Zinc Substitution of Cobalt in Vitamin B12 : Zincobyric acid and Zincobalamin as Luminescent Structural B12 -Mimics.

Replacing the central cobalt ion of vitamin B12 by other metals has been a long-held aspiration within the B12 -field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn-analogues of the natural cobalt-corrins cobyric acid and vitamin B12 , respectively. The solution structures of Znby and Znbl were studied by NMR-spectroscopy. Single crystals of Znby were produced, providing the first X-ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoII -corrins, making such Zn-corrins potentially useful for investigations of B12 -dependent processes. The singlet excited state of Znby had a short life-time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (ET =190 kJ mol-1 ) and was found to be an excellent photo-sensitizer for 1 O2 (ΦΔ =0.70).

The biological use of cobalt as the specific transition metal center in natural B12‐cofactors and the interaction between cobalt and the corrin ligand raise intriguing questions concerning the origins of its natural selection.1 Engineered B12‐biosynthesis2 has opened up a preparative route to hydrogenobyric acid (Hby),3 the metal‐free corrin ligand of vitamin B12, providing an excellent opportunity for the synthesis of transition‐metal analogues of the natural cobalt‐corrinoids.4 ZnII‐analogues of natural corrinoids have hardly been explored4b but are attractive, as Zn‐ and low‐spin CoII‐centers exhibit similar structural properties in small complexes and in metalloproteins.5

Fischli and Eschenmoser reported the synthesis and characterization of the first Zn‐corrin (ZnCor), when exploring the synthesis and chemistry of corrins in model studies towards the total synthesis of vitamin B12.4a, 6 Indeed, in the Eschenmoser7 and Woodward labs8 a 5,15‐nor‐zincobyrinate was an intermediate of the B12‐synthesis. UV/Vis‐spectroscopically characterized samples of zincobalamin (Znbl) and zincobyric acid (Znby), the Zn‐analogues of vitamin B12 (CNCbl) and cobyric acid (Cby) (Scheme 1), were first reported by Koppenhagen and Pfiffner.9

Scheme 1

Formulae of metal‐free, cobalt‐ and zinc‐corrinoids. Left: General formula of the cobalamins vitamin B12 (R=CN, CNCbl), coenzyme B12 (R=5′‐deoxyadenosyl, AdoCbl), cob(II)alamin (R=e−, Cbl) Center: Formulae of hydrogenobyric acid (Hby), CoII‐cobyric acid (Cby) and zincobyrate (Znby), where the axial solvent ligands for both the ZnII and CoII have been omitted. Right: formula of zincobalamin (Znbl) in its “base‐on” form.

Herein, we delineate an effective synthesis of Znby and of Znbl, starting from crystalline Hby,3 describe the pertinent spectroscopic and structural properties of these luminescent B12‐derivatives and report a kinetic study of the binding of ZnII‐ions to Hby. Znby was prepared at room temperature in 83 % yield from Hby 3 and ZnIIacetate (see Scheme 2 and the Supporting Information). ZnII‐ions bound to Hby readily under these conditions (Supporting Information, Figure S4), and over 20 times faster than CoII‐ions. Znby was resistant to removal of the ZnII‐ion in acidic aqueous solution, and Hby could not be efficiently (re)generated from Znby.

Scheme 2

Preparation of Znby and Znbl from Hby. i) 2 mg Hby in 2.3 mL aq. 0.5 mm Zn(OAc)2 at pH 6, 80 min, room temperature; ii) 5 mg Znby, 3.3 meq B12‐nucleotide moiety, 20 meq HOBt and 23 meq EDC*HCl in 1.9 mL H2O, 4 h, 0 °C (see the Supporting Information for details).

The UV/Vis spectrum of Znby in aqueous buffer, pH 5, displayed absorption maxima at 335 nm, 493 nm, and 518 nm9a (see Figure 1) and showed similar basic features as those recorded for ZnCor 6a and for a 5,15‐nor‐zincobyrinate,7, 8 but with maxima at roughly 20 nm longer wavelengths. The aqueous solution of Znby fluoresced with a maximum emission at 552 nm.

Figure 1

Absorption and fluorescence spectra of Znby and Znbl at 298 K. Left: UV/Vis absorption (black trace), CD (red trace), and fluorescence emission (blue trace) of Znby in H2O. Right: UV/Vis absorption (black trace), CD (red trace), and fluorescence emission (blue trace) of Znbl in 10 mm Na‐phosphate buffer, pH 5 (see the Supporting Information for details).

The solution structure of Znby (molecular formula C45H64N10O8Zn, see Supporting Information, Figure S3) was characterized by NMR spectroscopy, providing assignment of 52 H‐atoms and of all C‐atoms (Supporting Information, Table S1). A 500 MHz 1H‐NMR spectrum of Znby in D2O (Figure 2 a) featured eight methyl singlets, the singlet of HC10 at 5.51 ppm, and signals for HC19, HC3, HC8, and HC13 at intermediate field.

Figure 2

500 MHz 1H‐NMR spectra of Znby and Znbl (in D2O, 298 K). Top: Znby (c=1.1 mm). Bottom: Znbl (c=7.2 mm); residual water signal after pre‐saturation marked by an X.

Covalent attachment of the B12‐nucleotide moiety1a, 10 to Znby was achieved using a recently developed carbodiimide method.2d, 11 In brief, from 5.0 mg (4.8 μmol) of Znby 4.8 mg of Znbl (3.6 μmol, 75 %) were obtained, after chromatography and crystallization from aqueous acetonitrile (Scheme 2). An aqueous solution of Znbl exhibited a UV/Vis spectrum as previously reported9b (Figure 1). The absorption maxima of the α,β‐bands in the UV/Vis spectrum of Znbl occurred at 528 and 502 nm, suggesting intramolecular coordination of the nucleotide base.4b A fluorescence spectrum of Znbl showed an emission with a maximum at 560 nm, that is, about 8 nm longer wavelength than in the spectrum of Znby (Figure 1). The structure of Znbl (molecular formula C62H88N13O14PZn, see Supporting Information, Figure S5) was established by NMR spectroscopy (Figure 2 and Supporting Information, Table S2), providing assignment of 73 H‐atoms and all C‐atoms. The high‐field shifts of the signals of H3C1A (by about 0.5 ppm to δ=0.65 ppm) and of HN2 and HCN7 of the DMB‐moiety, by about 0.8 ppm to δ=7.55 ppm and δ=6.72 ppm, respectively, indicated a “base‐on” form, as in CoIIIcobalamins12 and in CoIIcobalamin (Cbl). The intramolecular Zn‐coordination of the DMB‐base was analyzed further using 1H,1H‐ROESY spectroscopy (see Supporting Information, Figure S3), characterizing Znbl as a roughly iso‐structural analogue of Cbl.13

Znby furnished orange‐red single crystals from aqueous acetonitrile (P212121), suitable for X‐ray crystal structure analysis (Figure 3 and Supporting Information, Table S3). The ZnII‐center is coordinated in an approximate pyramidal fashion, where the axial ligand is attached to the “top” (or β) face of the corrin‐bound Zn‐ion, lifting it by 0.624 Å from the best plane through the inner corrin N‐atoms (Figure 3). However, in the crystal the individual Znby‐molecules were part of a coordinative Znby polymer, generated by repetitive intermolecular axial ZnII‐coordination by the carboxylate function of a neighboring Znby molecule (see the Supporting Information).

Figure 3

Top: X‐ray crystal structure of Znby in two projections (color coding: red=corrin core carbons and oxygens; green=other carbons; blue=nitrogens; gray=Zn). Bottom: Simplified formulae with lengths of corrin π‐bonds and Zn−N bond lengths, coordination geometry around Zn‐center and “doming” of the corrin ligand (represented by the distances of the Zn‐ion from the best planes through the coordinating N‐atoms (red label), the adjacent C‐atoms (blue label), and the further C‐atoms (pink label)).

A comparison of the crystal structures of Znby and Hby 3 (Figure 4 and Supporting Information, Table S4) indicates a minor increase only in the radial size of the coordination hole on Zn‐binding. The average lengths of the N1–N3 and N2–N4 diagonals in Hby (d=3.82 Å) and in Znby (d=3.84 Å) are similar. Coordination of ZnII leads to a reduction of the corrin “helicity” h from h=12.9° in Hby 3 to h=8.0° in Znby (Supporting Information, Table S4). The major effects of the formal replacement of the penta‐coordinate CoII‐center in a vitamin B12 derivative by a ZnII‐ion are seen in a structural comparison of Znby and CoII‐heptamethyl‐cobyrinate perchlorate (Cbin)14 (Figure 4 and Supporting Information, Table S4). The Zn−N bonds in Znby (average length=2.03 Å) are longer than those found in Cbin (average Co−N bond length=1.90 Å). Likewise, the axial displacement of the metal‐ion from the mean plane through the four corrin N‐atoms in the Zn‐corrinate Znby (0.624 Å) is palpably greater than that of the CoII‐center of Cbin (0.048 Å). In Znby and Cbin, an axial ligand is bound at the β‐face with a long metal‐oxygen bond, and the four corrin N‐atoms are displaced slightly from a planar to a squashed tetrahedral arrangement (Supporting Information, Table S4). However, whereas the core of the corrin ligand is made nearly C2‐symmetrical by the coordination of a CoII‐center, in Znby the N2–N4 diagonal remains remarkably longer than its N1–N3 counterpart, with Δd=0.186 Å. Hence, about 60 % of Δd=0.297 Å in Hby are retained in the corrin ligand of Znby. This feature of Znby reflects a preferred mode of the conformational adaptation of the coordination hole of the flexible, unsymmetrical corrin ligand to the 5‐coordinate closed‐shell Zn‐ion. The “helicity” h(Znby)=8.0° is in line with a small directional effect of ZnII, compared to CoII‐ or CoIII‐binding, where h=6.1° in Cbin and h=4.1° in CNCbl.3 In Znby, the corrin ligand adapts to the skewed pyramidal arrangement around the ZnII‐center by an unprecedented conformational “doming” of the corrin ligand (Figure 4). Consequently, the corrin‐based inter‐planar angle ϕ 3 of the coordination polyhedral at the Zn‐center ϕ(Znby)=50.2° far exceeds ϕ(Cbin)=7.6° and ϕ(CNCbl)=4.6°.3

Figure 4

Left: Comparison of crystal structures of Znby, Hby, and Cbin. Right: Superposition of the cylindrical projections (top) of Znby (red) and Hby (black) and (bottom) of Znby (red) and Cbin (blue).

The fluorescence of Znby in EtOH at 296 K showed an emission maximum at 548 nm and an energy of the lowest singlet excited state of Znby of 225 kJ mol−1, close to the value observed with the metal‐free Hby (E S=223 kJ mol−1).3 Hence, the closed shell Zn‐ions do not appear to significantly perturb the π,π*‐transitions of the corrin ligand. However, the fluorescence of Znby (fluorescence lifetime τ f<0.4 nsec) decayed about an order of magnitude more rapidly at 23 °C than that of Hby (τ f=3.3 nsec), exhibiting a correspondingly lower quantum yield Φf=0.025 (for Hby Φf=0.18). The short fluorescence lifetime of photo‐excited Znby at 296 K is due to the efficient singlet‐triplet intersystem crossing with an estimated rate of more than 2×109 sec−1, boosted by the coordination of the Zn‐ion.15 At 77 K the solution of Znby in EtOH displayed an absorption maximum at 523 nm, and emitted both fluorescence (λ max=538 nm) and phosphorescence (first maximum at 628 nm, Figure 5, see the Supporting Information for details). Hence, at 77 K the lowest triplet state of Znby occurred at E T=190 kJ mol−1, furnishing the first such benchmark value for a natural corrin ligand. The phosphorescence of photo‐excited Znby decayed with a lifetime of 13±1 msec at 77 K. Znby sensitized the formation of 1O2 with a quantum yield ΦΔ=0.70. The Zn‐corrin ZnCor 6a emitted fluorescence with a maximum at 573 nm (Φf=0.09) at room temperature in EtOH,16 and was an efficient triplet sensitizer in the legendary photo‐induced A/D‐secocorrin to corrin cycloisomerization.4a, 7, 16

Figure 5

Phosphorescence excitation (left) and emission spectra (right) of Znby in EtOH at 77 K. The excitation spectrum (left) was recorded by monitoring phosphorescence at 628 nm. The steady‐state emission spectrum (right, blue line) was recorded with excitation at 515 nm, featuring both, the fluorescence and phosphorescence of Znby. The time‐resolved phosphorescence spectrum of Znby (right, red line) was recorded 2–12 msec after the pulsed excitation at 528 nm.

To shed further light on the structure of Znby, the gas‐phase structure of the hypothetical 4‐coordinate analogue Znby(4) was calculated, using DFT, from the crystal structures of Hby, as well as of the heptamethyl ester Cbin, the latter providing computational Znby models in which the polar side chain functions are replaced by methyl ester groups (for details, see the Supporting Information). Ligation of an acetate ligand at the “upper” (β) or at the “lower” (α) side of the latter Znby(4) structure, furnished models of Znby and of its coordination isomer Znby(α). The calculated structure of Znby closely reflected the observed crystallographic structural peculiarities of Znby, such as the longer N2–N4 diagonal (Δd calc=0.22 Å), the long Zn−N‐bonds (Zn−Nav=2.06 Å), the out‐of‐plane position of the 5‐coordinate Zn‐ion (0.65 Å), and the doming of the corrin ligand. In Znby(α), the calculations generated a model with comparably long Zn−N‐bonds (Zn−Nav=2.06 Å), an N2–N4 diagonal shorter than N1–N3 (Δd calc=−0.10 Å), a profound out‐of‐plane position of the 5‐coordinate Zn‐ion (−0.62 Å) and an “inverted” doming of the corrin ligand. A structure of Znbl was calculated (Figure 6) starting from a previously optimized gas‐phase structure of Cbl. It showed a pronounced out‐of‐plane movement of the 5‐coordinate Zn‐ion (−0.46 Å), exceeding that of CoII in Cbl (−0.13 Å), but compensated in part by the slightly shorter Zn−NDMB‐bond (2.07 Å) in Znbl than the Co−NDMB bond (2.11 Å) in Cbl. The structure of Znbl showed a downward movement of the DMB‐base, compared to Cbl, but was similar in its overall architecture. Hence, Znbl can be considered as a good structural mimic of the non‐luminescent Cbl.

Figure 6

The “base‐on” structure of Znbl, calculated by DFT (left), and from NMR‐derived correlations between corrin and DMB‐ moieties (right).

As an iso‐structural analogue of some Cbls that is inactive in the organometallic processes typical of B12‐dependent enzymes, Znbl may represent an “antivitamin B12”17 and be a useful fluorescent molecular probe in B12‐biology and biomedicine.18 The structure analysis of Znby has indicated that the closed shell d10‐ion of ZnII lacks the precise fit of the similarly sized low‐spin CoII‐centers (d7‐ions),19 where an empty dx 2−y 2‐orbital provides an excellent electronic complement for the four corrin N‐atoms.20 Hence, the basic fit of low spin CoII‐ and diamagnetic CoIII‐ions to the ring size of the corrin ligand3 is not extended to the 5‐coordinate ZnII‐ion. A similar (but less pronounced) difference is seen in ZnII‐ and CoII‐porphyrins, where porphyrin “doming” and axial displacement of 5‐coordinate ZnII‐centers towards the axial ligand exceed the effect of the 5‐coordinate CoII‐ions.21

The lack of out‐of‐plane displacement of the 5‐coordinate CoII‐centers in CoII‐corrins appears to be a consequence of the partially occupied valence shell of this electronically adaptable d7‐ion. Indeed, the 15‐membered equatorial perimeter of the “ring contracted” corrin ring is able to accommodate the size of both low‐spin CoII‐ and diamagnetic CoIII‐ions, which have the capacity to fit their electronic configuration to favorable interactions with the ligand.4a, 5a, 5b, 22 In contrast, when binding a 5‐coordinate closed shell d10 Zn2+‐ion, the corrin ligand undergoes doming and further conformational relaxations. In spite of the structural differences between Znby and Cbin, as well as those deduced for Znbl and Cbl, the redox‐inactive Zn‐complexes of natural corrins may be useful as luminescent (inactive) mimics of corresponding B12‐derivatives.

The work reported here describes a rational avenue to the construction and characterization of Znbl, promising to be useful in biological and biomedical experiments. Significantly, the engineering of bacterial strains for the production Hby 3 has unlocked the gateway to the direct generation of a range of other Metbls and Metbys, the transition‐metal analogues of the Cbls and Cbys, respectively. The helical, un‐symmetric natural corrin‐ligand is a unique binding partner for transition‐metal ions, providing an exciting opportunity to construct a diverse range of metal analogues of vitamin B12, investigate their structural behavior, examine their reactivity, and to test biological effects.

Experimental Section

Crystallographic Data. X‐ray crystal data of Znby have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under the reference number CCDC https://www.ccdc.cam.ac.uk/services/structures?id=doi:10.1002/anie.201908428.

Conflict of interest

The authors declare no conflict of interest.

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Supplementary

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