partial synthesis of acetyl-Co(III)-corrins, [15][16][17][18] was employed here for the synthesis of the acetylrhodibyrate AcRhby from aquochlororhodibyrate ((H 2 O)ClRhby).The raw (H 2 O)ClRhby was prepared in high yield from the metal-free corrin Hby, using a methodology that employs the dimeric Rh(I)-complex [ClRh(CO) 2 ] 2 under inert gas, as developed for the synthesis of model Rhcorrins by Blaser and Eschenmoser, [24] and later adapted by Koppenhagen et al. for Rh-incorporation into biologically available metal-free corrins. [25,26]erendipitously, under the precise original conditions employed in our lab for Rh(I)-incorporation into the metal-free corrin Hby, when acetic acid distilled from phosphorous pentoxide was used as a dry solvent, puzzlingly AcRhby was detected as a side product.In order to explore the suspected role of acetic anhydride, generated inadvertently in the distillate, it was found that the deliberate inclusion of a small amount of acetic anhydride in a control experiment led to AcRhby as a major product, indicating efficient acetylation of Rh(I)-rhodibyrate.This result also suggested the significance of this potential intermediate from the metallation of Hby by [ClRh(CO) 2 ] 2 .The attachment of the acetyl group of AcRhby occurred with high face-selectivity at the 'upper' β-face of the metal-center, so that covalent conjugation with the B 12 -nucleotide directly led to the Cbl-homolog AcRhbl.
The molecular formula C 64 H 91 N 13 O 15 PRh of the total-synthetic organometallic Rh-corrin AcRhbl was verified by mass spectrometric analysis.Further spectral and crystallographic characterization of AcRhbl established its detailed properties (see below and Supporting Information).The UV/Vis-spectrum of AcRhbl (see Figure 2) displays the typical features of the spectra observed with most metallo-corrins through the presence of pronounced α, β and χ-bands with maxima at 502, 482 and 344 nm, respectively.In contrast, acetylcobalamin (AcCbl) features an 'atypical' absorption spectrum with broader maxima and lacking a corrin-type band structure (see Figure 3).The UV/Visspectra of the B 12 -cofactors methylcobalamin (MeCbl) and coenzyme B 12 (AdoCbl) are similarly 'atypical', as are those of most organometallic Co(III)-corrins. [5]The puzzling spectral characteristics of the organocobalamins are a particular sign of the exceptional electronic interactions between the Co(III)-center and the corrin ligand. [27,28]Indeed, the deductions from the UV/Visspectral comparison of AcRhbl and AcCbl reported herein have precedence with the AdoRhbl and AdoCbl pair of compounds. [9]he solution structure of AcRhbl in water was studied by homo-and hetero-nuclear NMR-spectroscopy at 700 MHz.The data from 1 H, 1 H-COSY and ROESY spectra, as well as from 1 H, 13 S6 and S7).The methyl group singlet of the Rh-bound acetyl ligand of AcRhbl was observed at 0.77 ppm in the 700 MHz 1 H-NMR spectrum (see Figure 4), shifted up-field by 1.46 ppm from the resonance of the acetone methyl groups.As deduced from a 1 H, 13 C-HMBC-spectrum, it is spin-coupled with a carbonyl 13 C-atom at 235.3 ppm.Analysis of 1 H, 1 H-NOE-spectra verified the base-on nature of AcRhbl and the β-configuration of the Rh-bound acetyl group (see Supporting Information Figure S24).Furthermore, it suggested an extensive re-orientational mobility of the organometallic acetyl group, primarily residing in an East-West orientation with the acetyl-methyl close to the hydrophobic top face of the ring C-moiety.However, according to the observed NOE-correlations, the acetyl group also explored orientations that placed its methyl group close to H 2 C(71) at ring B and, in the opposite direction, towards H 3 C(17B) and HC (19) at ring D, but not close to ring A.
Acetylcobalamin (AcCbl) was prepared from aquacobalamin chloride (H 2 OCbl•Cl) applying the previously established method of acetylation of the reduced cob(I)alamin, [15,16,18] using acetic anhydride.AcCbl was fully characterized, including homo-and heteronuclear NMR spectroscopy in H 2 O/D 2 O (9 : 1).Based on the NMR data of AcCbl the signals of all of its 64 C-atoms could be assigned, as well as of all of its 76 non-(exchange)-labile H-atoms, in addition to the 13 amide protons (see Supporting Information, Tables S3 and S4).In a 700 MHz 1 H-NMR spectrum of AcCbl, the acetyl group gave rise to a methyl group singlet at 1.37 ppm, downfield by 0.60 ppm from the position of the acetyl methyl in AcRhbl (see Figures 4  and 5).A 1 H, 13 C-HMBC-spectrum revealed it to spincouple with a carbonyl 13 C-atom at 241.7 ppm.
The comparison of the chemical shifts of corresponding nuclei in AcRhbl and AcCbl (see Supporting Information, Figures S25 and S26) confirmed the very similar solution structure of the two homologous metal-corrins.However, significantly different electronic interactions of the metal cores with their directly bonded ligand-atoms were indicated.Further analysis has also revealed minor structural differences located     S9).However, when comparing the 1 H-NMR data for the 'incomplete' Rh-corrin AcRhby and the protonated base-off form HAcCbl + of the cobalt-corrin AcCbl signals of several corresponding H-atoms at the cobalt-corrin moiety display more pronounced chemical shift differences.These could relate, for instance, to different axial coordination in 5-coordinate protonated HAcCbl + and 6-coordinate AcRhby due to the absence or presence of ligated solvent molecules at the lower face of the metal-centers.Alternatively, the protonated and de-coordinated DMB-nucleotide of HAcCbl + might still exert significant anisotropic effects at the lower face of the corrin moiety, as found in 1 H-NMR spectra of hydrogenobalamin, the complete metal-free ligand of vitamin B 12 . [11]n the UV/Vis-spectrum of the 'incomplete' organometallic Rh-corrin AcRhby, the α, β and χ-bands occur at 481, 462 and 336 nm, respectively (see Supporting Information, Figure S19), i. e., at wavelengths significantly shorter than in the spectrum of AcRhbl, where the coordination of the DMB caused bathochromic shifts.The calculated molecular formula of AcRhby (C 47 H 67 N 10 O 9 Rh) was confirmed by an ESI-mass spectrum, in which the pseudo-molecular ion [M + H] + was at m/z = 1019.42(see Supporting Information, Figure S15).In a 700 MHz 1 H-NMR spectrum of AcRhby in D 2 O, the chemical shifts of the singlets at 6.42 ppm and 0.74 ppm allowed their assignment to the corrin methine HC (10) and to the acetyl methyl, respectively.The up-field shift of the signal of the acetyl methyl group of AcRhby was merely 0.03 ppm greater than for AcRhbl (see below and Supporting Information Figures S20 and S23).
The isolate from the incorporation of rhodium into the metal-free corrin Hby behaved as a slowly interconverting mixture of two only partially characterized chlororhodibyrates.When separated by HPLC the two main fractions appeared to contain a (apparently more polar) dichlororhodibyrate (Cl 2 Rhby) and a single (apparently less polar) aquochlororhodibyrate isomer, (H 2 O)ClRhby.Both were provisionally characterized on the basis of their UV/Vis-spectra.Consistent with this tentative assignment, the isolated (H 2 O)ClRhby slowly coordinated a second chloride ion in 5 M aqueous NaCl at room temperature, which was selectively lost at ambient temperature in dilute chloride free aqueous solution (see Supporting Information, Figures S10-S13).
Single crystals of AcRhbl and of AcCbl, were grown from water/acetonitrile mixtures at room temperature, allowing for well-resolved, cryotemperature X-ray crystal structures of both compounds (see Supporting Information).The two homologous 'complete' corrins, AcRhbl and AcCbl, crystallized in the orthorhombic space group P2 1 2 1 2 1 , with four solvent separated corrinoid molecules per unit cell.The crystal structures of AcRhbl and AcCbl confirmed their NMR-derived chemical constitutions and furnished precise insights into the bonding around both metal-centers, the corrin ligand conformation and the preferred orientation of the acetyl group with respect to the corrin ring (Figures 6 and 7).
The homologous Rh-and Co-corrins AcRhbl and AcCbl have very similar qualitative structural properties, including the unprecedented predominant East/ West-orientation of their organometallic acetyl ligands.In fact, in the crystal of AcCbl the acetyl group and the acetamide side chain attached to C(2) at ring A, both show some disorder (see Supporting Information, Figure S41).The average orientation of the acetyl-acylplane, close to the N(1)À CoÀ N(3) diagonal results from an orientational disorder by an angle of about 49°, suggesting dominating contributions of non-bonding interactions as external factors, in addition to weak inherent conformational preferences.For AcRhbl the orientation of the acetyl group in an approximate  East/West orientation is better defined crystallographically (see Supporting Information, Figure S40).
The hexacoordinate Rh(III)-and Co(III)-centers of AcRhbl and AcCbl display a common coordination pattern.Both metal-ions sit in the center of an unsymmetrical, deformed octahedron, spanned by the four inner N-atoms of the helical corrin ligand (arranged as a squashed tetrahedron) and the two axial groups.The six metal-coordinating bonds in AcRhbl were roughly 72 mÅ longer (onaverage) than in AcCbl (see Figure 8).This bond length difference is remarkably close to the difference of 60 mÅ of the covalent radii of Rh(III) and Co(III), [29] suggesting little differential strain in the two organometallic acetyl-corrins.All the equatorial RhÀ N bonds and the longer axial bonds had remarkably comparable lengths as in AdoRhbl. [9]The CoÀ C sp2 bond in AcCbl is slightly longer than in vinyl-cobalamin, [30] but shorter than in an aryl-cobalamin, [20] suggestive of insignificant conjugative or steric effects on the length of the CoÀ C sp2 bond of AcCbl.
The mutual structural adaptation of the ligand and the metal-centers has been studied in order to understand better the critical 'fit' of the bound metal-ions with respect to the tetrapyrrolic equatorial ligands. [31]or cobalt-corrins and transition metal analogues, the corrin fold angle, [32] corrin helicity [7] and the deviation from an in-plane arrangement of the inner corrin Natoms around the metal-centers [11] have been established as important parameters.Their values from the crystal structures of AcRhbl and AcCbl (see Table 1) were all slightly smaller for the Rh(III)-corrin than for the Co(III)-corrin.The slightly smaller corrin-fold angle observed in AcRhbl compared to AcCbl (13.4°vs.15.6°) may be considered an indication of a somewhat 'better fit' of the Rh(III)-center that is also better able to force the six coordinating atoms into a position closer to the preferred octahedral pattern.However, the interdependence between the corrin-fold angle and the length of the axial bond from the metal center to the DMB-nitrogen, known from extensive correlation in Cbls, [33 -35] would also be in line, qualitatively, with a larger fold angle in AcCbl, where the CoÀ N bond is significantly shorter than in AcRhbl (see Figure 8).
In order to gain further insights into the exceptional conformational characteristics of the acetyl-corrins AcCbl and AcRhbl and for a better understanding of their structural differences, computational studies of the  two organometallic acyl-metal complexes were carried out with density functional theory (BP86/def2-SVP/D3) in implicit water.The calculations produced structures of AcCbl and of AcRhbl, remarkably similar to each other and to the structures from the respective X-ray crystal analyses (see Figures 9 and 10).First-of-all, this similarity included the calculated preferred orientation of the axial acetyl-ligand in both structures.Indeed, the calculated bond lengths also followed the crystallographically observed values, all reflecting the basic differences expected for a Co(III)-corrin and a homologous Rh(III)-corrin due to the differing size of these group-IX metal ions [29] (see Figure 8).The corrin helicity, [7] 'non-planarity' [11] at the 6-coordinate metal centers and the tetrahedral distortion of the coordinating four N-atoms of AcRhbl and AcCbl were all calculated as slightly smaller for the Rh(III)-corrin AcRhbl than for the Co(III)-corrin AcCbl (Tables 1 and  2), in line with the crystallographic results.
A small difference was also derived for the corrin fold angle, [32] which was calculated as slightly larger in
Remarkably, the calculations reproduced the preferred approximate 'West' orientation of the (O-atom of the) acetyl group in both compounds, while also revealing local energy minima for AcRhbl and AcCbl in the opposite 'East' orientation and, for AcRhbl, an additional one in the 'South'.Furthermore, they furnished low global and local conformational energy barriers with respect to the rotation of the acetyl group around its organometallic bond.These were calculated to amount to less than about 28 kJ/mol (or 7 kcal/mol) and18 kJ/mol (or 4.5 kcal/mol) for AcCbl and AcRhbl, respectively (see Figure 11).Hence, the conformational dynamics and orientational preferences of the acetyl-group deduced from the NOEinterproton correlations in MHz 1 H, 1 H-ROESY spectra (see Supporting Information, Figures S7 and S24) were fully corroborated by the calculations.
The calculations dismiss the relevance of orientational preferences due to large π-conjugative interactions between (either of) the transition metal centers and the organometallic π-acceptor acyl groups.In fact, further computational investigations of the non-covalent interactions [36,37] between the metal-corrin and acetyl moieties revealed weak attractive interactions in both compounds between the acetyl oxygen and the a-acetamide methylene-group (H 2 C( 21)), as well as between the acetyl H 3 C-group and the π-system of the corrin ring (see Supporting Information, Figures S42ff).In the case of AcCbl, minor repulsive (steric and closed shell) interactions are indicated at N(2) and N(4) (see Supporting Information, Figures S42 and S47).The weak non-covalent interactions and steric effects exerted by the helical and folded corrin ligand and its 'upper' substituents appear to be the main determinants for the preferred conformation(s) of the acetyl group.In AcCbl and AcRhbl, such interactions may specifically destabilize the 'North/South' orientation of the organometallic ligand that has previously been observed for aryl-Cbls [20,38] and vinyl-Cbls. [30]Furthermore, the preferred orientation of the 'dipolar acetyl rotor', carbonyl oxygen near ring A and methyl group near ring C, reflects the complementary interfacial polarity at the 'upper' corrin surface, where ring C carries a methyl and ring A the a-acetamide group.

Conclusions
The enduring quest for rationalizing Nature's selection of cobalt for the biologically important corrinoid B 12cofactors [2,3,8] has stimulated a fundamental interest in preparing and studying transition metal analogues of B 12 , [9 -12] a long-standing challenge in the B 12field. [26,39,40]Here the specific case of a total biological and chemical synthesis of acetylrhodibalamin (AcRhbl) is described, representing the novel rhodium homologue of the known B 12 -derivative acetylcobalamin (AcCbl), together with a detailed structural comparison between this pair of Rh(III)-and Co(III)-corrins.Taken together, the comparison of AcRhbl and AcCbl, along with two other pairs of corresponding Rhbls and Cbls, [9,12] has suggested a slightly 'better fit' of Rh(III)ions for the corrin ligand in comparison to Co(III)-ions.However, such comparisons have also shown that rhodium is no match for cobalt, when bound to the natural corrin ligand, [2,7] in terms of providing the biologically essential organometallic reactivity and redox properties of the B 12 -cofactors. [23]Indeed, the exceptional chemical features of the cobalt-corrins are due to the particular electronic interactions between the bound cobalt-ions and the natural corrin ligand, which also manifest themselves in the 'atypical' absorption spectra of the organometallic B 12 -cofactors and in their specific photochemical reactivity. [41,42]he acetyl-corrins discussed herein were selected for the expected σand π-bonding properties of the organometallic acetyl group.In fact, AcCbl likely lacks any biological catalytic function, and therefore, is a poorly investigated B 12 -analogue.Earlier hypotheses considered that AcCbl may be a key intermediate in the important acetyl-CoA pathway of carbon dioxide fixation of acetogenic and methanogenic microorganisms, [16,[43][44][45][46] but these ideas have found no experimental support. [47,48]However, the capacity of AcCbl and related acyl-cobalamins to serve as lightactivated sources of the nucleophilic acyl radicals, [49] may qualify such vitamin B 12 -derivatives as bio-similar initiators of olefin-polymerization, which is a technology applied nowadays in dental medicine. [50]Preliminary photochemical experiments with AcRhbl, indicated that this Rh-homologue of AcCbl decomposes very slowly in the presence of air and provided no evidence for photo-induced RhÀ C bond cleavage (see Supporting Information and Figures S28-S32).
Rhodibalamins (Rbls) hold significant potential as antivitamins B 12 , [13,14,51] or as specific B 12antimetabolites, [26,52,53] and, in this regard, the function of AcRhbl requires further investigation within a biological context.In this respect, the biological activity of AcRhbl underwent exploratory tests using a Salmonella enterica B 12 auxotroph that relies on a B 12dependent methionine synthase for growth on minimal media. [54]As observed with AdoRhbl, [9] the bioassay indicated that AcRhbl was actively imported by the bacteria and produced inhibitory growth effects.These observations indicate that AcRhbl interferes with bacterial metabolism, most likely by impairing the B 12 -riboswitch regulated import of CNCbl. [55,56]In this scenario AcRhbl may regulate the riboswitch either directly, or indirectly as a possible metabolic precursor [57] to AdoRhbl, the structural AdoCbl-mimic.Further studies are planned to elucidate more precisely the inhibitory mechanism considered here.[63] Whereas the acetyl-group of the photo-labile AcCbl is rather reactive towards (even mild) nucleophiles, [64] impairing resilience in biological environments, the more photo-stabile AcRhbl may also be more resistant to the removal of its acetyl group, since RhÀ C bonds are comparatively strong, as seen, e. g., in methyl-Rh(III)-porphyrins. [65] In consequence, AcRhbl may be specifically suitable for targeting, e. g., the B 12 -tayloring glutathione-dependent enzyme CblC in humans and animals that Cblbased antivitamins B 12 characteristically inhibit, [20,51,66,67] thus, undercutting the removal of the 'upper' Cbl-ligands that is critical for the metabolic formation of the B 12 -cofactors. [60,68]ur structural work has further verified the predicted strong similarity of the coordination-chemical features of the Rh(III)-and Co(III)-centers of corresponding Rhbls and Cbls, respectively, including the unique specific structural trans-effect characteristic of the Co(III)-ions, which have been studied in a range of synthetically accessible, inorganic and organometallic B 12 -derivatives. [3,33]As was observed here for the case of the chlororhodibyrates, and as far as can be deduced more broadly, axial ligand exchange is much slower in Rh(III)-corrins compared to corresponding Co(III)-corrins, which feature fast exchange kinetics of their 'inorganic' axial ligands. [5]Consistent with their evidently stronger bonding, in contrast to the corresponding Co(III)-corrins, Rh(III)-centers appear to be remarkably more effective in imposing a nearly octahedral arrangement on the directly metal-binding atoms of the skewed, helical corrin ligand.As with other antivitamins B 12 , [66] the Rh-mimics of the B 12cofactors hold promise as useful dopplegangers in B 12related biological and bio-structural studies.
Our concerted biological and chemical synthesis approaches to AcRhbl and to other transition metal analogues of the cobalamins, or metbalamins (Metbls), [9 -12] provides an efficient path to the exciting though still hardly explored territory of metallocorrinoids [26,40] and to promising novel biological and biomedical applications. [51,53]As close structural B 12mimics with significantly altered chemical reactivity, the rhodium analogues of the vitamin B 12 derivatives deserve particular attention as remarkable chemicalbiological tools in the multifaceted B 12 -field.As potential antivitamins B 12 [14] they are expected to impair the known physiological effects of the vitamin B 12 forms -as well as still elusive ones -in the metabolism of mammals, potentially giving them key roles in B 12 -related medical diagnosis and treatment.Their growth-inhibiting effects in B 12 -dependent organisms, from microorganisms to humans, classify antivitamins B 12 as antibiotics and as potential anticancer agents, highlighting them as promising pharmaceuticals with a precise, activity targeting structurebased design.

General
All experimental work was carried out with extensive protection from light.RV: rotatory evaporator (operated at a bath temperature of 40 °C, if not stated otherwise).Desalting: the (mostly aqueous) solution of an isolate was absorbed on RP18 cartridge and washed with 10 ml of water, followed by MeOH to elute the colored compound(s).See the Supporting Information for further experimental details, abbreviations, chromatography, spectroscopy, X-ray crystallography, computation and bioactivity tests, as well as specifications for solvents and reagents.
Synthetic Procedures and Spectral Characterization (for more details, see Supporting Information) Preparation of Acetylcobalamin (AcCbl) AcCbl was prepared from aquocobalamin following the method by Müller and Müller, [15] but using AcOH/H 2 O (1 : 9 v/v) as solvent and an RP18 cartridge for purifying raw AcCbl (see Supporting Information, S4 for details).
For crystallization, a solution of the purified AcCbl in about 1 ml of water was treated with about 3 ml of acetone to give a slightly turbid mixture, which was placed into a refrigerator (10 °C).Red crystals grew overnight and were separated from the mother-liquor, washed with a copious amount of acetone and dried under HV, furnishing 14.4 mg (10.5 μmol, 72 %) of red crystalline AcCbl.The sample of AcCbl was identified by UV/Vis-, ESI-MS-and 1 H-NMR spectra, and characterized further by high field homo-and hetero-nuclear NMR of solutions at pH 6 (base-on form) and pH 2 (protonated base-off form), as well as by CD-and IRspectra.

Synthesis of Acetylrhodibyrate (AcRhby) from the Chlororhodibyrate Mixture
A methanolic solution of 1.3 μmol of the roughly 1 : 1 mixture of the chlororhodibyrates synthesized and isolated through the procedure described above, was dried using an RV.The residue was dissolved in 2.5 ml KP6-buffer to be transferred into a 5 ml flask that was fitted with a 1 mm UV-Vis cell.The orange-yellow solution was degassed through three cycles of freeze, evacuate (HV) and thaw under Ar.Then 5.1 mg (100 equiv.) of NaBH 4 were added under inert gas and bubbling as well as a slight color change of the solution to yellow-orange was observed.As a UV-Vis spectrum recorded 28 min after the NaBH  The solution of AcRhby from the previous experiment (1.3 μmol) in a 10 ml round bottom flask was concentrated to 2 ml using a RV, and cooled to about 0 °C by an ice bath.Then 1.7 mg (3 equiv.) of B 12nucleotide [2,21] and 1.0 mg (5 equiv.) of HOBt were added and the mixture was stirred to dissolve the solids.Subsequently    S32).

Exploratory Studies of Biological Activity of AcRhbl
The biological activity of AcRhbl was investigated by the use of a bioassay, using a Salmonella enterica B 12 auxotroph that relies on a B 12 -dependent methionine synthase for methionine production as previously described. [54]The use of this strain on a plate-based microbiological bioassay allows not only for the quantitative detection of cobalamin by relating the size of growth circles around an application point on the Salmonella-embedded agar, but also allows for the determination of inhibitory molecules by either mixing with specific quantities of cyanocobalamin (CNCbl), producing reduced growth circles, or by looking for zones of inhibition when the inhibitor is placed next to the CNCbl application point. [9]In the event, application of AcRhbl to the bioassay plate failed to promote any growth by itself and competition experiments with CNCbl support the conclusion that AcRhbl is actively imported by the bacteria resulting in growth inhibition (for further details see Supporting Information and Figure S48).

Crystallographic Work
Crystals of AcCbl for X-ray crystallography were obtained upon slow addition of about 1 ml MeCN to a light protected solution of 1.3 mg of AcCbl in about 100 μl of water (until the solution became slightly turbid).The crystals grew upon storage at room temperature (2 h) followed by overnight storage in a refrigerator.Crystals of AcRhbl suitable for X-ray crystallography were obtained in a similar way by slow addition of MeCN to a sample of roughly 0.3 mg of AcRhbl in about 100 μl of water, till the solution became slightly turbid.
Single-crystals were selected for measurements by a Bruker D8Quest diffractometer, equipped with a Photon 100 CMOS detector, using molybdenum radiation at temperatures of 183 K for AcCbl and 173 K for AcRhbl.The crystal structure data for AcCbl (CCDC-2217050) and for AcRhbl (CCDC-2217051) are available at the Cambridge Data System (see Supporting Information for more details).

Quantum Chemical Studies
Initial structures of AcRhCbl and AcCbl were adapted from the available X-ray crystal structures.These structures were fully optimized with density functional theory (BP86/def2-SVP) [69][70][71] including empirical dispersion corrections of the D3 type with Becke-Johnson damping [72,73] in water.Water was modelled as implicit solvent using the Conductor-Like Screening Model as implemented in Turbomole, [74,75] where the solute is placed in a cavity and the solvent is described by a dielectric continuum, here a permittivity of ɛ = 78.5 is used.To speed up the calculation time, the resolutionof-identity technique was utilized. [76]r the conformer search, a pre-final version of the crystal structure served as input structure.The structures calculated starting from the pre-final or the final crystal structures of AcRhCbl and AcCbl were rather similar: the major difference was the orientation of the nucleotide loop.An overlay of the two structures showed that the geometries at the upper face of the corrin were almost identical (see Supporting Information, Figures S43-S44).To generate the conformers, the orientation of the acetyl group with respect to the corrin ring, that is the dihedral between N1À MÀ C1-LÀ O2-L, was modified by increments of 15°.Each resulting structure was partially optimized with the respective dihedral angle being kept fixed.Final single www.helv.wiley.com(13 of 16) e202200158 © 2022 The Authors.Helvetica Chimica Acta published by Wiley-VHCA AG point energies on the partially optimized structures were calculated with the range-separated hybrid functional ωB97xd [77] in conjunction with the triple-zeta basis set def2-TZVP [71] and water treated by an implicit solvent model.All of the above calculations were performed with Turbomole 7.5. [78](TURBOMOLE V7.5 2020, a development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH, 1989-2007, TURBOMOLE GmbH, since 2007; available from http://www.turbomole.com).To visualize non-covalent interactions, the approach of Johnson et al. [36] was used (see Supporting Information for details).
Helv.Chim.Acta 2023, 106, e202200158 www.helv.wiley.com(4 of 16) e202200158 © 2022 The Authors.Helvetica Chimica Acta published by Wiley-VHCA AG 15222675, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/hlca.202200158 by Test, Wiley Online Library on [17/02/2023].See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions)on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License at the 'lower' face, which we ascribe to a slightly different nucleotide loop architecture in the cobaltcorrin AcCbl vs. the Rh-analogue AcRhbl.Hence, the NMR data suggest closely related structural properties of the acetyl-Co(III)-and acetyl-Rh(III)-corrins AcCbl and AcRhbl, including an exceptional preferred East/ West-orientation of their organometallic acetyl ligand (see Supporting Information, Figures S4 and S24, Table

Figure 8 .
Figure 8. Lengths of coordinating bonds (in Å) around the Rh(III)-center in AcRhbl (left), around the Co(III)-center in AcCbl (right), as derived from the crystal structures, and differences of these bond lengths in mÅ (middle).

Figure 11 .
Figure 11.Top: Energy diagrams of the orientation of the acetyl group with respect to the corrin ring in AcRhbl (left) and in AcCbl (right), depicting global minima for AcRhbl and AcCbl as 'Western' orientations of the acetyl group, as similarly observed in the respective crystal structures.The dihedral step-size of 15°resulted in 24 structures; each structure was partially optimized with BP86/def2-SVP/D3 in water, while final single point energies were obtained with ω-B97XD3/def2-TZVP, again in water.The dihedral of the orientation 'North' (of the O-atom of the acetyl group) was chosen as reference and set to zero.Bottom: structural models of (local) minimum energy conformers of AcRhbl (left) and AcCbl (right).
4 addition indicated significant adventitious re-oxidation, another portion of 5.1 mg (100 equiv.)NaBH 4 was added under inert gas.After 5 min a UV-Vis spectrum indicated reduction with formation of a Rh(I)-corrin (see Supporting Information, FigureS16) and 5 min later 40 μl (21.2 μmol, 15.8 equiv.) of a solution of 200 μl acetic acid anhydride in 3.8 ml MeOH, chilled to À 20 °C and saturated with Argon was added under inert gas.The solution instantly became light yellow and 3 min after the addition of acetic acid anhydride a UV-Vis spectrum showed the characteristics of an organometallic Rh-corrin.The solution was diluted with 15 ml KP6-buffer to be loaded onto a RP18 cartridge.The adsorbate was washed with 10 ml KP6-buffer and 20 ml of water and was eluted with 8 ml of MeOH.This solution was evaporated to dryness on an RV and the orange colored residue was dissolved in 5 ml water.HPLC-Analysis indicated a single corrin fraction, which was characterized as AcRhby by an ESI-MS spectrum (see Supporting Information, FigureS15) and UV-Vis quantified as about 1.3 μmol.The AcRhby solution was concentrated to 2 ml on an RV at T < 40 °C to give the sample of acetylrhodibyrate (AcRhby) directly used for the synthesis of AcRhbl.UV-Vis (c � 1.7 × 10 À 4 M, H 2 O, rel.ɛ): 481.0 (0.422), 462.0 (0.415), 410.0 (0.178), 398.0 (0.125), 336.5 (1.00), 288.5 (0.476), 265.5 (0.514), 241.5 (0.410) (see Supporting Information, FigureS19).
5.1 mg (20 equiv.) of EDC•HCl were added, and the solution was stirred at 0 °C for 50 min, when HPLC-analysis of a sample of the mixture indicated roughly 25 % conversion.After a reaction time of one hour, the ice bath was removed and the stirred solution was warmed up to r.t.After 90 min reaction at r.t.roughly 95 % conversion to AcRhbl was indicated by HPLC.After a total reaction time of 180 min at r.t., the mixture was diluted with 10 ml KP6-buffer and loaded onto an RP18 cartridge.The adsorbate was washed with 10 ml KP6-buffer and 10 ml of water and then eluted with 2 ml of MeOH.UV-Vis and HPLC-analysis of the eluate showed AcRhbl and B 12 -nucleotide left (see Supporting Information, Figures S22).
The collected eluate was evaporated on an RV and the residue was re-dissolved in 100 μl of water, for separation through HPLC in two similar sized portions.The fractions of AcRhbl were combined, desalted, eluted with 2 ml of MeOH and found pure by HPLC.The solution was evaporated on a RV and the residue was dried applying HV, yielding 1.1 mg of solid AcRhbl.The raw AcRhbl was dissolved in 80 μl water and 1.0 ml acetone was added slowly in small portions under mixing giving a slightly turbid mixture.Upon storage at r.t. for 15 min, first crystals began to form.The crystallization was completed overnight in a refrigerator at 8 °C.The orange crystals were separated from the mother-liquor, washed twice with about 1 ml of acetone and then dried under high vacuum for 1 hour.1.1 mg of orange needle-like crystals of AcRhbl (0.78 μmol, 58.0 % over all from the chlororhodibyrate mixture) were obtained, characterized by the set of spectra displayed aboveand in the Supporting Information, S20.UV-Vis (c = 5.23 × 10 À 5 M, H 2 O): 502.5 (3.96), 482.5 (3.93), 415.0 (3.53), 392.5 (3.44), 344.0 (4.33), 289.0 (4.14), 280.5 (4.16), 268.5 (4.15), 249.0 (4.10) (see Figure 2,A

Table 1 .
Comparison of key structural parameters of AcCbl and AcRhbl calculated from X-ray crystal structure data.

Table 2 .
Comparison of calculated structural parameters of AcCbl and AcRhbl.Optimizations were performed with BP86/ def2-SVP/D3 in water.