Replacement of the Cobalt Center of Vitamin B12 by Nickel: Nibalamin and Nibyric Acid Prepared from Metal‐Free B12 Ligands Hydrogenobalamin and Hydrogenobyric Acid

Abstract The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transition‐metal analogue of vitamin B12. Described here is Nibl, synthesized by incorporation of a NiII ion into the metal‐free B12 ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiII corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal‐free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X‐ray analysis of Niby shows the structural adaptation of the corrin ligand to NiII ions and the coordination behavior of NiII. The diamagnetic Niby and Nibl, and corresponding isoelectronic CoI corrins, were deduced to be isostructural. Nibl is a structural mimic of four‐coordinate base‐off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase.


Introduction
Biologically active vitamin B 12 derivatives exclusively utilize cobalt as their specific transition metal center, which is bound and activated exquisitely by ah elical corrin macrocycle. [1] Them etal-free corrin ligand of vitamin B 12 ,h ydrogenobyric acid (Hby), has recently been made available as ac onsequence of engineered B 12 biosynthesis in E. coli. [2,3] Thea vailability of Hby has provided an unparalleled opportunity for the effective synthesis of metal-free and transition metal analogues of the natural cobalt-corrinoids ap reviously intractable challenge in bioinorganic and B 12 chemistry. [4] We have recently used Hby for the synthesis of the corresponding zinc-corrin zincobyric acid (Znby)and the Zn analogue of vitamin B 12 zincobalamin (Znbl), of interest as luminescent structural B 12 mimics. [5] Herein, we report on the first nickel-complexes of natural corrin ligands,i ncluding nibalamin (Nibl). We also describe the syntheses of crystalline nibyric acid (Niby), the novel Ni II complex of Hby, [3] and hydrogenobalamin (Hbl), the metalfree complete B 12 ligand (see Scheme 1a nd Scheme 2). Koppenhagen and co-workers,b ack in the 1970s, reported the isolation of Hbl from a Chromatium strain supplemented with 5,6-dimethylbenz-imidazole (DMB). They were able to characterize Hbl by UV/Vis-spectroscopy and demonstrated that it could be converted into vitamin B 12 by insertion of cobalt, [4e, 7] and later reported its mass spectrum. [8] ANi II -corrin, the NiCor (see Scheme 1), was prepared in the Eschenmoser labs as the first synthetic corrin, making use of the Ni II ion as a" template" for the assembly of the corrin macro-ring. [6] NiCor also became the object of the first X-ray crystallographic investigation of the structure of anon-cobalt corrin. [9] Four coordinate Ni II complexes prefer to adopt aplanar geometry and therefore are more structurally related to the corresponding Co I complexes. [10] Indeed, recently,there has been ar esurgence in the quest for close Ni analogues of the B 12 cofactors. [4c,f] Theplanar ligand set of Nibl potentially represents as tructural B 12 mimic that is inert to the organometallic transformations typical of B 12 -dependent enzymes,assuggested by its expected coordination chemistry and structural properties.S pecific interest in Nibl,t he Ni IIanalogue of vitamin B 12 and of other cobalamins (Cbls)( see Scheme 1), is thus aconsequence not only of its chemistry,but also of its possible use as amolecular probe in B 12 biology and biomedicine,h elpful for the investigation of cobalamindependent processes and their physiological effects. [11] Results and Discussion Nibyric acid (Niby)w as prepared by dissolving 1.40 mg (1.6 mmol) of crystalline hydrogenobyric acid (Hby) [3] in 3.5 mL of deoxygenated 0.5 m aqueous Ni II acetate,pH6,with stirring at 90 8 8Cf or 75 min. Separation on as hort reverse phase column, evaporation and crystallization from aqueous acetonitrile yielded 0.90 mg (0.97 mmol, 61 %) of Niby,which was isolated as yellow crystals (see Scheme 2, Exptl. Part and Supporting Information (SI). TheU V/Vis absorption spectrum of an aqueous solution of Niby displayed bands at 464 nm (shoulder), 448 nm and 334 nm (Figure 1), and exhibited similar gross features to those observed in an absorption spectrum of the Ni-corrin NiCor (but with as lightly red-shifted maxima). [6a,c] Thes olution structure of the diamagnetic Ni II -corrin Niby (molecular formula C 45 H 64 N 10 O 8 Ni, for HR mass spectra see SI, Figure S3) was analyzed by NMR spectroscopy,p roviding assignment of all 52 non-exchangeable Ha toms and 44 Ca toms (see SI, Figure S4 and Table S1). A5 00 MHz 1 HNMR spectrum of Niby in D 2 Od isplayed five high field singlets for the six methyl groups,asinglet of HC10 at 6.30 ppm, as well as several signals at intermediate field for HC19, HC3, HC8 and HC13 (see Figure 2). Thed ata from homonuclear and heteronuclear correlations confirmed the stereostructure of Niby (see SI, Figure S5). Scheme 2. Preparation of the Ni II -corrins Niby and Nibl from Hby. i) 0.5 m Ni(OAc) 2 pH 6, 1h,9 08 8C, Ar.ii) 3equiv B 12 nucleotide moiety, [1a, 12] HOBt, EDC*HCl, H 2 O, 0 8 8C, 4d.i ii)0.5 m Ni(OAc) 2 pH 6, 1h, 90 8 8C, Ar (see the SI for details). Scheme 1. Structural formulae of cobalt, zinc, nickel and metal-free corrinoids. Left:cobalamins with "base-on"structures:v itamin B 12 (R = CN, CNCbl), coenzyme B 12 (R = 5'-deoxyadenosyl, AdoCbl), methylcobalamin (R = CH 3 , MeCbl), cob(II)alamin (R = e À , Cbl II ). Center, top:the Ni II corrin nibyrate (Niby), hydrogenobyric acid (Hby), Co IIcobyric acid (Cby II )a nd zincobyricacid (Znby), where Co II and Zn II carry an unspecified axial ligand (e.g.,s olvent molecule).C enter, bottom:Eschenmoser's synthetic racemic Ni II -corrin NiCor.  Thecomplete metal-free ligand of the cobalamins,hydrogenobalamin (Hbl), was assembled by attaching the B 12 nucleotide moiety [1a, 12] to the propionate moiety of Hby at 0 8 8Ct hrough application of the carbodiimide method (Scheme 2). [4d, 13] In brief,a na queous solution of 9.12 mg (10.4 mmol) of Hby and of 14.71 mg (33.4 mmol) of the B 12nucleotide was treated with 9.4 moleq of HOBt and degassed. To the frozen reaction mixture 4.4 moleq of EDC*HCl were added under Ar. Upon subsequent warm-up of the reaction mixture to 0 8 8C, 16 moleq EDC*HCl were added and stirring was continued for 4d (see SI). Work-up,u sing RP18chromatographic purification, precipitation with MeCN and drying, furnished 11.3 mg (8.89 mmol, 85 %yield) of Hbl as an orange powder.Anaqueous solution of Hbl at pH 5exhibited UV/Vis [4e] and CD spectral features (SI, Figure S8 and S9) similar to those of Hby. [3] TheUV/Vis absorption maximum at 525 nm of the a-band of Hbl and the fluorescence emission maximum at 554 nm (SI, Figure S10) located the first excited singlet state of Hbl at E S near 221 kJ mol À1 ,marginally lower than for Hby. [3] Thes tructure of Hbl (molecular formula C 62 H 90 N 13 O 14 P, see HR-MS data in SI, Figure S11) in H 2 Owas characterized by NMR spectroscopy (600 MHz 1 HNMR spectrum in SI, Figure S12), providing assignment of 89 H atoms and of all 62 Ca toms (see SI, Table S2). Thet wo "inner" Ha toms gave rise to singlets at d = 12.32 and d = 12.57 ppm, which were assigned to H(N4) and to H(N2), respectively,indicating aminor up-field shift of both of them when compared to Hby. [3] Themethyl group singlet of H 3 C1 at d = 0.81 ppm occurred at 0.47 ppm to higher field, compared to Hby,s uggesting at emporary residence of the heteroaromatic DMB unit of Hbl near to its corrin moiety,aconclusion that was further supported by weak inter-residual correlations in the 1 H, 1 HROESY spectra (see SI, Figure S13). However, the signals of the DMB moiety (HN2 at d = 8.35 ppm, HN4 at d = 7.31 ppm, HCN7 at d = 7.30 ppm) were found at similar chemical shift values to those of the free B 12 nucleotide, [12] effectively incompatible with at ime-averaged positioning of the DMB part close to the corrin chromophore,a sfound for zincobalamin (Znbl) [5] and for typical "base-on" Co III Cbls. [14] TheNi II -corrin nibalamin (Nibl)was prepared by heating adeoxygenated aqueous solution of Hbl and Ni(OAc) 2 for 1h at 90 8 8C(Scheme 2), furnishing Nibl in 77 %yield as ayellow powder.A nu nbuffered aqueous solution of Nibl exhibited aUV/Vis spectrum that is incompatible with coordination by the DMB base and nearly indistinguishable (at > 300 nm) from the spectrum of Niby,and similar to the spectrum of the Ni II -corrin NiCor [6a,c] (see Figure 1). However,the absorption maxima of Nibl occurred at characteristically longer wavelengths when compared to the spectrum of the recently described vitamin B 12 -derived 5,6-dihydroxy-5,6-dihydronibalamin, which features an interrupted corrin p-system. [4f] Lower pH values affected only the short wavelength part of the Nibl UV/Vis-absorption spectrum, which was altered by DMB-protonation, consistent with ap K a = 4.35 AE 0.06 for protonated Nibl-H + (see SI, Figure S20).
Thes tructure of Nibl (molecular formula C 62 H 88 N 13 O 14 PNi, SI, Figure S17) was characterized in aqueous solution by heteronuclear NMR spectroscopy (see 500 MHz 1 HNMR spectrum in Figure 3), providing assign-ment of all 73 non-exchangeable Ha toms and of all 62 C atoms (SI , Table S3). Thep ositions of the singlets of H 3 C1A (d = 1.10 ppm), of HCN2 (d = 8.51 ppm), HN4 (d = 7.36 ppm) and HCN7 (d = 7.39 ppm) of the DMB moiety all indicate abase-off form with afour-coordinate Ni II center.Hence,the UV/Vis and NMR spectral features of Nibl characterize it as an isoelectronic and, roughly,i sostructural analogue of the diamagnetic cob(I)alamin (Cbl I ), which is considered to feature a" base-off" structure with af our-coordinated Co I center. [15,16] Then ickel corrin Niby was crystallized from aqueous acetonitrile,furnishing yellow single crystals (P 2 1 2 1 2 1 )suitable for X-ray analysis (see Figure 4). Thei ncorporation of aN i II ion into the corrin macrocycle of the metal-free Hby increased the effective symmetry of the corrin ligand as revealed by acomparison of the crystal structures of Niby and  of Hby (see SI for details). Coordination of the Ni II -ion largely equalizes the lengths of the two diagonals,w here the N2-N4 diagonal of Niby exceeds its N1-N3 counterpart by only Dd = 0.047 ,far less than in Hby (Dd = 0.297 ) [3] or in Znby (Dd = 0.197 [5] ). Thes omewhat longer N2-N4 diagonals in the metal corrins Niby and Znby appear to reflect the preferred mode of the conformational adaptation of the coordination hole of the flexible,unsymmetrical corrin ligand to bound metal ions.T he radial size of the coordination hole also shrank upon Ni II coordination as the average length of the N1-N3 and N2-N4 diagonals of Hby (d = 3.82 )w as reduced to d = 3.71 in the complex Niby.H ence,t he coordination of the Ni II ion in Niby contracts the corrin ligand and makes it more symmetrical. This latter effect is also expressed by the regularly alternating bond lengths of the corrin p-system in Niby,o bservations that are compatible with the model Ni-corrin NiCor. [9] Thefour-coordinate Ni II ion sits very close to the plane of the four inner corrin Natoms,comparable to the situation in the Ni II -corrin NiCor, [9] and in typical Co III -corrins, [16a] but contrasting somewhat with the out of plane distance of 0.048 of the five-coordinate Co II center of heptamethylcob(II)yrinate perchlorate (Cbin II ) [17] (SI, Table S5). As expected, [9,18] the metal À Nb onds in Niby (average Ni À N bond length = 1.86 )are shorter than those found in the Co II analogue Cbin II and in the Co III -corrin coenzyme B 12 (AdoCbl), where average (Co II -N) and (Co III -N) bond lengths of 1.89 [19] and of 1.90 , [20] respectively,were observed.
Thec oordination of the Ni II ion barely affects the conformational properties of the metal-free corrin ligand (Figures 5,6). Only as light reduction of the helicity, h (Figure 7), of the inner corrin Na toms from h = 12.98 8 in Hby to h = 10.18 8 is seen in Niby.I ndeed, the effect of the binding of the Ni II ion on the corrin helicity is comparable to the situation in the enzyme-bound four-coordinate cob-(II)alamin (4c-Cbl II (ACA)) of the adenosyltransferase ACA, [21] for which h = 88 8. [3] In contrast, in five-coordinate Co II -corrins the corrin helicity is significantly smaller, for example, h = 6.18 8 in the Co II -corrin Cbin II ,and in typical Co IIIcorrins planarization of the corrin ligand is still more pronounced, leading,f or example,t oh = 3.58 8 in AdoCbl. [3] Theobserved lower drive of the four-coordinate d 8 Ni II ion to planarize the corrin macrocycle is similarly reflected by its own coordination geometry,w hich deviates strongly in Niby from the coplanar arrangement of the coordinating ligand atoms in typical four-coordinate low-spin Ni II complexes. [9,10,18] In Niby ar emarkably large interplanar angle f (Figure 7) at Ni II (f = 11.18 8)results from extensive directional coordinative adaptation of the Ni II -center to the geometric requirements imposed by the helical corrin ligand (see Figure 6a nd SI). f is significantly larger in Niby than in Co III -corrins,w hich exhibit f'sa round 58 8 or less, [3] and is comparable to the situation in five-coordinate Co II -corrins Cbl II (f = 128 8)a nd Cbin II (f = 7.68 8).
Thec orrin helicity h and the inter-planar angle f (Figure 7), were introduced recently as two complementary parameters characterizing inner conformational effects of the mutual structural adaptation of the corrin macrocycle and of the coordination geometry at the bound metal ion. [3] Theso called corrin fold of the helical corrin macrocycle, [22] aclassic parameter characterizing the nonplanar corrin ring in Cbls and in other "complete" cobamides ( Figure 7), was not used in this current study.C onceived as am easure of the major conformational adaptation of the corrin ring to the cobalt coordination of the (bulky) DMB moiety in "base-on" Cbls,it runs roughly along the Co-C10 (east-west) axis. [22] However, in four-a nd five-coordinate metal-corrins lacking the DMB unit, like Cbin II , Niby and Znby,t he calculated corrin-fold is dominated by the effects of the corrin helicity and the intersection between the two relevant planes adheres to anorth-south direction (see SI Table S5 and Figure S22).
DFT calculations were carried out to test further the proposed close structural similarity between Ni II -corrins and their analogues with four-coordinate cobalt centers (see Figure 8a nd SI for further details). In order to minimize the relevance of peripheral H-bonds between the amide functions in the implicit solvent calculations,t he five-coordinate lip-

Angewandte Chemie
Research Articles 20132 www.angewandte.org ophilic heptamethyl-cob(II)yrinate perchlorate (Cbin II ) [17] was used as as tarting model. Calculations of the structures of the related unknown corrins heptamethyl-nibyrinate (Nibin), four-coordinate heptamethyl-cob(II)yrinate (4 c-Cob II in)a nd heptamethyl-cob(I)yrinate (Cob I in)w ere carried out. They indicate extensive structural similarities,b ut the equatorial metalÀNb onds are shorter (by roughly 0.01-0.03 )i nt he Co I -a nd Ni II -corrins Cob I in and Nibin in comparison to the four-coordinate Co II -corrin 4 c-Cob II in and 5-coordinate Cbin II .The N1-N3 diagonal was calculated to be shorter than its N2-N4 by roughly 0.01-0.02 ,which is also in good qualitative agreement with the crystallographic data for Niby and Cbin II . [17] All 4-coordinate metal centers (Ni II ,C o I and Co II )w ere calculated to be located virtually in the best plane through the four corrin N-atoms.The latter is arranged in ahelix with acalculated value of h slightly decreasing from Nibin (7.68 8)t oCob I in to 4c-Cob II in (6.68 8), and induced an interplanar angle f that slightly decreased in the same order (from 8.48 8 to 7.58 8). Thecalculations for five-coordinate Cbin II also reproduced, qualitatively,t he still smaller value for h (5.48 8), al arger value for f (11.38 8)a nd as ignificant displacement of the Co II center towards the axial b-ligand (calculated as 0.112 ).
Thed educed utility of Nibl as as pecific new structural mimic of four-coordinate base-off Cbls was initially tested in binding studies of Nibl to an adenosyltransferase enzyme (ATP:Co I rrinoid adenosyltransferase), which catalyzes the biosynthetic construction of AdoCbl by Co b -adenosylation of bound Cbl IÀ .S uch bacterial [23] and human adenosyltransferases, [24] for example,B tuR, [23a] CobA, [23b] ACA, [21] and CblB, [24] have been shown to facilitate the adenosylation process by first inducing the corrin substrate to adopt afourcoordinate structure,t hus raising the redox potential of the Co II /Co I couple by around 250 mV, [23,24] thereby allowing the physiologically difficult reduction. We,t hus,i nvestigated the effect of Nibl on the adenosylation process by incubating the Brucella melitensis BtuR [23a] and the structural Cbl mimic Nibl in the presence of an excess of Cbl I (see SI, Figures S27-S29). As expected, Nibl itself was not as ubstrate for the enzyme and was not adenosylated. However,t he presence of Nibl within the incubation did effectively inhibit the adenosyltransferase,r educing the activity of the enzyme by 38 %a t ac oncentration of 1 mm,a nd by 60 %a t5mm ( Figure S29). Thus,t he four-coordinate Ni II center of Nibl affords it the ability to bind within the active site of the adenosyltransferase and to prevent the productive binding of the natural substrate Cbl.

Conclusion
Herein, we have described the first Ni II analogues of natural cobalt corrins vitamin B 12 (CNCbl)a nd cobyric acid (Cby). Then ew Ni-corrins Nibl and Niby display the same basic structural features and lack of coordinative activity as synthetic model Ni II -corrins,s uch as NiCor. [1a, 6a,c] TheN i IIcorrins are known to be exceptionally stable in regard to the chemical removal of their metal center, [25] and to exhibit no affinity for axially coordinating ligands. [1a, 6c, 7] This latter feature has been rationalized by the extraordinary stabilization of the low-spin d 8 configuration by the ligand field of the ring-contracted corrin ligand. [1a, 9] As shown here,the natural corrin ligand undergoes only as mall contraction of its coordination hole by about 0.03 to 0.04 to accommodate the low-spin Ni II ion. Indeed, the 15-membered inner perimeter provided by the natural corrin macrocycle,selected for binding low-spin cobalt ions,also binds Ni II consistently in Figure 7. Geometrical parameters describing conformational effects in corrins. Left:corrin helicity (h = the dihedral angle N1-N2-N3-N4 around avirtual bond between N2 and N3 of the corrin ligand), revealing the effect of bound metal-ions on the corrin helix ( Figure 5). [3] Center:interplanar angle (f = angle between the two planes through the metal ion and corrin N'sN 2/N4 or N1/N4, respectively)c haracterizing the deviation of the coordination environmentatt he metal center due to the helical corrin ligand (Figure 6). [3] Right:c orrin fold (angle between the best two planes through the inner corrin atoms from N1 to C10 and from C10 to N4, respectively)d escribing the main conformational response of the corrin macrocycle to the binding of the DMB base in Cbls. [22] . its low-spin state.Incontrast, in the porphyrinoid B 12 -related nickel complex coenzyme F 430 [1a, 18,26] the 16-membered porphyrinoid macrocycle is ak ey player in the active,s pecific adjustment of the spin state and coordinative activity of the nickel center to its function in the enzyme catalyzed methane formation. [27] Indeed, the discovery of coenzyme F 430 provoked an entirely new look at the structural effect of the tetrapyrrolic macrocycle on the coordination chemistry of bound first-row transition metals. [18] Ac ommon feature of the valence shell of the low spin states of the transition metal ions Ni II ,C o III ,C o II and Co I is their unoccupied d x 2 Ày 2 orbital, ak ey factor responsible for their strong sigma bonding interactions with the four inner corrin Na toms,l eading to similar radial characteristics of their corrin complexes.Adiffering number of valence shell electrons in Ni II -, Co III -, Co II -a nd Co I -corrins is transduced primarily into characteristically different reactivity of the metal centers in the axial direction, strongly affecting their potential binding sites there.Consequently,Ni II -corrins are to be considered particularly well-suited structural mimics of corresponding isoelectronic Co I -corrins,t he critical intermediates in heterolytic organometallic transitions in B 12dependent enzymes. [28] Crystallographic insights and DFTbased structure calculations also indicate as tructural similarity between Ni II -corrins and the exceptional four-coordinate Co II -corrins.T his result contrasts strikingly with the mutually different structures of the typical five-coordinate Co II -corrins and their Zn II -analogues [5] with similarly sized metal ions [29] that differ by the number of electrons in the valence shell.
Thestructural analysis of the Nibyrinates predicts that the constitutively robust Nibl would likely be an excellent redoxresistant structural mimic for the elusive cob(I)alamin (Cbl I ), ahighly reactive redox-active intermediate [30] that is found in B 12 -dependent methyl group transferases,such as methionine synthase, [28a] as well as in the biosynthesis of AdoCbl from Cbl II (via Cbl I )b yC bl-adenosyltransferases. [23,24] Nibl may, likewise,a ct as ag ood structural mimic of the recently described natural four-coordinate Co II -corrins,p roposed as key intermediates in the enzymatic transformations catalyzed by the vitamin B 12 tailoring enzyme CblC, [31] in corrinoid dehalogenases, [32] or as substrates for the reduction to Co Ispecies in enzymatic cobalt alkylation. [14,33] Indeed, as verified here, Nibl is av ery effective inhibitor of the bacterial Cbladenosyltransferase BtuR.
We have developed ar ational and direct synthetic path from hydrogenobyric acid (Hby)via hydrogenobalamin (Hbl) to nibalamin (Nibl), anovel transition-metal analogue of the Cbls.Our recent studies with the Rh III analogue AdoRhbl of AdoCbl, [4d] with the Zn II analogue Znbl of Cbl II , [5] and now the Ni II analogue Nibl of Cbl I ,have furnished avaluable suite of cobalamin mimics for use in the study of B 12 -dependent enzymatic processes, [16a, 28,30, 34, 35] and in B 12 -dependent biological regulation. [36] Well-characterized and adequately accessible transition metal analogues (Metbls)ofthe Cbls provide ap romising small-compound platform that may contribute significantly to the ongoing quest for innovative B 12 -based biological and biomedical applications. [11,37] Along these lines, some Metbls may find applications as effective antivita-mins B 12 . [4d, 11] Thea vailability of selected Metbls and of related metal corrins (MetCors)will also allow more detailed experimental investigations into the chemical relevance of the coordination of transition metal ions by the uniquely skewed, strongly helical and unsymmetric natural corrin ligands. [3] Such studies will endow am ore informed understanding of the specific evolutionary selection of cobalt rather than any other transition metal [1] for the task of complex organometallic catalysis achieved by the B 12 cofactors.