Antivitamins B12—Some Inaugural Milestones

Abstract The recently delineated structure‐ and reactivity‐based concept of antivitamins B12 has begun to bear fruit by the generation, and study, of a range of such B12‐dummies, either vitamin B12‐derived, or transition metal analogues that also represent potential antivitamins B12 or specific B12‐antimetabolites. As reviewed here, this has opened up new research avenues in organometallic B12‐chemistry and bioinorganic coordination chemistry. Exploratory studies with antivitamins B12 have, furthermore, revealed some of their potential, as pharmacologically interesting compounds, for inducing B12‐deficiency in a range of organisms, from hospital resistant bacteria to laboratory mice. The derived capacity of antivitamins B12 to induce functional B12‐deficiency in mammalian cells and organs also suggest their valuable potential as growth inhibitors of cancerous human and animal cells.


Introduction
Vitamin B 12 ,t he Co III -corrin cyanocobalamin (CNCbl), is am ost fascinating and intriguing natural product, [1] that was discovered as the originali solation form of the life-saving 'extrinsic' anti-pernicious anemia factor. [2] An exceptional 5,6-dimethylbenzimidazole pseudonucleotide appendage to the corrin core coordinates to the cobalt-centre of CNCbl, establishing the unique and characteristict hree-dimensional architectureo ft he cobalamins (Cbls). Cbls belongt ot he larger family of the cobamides (Cbas), also including the related natural 'complete' corrinoids [3] with other pseudonucleotide heterocycles [3a, 4] or linker units. [5] These complex cobalt-corrins are all generated in Nature by intricateB 12 -biosynthetic paths, [6] an exclusive capacity of some bacterial procaryotes and archaea. [6b] Indeed, according to Eschenmoser's proposal, the natural B 12 -derivatives may originate from structurallys implerc obalt-corrinoid precursors, presumed to have developed in early forms of life. [7] In spite of many years of intense medicinal, [8] molecular biological and biochemical [9] research, new physiological roles of B 12 in humans keep emerging, [8e, 10] while some furtherC bl-related medical findings remainp uzzling, [11] so that B 12 has been classified as a' moonlighting' vitamin. [12] The association of the B 12 's own cobalt with a' Kobold', the German word for goblin, appearst of it the occasionally puzzlings ituation. In fact, vitamin B 12 (CNCbl) itself is not ad irectly physiologically active vitamin in humans and other mammals. [8e, 13] In order to set free its functional capacity,C NCbln eeds to be converted by the mammalian metabolism, [10c, 13] into the organometallic B 12 -cofactors methylcobalamin (MeCbl) and coenzyme B 12 (adenosyl-cobalamin, AdoCbl). [3c] CNCblh as, thus, the role of a' provitamin'. [14] In fact, various Cbls, more directly functional physiologically than CNCbl, among them AdoCbl, are preferred B 12 -vitamers forthe treatment of some patients (Figure 1). [15] The possible physiological effects of artificial intact Cbls designedt oclosely mimicthe molecular shape of vitaminB 12 and to resist metabolic conversion into the B 12 -cofactors, have begun to attract our interest. [16] The highly efficient and complex B 12 -uptakea nd transport system in humans [17] and higher animals [18] shouldb ind such inactive vitamin B 12 analogues rather indiscriminately (as would, typically,a lso be the case for B 12 -using bacteria [19] ), with the consequence of the cellular import of (inactive) B 12 -dummies competing with the natural cobalamins and effectively impairing B 12 -metabolism. In consequence, B 12 -analogues designed accordingt ot hese criteria, would act as antivitaminsB 12 that induce functional Cbl-deficiency in humans and other mammals in vivo;aconceptp resented in this Journal about5years ago. [16] Antivitamins B 12 relate to the broader class of the B 12 -antimetabolites and were discussed in this context. [14,20] Typical B 12 -baseda ntimetabolites, which are not covered in this Minireview,a re Cbls (or other Cbas), modified at their periphery,t hat counteract, or fail to fulfil adequately,the physiological roles of natural B 12 -derivatives in various B 12 -dependento rganisms, including many microorganisms. B 12 -deficiency deprives someb acteria, animal Figure 1. General structural formula of the cobalamins (centre), symbolicformulaeo fsomeimportant B ,methylcobalamin (MeCbl)and coenzyme B 12 (AdoCbl)), and of (potential)Cbl-based antivitamins B 12 (right:t he aryl-Cbl EtPhCbla nd the alkynyl-Cbls PhEtyCbland F2PhEtyCbl). and human cells of vital metabolic processes, which is ad esirable consequence of the administration of metabolism based antibioticsa nd anti-cancer agents. [14a, 16, 20a] Hence, broad biological [3] andb iomedical research interests [8,12,14,16,20,21] are exploring meanso fi nducing (functional)B 12 -deficiencya nd are devotedt ostudies of its pathological effects. [8] From vitamin B 12 to antivitamins B 12 -the cobalamin strategy In line with the original concept, [16] the complete Cbl-scaffold of vitaminB 12 was used as starting point for a( moste fficient) preparation of antivitaminsB 12 .T he aryl-Cbl 4-ethylphenyl-cobalamin (EtPhCbl), an ovel type of organometallic B 12 -derivative, was generated as af irst such Cbl-based antivitamin B 12 ( Figure 1). [22] The critical design criteria for EtPhCbl were (i)its predicted (andv erified) structural similarity with for example, CNCbl and (ii)its expected resistance against the metabolic removal of its aromatic capping group by the cellular 'B 12 -tailoring' enzymeCblC, [10c] thus inhibiting al ater conversion into the organometallic B 12 -cofactors. [16,22] The aryl-Cbl EtPhCbl bound well to the human B 12 -transporter proteins, intrinsic factor, transcobalamin and holocobalamin, and was resistant against its tailoringb yt he enzymeC blC, as postulated. [22] Most critically,E tPhCbl also led to functional Cbl-deficiency in experiments with laboratory mice. [23] However,w hile fulfillingt he criteria of an antivitamin B 12 ,E tPhCbl is photosensitive and visible light degrades it into the B 12 -vitamer hydroxocobalamin (HOCbl), [22] althoughw ith al ow quantum yield. [24] Hence, since EtPhCbl has the (often undesirable) property of a' photo-conditional antivitamin B 12 ', [24] our interest has turned to light stable Cblbased B 12 -dummies. Suitable variants of the barely explored alkynylcobalamins [25] with as trong organometallicC o ÀC sp bond appeared attractive as presumed light stable potential antivitamins B 12 . [26] The previously unknown phenylethynyl-cobalamin (PhEtyCbl) was prepared, which turned out to be slightly hydrolysis-sensitive,b ut was light stable and thermally robust and exhibited similarb inding-affinity as CNCbl for the human proteins of B 12 -transport. [26a] Furthermore, the fluorinated 2,4difluorophenyl-derivative F2PhEtyCbl was not only lightstable, [27] but also rather inert against acid-induced hydrolytic cleavage of its CoÀCb ond, as expected. [26b] F2PhEtyCbl bound and inhibitedt he holoenzyme CblC loaded with the co-substrate glutathione, allowing for af irst crystal-structure analysis of fully assembled human CblC. [26b] Investigations, not only from our laboratory,b ut also from the Gryko group, [28] have meanwhilee xpanded the methodology for the preparation of organometallic alkynyl-cobalt-corrinoids. Indeed, the robust alkynyl-Cblsh ave becomea ttractive potentialc ellular import vehicles (,Trojan Horses') with ar ange of biological and biomedical applications. [28b, 29] Engineered B 12 -biosynthesis opens direct non-cobalt synthesis-paths to antivitaminsB 12 The possible conversion of aryl-and alkynyl-Cbls into the B 12 vitamersh ydroxocobalamin (HOCbl) or aquocobalamin (H 2 OCbl), by light or acid, respectively,w as seen as ad rawback as to their use as antivitamins B 12 , [22, 26a] prompting us to look out for strategic alternatives. Indeed, our simple structurebased design criteria for the antivitaminsB 12 ,t hat is, structural similarity with CNCbl and resistance against metabolic tailoring by the enzymeC blC, [16] would not only be an inbuiltf eature of some inert Cbls, but as elect and suitably designed group of metbalamins (Metbls), [14b, 30] transition metal analogues of the Cbls, might also serve this purpose. In this respect, rhodium, the group IX homologue of cobalt, appeared to offer am ost promising access to effective potential antivitamins B 12 ,b yf urnishing rhodibalamins (Rhbls), the Rh-based Cbl-analogues, presumed to be largely iso-structural to corresponding Cbls. [16] ( Figure 2).
In the 1970s Koppenhagen and co-workers reported the preparation of partially characterized Rhbls. [30] In their exploratory tests with microorganisms andh uman cell cultures, adenosylrhodibalamin (AdoRhbl), [31] the Rh-homologue of AdoCbl, was indicated to behavea saB 12 antimetabolite. [32] We have recently developed an intricatec hemical-biological total synthesis of AdoRhbl in at eam with the Warren group in Canterbury (UK). AdoRhblw as first synthesized using the biotechnologically prepared naturalm etal-free B 12 -ligandh ydrogenobyrinic acid  a,c-diamide (Hbad) [6b, 33] as starting material, followed by an adequate cocktail of further chemical and enzymatic transformations of Hbad. [34] AdoRhblw as fully characterized in detail as a close structural, non-functional AdoCbl mimic that efficiently inhibiteda nA doCbl-dependente nzyme diol dehydratase, as well as the growth of the bacterial pathogen Salmonella enterica. [34] In an additional welcomec ontrast to the antivitamin B 12 EtPhCbl and to the coenzyme AdoCbl, the relatedA doRhbl provedstable when irradiated with sunlight. [31,34] As Rhbls, the Rh-analogues of the Cbls, appeared to constitute ag roup of promisinga ntivitamins B 12 ,asystematic and more directs ynthesis methodology of Rhbls was developed. Its basis was an ewly bioengineered preparative route to the now thoroughly characterized metal-free B 12 -ligand hydrogenobyric acid (Hby). [35] The metal-free Hby also constituted an excellent basis for the partial synthesis of hydrogenobalamin (Hbl), the complete metal-free ligand of the Cbls (Figure 3). [36] The metal-free Hbl, in turn, is arational general startingm aterial for the synthesis of specific Metbls,along-standing dream and topicals ubjecti nt he B 12 -field, [14b, 30, 37] and in bioinorganic chemistry. [38] The biosynthetically availabile Hbl has meanwhile served in our hands for the one-step synthesis of chlororhodibalamin (ClRhbl), [39] and from there, of methylrhodibalamin (MeRhbl), [31,39] that is, of the Rh-analogues of chlorocobalamin (ClCbl) [40] and of MeCbl, [41] respectively (see Figure 2). As revealed by the crystal structures of the organometallic AdoRhbl [34] and of the 'inorganic' ClRhbl [39] Rh III -corrins and Co III -corrins are closely isostructural and the slightly larger Rh IIIion appears to fit strikingly better into the corrin ligand of the Cbls than the 'natural' Co III -ions. [34,39] The metal-free B 12 -ligands Hby and Hbl are starting materials, not only for the syntheses of Rhbls, but, obviously,a lso of other Metbls. So far,w eh ave reported on the synthesis and on the detaileds tructuralc haracterization of zincobalamin (Znbl), the Zn II -analogue of vitamin B 12 , [42] and of the novel Ni II -analogue, nibalamin (Nibl) [36] (see Figure3). According to detailed structural and computational studies, the redox-inactive pentacoordinate ('base-on') Znbl constitutes al uminescents tructural mimic [42] of the penta-coordinate 'base-on' Co II -cobalamin (Cbl II ). [43] The tetra-coordinate diamagnetic 'base-off' Ni II -corrin Nibl represents al argely redox-inactives tructural mimic of the highly activated tetra-coordinate 'base-off' Co II -a nd Co I -Cbls. [36,45] The reduced Cbls represent the often cryptic highenergy intermediates in many Cbl-dependent enzymatic reactions, [3c, 44, 45] as well as in some essential B 12 -biosynthetic organometallic transformation,for example, as catalysed by adenosyl transferases. [46] To gether with the newly availableh exa-coordinate Rhbls, penta-coordinate ('base-on') Znbl and tetra-coordinate ('baseoff') Nibl constitute ac omplete suite of structuralt ransition metal mimics of the Cbls in their biologically accessible redox states, that is, hexa-coordinate 'base-on' Co III -Cbls, penta-coordinate 'base-on' Co II -a nd tetra-coordinate 'base-off' Co II -o r Co I -Cbls, providingu sw ith as tructurally 'complete' small set of biochemically inactive B 12 -antimetabolites, inhibitors of B 12enzymes and (some of them) potentiala ntivitamins B 12 [36] ( Figure 4). The 'base-on' Metbls Rhbls and Znbl are likely to functiona sg enuine antivitamins B 12 ,t he 'base-off' Ni II -analogue Nibl as aB 12 -antimetabolite that inhibits some B 12 -dependente nzymes butm ay not be bound well by the mammalian B 12 -transporter proteins.H ence, in order to clarify the capacity of Metbls to serve as antivitamins B 12 according to our concept, [16] their ability to mimic the Cbls with respect to highaffinity binding to the very structure-selective B 12 -uptake and transport system of humans (and other mammals) needs to be analysed.

Application of antivitamins B 12 induces functional B 12 -deficiency
As delineated above,a ntivitaminsB 12 are structural Cbl-mimics designed to counteract the effect of CNCbl (and of its B 12 vitamer forms) in humans and animalsb yc ausing (functional) B 12 -deficiency upon their cellular uptake, [16] ad eadly metabolic defect. Such an uptake of antivitamins B 12 leads, first of all, to the inactivity of the mammalian B 12 -dependent enzymesm ethionine synthase (MetH) [47] and methylmalonyl-CoA-mutase (MCM) [44,48] due to functional B 12 -deficiency,d etectable in the accumulation of homocysteine and methylmalonic acid,t wo  biomarkers of B 12 -deficiency. [10d, 49] Functional B 12 -deficiency,i nduced by antivitaminsB 12 in humans and in other mammals, results, on the one hand, from the inability of these B 12 -dummies to assume the specific 'canonical' roles of the B 12 -cofactors of MetH and MCM,which are based on the organometallic reactivityo fM eCbla nd of AdoCbl, respectively. [3c, 44, 45] However, antivitamins B 12 will, on the other hand, extensively mimic the (merely) structure-based ('non-canonical') regulatory functions of the Cbls, giving fake signals for the availability of genuine B 12 -cofactors by imitating effectively their binding capacity to natural bio-macromolecular targets, such as B 12 -responsive regulatory proteins and RNA. [16,34] As described below,amultitude of gene-regulatory roles of the natural B 12 -cofactors have been discoveredi nm icroorganisms. [50] However,s of ar,i nh umans only two such bio-macromolecular binding interactions have been detected. [10a, 51] Further 'non-canonical' roles of Cbls in humans and in other mammals are suggested, fore xample, by the observation of ac ytokine and growth-factor imbalance in the central nervous system in laboratory rats due to Cbl-deficiency, [8d, 12] as well as of irregular melanocyte homeostasis induced by B 12 -deficiency in human cell cultures. [52] Antivitamins B 12 may be particularly helpful in imitatinga nd identifying such puzzling roles, as well as in discovering new 'non-canonical' ones.

Antivitamins B 12 as molecular probes
Ar ange of remarkable recent discoveries in the B 12 -field has put Vitamin B 12 in the spotlight again. [53] Indeed, B 12 -derivatives play essential roles as organometallic biocatalysts, [45] not only in humans,a nimals, bacteria and archaea but, surprisingly,i na range of algae, as well. [54] Some forms of bacterial photo-regulation involve natural cobamides, [55] as do criticals teps of the biosynthesis of photosynthetic tetrapyrroles [6b] and of other complexm etabolites, [56] including the anaerobic metabolism of hydrocarbons. [56d] Mechanistic insights into the exceptional biochemistry of the involved B 12 -dependente nzyme reactions or means of the B 12 -based control of essential cellular processes are areas of continuous interest. Studies with antivitaminsB 12 and other structurally characterized Metbls mayp otentially contribute to this subject, [36] relying on two key structurebased factors: (i)Byi mitating the structureso ft he B 12 -cofactors or of reactivei ntermediate B 12 -species in the courseo fe nzyme reactions, suitably structured (inactive) B 12 -mimicsh ave an excellent capacity to inhibitt he corresponding enzymatic steps. Hence, for example, the Ni II -analogueo ft he cryptic intermediate Co I -form cob(I)alamin inhibits an AdoCbl-generating Adotransferasei na ni nv itro study [36] (see above for corresponding pertinent findings with the alkynyl-Cbl F2PhEtyCbl [26b] and with AdoRhbl [34] ). (ii)Bym imicking the structures of the B 12 -type ligands in B 12 -dependent regulatory functions in variouso rganisms, antivitaminsB 12 are, on the other hand, presumed to simulate the availability of the corresponding physiologically active B 12 -derivatives, for example, via B 12 -riboswitches [57] and in B 12 -responsive regulatory proteins. [51,58] The observed strong growth-inhibition of Salmonella enterica by AdoRhbl was, hence,a scribed to its specific binding to the BtuB B 12 -ribo-switch as as tructural AdoCbl-mimic, inhibiting the expression of aB 12 -uptakep rotein in this microorganism. [34] Similarf urther in vitro and in vivo experiments with AdoRhbla nd some Cblbased antivitamins B 12 have recently been carried out, [59] signifying the ability of structurally competent antivitaminsB 12 to simulatet he presenceo fp hysiologically functional Cbls. Indeed, as long as the cellular and organismal import of antivitamins B 12 and of other Metbls by the natural pathways would be feasible, as expected, their capacity for generating functional B 12 -deficiency should also be maintained in vivo, even in living animals. [23] Antivitamins B 12 as antibiotics and as cellular growth-inhibitors for human and animals Antivitamins B 12 [16,60] ando ther B 12 -antimetabolites [14, 20, 61] may functiona sB 12 -dummies and act as inhibitors of B 12 -dependent enzymes,i mpairing the growth and reproductiono fb acteria and of other microorganisms. This early explored effect of modified vitamin B 12 -derivatives as B 12 -antimetabolites (see for example [3a, 20] )c ould recently be extendedt ot he criticalc ase of hospital-resistant Gram-negativeb acteria, where the broad antibiotic activity of sulfonamides was boosted decisively by the addition of the antivitamin B 12 EtPhCbl to the bactericidals ulfonamide cocktail. [60] Addition of the antivitamin B 12 was proposed to result in an effective methylfolate trap, [60] by blocking the formation of free tetrahydrofolate by methionine synthase. In addition to their proposed role in impairing the biosynthetic formation and in reducing the cellular availability of the (active) B 12 -cofactors, [16,22,23,60] antivitaminsB 12 may also intercept the uptake of the essential B 12 -derivatives by B 12 -dependent microorganisms due to their B 12 -mimetic regulatory activity as ligandso f( for example) B 12 -riboswitches. [59] Indeed, the response of B 12 -regulatory elements to binding of aB 12 -type ligand is expected not to differentiateb etween the functional classification of the latter as 'vitamin' or as 'antivitamin'. In consequence, both the 'canonical' bio-catalytic and the 'non-canonical' B 12 -regulatory roles playedb yt he natural cobamides bestowa ntivitaminsB 12 with ap otentially very effective twopronged bactericidal activity,a sv erified recently with AdoRhbl, the rhodium analogue of AdoCbl. [34] Since the deactivation of the B 12 -dependent enzymaticp rocessesi nh umans and other mammals leads to an impaired metabolism, disrupting physiological function [8a, 21a, 62] and also causing fundamental neuropathological deficiencies, [63] regular cellular growth is inhibited as consequenceo fa(functional) B 12 -deficiency.A ntivitamins B 12 may,h ence, be useful as anticancer agents. [14b, 16] As already explored in early in vitro investigations, B 12 rhodiuma naloguesw ere observed to inhibita sd iversely active B 12 -antimetabolites, the growth of human normo-and megalo-blastic bone marrow cells. [30,32] It will be of interestt ol earn more aboutt he diagnostic and therapeutic applicationso fw ell-characterized,p ure antivitamins B 12 as agents for anti-cancerd iagnosis and treatment in humans and other mammals. Indeed,s uitably fluorescencel abelled, radiolabelled and other bio-conjugated B 12 -derivatives have proved useful,o ver the recent years, as 'Trojan Horses' for the cellular Chem. Eur.J.2020, 26,15438 -15445 www.chemeurj.org 2020 The Authors. Published by Wiley-VCH GmbH import of diagnostic loads and for targeted drug delivery, [20a, 64] helpful in inhibiting the growth and the detection of malignant cells, [64a, 65] and useful for ar ange of other biomedical applications. [66] Summary and Outlook Our original interesti nt he subjecto fa ntivitamins B 12 wask indled by the expectation that these B 12 -dummies would offer insights into functional B 12 -deficiency in animalsbya ne ffective alternative methodology [23] replacing total gastrectomy. [67] This work has led to fruitful research collaborations, discovering new organometallic Cbl-chemistry,p hotochemistry and biochemistry. [22,24,68] It has, likewise, opened up new avenues in the field of the fascinating transition metal analogues of the Cbls and of other natural corrinoids. [34,36,39,42] The helical, ringcontracted natural corrinl igand has been characterized as an exceptional 'Procrustean Bed' for bound transition metal ions, important for tightly binding and specifically activating the bound cobalt-ions in their low-spins tates. [35] As discovered with synthetic Ni II -corrins, [69] the naturalc orrin ligand also imposes the diamagnetic low-spins tate on bound Ni II -ions, [36] contrasting with the situation in related porphyrin-type Ni II -corphinoids. [7,70] Interestingly,t he 5,6-dihydroxy-corrin variant of a 'B 12 -type' Ni II -complex, recently prepared and studied in the Zelder group, also features al ow-spin4 -coordinateN i II -centre. [37b] Cbl-based antivitamins B 12 promise to represent exceptional antibiotics, [60] an important area to be developedf urtheri n view of the acute problemo fh ospital-resistant bacteria. As some bacteria use preferentially cobamides (Cbas) other than Cbls, [71] the eventual adaptation of the methodology fort he synthesis of Cbl-based antivitamins B 12 to the generation of correspondingC ba-forms is expected to enhancet heir selective bacterial import as antibiotics, while simultaneously reducing the likelihood of the undesired uptake in human cells by their B 12 -transporters. [17a, 72] In ongoing collaborative studies, antivitamins B 12 and some other metbalaminsa re used as specifically targeted B 12 -antimetabolites, under investigationw ith respect to their capacityt os erve as, for example, enzyme inhibitors, as ligandso fr egulatory proteins and of B 12 -riboswitches, as antibiotics, and as potentially usefula nti-cancera gents. Having now set up some inaugural milestones,abroad further impact of studies on antivitaminsB 12 and (further) B 12 -transition metal analogues in the bio-structural, biological and biomedicalfields can be foreseen. ful to ChristophK reutz, Thomas Müller,K laus Wurst and Maren Podewitzf or their spectroscopic, crystallographic and computationals upport. Our work in the B 12 -area has been supported generously by the AustrianS cienceF und (FWF), notably by the two recent and ongoing projects P-28892 and P-30359.

Conflict of interest
The authordeclares no conflict of interest.