Vitamin B12 total synthesis

The biomolecule vitamin B12 (molecular formula C63H88CoN14O14P), is the most complex of all known vitamins. Its chemical structure was previously elucidated by X-ray crystallography in 1956 by the research group of Dorothy Hodgkin (Oxford), in collaboration with Kenneth N. Trueblood (UCLA) and John G. White (Princeton). At the core of this molecule is the corrin structure, a nitrogenous tetradentate ligand system. The corrin system is biogenetically related to porphyrins and chlorophylls, yet differs from them structurally. Its carbon skeleton lacks a meso carbon atom “bridge” otherwise linking the five-membered pyrrole-like rings; two of these rings (A and D, Figure 1) are directly fused by a C–C single bond. Lined up along the periphery of the B12 corrin ring are eight methyl groups, three propanamide, and three acetamide side chains. The periphery also contains nine stereocenters. The monobasic corrin ligand is equatorially coordinated with a trivalent Co3+ cobalt ion, which bears two additional axial ligands. Several natural variants of the B12 structure exist that differ in the identity of these axial ligands. In vitamin B12 itself, the cobalt is coordinated to a cyano group on the top side of the corrin plane (cyanocobalamin), and a nucleotide loop on the opposite side. This nucleotide loop is connected on its other end to the peripheral propanamide group located at ring D (Figure 1) and consists of structural elements derived from aminopropanol, phosphate, ribose, and 5,6-dimethylbenzimidazole. The imidazole derivative is axially coordinated to the cobalt, closing the loop. Cobyric acid, one of the natural derivatives of vitamin B12, lacks this nucleotide loop. Depending on the nature of the two axial ligands at the cobalt center, it instead displays propionic acid function at ring D as a carboxylate (as shown in Figure 1), or as the carboxylic acid, in the case of two cyanide ligands at cobalt. ==The two syntheses==

The structure of vitamin B12 was the first low-molecular weight natural product to be determined by X-ray analysis rather than by chemical degradation. As a result, though the structure of this novel type of complex biomolecule was established, its chemistry remained essentially unknown. Exploration of this chemistry became one of the tasks of the vitamin's chemical synthesis. In its time, the synthesis of such an exceptionally complex and unique structure presented a major challenge at the frontier of research in organic natural product synthesis. In 1960, the research group of biochemist in Stuttgart had reconstituted vitamin B12 from one of its naturally occurring derivatives, cobyric acid. This was achieved by stepwise construction of the vitamin's nucleotide loop. This particular work amounted to a partial synthesis of vitamin B12 from a natural product containing all the structural elements of vitamin B12, except the nucleotide loop. As a result, cobyric acid was chosen as the target molecule for a total synthesis of vitamin B12. Collaborative work between research groups at Harvard and at ETH resulted in two cobyric acid syntheses (Figure 3), concomitantly accomplished in 1972, one by Harvard and the other by ETH. The described "competitive collaboration" of that size (totaling 103 graduate students and postdoctoral researchers for a time of almost 177 person-years) was then unheard of in the history of organic synthesis. The two syntheses are intricately intertwined chemically, though differ in the way the central macrocyclic corrin ligand system is constructed. Both strategies are patterned after two model corrin syntheses developed at ETH. The first of these syntheses, published in 1964, achieved the construction of the corrin chromophore by combining an A–D component with a B–C component via iminoester/enamine C–C condensations; the final ring closure was attained between rings A and B. The second model synthesis, published in 1969, explored a novel photochemical cycloisomerization process to create the direct A/D ring junction, with final ring closure occurring between rings A and D. The A/B approach to the cobyric acid syntheses was collaboratively pursued and accomplished in 1972 at Harvard. It combined a bicyclic Harvard A–D component with an ETH B–C component, and closed the macrocyclic corrin ring between rings A and B. The A/D approach to the synthesis, accomplished at ETH and finished at the same time as the Harvard A/B approach, successively adds rings D and A to the B–C component of the A/B approach and attains the corrin ring closure between rings A and D. The paths of the two syntheses met in a common corrinoid intermediate. The final steps from this intermediate to the cobyric acid target were also carried out collaboratively; each group working with material prepared via their own respective approach. ==Synopsis of the Harvard/ETH collaboration==

The beginnings Woodward and Eschenmoser embarked on the project of a chemical synthesis of vitamin B12 independently from each other. The ETH group started with a model study on how to synthesize a corrin ligand system in December 1959. In August 1961, the Harvard group began attacking the buildup of the B12 structure directly by aiming at the most complex part of the B12 molecule, the so-called "western half" that contains the direct junction between rings A and D (the A–D component). By October 1960, the ETH group had commenced the synthesis of a ring B precursor of vitamin B12. The progress at Harvard was rapid from the start, until an unexpected stereochemical course of a central ring formation step interrupted the project. Woodward's recognition of the stereochemical enigma (which came to light by the irritating behavior of one of his meticulously planned synthetic steps) became, according to his own writings, part of the developments that led to the orbital symmetry rules. After 1965, the Harvard group continued work towards an A–D component along a modified plan, using (−)-camphor as the source of ring D. Joining forces: the A/B approach to cobyric acid synthesis By 1964, the ETH group had accomplished the first corrin model synthesis, and also the preparation of a ring B precursor as part of a construction of the B12 molecule itself. Since the independent progress of the two groups towards their long-term objective was so clearly complementary, Woodward and Eschenmoser decided in 1965 to pursue the project of a B12 synthesis collaboratively, planning to utilize the ligand construction (ring coupling of components) strategy of the ETH model system. By 1966, the ETH group had succeeded in synthesizing the B–C component (the analogous "eastern half") by coupling their ring B precursor to the ring C precursor. This ring C precursor had also been prepared at Harvard from (−)-camphor by employing a strategy conceived and used earlier by A. Pelter and J. W. Cornforth in 1961. At ETH, the synthesis of the B–C component involved the implementation of the C–C condensation reaction via sulfide contraction. This newly-developed method turned out to provide a general solution to the problem of constructing the characteristic structural elements of the corrin chromophore, the vinylogous amidine systems bridging the four peripheral rings. Early in 1967, the Harvard group accomplished the synthesis of the model A–D component, with the side chain "f" (Figure 4) undifferentiated, bearing a methyl ester like all the other side chains. From then on, the two groups systematically exchanged samples of their respective halves of the corrinoid target structure. By 1970, they had collaboratively connected Harvard's undifferentiated A–D component with ETH's B–C component, producing dicyano-cobalt(III)-5,15-bisnor-heptamethyl-cobyrinate (1, Figure 4). This synthetic corrinoid intermediate was identified by the ETH group via direct comparison with a sample produced from natural vitamin B12. In this advanced model study, reaction conditions for the demanding processes of the C/D coupling and the A/B cyclization via sulfide contraction method were established. The reaction conditions for the C/D coupling were successfully explored in both laboratories, with the superior conditions being found at Harvard. The ideal reaction conditions for the A/B ring closure via an intramolecular version of the sulfide contraction were developed at ETH. Later, it was shown at Harvard that the A/B ring closure could also be achieved by thio-iminoester/enamine condensation. By early 1971, the Harvard group had accomplished the synthesis of the final A–D component, containing a nitrile group as part of side chain "f" located on ring D, different from the remaining carboxyl groups (2, Figure 4; see also Figure 3). The A/D part of the B12 structure in carboxyl groups represents, constitutionally and configurationally, the most intricate part of the vitamin molecule; its synthesis is regarded as the apotheosis of the Woodwardian art seen in natural product total synthesis. The alternative approach to cobyric acid synthesis As far back as 1966, the ETH group started to explore a model alternative strategy of corrin synthesis in which the corrin ring would be closed between rings A and D. The project was inspired by the conceivable existence of an unknown bond reorganization process. This reorganization, if existing, would make the construction of cobyric acid from a single starting material a possibility. Importantly, this hypothetical process, being interpreted as implying two sequential rearrangements, was recognized to be formally covered by the new reactivity classifications of sigmatropic rearrangements and electrocyclizations propounded by Woodward and Hoffmann in the context of their orbital symmetry rules! By May 1968, the ETH group had demonstrated in a model study that the envisaged process, a photochemical A/D-seco-corrinate→corrinate cycloisomerization, does in fact exist. This process was first found to proceed with the palladium(II) complex, but not at all with corresponding nickel(II)- or cobalt(III)-A/D-seco-corrinate complexes. The cycloisomerization also proceeded smoothly in complexes of metal ions such as zinc and other photochemically inert and loosely bound metal ions. These metal ions could, after ring closure, easily be replaced by cobalt. These observations opened the door to what eventually became the photochemical A/D approach of cobyric acid synthesis. Starting in fall of 1969, with the B–C component of the A/B approach and a ring D precursor prepared from the enantiomer of the starting material leading to the ring B precursor, it took PhD student Walter Fuhrer less than one and a half years to translate the photochemical model corrin synthesis into a synthesis of 2 (Figure 4), the common corrinoid intermediate on the synthetic pathway to cobyric acid. At Harvard, intermediate 2 was obtained around the same time by coupling the ring D-differentiated Harvard A–D component (available in spring 1971) with the ETH B–C component, applying the condensation methods developed earlier using the undifferentiated A–D component. In the spring of 1971, two different routes to the common corrinoid intermediate 2 (Figure 4) had become available. The Harvard/ETH A/B approach required 62 synthetic steps, while the ETH A/D approach required 42. In both approaches, the four peripheral rings were derived from enantiopure precursors possessing the correct sense of chirality, thereby circumventing major stereochemical problems in the buildup of the ligand system. In the construction of the A/D junction by the A/D-seco-corrin→corrin cycloisomerization, the formation of two A/D-diastereomers was expected. The use of cadmium(II) as the coordinating metal ion led to a very high diastereoselectivity in favor of the natural A/D-trans-isomer. Once the corrin structure was formed by either approach, the three C–H stereocenters at the periphery adjacent to the chromophore system turned out to be prone to epimerization. This required a separation of diastereomers in an advanced stage of the syntheses. Coincidentally, the technique of high pressure liquid chromatography (HPLC) had been newly-developed; the technique became an indispensable tool in both laboratories. The use of HPLC in the B12 project, pioneered by Jakob Schreiber at ETH, was the earliest application of the technique in natural product synthesis. The joint final steps The final conversion of the common corrinoid intermediate 2 (Figure 6) from the two approaches into the target cobyric acid required the introduction of the two missing methyl groups at the meso C-5 and C-15 positions of the corrin chromophore between rings A/B and C/D, as well as the conversion of all peripheral carboxyl functions into their amide form, except at the critical carboxyl at side chain "f" of ring D (Figure 6). These steps were explored collaboratively in parallel fashion, with the Harvard group using material produced via the A/B approach and the ETH group using material prepared by the photochemical A/D approach. The first decisive identification of a totally synthetic intermediate on the synthetic pathway to cobyric acid was carried out in February 1972 with a crystalline sample of synthetic dicyano-cobalt(III)-hexamethyl-cobyrinate-f-amide 3 (Figure 6), found to be identical in all data with a crystalline relay sample made from vitamin B12 by methanolysis to "cobester" 4, followed by partial ammonolysis and separation of the resulting mixture. At the time when Woodward announced the "Total Synthesis of Vitamin B12" at the IUPAC conference in New Delhi in February 1972, the totally synthetic sample of the f-amide was one that had been made at ETH by the photochemical A/D approach, while the first sample of synthetic cobyric acid, identified using natural cobyric acid, had been obtained at Harvard by partial synthesis from B12-derived f-amide relay material. The Woodward/Eschenmoser achievement around this time had been, strictly speaking, two formal total syntheses of cobyric acid, as well as two formal total syntheses of the vitamin. In the later course of 1972, two crystalline epimers of 3, as well as two crystalline epimers of the totally synthetic f-nitrile (all prepared using both synthetic approaches) were stringently identified chromatographically and spectroscopically with the corresponding B12-derived substances. At Harvard, cobyric acid was then made also from totally synthetic f-amide 3 prepared via the A/B approach. Finally, in 1976 at Harvard, totally synthetic cobyric acid was converted into vitamin B12 via the pathway pioneered by . The publication record Over the almost 12 years it took the two groups to reach their goal, both Woodward and Eschenmoser periodically reported on the stage of the collaborative project in lectures, some of which appeared in print. Woodward discussed the A/B approach in lectures published in 1968 and 1971, culminating in the announcement of the "Total Synthesis of Vitamin B12" in New Delhi in February 1972 which was published in 1973. This publication, and other lectures under the same title Woodward delivered in the later part of the year 1972, are confined to the A/B approach of the synthesis and do not discuss the ETH A/D approach. Eschenmoser had discussed the ETH contributions to the A/B approach at the 1968 Robert A. Welch Foundation conference in Houston, as well as in his 1969 RSC Centenary Lecture titled "Roads to Corrins", published in 1970. He presented the ETH photochemical A/D approach to the B12 synthesis at the 23rd IUPAC Congress in Boston in 1971. The Zürich-based group announced the accomplishment of the synthesis of cobyric acid via the photochemical A/D-approach in two lectures delivered by PhD students Hans Maag and Walter Fuhrer at the Swiss Chemical Society Meeting in April 1972. Eschenmoser presented a lecture titled "Total Synthesis of Vitamin B12: the Photochemical Route" for the first time as a Wilson Baker Lecture at the University of Bristol on May 8, 1972. By 1977, a joint, collaborative publication of the syntheses by the Harvard and ETH groups had yet to appear in scientific record. Rather, an article describing the final version of the photochemical A/D approach (previously achieved in 1972) was published in 1977 in Science. This article is an extended English translation of one that had already appeared 1974 in Naturwissenschaften, based on a lecture given by Eschenmoser on January 21, 1974, at a meeting of the Zürcher Naturforschende Gesellschaft. Four decades later, in 2015, Eschenmoser published a series of six full papers describing the work of the ETH group on corrin synthesis. Part I of the series contains a chapter entitled "The Final Phase of the Harvard/ETH Collaboration on the Synthesis of Vitamin B12", in which the contributions of the ETH group to the collaborative work on the synthesis of vitamin B12 between 1965 and 1972 are recorded. The entirety of the ETH work is documented, in full experimental detail, in publicly accessible PhD theses, totalling almost 1,900 pages, all in German. Contributions of the 14 postdoctoral ETH researchers involved in the cobyric acid syntheses are mostly integrated in these theses. The detailed experimental work at Harvard was documented in reports by the 77 postdoctoral researchers involved, a total volume containing more than 3,000 pages. Representative reviews of the two approaches to the chemical synthesis of vitamin B12 have been published in detail by A. H. Jackson and K. M. Smith, T. Goto, R. V. Stevens, K. C. Nicolaou & E. G. Sorensen, summarized by J. Mulzer & D. Riether, and G. W. Craig, in addition to many other publications where these epochal syntheses are discussed. ==The Harvard/ETH synthesis of cobyric acid: the path to the common corrinoid intermediate via A/B-corrin ring closure==

In the A/B approach to cobyric acid, the Harvard A–D component was coupled to the ETH B–C component between rings D and C, and then closed to the corrin between rings A and B. Both these critical steps were accomplished by C–C coupling via sulfide contraction, a new reaction type developed in the synthesis of the B–C component at ETH. The A–D component was synthesized at Harvard from a ring A precursor (prepared from achiral starting materials), and a ring D precursor prepared from (−)-camphor. A model A–D component was used to explore the coupling conditions. This structure differed from the A–D component used in the final synthesis by having a methyl ester group on ring D side chain "f" instead of a nitrile group. ==The ETH synthesis of cobyric acid: the path to the common corrinoid intermediate via A/D-corrin-ring closure==

In the A/D approach to the synthesis of cobyric acid, the four ring precursors (ring C precursor only formally so) derive from the two enantiomers of one common chiral starting material. All three vinylogous amidine bridges that connect the four peripheral rings were constructed by the sulfide contraction method, with the B–C component–already prepared for the A/B approach–serving as an intermediate. The photochemical A/D seco-corrin→corrin cycloisomerization, by which the corrin ring was closed between rings A and D, is a novel process, targeted and found to exist in a model study (Figure 2). ==ETH/Harvard – the jointly executed final steps from the common corrinoid intermediate to cobyric acid==

The final steps from the common corrinoid intermediate E-37/HE-44 to cobyric acid E-44/HE-51 were carried out by the two groups collaboratively and in parallel, with the ETH group working with material produced by the A/D approach, and the Harvard group with that from the A/B approach. What the two groups in fact accomplished thus were the common final steps of two different syntheses. The tasks in this end phase of the project were the regioselective introduction of methyl groups at the meso C-5 and C-15 positions of E-37/HE-44, followed by conversion of all its peripheral carboxyl functions into primary amide groups, excepting that in side chain "f" at ring D, which had to end up as free carboxyl. These conceptually simple finishing steps turned out to be rather complex in aon, including unforeseen pitfalls, including a dramatic loss of precious synthetic material on July 9, 1971 in what came to be known as "Black Friday". ==Notes==