Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B12 Dependent Mutases

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

Experimental Study of Hydrogen Bonding Potentially Stabilizing the 5′‐Deoxyadenosyl Radical from Coenzyme B12

Coenzyme B₁₂ can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co? C bond homolysis of coenzyme B₁₂ to cob(II)alamin and the 5′‐deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19? H to the 3′‐O of the ribose moiety of the incipient 5′‐deoxyadenosyl radical, which is stabilized by 30 kJ mol^?1 (B. Durbeej et al., Chem. Eur. J. 2009 , 15, 8578–8585). The diastereoisomers (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin were used as models for coenzyme B₁₂. A downfield shift of the NMR signal for the C19? H proton was observed for the (R)‐isomer (δ=4.45 versus 4.01 ppm for the (S)‐isomer) and can be ascribed to an intramolecular hydrogen bond between the C19? H and the oxygen of CHOH. Crystal structures of (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin showed C19? H???O distances of 3.214(7) Å (R‐isomer) and 3.281(11) Å (S‐isomer), which suggest weak hydrogen‐bond interactions (?ΔG<6 kJ mol^?1) between the CHOH of the dihydroxypropyl ligand and the C19? H. Exchange of the C19? H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3–11. No exchange was found for any of the cobalt oxidation states. 3′,5′‐Dideoxyadenosylcobalamin, but not the 2′,5′‐isomer, was found to act as a coenzyme for glutamate mutase, with a 15‐fold lower k_cat/K_M than 5′‐deoxyadenosylcobalamin. This indicates that stabilization of the 5′‐deoxyadenosyl radical by a hydrogen bond that involves the C19? H and the 3′‐OH group of the cofactor is, at most, 7 kJ mol^?1 (?ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2′‐OH and 3′‐OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19? H and the 3′‐O of the ribose moiety of the 5′‐deoxyadenosyl group is concluded not to exceed 6 kJ mol^?1 (?ΔG).     

Mechanistic Insights into the Radical S‐adenosyl‐l‐methionine Enzyme NosL From a Substrate Analogue and the Shunt Products

The radical S‐adenosyl‐l ‐methionine (SAM) enzyme NosL catalyzes the transformation of l ‐tryptophan into 3‐methyl‐2‐indolic acid (MIA), which is a key intermediate in the biosynthesis of a clinically interesting antibiotic nosiheptide. NosL catalysis was investigated by using the substrate analogue 2‐methyl‐3‐(indol‐3‐yl)propanoic acid (MIPA), which can be converted into MIA by NosL. Biochemical assays with different MIPA isotopomers in D₂O and H₂O unambiguously indicated that the 5′‐deoxyadenosyl (dAdo)‐radical‐mediated hydrogen abstraction is from the amino group of l ‐tryptophan and not a protein residue. Surprisingly, the dAdo‐radical‐mediated hydrogen abstraction occurs at two different sites of MIPA, thereby partitioning the substrate into different reaction pathways. Together with identification of an α,β‐unsaturated ketone shunt product, our study provides valuable mechanistic insight into NosL catalysis and highlights the remarkable catalytic flexibility of radical SAM enzymes.     

Sulfonium‐Based Homolytic Substitution Observed for the Radical SAM Enzyme HemN

Sulfur‐based homolytic substitution (S_H reaction) plays an important role in synthetic chemistry, yet whether such a reaction could occur on the positively charged sulfonium compounds remains unknown. In the study of the anaerobic coproporphyrinogen III oxidase HemN, a radical S‐adenosyl‐l ‐methionine (SAM) enzyme involved in heme biosynthesis, we observed the production of di‐(5′‐deoxyadenosyl)methylsulfonium, which supports a deoxyadenosyl (dAdo) radical‐mediated S_H reaction on the sulfonium center of SAM. The sulfonium‐based S_H reactions were then investigated in detail by density functional theory calculations and model reactions, which showed that this type of reactions is thermodynamically favorable and kinetically competent. These findings represent the first report of sulfonium‐based S_H reactions, which could be useful in synthetic chemistry. Our study also demonstrates the remarkable catalytic promiscuity of the radical SAM superfamily enzymes.     

Antivitamins B12—A Structure‐ and Reactivity‐Based Concept

B₁₂‐antimetabolites are compounds that counteract the physiological effects of vitamin B₁₂ and related natural cobalamins. Presented here is a structure‐ and reactivity‐based concept of the specific ′antivitamins B₁₂′: it refers to analogues of vitamin B₁₂ that display high structural similarity to the vitamin and are ′locked chemically′ to prevent their metabolic conversion into the crucial organometallic B₁₂‐cofactors. Application of antivitamins B₁₂ to healthy laboratory animals is, thus, expected to induce symptoms of B₁₂‐deficiency. Antivitamins B₁₂ may, hence, be helpful in elucidating still largely puzzling pathophysiological phenomena associated with B₁₂‐deficiency, and also in recognizing physiological roles of B₁₂ that probably still remain to be discovered.     

Expanding the Chemistry of the Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM

The radical S‐adenosylmethionine (SAM) superfamily enzymes cleave SAM reductively to generate a highly reactive 5′‐deoxyadenosyl (dAdo) radical, which initiates remarkably diverse reactions. Unlike most radical SAM enzymes, the class C radical SAM methyltransferase NosN binds two SAMs in the active site, using one SAM to produce a dAdo radical and the second as a methyl donor. Here, we report a mechanistic investigation of NosN in which an allyl analogue of SAM (allyl‐SAM) was used. We show that NosN cleaves allyl‐SAM efficiently and the resulting dAdo radical can be captured by the olefin moieties of allyl‐SAM or 5′‐allylthioadenosine (ATA), the latter being a derivative of allyl‐SAM. Remarkably, we found that NosN produced two distinct sets of products in the presence and absence of the methyl acceptor substrate, thus suggesting substrate‐triggered production of ATA from allyl‐SAM. We also show that NosN produces S‐adenosylhomocysteine from 5′‐thioadenosine and homoserine lactone. These results support the idea that 5′‐methylthioadenosine is the direct methyl donor in NosN reactions, and demonstrate great potential to modulate radical SAM enzymes for novel catalytic activities.     

Homocoenzyme B12 and Bishomocoenzyme B12: Covalent Structural Mimics for Homolyzed,Enzyme‐Bound Coenzyme B12

Efficient electrochemical syntheses of “homocoenzyme B₁₂” ( 2 , Co_β‐(5′‐deoxy‐5′‐adenosyl‐methyl)‐cob(III )alamin) and “bishomocoenzyme B₁₂” ( 3 , Co_β‐[2‐(5′‐deoxy‐5′‐adenosyl)‐ethyl]‐cob(III )alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in‐depth investigations of the structures of 2 and 3 in solution were carried out and a high‐resolution crystal structure of 2 was obtained. The two homologues of coenzyme B₁₂ ( 2 and 3 ) are suggested to function as covalent structural mimics of the hypothetical enzyme‐bound “activated” (that is, “stretched” or even homolytically cleaved) states of the B₁₂ cofactor. From crude molecular models, the crucial distances from the corrin‐bound cobalt center to the C5′ atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 Å, respectively. These values are roughly the same as those found in the two “activated” forms of coenzyme B₁₂ in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2 , the cobalt center was observed to be at a distance of 2.99 Å from the C5′ atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B₁₂. The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

Direct Participation of a Peripheral Side Chain of a Corrin Ring in Coenzyme B12 Catalysis

The crystal structures of the B₁₂‐dependent isomerases (eliminating) diol dehydratase and ethanolamine ammonia‐lyase complexed with adenosylcobalamin were solved with and without substrates. The structures revealed that the peripheral a‐acetamide side chain of the corrin ring directly interacts with the adenosyl group to maintain the group in the catalytic position, and that this side chain swings between the original and catalytic positions in a synchronized manner with the radical shuttling between the coenzyme and substrate/product. Mutations involving key residues that cooperatively participate in the positioning of the adenosyl group, directly or indirectly through the interaction with the a‐side chain, decreased the turnover rate and increased the relative rate of irreversible inactivation caused by undesirable side reactions. These findings guide the engineering of enzymes for improved catalysis and producing useful chemicals by utilizing the high reactivity of radical species.     

Enzymatic Cascade Reactions in Biosynthesis

Enzyme‐mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron‐based enzymes use O₂ to generate high valent iron‐oxo species to homolyze unactivated C?H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S‐adenosylmethionine (SAM) to generate the 5′‐deoxyadenosyl radical as a powerful oxidant to initiate C?H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as “thwarted oxygenases”.     

Crystal Structure of Tryptophan Lyase (NosL): Evidence for Radical Formation at the Amino Group of Tryptophan

Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3‐methyl‐2‐indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan Cα? Cβ bond and recombination of the amino‐acid‐derived ‐COOH fragment at the indole ring. Reported herein is the 1.8 Å resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for H abstraction by the SAM‐derived 5′‐deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Cα? Cβ bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a ^.NH tryptophanyl radical. A structure‐based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent ^.NH radical operates in the latter enzymes.     

Unusual kinetic role of a water‐soluble iron(III) porphyrin catalyst in the oxidation of 2,4,6‐trichlorophenol by hydrogen peroxide

The oxidation of 2,4,6‐trichlorophenol (TCP) to 2,6‐dichloro‐1,4‐benzoquinone (DCQ) by hydrogen peroxide using iron(III) meso‐tetra(4‐sulfonatophenyl) porphine chloride, Fe(TPPS)Cl, as a catalyst was studied with stopped‐flow UV–vis spectrophotometry and potentiometry using a chloride ion selective electrode. The observations are interpreted by a three‐step kinetic model: the initial reaction of the catalyst with the oxidant (Fe(TPPS)⁺ + H₂O₂ → Cat′) produces an active intermediate, which oxidizes the substrate (Cat′ + TCP → Fe(TPPS)⁺ + DCQ + Cl^?) in the second step. The third step is the transformation of the catalyst into a much less active form (Cat′ → Cat″) and is responsible for the unusual kinetic phenomena observed in the system. © 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36:449–455, 2004.     

Pseudocoenzyme B12 and Adenosyl‐Factor A: Electrochemical Synthesis and Spectroscopic Analysis of Two Natural B12 Coenzymes with Predominantly ‘Base‐off' Constitution

Pseudocoenzyme B₁₂ (=Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(adenin‐7‐yl)cobamide; 1 ) and adenosyl‐factor A (=Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(2‐methyladenin‐7‐yl)cobamide; 3 ) are two natural analogues of coenzyme B₁₂ (=adenosylcobalamin‐Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(5,6‐dimethyl‐1H‐benzimidazolyl)cobamide; 2 ), where the Co‐coordinating 5,6‐dimethyl‐1H‐benzimidazole nucleotide base of 2 is replaced by the purine bases adenine and 2‐methyladenine. In contrast to 2 , which exists solely in the ‘base‐on' form, UV/VIS spectroscopy qualitatively indicates ‘base‐off' constitution for 1 and 3 in aqueous solution. (cf. the established ‘base‐off' form as unexpected binding mode of B₁₂ cofactors in several B₁₂‐dependent enzymes, such as in methionine synthase from Escherichia coli and in glutamate mutase from Clostridium cochlearium). In the present work, pseudocoenzyme B₁₂ ( 1 ) was synthesized in 85% yield by alkylation with 5′‐O‐tosyladenosine of (adenin‐7‐yl)cob(I)amide, which was produced electrochemically from pseudovitamin B₁₂ (Coβ‐cyano‐(adenin‐7‐yl)cobamide). Likewise, adenosyl‐factor A ( 3 ) was prepared in ca. 70% yield from factor A (=Coβ‐cyano‐(2‐methyladenin‐7‐yl)cobamide; 5 ). All the spectroscopic properties of 1 and 3 in aqueous solution indicated that these two Coβ‐(5′‐deoxy‐5′‐adenosyl)‐(adenin‐7‐yl)cobamides exist predominantly in a ‘base‐off' constitution, with minor but significant contributions of the ‘base‐on' form. From the UV/VIS spectra, the temperature‐dependent equilibrium constants of the ‘base‐off'/‘base‐on' reconstitution reaction were determined as K_on ( 1 )=0.30 and K_on ( 3 )=0.48 at 25°, corresponding to a contribution of the ‘base‐on' forms of 23% for 1 and of 32% for 3 .     

Expanding Radical SAM Chemistry by Using Radical Addition Reactions and SAM Analogues

Radical S‐adenosyl‐l ‐methionine (SAM) enzymes utilize a [4Fe‐4S] cluster to bind SAM and reductively cleave its carbon–sulfur bond to produce a highly reactive 5′‐deoxyadenosyl (dAdo) radical. In almost all cases, the dAdo radical abstracts a hydrogen atom from the substrates or from enzymes, thereby initiating a highly diverse array of reactions. Herein, we report a change of the dAdo radical‐based chemistry from hydrogen abstraction to radical addition in the reaction of the radical SAM enzyme NosL. This change was achieved by using a substrate analogue containing an olefin moiety. We also showed that two SAM analogues containing different nucleoside functionalities initiate the radical‐based reactions with high efficiencies. The radical adduct with the olefin produced in the reaction was found to undergo two divergent reactions, and the mechanistic insights into this process were investigated in detail. Our study demonstrates a promising strategy in expanding radical SAM chemistry, providing an effective way to access nucleoside‐containing compounds by using radical SAM‐dependent reactions.     

Protic Anions [H(B12X12)]− (X=F,Cl, Br,I) That Act as Brønsted Acids in the Gas Phase

The acidity of protic cations and neutral molecules has been studied extensively in the gas phase, and the gas‐phase acidity has been established previously as a very useful measure of the intrinsic acidity of neutral and cationic compounds. However, no data for any anionic acids were available prior to this study. The protic anions [H(B₁₂X₁₂)]^? (X=F, Cl, Br, I) are expected to be the most acidic anions known to date. Therefore, they were investigated in this study with respect to their ability to protonate neutral molecules in the gas phase by using a combination of mass spectrometry and quantum‐chemical calculations. For the first time it was shown that in the gas phase protic anions are also able to protonate neutral molecules and thus act as Brønsted acids. According to theoretical calculations, [H(B₁₂I₁₂)]^? is the most acidic gas‐phase anion, whereas in actual protonation experiments [H(B₁₂Cl₁₂)]^? is the most potent gas‐phase acidic anion for the protonation of neutral molecules. This discrepancy is explained by ion pairing and kinetic effects.     

Quantum chemistry studies of adenosine 2503 methylation by S‐adenosylmethionine‐dependent enzymes

Ribosome methylation is important for life processes and is mainly catalyzed by radical S‐Adenosylmethionine (SAM) enzymes. Two SAM molecules serve as the cofactor by providing the 5 ′‐deoxyadenosyl radical for substrate activation and the methyl. Recently, Booker and coworkers (Science 2011, 332, 604) proposed an alternative mechanism for a pair of radical SAM enzymes, RlmN and Cfr, which respectively methylate the C2 and C8 of adenosine 2503. Their deuterium labeling experiments reveal that methyl group does not transfer directly from SAM to adenosine, instead it passes to Cys³⁵⁵ first, then onto adenosine. In this article, this new reaction mechanism is studied using density functional theory with B3LYP hybrid functional. The reaction system is simulated using small model compounds in the gas phase, and the protein environment is approximated using polarizable continuum model. The structures of the transition states and the intermediates are identified, and their free energies are calculated. The activation barriers indicate that the proposed reaction mechanism is plausible. The formation of a disulfide bond is found to be the rate‐limiting step. © 2012 Wiley Periodicals, Inc.     

Intrinsic Peroxidase‐like Activity of Porous CuO Micro‐/nanostructures with Clean Surface

Porous CuO micro‐/nanostructures with clean surface, prepared through Cu₂(OH)₂CO₃ precursor followed by calcination in air, were proven to be an effective peroxidase mimic. They can quickly catalyze oxidation of the peroxidase substrate 3,3′,5,5′‐tetramethylbenzidine (TMB) in the presence of H₂O₂, producing a blue color. The obtained porous CuO micro‐/nanostructure have potential application in wastewater treatment. The apparent steady‐state kinetic parameter was studied with TMB as the substrate. In addition, the potential application of the porous CuO in wastewater treatment was demonstrated with phenol‐containing water as an example. Such investigation not only confirms the intrinsic peroxidase‐like activity of micro‐/nanostructured CuO, but also suggests its potential application in wastewater treatment.     

Substrate‐Tuned Catalysis of the Radical S‐Adenosyl‐L‐Methionine Enzyme NosL Involved in Nosiheptide Biosynthesis

NosL is a radical S‐adenosyl‐L ‐methionine (SAM) enzyme that converts L ‐Trp to 3‐methyl‐2‐indolic acid, a key intermediate in the biosynthesis of a thiopeptide antibiotic nosiheptide. In this work we investigated NosL catalysis by using a series of Trp analogues as the molecular probes. Using a benzofuran substrate 2‐amino‐3‐(benzofuran‐3‐yl)propanoic acid (ABPA), we clearly demonstrated that the 5′‐deoxyadenosyl (dAdo) radical‐mediated hydrogen abstraction in NosL catalysis is not from the indole nitrogen but likely from the amino group of L ‐Trp. Unexpectedly, the major product of ABPA is a decarboxylated compound, indicating that NosL was transformed to a novel decarboxylase by an unnatural substrate. Furthermore, we showed that, for the first time to our knowledge, the dAdo radical‐mediated hydrogen abstraction can occur from an alcohol hydroxy group. Our study demonstrates the intriguing promiscuity of NosL catalysis and highlights the potential of engineering radical SAM enzymes for novel activities.     

Glycerol as a Substrate and Inactivator of Coenzyme B12-Dependent Diol Dehydratase

Diol dehydratase, dependent on coenzyme B₁₂ (B₁₂-dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B₁₂-independent glycerol dehydratase (B₁₂-iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B₁₂-dDDH and that of resistance of B₁₂-iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B₁₂-dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B₁₂-iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.     

Carborane Substituents Promote Direct Electrophilic Insertion over Reduction–Metalation Reactions

Two‐electron reduction of 1,1′‐bis(o‐carborane) followed by reaction with [Ru(η‐mes)Cl₂]₂ affords [8‐(1′‐1′,2′‐closo‐C₂B₁₀H₁₁)‐4‐(η‐mes)‐4,1,8‐closo‐RuC₂B₁₀H₁₁]. Subsequent two‐electron reduction of this species and treatment with [Ru(η‐arene)Cl₂]₂ results in the 14‐vertex/12‐vertex species [1‐(η‐mes)‐9‐(1′‐1′,2′‐closo‐C₂B₁₀H₁₁)‐13‐(η‐arene)‐1,13,2,9‐closo‐Ru₂C₂B₁₀H₁₁] by direct electrophilic insertion, promoted by the carborane substituent in the 13‐vertex/12‐vertex precursor. When arene=mesitylene (mes), the diruthenium species is fluxional in solution at room temperature in a process that makes the metal–ligand fragments equivalent. A unique mechanism for this fluxionality is proposed and is shown to be fully consistent with the observed fluxionality or nonfluxionality of a series of previously reported 14‐vertex dicobaltacarboranes.     

Partial Synthesis of Coenzyme B12 from Cobyric Acid

Here we report the direct chemical synthesis of coenzyme B₁₂ (AdoCbl) from Co_β ‐5′‐deoxyadenosylcobyric acid (AdoCby) and the preparation of the latter from crystalline CN ,H₂O‐cobyric acid (CN ,H₂OC by). AdoCby is a suggested common key intermediate in the biosynthesis of AdoCbl and of other cobamides in microorganisms. AdoCby was thoroughly characterized by spectroscopic means, including homo‐nuclear and hetero‐nuclear NMR , as such data are not available in published work. AdoCbl was prepared from AdoCby in one‐step in over 85% yield, by covalent attachment in aqueous solution of the integral B₁₂‐nucleotide moiety using 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC ·HC l) and N‐hydroxybenzotriazole (HOB t) as coupling reagents. By the same procedure crystalline vitamin B₁₂ (CNC bl) was also prepared in 92% yield from CN ,H₂OC by. Coordination of the B₁₂‐nucleotide base at the Co_α ‐face of AdoCby or of CN ,H₂OC by was indicated to assist in the efficient covalent coupling at the activated f‐side chain function to furnish the complete corrinoids AdoCbl and CNC bl.