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What's unique for vitamin B-12 group?

What's unique for vitamin B-12 group?


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I've been trying to figure out what makes the vitamin B group really big with 8 vitamins. Chemically they have different structures. Solubility: not only vitamins from this group are water soluble vitamins (also vitamin C). Function: not only this group of vitamins are cofactors for metabolic processes.

So what's the reason for this grouping, or why we can't include other vitamins in this group? Also, what does the letter B stands for?


The common scientific belief around 1900 was that the only dietary components needed to support life were protein, fats, carbohydrates, and salts.[1] In 1912 Hopkins reported on experiments showing that "accessory factors" seemed to be needed.[1] In 1916, some of the factors began to be characterized in the paper "The Relation of the Unidentified Dietary Factors, the Fat-Soluble A, and Water-Soluble B, of the Diet to the Growth Promoting Properties of Milk."[2] The title itself designates two factors as A and B. Lack of factor A seemed to be associated with various problems particularly with the eyes, and factor B with a condition similar to beriberi. Before long, another factor was found that was associated with scurvy. In 1920, the literature had a confusion of terminology in use, and Drummond, using a variant of Funk's word, proposed:

that the substances be spoken of as Vitamin A, B, C, etc. This simplified scheme should be quite sufficient until such time as the factors are isolated, and their true nature identified.[3]

It was only later that the factors became well characterized. Vitamin B came to be known to be a complex mixture, and its components took decades to be characterized.(Wikipedia) As components of Vitamin B came to be known, subscripts were assigned. Some of the subscripts were assigned to compounds that later turned out to not to be fundamental and essential, and so are no longer used. Vitamin B12 took a particularly long time to characterize.(Wikipedia)

The vitamins are now well understood, and each has a specific name unrelated to the vitamin naming scheme, yet the vitamin terminology continues to be used because it is well known and useful to people at many different levels of technical knowledge. The significance of the name of the B group of vitamins is that "B" follows "A" and precedes "C" in the alphabet, and the history of discovery is still embedded in the names we use.

[1] Hopkins FG. Feeding experiments illustrating the importance of accessory factors in normal dietaries. J Physiol. 1912 Jul 15;44(5-6):425-60. doi: 10.1113/jphysiol.1912.sp001524. PMID: 16993143; PMCID: PMC1512834.

[2] EV McCollum, N Simmonds, W Pitz. The Relation of the Unidentified Dietary Factors, the Fat-Soluble A, and Water-Soluble B, of the Diet to the Growth Promoting Properties of Milk - Journal of Biological Chemistry, 1916

[3] Drummond JC. The Nomenclature of the so-called Accessory Food Factors (Vitamins). Biochem J. 1920 Oct;14(5):660. doi: 10.1042/bj0140660. PMID: 16742922; PMCID: PMC1258930.


According to PeaceHealth:

The vitamin B-complex refers to all of the known essential water-soluble vitamins except for vitamin C.

Vitamin B was once thought to be a single nutrient. Researchers later discovered these extracts contained several vitamins.

Each member of the B-complex has a unique structure and performs unique functions in the human body. Vitamins B1, B2, B3, and biotin participate in different aspects of energy production, vitamin B6 is essential for amino acid metabolism, and vitamin B12 and folic acid facilitate steps required for cell division.

So, vitamins B have different functions, but most of them appear to be involved in the metabolism, especially of carbohydrates (Medical Libre Texts).


B doesn't stand for anything. Originally, the vitamins were name alphabetically which can be traced to Cornelia Kennedy's 1916 Master's Thesis. What separates B-vitamins (and all vitamins) is there function. The function of B-vitamins is in metabolism and certain metabolic cycles, such as the Citric Acid Cycle.

Citations: http://vc.bridgew.edu/cgi/viewcontent.cgi?article=1029&context=mahpls_fac https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772032/


What's unique for vitamin B-12 group? - Biology

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Sources of Vitamin B12

Vitamin B12 relates to a group of water-soluble cobalamin compounds. This signifies that such vitamins can be dissolved in water and transported through the tissues, but cannot be stored in the body itself. Surprisingly, there are no ways to produce this unique vitamin directly in the body.

A good source of vitamin B12 is generally obtained from animal-based foods. These foods promote microbial growth responsible for the production of vitamin B12. Here are some of the familiar sources of vitamin B12 according to the different diets:

  • Non-vegetarian sources: Include fish, meat, egg, and poultry-based products.
  • Vegetarian sources: Items mainly derived from milk and dairy items.
  • Vegan food sources: Food items obtained through fortified cereals and nutritional yeast.

Vitamin B2 ( Ribolflavin)

Vitamin B2 is a proven antioxidant, it’s known to boost energy, improve healthy metabolism and protects the skin.

One of the essential B vitamins, every cell in your body needs B2.

In fact the other B vitamins cannot function without it being present in ample quantities.

Regarding testosterone boosting, B2 is a major part of the process.

It plays a crucial role by inhibiting 5 alpha-reductase.

This is an enzyme that converts testosterone into DHT in the testicles.

This is required for a number of bodily functions but it can also have detrimental health effects too if left unchecked.

Sources Of Vitamin B2

Our diet is packed with vitamin B2, and so deficiency is rare in the western world.

Most leafy greens, eggs, mushrooms, grains, soybeans as well as other dairy products, almonds and organic meats contain it at good levels.

Should I Supplement with B2?

As with Vitamin B1, unless your diet is really poor, it’s very unlikely that taking B2 in supplement form will have any real impact on your testosterone production.


Vitamin B12: a tunable, long wavelength, light-responsive platform for launching therapeutic agents

Light-responsive agents offer the promise of targeted therapy, whose benefits include (i) prolonged action at the target site, (ii) overall reduced systemic dosage, (iii) reduced adverse effects, and (iv) localized delivery of multiple agents. Although photoactivated prodrugs have been reported, these species generally require short wavelengths (<450 nm) for activation. However, maximal tissue penetrance by light occurs within the "optical window of tissue" (600-900 nm), well beyond the wavelength range of most existing photocleavable functional groups. Furthermore, since multidrug therapy holds promise for the treatment of complex diseases, from cancer to neurological disorders, controlling the action of multiple drugs via wavelength modulation would take advantage of a property that is unique to light. However, discrimination between existing photoresponsive moieties has thus far proven to be limited. We have developed a vitamin B12/light-facilitated strategy for controlling drug action using red, far-red, and NIR light. The technology is based on a light-triggered reaction displayed by a subset of B12 derivatives: alkyl-cob(III)alamins suffer photohomolysis of the C-Co(III) bond. The C-Co(III) bond is weak (<30 kcal/mol), and therefore all wavelengths absorbed by the corrin ring (330-580 nm) induce photocleavage. In addition, by appending fluorophores to the corrin ring, long wavelength light (>600 nm) is readily captured and used to separate the Co-appended ligand (e.g., a drug) from B12. Consequently, it is now feasible to preassign the wavelength of homolysis by simply installing a fluorescent antenna with the desired photophysical properties. The wavelength malleability inherent within this strategy has been used to construct photoresponsive compounds that launch different drugs by simply modulating the wavelength of illumination. In addition, these phototherapeutics have been installed on the surface and interior of cells, such as erythrocytes or neural stem cells, and released upon expoure to the appropriate wavelength. We have shown that cytotoxic agents, such as doxorubicin, anti-inflammatories, such as dexamethasone, and anti- and pro-vascular agents are readily released from cellular vehicles as biologically active agents. We have also demonstrated that the concept of "optical window of tissue" phototherapeutics is not just limited to prodrugs. For example, stem cells have received considerable attention in the area of regenerative medicine. Hydrogels serve as scaffolds for stem cell growth and differentiation. We have shown that the formation of hydrogels can be triggered, in the presence of cells, using appropriately designed alkyl-cob(III)alamins and long wavelength light. The potential applications of phototherapeutics are broad and include drug delivery for a variety of indications, tissue engineering, and surgery.

Figures

Photosurgery in the 24th century?…

Photosurgery in the 24th century? Wavelength-modulated illumination at the base of the brain…

Light-induced (650 nm) translocation of…

Light-induced (650 nm) translocation of BODIPY 650 in HeLa cells. (a) BODIPY–Cbl, 7,…

Light-triggered cAMP-Cbl ( 6 )-mediated…

Light-triggered cAMP-Cbl ( 6 )-mediated rearrangement of actin cytoskeleton in REF52 cells. Cells…

Self-assembly of a functional light-responsive…

Self-assembly of a functional light-responsive drug release array on the surface of erythrocytes.…

Impact of the light-triggered release…

Impact of the light-triggered release of drugs from erythrocyte carriers on co-incubated cells.…

Light-triggered gelation of (a) acrylamide…

Light-triggered gelation of (a) acrylamide and bis-(acrylamide) or (b) PEG-monoacrylate and PEG-diacrylate in…

Structure of Vitamin B 12…

Structure of Vitamin B 12 (R = CN, CH 3 , Adenosyl), 1,…

Photohomolysis of 14 or 15…

Photohomolysis of 14 or 15 at Wavelengths 520 or 660 nm, respectively, Generates…

Wavelength-Distinguishable Photoresponsive Moieties 3–5

Wavelength-Distinguishable Photoresponsive Moieties 3–5

Cbl-Linked cAMP (6), BODIPY (7),…

Cbl-Linked cAMP (6), BODIPY (7), and Dox (8)a a Photolysis generates a membrane…

Photohomolysis of Fluorophore-Cbl Conjugates Where…

Photohomolysis of Fluorophore-Cbl Conjugates Where the Fluorophores Serve as Antennas To Capture 546…

Photohomolysis (520 nm) of 14…

Photohomolysis (520 nm) of 14 Is Compromised by Filters Mimicking Skin Types That…

Drug–Cbl–Lipid Conjugates (16–18) and Lipidated…

Drug–Cbl–Lipid Conjugates (16–18) and Lipidated Fluorophores (19–22) Self-Assemble on the Surface of Erythrocytes…


Unique B-12 - 90 Vegetarian Tablets

Taking Unique B-12 by Dee Cee Laboratories to recover after surgery.

Description

Unique B-12 90 Vegetarian Tablets from Dee Cee Laboratories&sbquo Inc.® is designed to provide you with vitamin B-12&sbquo B-6&sbquo folic acid&sbquo and biotin to support the nervous and cardiovascular systems.

Most of the vitamins comprising the B complex group are involved in the synthesis of food and the production of energy B-12 too is part of this process. Apart from the production of energy&sbquo vitamin B-12 might also help with the production of red blood cells and nerve tissues. The supplement from Dee Cee Laboratories&sbquo Inc.® combines the benefits of B-12&sbquo B-6&sbquo folic acid&sbquo and biotin for top wellness.

Studies show that when taken with folic acid&sbquo B-12 could help support healthy homocysteine levels that are within a normal range. Vitamin B-12 might also help maintain healthy nerve cells&sbquo support the production of DNA&sbquo and the synthesis of fats and proteins. Vitamin B-6 or pyridoxine&sbquo present in this supplement might be useful for the functioning of the cardiovascular&sbquo digestive&sbquo immune&sbquo muscular&sbquo and nervous systems. It may also support the development and functioning of the brain.

Vitamin B-6 may be involved in the production of melatonin&sbquo the hormone that controls the internal clock. The benefits of B-6 include prevention and relief from deficiency. Folic acid&sbquo a type of B vitamin might help in the production of red blood cells.

Biotin present in Unique B-12 90 Vegetarian Tablets from Dee Cee Laboratories&sbquo Inc.® might help with brittle nails as well.

These statements have not been evaluated by the Food and Drug Administration (FDA). These products are not meant to diagnose‚ treat or cure any disease or medical condition. Please consult your doctor before starting any exercise or nutritional supplement program or before using these or any product during pregnancy or if you have a serious medical condition.

Ingredients

Vitamin B-6
(as pyridoxine HCl) 5 mg 250%
Folic Acid 400 mcg 100%
Vitamin B-12
(as methylcobalamin) 1000 mcg 16&sbquo666%
Biotin 25 mcg 8.3%

Other ingredients: Mannitol&sbquo natural cherry flavor&sbquo vegetable stearic acid&sbquo vegetable cellulose&sbquo L-ascorbic acid&sbquo vegetable magnesium stearate&sbquo silica&sbquo stevia.

Dosage

As a dietary supplement&sbquo take 1 or 2 tablets daily&sbquo Allow tablets to dissolve under tongue.


No, you cannot overdose on B12

It is highly unlikely that someone could take too much B12, says Natalie Allen, RD, a clinical assistant professor of biomedical sciences at Missouri State University.

The Institute of Medicine has not established a tolerable upper intake level of B12 because studies show no adverse health effects when taking excess levels of B12, either through food or supplements.

Medical term: A tolerable upper intake level is the highest level of nutrient intake that does not pose any adverse health effects for the majority of people.

Vitamin B12 is a water-soluble vitamin, meaning it dissolves in water and is quickly absorbed by the body. It is stored in the liver and whatever your body doesn't use is excreted through urine, Allen says. Even in high doses, your body can only absorb a fraction of B12 supplements. For example, a healthy person taking a 500 mcg oral B12 supplement will only absorb about 10 mcg.

Although uncommon, it is possible to have elevated B12 levels in a blood test, says Sheri Vettel, a registered dietitian nutritionist (RDN) with the Institute for Integrative Nutrition.

A serum B12 level between 300 pg/mL and 900 pg/mL is considered normal and levels above 900 pg/mL are considered high.

Elevated levels of B12 could be indicative of certain health concerns, Vettel says, including:

If you have elevated levels of B12, your doctor may run other tests to determine the underlying cause.


Contents

Vitamin B12, C63H88CoN14O14P, is the most complex of all known vitamins. Its chemical structure had been determined by x-ray crystal structure analysis in 1956 by the research group of Dorothy Hodgkin (Oxford University) in collaboration with Kenneth N. Trueblood at UCLA and John G. White at Princeton University. [24] [25] Core of the molecule is the corrin structure, a nitrogenous tetradentate ligand system. [note 1] This is biogenetically related to porphyrins and chlorophylls, yet differs from them in important respects: the carbon skeleton lacks one of the four meso carbons between the five-membered rings, two rings (A and D, fig. 1) being directly connected by a carbon-carbon single bond. The corrin chromophore system is thus non-cyclic and expands over three meso positions only, incorporating three vinylogous amidine units. Lined up at the periphery of the macrocylic ring are eight methyl groups and four propionic and three acetic acid side chains. Nine carbon atoms on the corrin periphery are chirogenic centers. The tetradentate, monobasic corrin ligand is equatorially coordinated with a trivalent cobalt ion which bears two additional axial ligands. [note 2]

Several natural variants of the B12 structure exist that differ in these axial ligands. In the vitamin itself, the cobalt bears a cyano group on the top side of the corrin plane (cyanocobalamin), and a nucleotide loop on the other. This loop is connected on its other end to the peripheral propionic amide group at ring D and consists of structural elements derived from aminopropanol, phosphate, ribose, and 5,6-dimethylbenzimidazole. One of the nitrogen atoms of the imidazole ring is axially coordinated to the cobalt, the nucleotide loop thus forming a nineteen-membered ring. All side chain carboxyl groups are amides.

Cobyric acid, one of the natural derivatives of vitamin B12, [26] lacks the nucleotide loop depending on the nature of the two axial ligands, it displays instead its propionic acid function at ring D as carboxylate (as shown in fig. 1), or carboxylic acid (with two cyanide ligands at cobalt).

The structure of vitamin B12 was the first low-molecular weight natural product determined by x-ray analysis rather than by chemical degradation. Thus, while 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. [12] : 1411 [18] : 1488-1489 [27] : 275 In the 1960s, synthesis of such an exceptionally complex and unique structure presented the major challenge at the frontier of research in organic natural product synthesis. [17] : 27-28 [1] : 519-521

Already in 1960, the research group of the biochemist Konrad Bernhauer [de] in Stuttgart had reconstituted vitamin B12 from one of its naturally occurring derivatives, cobyric acid, [26] by stepwise construction of the vitamin's nucleotide loop. [note 4] This 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. Therefore, cobyric acid was chosen as the target molecule for a total synthesis of vitamin B12. [6] : 183-184 [1] : 521 [8] : 367-368

Collaborative work [3] : 1456 [17] [30] : 302-313 of research groups at Harvard and at ETH resulted in two cobyric acid syntheses, both concomitantly accomplished in 1972, [31] [32] one at Harvard [3] , and the other at ETH. [10] [11] [12] A "competitive collaboration" [17] : 30 [33] : 626 of that size, involving 103 graduate students and postdoctoral researchers for a total almost 177 man-years, [13] : 9-10 is so far unique in the history of organic synthesis. [4] ( 0:36:25-0:37:37 ) The two syntheses are intricately intertwined chemically, [18] : 1571 yet they differ basically in the way the central macrocylic corrin ligand system is constructed. Both strategies are patterned after two model corrin syntheses developed at ETH. [8] [18] : 1496,1499 [34] : 71-72 The first, published in 1964, [28] 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 corrin-ring closure being attained between rings A and B. [35] The second model synthesis, published 1969, [36] explored a novel photochemical cycloisomerization process to create the direct A/D-ring junction as final corrin-ring closure between rings A and D. [37]

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. [3] : 145,176 [4] ( 0:36:25-0:37:37 ) The A/D approach to the synthesis, accomplished at ETH and finished at the same time as the A/B approach also in 1972, 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. [10] [11] [12] The paths of the two syntheses met in a common corrinoid intermediate. [11] : 519 [38] : 172 The final steps from this intermediate to cobyric acid were carried out in the two laboratories again collaboratively, each group working with material prepared via their own approach, respectively. [17] : 33 [18] : 1567

The beginnings Edit

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. [18] : 1501 In August 1961, [17] : 29 [13] : 7 the Harvard group began attacking the buildup of the B12 structure directly by aiming at the most complex part of the B12 molecule, the "western half" [1] : 539 that contains the direct junction between rings A und D (the A-D-component). Already in October 1960, [17] : 29 [13] : 7 [39] : 67 the ETH group had commenced the synthesis of a ring-B precursor of vitamin B12.

At the beginning, [40] progress at Harvard was rapid, until an unexpected stereochemical course of a central ring formation step interrupted the project. [41] [17] : 29 Woodward's recognition of the stereochemical enigma that came to light by the irritating behavior of one of his carefully planned synthetic steps became, according to his own writings, [41] 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 [42] as the source of ring D. [17] : 29 [18] : 1556

Joining forces: the A/B approach to cobyric acid synthesis Edit

By 1964, the ETH group had accomplished the first corrin model synthesis, [28] [27] : 275 and also the preparation of a ring-B precursor as part of a construction of the B12 molecule itself. [39] [43] Since independent progress of the two groups towards their long-term objective was so clearly complementary, Woodward and Eschenmoser decided in 1965 [18] : 1497 [17] : 30 to join forces and to pursue from then on the project of a B12 synthesis collaboratively, planning to utilize the ligand construction (ring coupling of components) strategy of the ETH model system. [2] : 283 [18] : 1555-1574

By 1966, the ETH group had succeeded in synthesizing the B-C-component ("eastern half" [1] : 539 ) by coupling their ring-B precursor to the ring-C precursor. [18] : 1557 The latter had also been prepared at Harvard from (−)-camphor by a strategy conceived and used earlier by A. Pelter and J. W. Cornforth in 1961. [note 6] 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. [18] : 1499

Early in 1967, the Harvard group accomplished the synthesis of the model A-D-component, [note 7] with the f-side chain undifferentiated, bearing a methyl ester function like all other side chains. [18] : 1557 From then on, the two groups systematically exchanged samples of their respective halves of the corrinoid target structure. [17] : 30-31 [18] : 1561 [32] : 17 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 (fig. 4). [note 2] The ETH group identified this totally synthetic corrinoid intermediate by direct comparison with a sample produced from natural vitamin B12. [2] : 301-303 [18] : 1563

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. Those for the C/D-coupling were successfully explored in both laboratories, the superior conditions were those found at Harvard, [2] : 290-292 [18] : 1562 while the method for the A/B-ring closure via an intramolecular version of the sulfide contraction [46] [36] [47] was developed at ETH. [2] : 297-299 [48] [18] : 1562-1564 Later it was shown at Harvard that the A/B-ring closure could also be achieved by thio-iminoester/enamine condensation. [2] : 299-300 [18] : 1564

By early 1971, the Harvard group had accomplished the synthesis of the final A-D-component, [note 8] containing the f-side chain carboxyl function at ring D differentiated from all the carboxyl functions as a nitrile group (as shown in 2 in fig. 4 see also fig. 3). [3] : 153-157 The A/D-part of the B12 structure incorporates the constitutionally and configurationally most intricate part of the vitamin molecule its synthesis is regarded as the apotheosis of the Woodwardian art in natural product total synthesis. [11] : 519 [12] : 1413 [18] : 1564 [33] : 626

The alternative approach to cobyric acid synthesis Edit

As far back as 1966, [37] : 1946 the ETH group had started to explore, once again in a model system, an 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 a thus far unknown bond reoganisation process. [37] : 1943-1946 This – if existing – would make possible the construction of cobyric acid from one single starting material. [6] : 185 [8] : 392,394-395 [33] Importantly, the 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! [8] : 395-397,399 [11] : 521 [49] [18] : 1571-1572

By May 1968, [18] : 1555 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 Pd complex, but not at all with corresponding Ni(II)- or cobalt(III)-A/D-seco-corrinate complexes. [36] [50] : 21-22 It also went smoothly in complexes of metal ions such as zinc and other photochemically inert and loosely bound metal ions. [8] : 400-404 [12] : 1414 These, after ring closure, could easily be replaced by cobalt. [8] : 404 These discoveries opened the door to what eventually became the photochemical A/D approach of cobyric acid synthesis. [7] : 31 [9] : 72-74 [37] : 1948-1959

Starting in fall of 1969 [51] : 23 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 [51] less than one and a half years [17] : 32 to translate the photochemical model corrin synthesis into a synthesis of dicyano-cobalt(III)-5,15-bisnor-a,b,d,e,g-pentamethyl-cobyrinate-c-N,N-dimethylamide-f-nitrile 2 (fig. 4), the common corrinoid intermediate on the way to cobyric acid. At Harvard, the very same intermediate 2 was obtained around the same time by coupling the ring-D differentiated Harvard A-D-component (available in spring 1971 [18] : 1564 footnote 54a [3] : 153-157 ) with the ETH B-C-component, applying the condensation methods developed earlier using the undifferentiated A-D-component. [1] : 544-547 [2] : 285-300

Thus, in spring 1971, [33] : 634 two different routes to a common corrinoid intermediate 2 (fig. 4) along the way to cobyric acid had become available, one requiring 62 chemical steps (Harvard/ETH A/B approach), the other 42 (ETH A/D approach). In both approaches, the four peripheral rings derived from enantiopure precursors possessing the correct sense of chiral, thereby circumventing major stereochemical problems in the buildup of the ligand system. [1] : 520-521 [7] : 12-13 [11] : 521-522 In the construction of the A/D-junction by the A/D-secocorrin→corrin cycloisomerization, formation of two A/D-diastereomers had to be expected. Using cadmium(II) as the coordinating metal ion led to a very high diastereoselectivity [51] : 44-46 in favor of the natural A/D-trans-isomer. [12] : 1414-1415

Once the corrin structure was formed by either approach, the three C-H-chirogenic centers at the periphery adjacent to the chromophore system turned out to be prone to epimerizations with exceptional ease. [2] : 286 [9] : 88 [3] : 158 [4] ( 1:53:33-1:54:08 ) [18] : 1567 This required a separation of diastereomers after most of the chemical steps in this advanced stage of the syntheses. It was fortunate indeed that, just around that time, the technique of high pressure liquid chromatography (HPLC) had been developed in analytical chemistry. [52] HPLC became an indispensable tool in both laboratories [32] : 25 [9] : 88-89 [3] : 165 [4] ( 0:01:52-0:02:00,2:09:04-2:09:32 ) its use in the B12 project, pioneered by Jakob Schreiber at ETH, [53] was the earliest application of the technique in natural product synthesis. [18] : 1566-1567 [38] : 190 [54]

The joint final steps Edit

The final conversion of the common corrinoid intermediate 2 (fig. 6) from the two approaches into the target cobyric acid required the introduction of the two missing methyl groups at the meso 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 the critical carboxyl at the ring-D f-side chain (see fig. 6). These steps were collaboratively explored in strictly parallel fashion in both laboratories, the Harvard group using material produced via the A/B approach, the ETH group such prepared by the photochemical A/D approach. [17] : 33 [18] : 1567

The first decisive identification of a totally synthetic intermediate on the way to cobyric acid was carried out in February 1972 with a crystalline sample of totally synthetic dicyano-cobalt(III)-hexamethyl-cobyrinate-f-amide 3 (fig. 6 [note 2] ), found to be identical in all data with a crystalline relay sample made from vitamin B12 by methanolysis to cobester 4, [note 9] followed by partial ammonolysis and separation of the resulting mixture. [55] : 44-45,126-143 [3] : 170 [57] : 46-47 At the time when Woodward announced the "Total Synthesis of Vitamin B12" at the IUPAC conference in New Delhi in February 1972, [3] : 177 the totally synthetic sample of the f-amide was one that had been made at ETH by the photochemical A/D approach, [17] : 35 [58] : 148 [18] : 1569-1570 while the first sample of synthetic cobyric acid, identified with natural cobyric acid, had been obtained at Harvard by partial synthesis from B12-derived f-amide relay material. [57] : 46-47 [3] : 171-176 Thus, the Woodward/Eschenmoser achievement around that time had been, strictly speaking, two formal total syntheses of cobyric acid, as well as two formal total syntheses of the vitamin. [57] : 46-47 [18] : 1569-1570

In the later course of 1972, two crystalline epimers of totally synthetic dicyano-cobalt(III)-hexamethyl-cobyrinate-f-amide 3, as well as two crystalline epimers of the totally synthetic f-nitrile, all prepared via both synthetic approaches, were stringently identified chromatographically and spectroscopically with the corresponding B12-derived substances. [18] : 1570-1571 [55] : 181-197,206-221 [5] ( 0:21:13-0:46:32,0:51:45-0:52:49 ) [59] At Harvard, cobyric acid was then made also from totally synthetic f-amide 3 prepared via the A/B approach. [57] : 48-49 Finally, in 1976 at Harvard, [57] totally synthetic cobyric acid was converted into vitamin B12 via the pathway pioneered by Konrad Bernhauer [de] . [note 4]

The publication record Edit

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 them appearing in print. Woodward discussed the A/B approach in lectures published in 1968, [1] and 1971, [2] culminating in the announcement of the "Total Synthesis of Vitamin B12" in New Delhi in February 1972 [3] : 177 published in 1973. [3] This publication, and lectures with the same title Woodward delivered in the later part of the year 1972 [4] [5] 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 in 1968 at the 22nd Robert A. Welch Foundation conference in Houston, [7] as well as in his 1969 RSC Centenary Lecture "Roads to Corrins", published in 1970. [8] He presented the ETH photochemical A/D approach to the B12 synthesis at the 23rd IUPAC Congress in Boston in 1971. [9] The Zürich group announced the accomplishment of the synthesis of cobyric acid by the photochemical A/D-approach in two lectures delivered by PhD students Maag and Fuhrer at the Swiss Chemical Society Meeting in April 1972, [10] Eschenmoser presented a lecture "Total Synthesis of Vitamin B12: the Photochemical Route" for the first time as Wilson Baker Lecture at the University of Bristol, Bristol/UK on May 8th, 1972. [note 10]

As a joint full publication of the syntheses by the Harvard and ETH groups (announced in [10] and expected in [11] ) had not appeared by 1977, [note 12] an article describing the final version of the photochemical A/D approach already accomplished in 1972 [10] [51] [55] [63] was published 1977 in Science. [12] [58] : 148 This article is an extended English translation of one that had already appeared 1974 in Naturwissenschaften, [11] based on a lecture given by Eschenmoser on January 21, 1974 at a meeting of the Zürcher Naturforschende Gesellschaft. Four decades later, in 2015, the same author finally published a series of six full papers describing the work of the ETH group on corrin synthesis. [64] [18] [65] [66] [35] [37] Part I of the series contains a chapter entitled "The Final Phase of the Harvard/ETH Collaboration on the Synthesis of Vitamin B12", [18] : 1555-1574 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 entire ETH work is documented in full experimental detail in publicly accessible Ph.D. theses, [39] [43] [60] [46] [61] [56] [62] [44] [48] [51] [55] [63] almost 1'900 pages, all in German. [67] Contributions of the 14 postdoctoral ETH researchers involved in the cobyric acid syntheses are mostly integrated in these theses. [12] : 1420 [64] : 1480 [13] : 12,38 The detailed experimental work at Harvard was documented in reports by the 77 postdoctoral researchers involved, with a total volume of more than 3'000 pages. [13] : 9,38 [note 11]

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, [45] T. Goto, [68] R. V. Stevens, [38] K. C. Nicolaou & E. G. Sorensen, [15] [19] summarized by J. Mulzer & D. Riether, [69] and G. W. Craig, [14] [33] besides many other publications where these epochal syntheses are discussed. [note 13]

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 a 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 component differed from the A-D-component used in the final synthesis by having as the functional group at the ring-D f-side chain a methyl ester group (like all other side chains) instead of a nitrile group.

Synthesis of the ring-A precursor

Starting point for the synthesis of the ring-A precursor was methoxydimethyl-indol H-1 synthesized by condensation of the Schiff base from m-anisidine and acetoin. Reaction with the Grignard reagent of propargyl iodide gave racemic propargyl indolenine rac-H-2 ring closure to the aminoketone rac-H-3 was brought about by BF3 and HgO in MeOH through intermediate rac-H-2a (electrophilic addition) with the two methyl groups forced into a cis-relationship by kinetic as well as thermodynamic reasons. [1] : 521-522

Resolution of the racemic aminoketone into the two enantiomers. Reaction of rac-H-3 with (−)-ethyl isocyanate permitted isolation by crystallization of one of the two diastereomeric urea derivatives formed (the other does not crystallize). Treatment of racemic ketone rac-H-3 (or of mother liquors from the previous crystallization) with (+)-ethyl isocyanate gave the enantiomer of the first urea derivative. Pyrolytic decomposition of each of these urea derivatives led to enantiopure aminoketones, the desired (+)-H-3, and (−)-H-3. [1] : 524-525 The "unnatural" (−)-enantiomer (−)-H-3 was used to determine the absolute configuration in various later steps, (−)-H-3 and enantio-intermediates derived from it were used as model compounds in exploratory experiments. [38] : 173 Woodward wrote regarding the unnatural enantiomer "our experience has been such that this is just about the only kind of model study which we regard as wholly reliable". [1] : 529

Determination of the absolute configuration of ring-A precursor (+)-H-3. For this determination, the levo-rotatory ("unnatural") enantiomer of aminoketone (−)-H-3 was used in order to save precious material: Acylation of the amino group of (−)-H-3 with chloroacetyl chloride, followed by treatment of the product H-3a with potassium t-butoxide in t-butanol, afforded tetracyclic keto-lactame H-3b. Its keto carbonyl was converted to a methylene group by desulfurization of the dithioketal of H-3b with Raney nickel to give lactam H-3c. Destruction of the aromatic ring by ozonolysis, involving the loss of a carboxyl function by spontaneous decarboxylation, led to bicyclic lactam-carboxylic acid H-3d. This material was identified with a product H-3h derived from (+)-camphor, possessing the same constitution and the absolute configuration as shown in formula H-3d. [1] : 525-526

The material for this identification of H-3d was synthesized from (+)-camphor as follows: cis-isoketopinic acid H-3e, obtained from (+)-camphor by an established route described in the literature, [70] was converted via the correspondig chloride, azide, and isocyanate to methyl-urethane H-3f. When treated with potassium t-butoxide in t-butanol and subsequently with KOH, H-3f was converted to H-3h, clearly by way of the intermediate H-3g. The identity of the two samples of H-3d and H-3h obtained by the two routes described, established the absolute configuration of (+)-H-3, the enantiomer of the ring-A precursor. [1] : 525-526

Synthesis of the ring-D precursor from (-)-camphor

(−)-Camphor was nitrosated in the α-position of the carbonyl group to give oxime H-4, Beckmann cleavage afforded via the corresponding nitrile the amide H-5. Hofmann degradation via an intermediary amine and its ring closure led to lactam H-6. Conversion of its N-nitroso derivative H-7 gave diazo compound H-8. Thermal decomposition of H-8 induced methyl migration to give cyclopentene H-9. Reduction to H-10 (LiAlH4), oxidation (chromic acid) to aldehyde H-11, Wittig reaction (carbomethoxymethylenetriphenylphosphorane) to H-12 and hydrolysis of the ester group finally gave trans-carboxylic acid H-13. [1] : 527-528 [note 14]

Coupling of ring-A and ring-D precursors to "pentacyclenone"

N-acylation of tricyclic aminoketone (+)-H-3 with the chloride H-14 of carboxylic acid H-13 gave amide H-15, which on treatment with potassium t-butoxide in t-butanol stereoselectively produced pentacyclic keto-lactam H-16 via an intramolecular Michael reaction which directs the indicated hydrogen atoms in trans relationship to each other. In anticipation of the Birch reduction of the aromatic ring, protective groups for the two carbonyl functions of H-16 were required, one for the ketone carbonyl group as ketal H-17, and the other for the lactam carbonyl as the highly sensitive enol ether H-20. The latter protection was achieved by treatment of H-17 with Meerwein salt (triethyloxonium tetrafluoroborate) to give iminium salt H-18, followed by conversion to orthoamide H-19 (NaOMe/MeOH), and finally expelling one molecule of methanol by heating in toluene. Birch reduction of H-20 (lithium in liquid ammonia, t-butanol, THF) provided tetraene H-21. Treatment with acid under carefully controlled conditions led first to an intermediate dione with the double bond in β,γ position which moved to the conjugated position in dione H-22, dubbed pentacyclenone. [1] : 528-531 [14] : 5

From "pentacyclenone" to "corrnorsterone"

The ethylene ketal protecting group in pentacyclenone H-22 was converted to the ketone group of H-23 by acid-catalyzed hydrolysis. [1] : 531 The dioxime primarily formed by reaction of diketone H-23 with hydroxylammonium chloride was regioselectively hydrolysed (nitrous acid/acetic acid) to the desired mono-oxime H-24. This is the oxime of the sterically more hindered ketone group, the nitrogen atom of which is destined to become the nitrogen of the target molecule's ring D. Crucial for this purpose is the configuration at the monoxime double bond, the hydroxyl group occupying the sterically less hindered position. [1] : 532 The C,C double bonds of both the cyclopentene and the cyclohexenone ring in H-24 were then cleaved by ozonolysis (ozone at 80 °C in MeOH, periodic acid), and the carboxylic group formed esterified with CH2N2) to diketone H-25. An intramolecular aldol condensation of the 1,5-dicarbonyl unit in MeOH using pyrrolidine acetate as the base, followed by tosylation of the oxime's hydroxyl group, afforded the cyclohexenone derivative H-26. A second ozonolysis in wet methyl acetate, followed by treatment with periodic acid and CH2N2 gave H-27. Beckmann rearrangement (MeOH, sodium polystyrene sulfonate, 2 hrs, 170 °C) produced regioselectively [1] : 532 lactam H-27a (not isolated) which reacted further in an amine-carbonyl condensation → aldol condensation cascade to the tetracycle H-28, [1] : 533-534 called α-corrnorsterone, implicating it as a "cornerstone" [1] : 534 in the synthesis of the desired A-D-component. [1] : 531-537

This compound required strongly alkaline conditions in order to open its lactam ring, but it was discovered that a minor isomer, also isolated from the reaction mixture, β-corrnorsterone H-29, undergoes this lactam ring opening under alkaline condition with great ease. [1] : 536 Structurally, the two isomers differ only in the orientation of the propionic acid side chain at ring A: the β-isomer has the more stable trans-orientation of this chain relative to the neighboring acetic acid chain formed after opening of the lactam ring. Equilibration of α-corrnorsterone H-28 by heating in strong base, followed by acidification and treatment with diazomethane, led to the isolation of pure β-corrnorsterone H-29 in 90 % yield. [1] : 537 The correct absolute configuration of the six contiguous asymmetric centers in β-corrnorsterone was confirmed by an x-ray crystal structure analysis of bromo-β-corrnorsterone [71] [1] : 529 with the "unnatural" configuration. [1] : 538 [14] : 8 [4] ( 0:49:20-0:50:42 )

Synthesis of the A-D-component carrying the propionic acid function at ring D as methoxycarbonyl group (model A-D-component)

Treatment of β-corrnorsterone H-29 with methanolic HCl cleaved the lactam ring and produced an enol ether derivative named hesperimine [note 15] H-30u. Ozonolysis to aldehyde H-32u, reduction of the aldehyde group with NaBH4 in MeOH to the primary alcohol H-33u and, finally, conversion of the hydroxy group via the corresponding mesylate gave bromide H-34u. This constitutes the model A-D-component, the one with an undifferentiated propionic acid function at ring D (i.e., bearing a methyl ester group like all other side chains). [1] : 539-540

Synthesis of the A-D-component carrying the propionic acid function at ring D as nitrile group

Conversion of β-corrnorsterone H-29 to the proper A-D-component H-34 [1] : 538-539 containing the carboxyl function of the ring D propionic acid side chain as a nitrile group, differentiated from all the other methoxycarbonyl groups, involved the following steps: treatment of H-29 with a methanolic solution of thiophenol and HCl afforded phenyl-thioenolether derivative H-30, which upon ozonolysis at low temperature gave the corresponding thioester-aldehyde H-31 and, when followed by treatment with liquid ammonia, the amide H-32. Reduction of the aldehyde group with NaBH4 to H-33, mesylation of the primary hydroxy group with methanesulfonic anhydride under conditions that also convert the primary amide group into the desired nitrile group and, finally, replacement of the methansulfonyloxy group by bromide produced A-D-component H-34 with the propionic acid function at ring D as nitrile, differentiated from all other such side chains. [1] : 539-540 [4] ( 1:01:56-1:19:47 )

The construction of the corrin chromophore with its three vinylogous amidine units constitutes – besides the direct single bond connection between the rings A and D – the central challenge to any attempt to synthesize vitamin B12. The very first approach to a total synthesis of vitamin B12 launched by Cornforth [45] : 261-268 was discontinued when confronted with the task of coupling synthesized ring precursors. [18] : 1493,1496 Coupling the Harvard A-D-components with the ETH B-C-component required extensive exploratory work, this in spite of the knowledge gained in the ETH model syntheses of less complex (i.e., less peripherally substituted) corrins. What might be called an epic engagement for formally making just two C,C bonds lasted from early 1967 [18] : 1557 until June 1970. [2]

Both at ETH and Harvard, extensive model studies on the coupling of simplified enaminoid analogues of the A-D-component with the (ring C) imino- and thio-iminoester derivative of the full-fledged B-C-component had consistently shown that a coupling of the Harvard and the ETH components could hardly be achieved by the method that had been so successful in the synthesis of the simpler corrins, namely, by an intermolecular enamino-imino(or thio-imino)ester condensation [7] [8] [18] : 1561 [62] : 41-58 [1] : 544 [4] ( 1:25:02-1:26:26 ) The outcome of these model studies determined the final structure type of a Harvard A-D-component: a structure capable of acting as a component of a C/D-coupling by sulfide contraction via alkylative coupling, [8] : 384-386 [47] i.e., the bromide H-34u. [7] : 18-22 [62] : 47,51-52 This method had already been implemented by the ETH group in the synthesis of the B-C-component. [33] : 16-19 [37] : 1927-1941 [18] : 1537-1540

An extensive search for optimal conditions, first for a C/D-coupling of a A-D-component with the ETH B-C-component E-19, then for conditions of the subsequent intramolecular A/B-corrin-ring closure was pursued in both laboratories, using the f-undifferentiated model A-D-component [note 7] H-34u [1] : 540 as a model. [2] : 287-300 [18] : 1561-1564 As the result of work by Yoshito Kishi at Harvard, [2] : 290 [18] : 1562 [14] : 11-12 and Peter Schneider at ETH, [48] : 12,22-29 [18] : 1563-1564 optimal conditions for the C/D-coupling were eventually found at Harvard, while the first and most reliable method for the corrin-ring closure between rings A and B was developed at ETH. [18] : 1562 The procedures of C/D-coupling and A/B-corrin-ring closure developed in this model series were later applied to the corresponding steps in the f-differentiated series as parts of the cobyric acid synthesis.

Synthesis of dicyano-cobalt(III)-5,15-bisnor-a,b,c,d,e,f,g-heptamethyl-cobyrinate from the ring-D undifferentiated model A-D-component

D/C coupling. [7] : 22-23 [2] : 287-292 [48] : 12,22-28 [18] : 1561-1562

The key problem in this step was the lability of the primary coupling product, thioether HE-35u, isomerizing to other thioethers at first not amenable to sulfide contraction in a reproducible procedure with acceptable yields. [2] : 287-290 [4] ( 1:26:59-1:32:00 ) Induced by potassium t-butoxide in THF/t-butanol under rigorously controlled conditions with strict exclusion of air and moisture, the model A-D-component H-34u smoothly reacted with the B-C-component E-19 [48] : 53-58 to give the sulfur-bridged coupling product HE-35u, named "thioether type I", in essentially quantitative yield. [2] : 287-288 However, this product could be isolated only under very carefully controlled conditions, since it equilibrates with extreme ease (e.g., chromatography, or traces of trifluoroacetic acid in methylenechloride solution) to the more stable isomeric thioether HE-36u (thioether type II) which contains, in contrast to thioether type I, the π-system of a conjugatively stabilized vinylogous amidine. [2] : 289 Depending on conditions, still another isomer HE-37u (thiother type III) was observed. [2] : 290 Starting with such mixtures of coupling products, at ETH a variety of conditions (e.g. methyl-mercury complex, BF3, triphenylphosphine [48] : 58-65 [2] : 291 ) were found to induce (via HE-38u) the contraction step to HE-39u in moderate yields. [18] : 1562 [2] : 287-292 With the choice of the solvent found to be crucial, [4] ( 1:34:52-1:35:12 ) the optimal procedure at Harvard was heating thiother type II HE-36u in sulfolane in the presence of 5.3 equivalents trifluoroacetic acid and 4.5 equivalents of tris-(β-cyanoethyl)-phosphine at 60 °C for 20 hours, producing HE-39u in up to 85% yield. [2] : 292 [48] : 65-72 Later it was discovered that nitromethane could also be used as solvent. [4] ( 1:34:52-1:35:13 ) [48] : 28

A/B-ring closure. [2] : 293-300 [48] : 12,29-39 [18] : 1562-1564

The problem of corrin-ring closure between rings A and B was solved in two different ways, one developed at ETH, the other pursued at Harvard. [32] : 19 Both methods correspond to procedures developed before in the synthesis of metal complexes [72] as well as free ligands [73] of simpler corrins. [7] : 25-28 [8] : 387-389 [18] : 1563 In the explorations of ring-closure procedures for the much more highly substituted A/B-seco-corrinoid intermediate HE-39u, the ETH group focused on the intramolecular version of the oxidative sulfide contraction method, eventually leading to the dicyano-cobalt(III)-complex HE48u. [48] : 29-39 [2] : 297-299 This first totally synthetic corrinoid intermediate was identified with a corresponding sample derived from vitamin B12. [18] : 1563

At Harvard, it was shown that the closure to the corrin macrocycle could also be realized by the method of thioiminoester/enamine condensation. [2] : 299-300 All reactions described here had to be executed on a very small scale, with ". the utmost rigour in the exclusion of oxygen from the reaction mixtures" [2] : 296 , and most of them also under strict exclusion of moisture and light, demanding very high standards of experimental expertise. [2] : 304

The major obstacle in achieving an A/B-corrin-ring closure was the exposure of the highly unstable ring B exocyclic methylidene double bond, which tends to isomerize into a more stable, unreactive endocyclic position with great ease. [48] : 86,97-98 [2] : 293-294 [3] : 161 [18] : 1562

The problem was solved at ETH [18] : 1562-1563 [48] : 29-39,126-135 by finding that treatment of the thiolactone-thiolactam intermediate HE-40u (obtained from HE-39u by reacting with P2S5 [48] : 73-83 ) with dimethylamine in dry MeOH (room temperature, exclusion of air and light) smoothly opens the thiolactone ring at ring B, forming by elimination of H2S the exocyclic methylidene double bond as well as a dimethylamino-amide group in the acetic acid side chain. [48] : 32-34,96-99 These conditions are mild enough to prevent double bond tautomerization to the thermodynamically more stable isomeric position in the ring. Immediate conversion with a Zn-perchlorate-hexa(dimethylformamide) complex in methanol to zinc complex HE-41u, followed by oxidative coupling (0,05 mM solution of I2/KI in MeOH, 3 h) afforded HE-42u. [48] : 100-105 Sulfide contraction (triphenylphosphine, trifluoroacetic acid, 85 °C, exclusion of air and light) followed by re-complexation with Zn(ClO4)2 (KCl, MeOH, diisopropylamine) led to the chloro-zinc complex HE-43u. [48] : 105-116 The free corrinium salt formed when HE-43u was treated with trifluoroacetic acid in acetonitrile was re-complexed with anhydrous CoCl2 in THF to the dicyano-cobalt(III)-complex HE-44u. [48] : 117-125 [2] : 295 Conversion of the dimethylamino-amide group in the acetic acid side chain of ring B into the corresponding methylester group (O-methylation by trimethyloxonium tetrafluoroborate, followed by decomposition of the iminium salt with aqueous NaHCO3) afforded totally synthetic 5,15-bisnor-heptamethyl cobyrinate HE-48u. [48] : 11,117-125 A crystalline sample of HE-48u was identified via UV/VIS, IR, and ORD spectra with a corresponding crystalline sample derived from vitamin B12 [48] : 42,135-141 [55] : 14,64-71,78-90 [2] : 287,301-303 [3] : 146-150 [74]

Later at Harvard, [2] : 299-300 the A/B-corrin-ring closure was also achieved by converting the thiolactone-thiolactame intermediate HE-40u to thiolactone-thioiminoester HE-45u by S-methylation of the thiolactam sulfur (MeHgOi-Pr, then trimethyloxonium tetrafluoroborate). The product HE-45u was subjected to treatment with dimethylamine (as in the ETH variant), forming the highly labile methylidene derivative HE-46u, which then was converted with anhydrous CoCl2 in THF to dicyano-cobalt(III) complex HE-47u, the substrate ready to undergo the (A⇒B)-ring closure by a thioiminoester/enamine condensation. A careful search at Harvard for reaction conditions led to a procedure (KO-t-Bu, 120 °C, two weeks) that gave corrin Co complex HE-44u, identical with and in overall yields comparable with HE-44u obtained by the ETH variant of the sulfide contraction procedure. [2] : 300 Since in corrin model syntheses such a C,C-condensation required induction by a strong base, its application in a substrate containing seven methylester groups was not without problems [18] : 1562 in a, milder reactions conditions were applied. [3] : 162

Synthesis of dicyano-cobalt(III)-5,15-bisnor-a,b,d,e,g-pentamethyl-cobyrinate-c-N,N-dimethylamide-f-nitrile (the common corrinoid intermediate) from the ring-D-differentiated A-D-component

The A-D-component H-34 [note 8] with its propionic acid function at ring D differentiated from all the other carboxyl functions as nitrile group had become available at Harvard in spring 1971. [51] : 23 As a result of the comprehensive exploratory work that had been done with the model A-D-component at Harvard and ETH, [2] : 288-292 [48] : 22-28 [18] : 1561-1562 joining the proper A-D-component H-34 with the B-C-component E-19 by three operations H-34 + E-19 →→ HE-36HE-39. [3] : 158-159 [4] ( 1:19:48-1:36:15 )

Closing the corrin ring was achieved in the sequence HE-39 (P2S5, xylene, γ-picoline)→ HE-40 [4] ( 1:36:45-1:37:49 ) → HE-41 [4] ( 1:37:51-1:42:33 ) → HE-42 [4] ( 1:42:35-1:44:34 ) → HE-43 (overall yield "about 60 %" [4] ( 1:44:35-1:46:32 ) ), and finally to cobalt complex HE-44. [4] ( 1:46:34-1:52:51 ) [3] : 160-166 Reactions in this sequence were based on the procedures developed in the undifferentiated model series. [2] : 293-300 [48] : 29-39 [18] : 1562-1564 Two methods were available for the A/B-ring closure: oxidative sulfide contraction within a zinc complex, followed by exchange of zinc by cobalt (ETH [3] : 162-165 ), or the Harvard alkylative variant of a sulfide contraction, [3] : 160-162 thio-iminoester/enamine condensation of the cobalt complex (improved reaction conditions: diazabicyclononanone in DMF, 60 °C, several hours [3] : 162 ). Woodward preferred the former one: [3] : 165 ". the oxidative method is somewhat superior, in that it is relatively easier to reproduce, . ". [4] ( 1:52:37-1:53:06 )

The corrin complex dicyano-cobalt(III)-5,15-bisnor-pentamethyl-cobyrinate-c-N,N-dimethylamide-f-nitrile HE-44 took up the role of the common corrinoid intermediate in the two approaches to cobyric acid synthesis: HE-44E-37. Due to the high configurational lability of C-H chirogenic centers C-3, C-8 and C-13 [4] ( 1:21:49-1:23:42,1:35:43-1:36:14,1:51:51-1:52:30 ) at the ligand periphery in basic or acidic milieu, separation by HPLC was indispensable for isolation, purification and characterization of pure diastereomers of this and the following corrinoid intermediates. [3] : 165-166 [9] : 88-89 [4] ( 1:53:07-2:01:24 )

Starting material for the synthesis of a ring-C precursor was (+)-camphorquinone H-35 [note 16] which was converted to the acetoxy-trimethylcyclohexene-carboxylic acid H-36 by BF3 in acetic anhydride, a reaction pioneered by Manasse & Samuel in 1902, [75] , already successfully applied in a previous synthesis of the ring-C precursor by Pelter and Cornforth. [note 6] Conversion of H-36 to amide H-37 was followed by its ozonolysis to peroxide H-38 which was reduced to the keto-succinimide H-46 by zinc and MeOH. Treatment with methanolic HCl gave lactam H-40, followed by thermal elimination of methanol to the ring-C precursor H-41 [1] : 540-542 [48] : 49-50 [14] : 4-5,15 This was found to be identical with the ring-C precursor E-13 prepared by a different route [note 5] at ETH. [61] : 32 [44] : 30,33-34,81

In the A/D approach to the synthesis of cobyric acid, the four ring precursors (ring-C precursor only formally so [12] : ref. 22 ) 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. [12] [11] The photochemical A/D-secocorrin→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 (cf. fig. 2). [36] [37] : 1943-1948

Syntheses of the ring-B precursor

Two syntheses of ring-B precursor (+)-E-5 were realized the one starting from 2-butanone was used further. [6] : 188 Two pathways for the conversion of the ring-B precursor into the ring-C precursor (+)-E-5(−)-E-13H-41 were developed, one at ETH, [44] : 15-39 [1] : 544 , and one at Harvard. [6] : 193 [note 17] These conversions turned out to be inadequate for producing large amounts of ring-C-precursor. [46] : 38 [18] : 1561 However, the pathway developed at ETH served the purpose of determining the absolute configuration of the ring-B precursor. [6] : 193 [61] : 32 Bulk amounts of ring-C precursor to be used for the production of the B-C-component at ETH [44] : 40 [6] : 193 [33] : 631 were prepared at Harvard from (+)-camphor by a route originally developed by Pelter and Cornforth. [note 6]

Ring-B precursor from 2-butanone and glyoxylic acid. Aldol condensation between 2-butanone and glyoxylic acid by treatment with concentrated phosphoric acid) gave stereoselectively (trans)-3-methyl-4-oxo-2-pentenoic acid E-1. [39] : 11-20,45-45 Diels-Alder reaction of E-1 with butadiene in benzene in the presence of SnCl4 afforded the racemate of the chiral Diels-Alder adduct E-2 which was resolved into the enantiomers by sequential salt formation with both (−)- and (+)-1-phenylethylamine. [43] : 22,59-62 The chirogenic centers of the (+)-enantiomer (+)-E-2 possessed the absolute configuration of ring B in vitamin B12. [60] : 35 [6] : 191 Oxidation of this (+)-enantiomer with chromic acid in acetone in the presence of sulfuric acid afforded the dilactone (+)-E-3 of the intermediary tricarboxylic acid E-3a. [43] : 35,72-73 Thermodynamic control of dilactone formation leads to the cis-configuration of the ring junction. [43] : 32-34 Elongation of the acetic acid side chain of (+)-E-3 by the Arndt-Eistert reaction (via the corresponding acid chloride and diazoketone) gave dilactone (+)-E-4. [61] : 15-16,65-67 Treatment of (+)-E-4 with NH3 in MeOH at room temperature formed a dual mixture of isomeric lactam-lactones in a ratio of 2:1, with ring-B precursor (+)-E-5 predominating (isolated in 55% yield). [46] : 12-17,57-63 [6] : 186-188 [12] [1] : 542-543 The isomeric lactam-lactone could be isomerized to (+)-E-5 by treatment in methanolic HCl. [61] : 24-26,81-84

Alternative synthesis of racemic ring-B precursor from Hagemann's ester: implementation of the amidacetal-Claisen rearrangement. Five steps were needed to transform Hagemann's ester rac-E-6 into the racemate of the lactam-lactone rac-E-5 form of the ring-B precursor. [60] : 14-31 [6] : 188-190 The product of the C-methylation step rac-E-6rac-E-7 (NaH, CH3I) was purified via its crystalline oxime. The cis-hydroxy-ester (configuration secured by lactone formation [60] : 64 ) resulting from the reduction step rac-E-7rac-E-8 (NaBH4) had to be separated from the trans isomer. The thermal rearrangement rac-E-8rac-E-9 constitutes the implementation of the amidacetal-Claisen rearrangement in organic synthesis, [76] [60] : 36-49 a precedent to Johnson's orthoester-Claisen and Ireland's ester-enolate rearrangement. [77] Ozonolysis (O3/MeOH, HCOOH/H2O2) of the N,N-dimethylamide ester rac-E-9 afforded dilactone acid rac-E-10, from which two reactions led to lactam-lactone methylester rac-E-7, the racemate of ring-B precursor (+)-E-7. [60] : 57-67

Determination of absolute configuration of (+)-ring-B precursor via its conversion into the (+)-ring-C precursor

The conversion of ring-B precursor into the ring-C precursor was based on a reductive decarbonylation of thiolactone E-12 with chloro-tris-(triphenylphosphino)-rhodium(I). [44] : 14-32 [6] : 191-193 [12] Treatment of a methanolic solution of ring-B precursor (+)-E-5 with diazomethane in the presence of catalytic amounts of sodium methoxide, followed by thermal elimination of methanol, gave methylidene lactam E-11, which was converted to the thiolactone E-12 with liquid H2S containing a catalytic amount of trifluoracetic acid. [44] : 15-16,56-58 Heating E-12 in toluene with the Rh(I)-complex afforded ring-C precursor (−)-E-13 besides the corresponding cyclopropane derivative E-14. Ring-C precursors prepared via this route and from (+)-camphor at Harvard [1] : 540-542 were found to be identical: (−)-E-13H-41. [44] : 33-34

Ozonolysis of ring-C precursor (−)-E-13 gave succinimide derivative (−)-E-15. [44] : 33-35,88-89 This succinimide was found to be identical [6] : 193 [1] : 543-544 in constitution and optical rotation (i.e., configuration) with the corresponding succinimide derived from ring C of Vitamin B12, isolated after ozonolysis of crystalline heptamethyl-cobyrinate (cobester [note 9] ) prepared from Vitamin B12. [56] : 9-18,67-70

The approach pursued at Harvard for conversion of ring-B precursor into ring-C precursor was based on a photochemical degradation of the acetic acid side chain carboxyl group, starting from (+)-E-7 prepared at ETH. [note 17]

Coupling of ring-B and ring-C precursors to the B-C-component. Implementation of the sulfide contraction C,C-condensation method

The iminoester/enamine C,C-condensation method for constructing the vinylogous amidine system, developed in the model studies on corrin synthesis, [28] [35] failed completely in attempts to create the targeted C,C-bond between ring-B precursor (+)-E-5 with ring-C precursor (−)-E-13 to give the B-C-component E-18. [6] : 193-194 [8] : 379 [1] : 544 The problem was solved by "intramolecularization" of the bond formation process between the electrophilic (thio)iminoester carbon and the nucleophilic methylidene carbon of the enamine system through first oxidatively connecting these two centers by a sulfur bridge, and then achieving the C,C-bond formation by a now intramolecular thio-iminoester/enamine condensation with concomitant transfer of the sulfur to a thiophile. [6] : 194-197 [8] : 380-386 [18] : 1537-1538

Conversion of lactam (+)-E-5 into the corresponding thiolactam E-16 (P2S5), [46] : 20-23,74-75 oxidation of E-16 with benzoyl peroxide in the presence of ring-C precursor (−)-E-13 (prepared at Harvard by the Cornforth route [note 6] ), followed by heating the reaction product E-17 in triethylphosphite (as both solvent and thiophile) afforded B-C-component E-18 as a (not separated) mixture of two epimers (regarding the configuration of the propionic side chain at ring B) in up to 80 % yield. [46] : 38-43,96-102 [33] : 16-19 [8] : 381-383 [48] : 20-21,50-52

The bracketed formulae in the reaction scheme illustrate the type of mechanism operating in the process: E-16a = primary coupling of E-12 and E-10 to E-13 E-17a = extrusion of the sulfur atom (captured by thiophile) to E-14, where it is left open whether this latter process occurs at the stage of the episulfide. This reaction concept developed at this stage, dubbed sulfide contraction, [6] : 199 [47] [18] : 1534-1541 [37] : 1927-1941 turned out to make possible the construction of all three meso-carbon bridges of the vitamin's corrin ligand in both approaches of the synthesis. [12] [11] [2] : 288-292,297-300 [3] : 158-164

The conversion of bicyclic lactone-lactam E-18 into the corresponding thiolactone-thiolactam E-20 was brought about by heating with P2S5/4-methylpyridine in xylene at 130 °C milder condition produced thiolactam-lactone E-19, used for coupling with the Harvard A-D-components. [51] : 73-83

Synthesis of ring-D precursor for the A/D approach

The starting material for the ring-D precursor, [61] : 40-61 [63] : 17-22 [12] the (−)-enantiomer of the dilactone-carboxylic acid (−)-E-3, was prepared from the (−)-enantiomer of the Diels-Alder adduct (−)-E-2 [note 18] by oxydation with chromic acid/sulfuric acid in acetone. [43] : 35,72-73 Treatment of (−)-E-3 with NH3 in MeOH gave a lactone-lactam-acid which was esterified with diazomethane to the ester E-21, [61] : 104-110 the lactone ring of which was opened with KCN in MeOH to give E-22. [61] : 114-116 Conventional conditions of an Arndt-Eistert reaction (SOCl2: acid chloride, then CH2N2 in THF: diazoketone, treated with Ag2O in MeOH) led to an – unforeseen, yet useful – ring closure of the originally formed chain-elongated ester through participation of the cyano group as a neighboring electrophile, affording the bicyclic enamino-ester derivative E-23. [61] : 116-120 Hydrolysis with aqueous HCl, accompanied by decarboxylation, and re-esterification with diazomethane gave keto-lactam-ester E-24. [61] : 123-126 [63] : 40-41 Ketalization ((CH2OH)2, CH(OCH3)3, TsOH) of E-24 and conversion of this lactam-ester to thiolactam E-25 (P2S5) was followed by reductive removal of the sulfur with Raney nickel, acetylation of the amino group, and hydrolysis of the ketal (AcOH) to afford E-26. [63] : 42-59 This was converted by deacetylation of the amino group with HCl, and then by treatment with NH2OH/HCl, MeOH/NaOAc into oxime E-27. Beckmann fragmentation (HCl, SOCl2 in CHCl3, N-polystyryl-piperidine) of this oxime E-27 produced imino-nitrile E-28, [63] : 60-67 which, when treated with bromine (in MeOH, phosphate buffer pH 7.5, -10 °C) gave ring-D precursor E-29. [51] : 84-88

Conversion of the ring-B precursor into the ring-A precursor for the A/D approach

The ring-A precursor (−)-E-31 required in the A/D approach is a close derivative of ring-B precursor (+)-E-5. Its preparation from (+)-E-5 required opening of the lactone group (KCN in MeOH), followed by re-esterification with diazomethane to E-30, then conversion of the lactam group into a thiolactam group with P2S5 to yield (−)-E-31. [51] : 63-72 [12]

Coupling of the B-C-component with ring-D and ring-A precursors

The most efficient way of attaching the two rings D and A to the B-C-component E-18 was to convert E-18 directly into its thiolactam-thiolactone derivative E-20 and then to proceed by first coupling ring-D precursor E-29 to ring C, and then ring-A precursor E-31 to ring B, both by the sulfide contraction method. [51] : 26-31 [9] : 80-83 [12] The search for the reaction conditions for these attachments was greatly facilitated by exploratory work done on the two sulfide contraction steps in the A/B approach model study. [51] : 27 [48] : 22-39 [2] : 285-300

Attachment of ring-D precursor E-29 to the ring-C thiolactam in E-20 by sulfide contraction via alkylative coupling (t-BuOK in t-BuOH/THF, tris-(β-cyano-ethyl)-phosphin/CF3COOH in sulfolane) afforded the B/C/D-sesqui-corrinoid E-32. [51] : 89-97 To attach ring-A precursor E-31, the ring B of E-32 was induced to expose its exocyclic methylidene double bond by treatment with dimethylamine in MeOH (using the method [note 19] developed by Schneider [48] : 32-34 ) forming E-33 [51] : 108-115 which was subjected to the following cascade of operations: [51] : 130-150 iodination (N-iodosuccinimide, CH2Cl2, 0°), coupling with the thiolactam sulfur of the ring-A precursor E-31 [(CH3)3Si]2N-Na in benzene/t-BuOH), complexation (Cd(ClO4)2 in MeOH), treatment with triphenylphosphine/CF3COOH in boiling benzene (sulfide contraction) and, finally, re-complexation with Cd(ClO4)2/N,N-diisopropylethylamine in benzene/MeOH). These six operations, all carried out without isolation of intermediates, gave A/D-seco-corrin complex E-34 as mixture of peripheral epimers (separable via HPLC [51] : 143-147 ) in 42-46 % overall yield. [51] : 139

A/D-corrin-ring closure by the photochemical A/D-seco-corrin→corrin cycloisomerization to dicyano-cobalt(III)-5,15-bisnor-a,b,d,e,g-pentamethyl-cobyrinate-c-N,N-dimethylamide-f-nitrile (the common corrinoid intermediate)

The conditions and prerequisites for the final (A⇒D)-corrin-ring closure were taken over from extensive corrin model studies. [36] [78] [9] : 71-74,83-84 [18] : 1565-1566 [37] : 1942-1962 Problems specific to the cobyric acid synthesis that had to be tackled were: [9] : 84-88 the possible formation of two diastereomeric A/D-trans-junctions in the ring closure, [51] : 37-38 exposure of the methylidene double bond at ring A of the A/D-seco-corrin E-34 in a labile Cd complex, [51] : 35-36 [18] : 1566 and epimerizability of the peripheral stereogenic centers C-3, C-8 and C-13 before and after ring closure. [51] : 39 [3] : 148-150

In the application of this novel process in the A/D approach of the cobyric acid synthesis, [9] : 86-95 [51] : 39-53 [12] : 1419 the reaction proceeded most efficiently and with highest coil stereoselectivity in favor of the natural A/D-trans junction in an A/D-seco-corrin cadmium complex. [51] : 42-45 [3] : 166 Treatment of Cd-complex E-34 as mixture of peripheral epimers with 1,8-Diazabicyclo(5.4.0)undec-7-ene in sulfolane at 60 °C under strict protection against light to eliminate the cyano group at ring A, directly followed by re-treatment with Cd(ClO4)2, led to labile [51] : 172 A/D-seco-corrin complex E-35 as a mixture of peripheral epimers. This was directly subjected to the key step, the photochemical ring closure reaction under rigorous exclusion of air: [51] : 40 visible light, under Argon, MeOH, AcOH, 60° C. Product of the A/D-ring closure was the free corrin ligand E-36, as the originally formed Cd-corrinate – in contrast to the Cd-seco-corrinate E-35 – decomplexes in the reaction medium. [51] : 173 [12] : 1419 Corrin E-36 was immediately complexed (CoCl2, [18] : 1499-1500,1563-64 KCN, air, H2O, CH2Cl2) and finally isolated (thick-layer chromatography) as mixture of peripheral epimers in 45-50 % yield over four operations: [51] : 169-179 the common corrinoid intermediate dicyano-cobalt(III)-complex E-37HE-44. [note 20]

HPLC analysis of this mixture E-37 showed the presence of six epimers with natural ligand helicity (Σ 95%, CD spectra), among them 26% of natural diastereomer 3α,8α,13α, and an equal amount of its C-13 neo-epimer 3α,8α,13β. [51] : 46,179-186 [12] : 1414 Two HPLC fractions (Σ 5%) contained diastereomers with unnatural ligand helicity, as shown by inverse CD spectra. [51] : 42-43 Product mixtures from several such cycloisomerizations were combined for preparative HPLC separation and full characterization of the 14 isolated diastereomers of E-37 [51] : 207-251 (of 16 theoretically possible, regarding helicity and the epimeric centers C-3, C-8, C-13 [51] : 39 ).

In an analytical run, the mixture of cadmium-seco-complex epimers E-35 was separated by HPLC (in the dark) into the natural chloro-cadmium-3α,8α,13α-A/D-seco-corrinate diastereomer (ααα)-E-35 and four other epimer fractions [51] : 281-293 Upon irradiation [51] : 53 [12] and following cobaltation, (ααα)-E-35 produced E-37 in yields of 70-80% as an essentially dual mixture of mainly the 3α,8α,13α epimer, besides some 3α,8α,13β epimer. Less than 1% of fractions with unnatural coil were formed (HPLC, UV/VIS, CD). [51] : 293-300

Mechanistically, the photochemical A/D-seco-corrin corrin cycloisomerization involves an antarafacial sigmatropic shift of the α-hydrogen of the CH2 position C-19 at ring D to the CH2 position of the methylidene group at ring A within a triplet excited state, creating a transient 15-center-16-electron π-system (see E-35a in fig. 27) that antarafacially collapses between positions C-1 and C-19 to the corrin system. [36] [37] : 1946,1967-1993 [79] The coil selectivity of the ring closure in favor of the corrin ligand's natural helicity is interpreted as relating to the difference in steric hindrance between the g-methoxycarbonyl acetic acid chain at ring D and the methylidene region of ring A in the two possible helical coil configurations of the A/D-seco-corrin complex (fig. 28). [51] : 38 [37] : 1960-1962

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, the ETH group working with material produced by the A/D approach, and the Harvard group with that from the A/B approach. [63] : 15 [55] : 22 [57] : 47 [14] : 12 [18] : 1570-1571 What the two groups in fact accomplished thus were the common final steps of two different syntheses. [11] [12]

The tasks in this end phase of the project were the regioselective introduction of methyl groups at the two meso positions C-5 and C-15 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 execution, including unforeseen pitfalls like a dramatic loss of precious synthetic material in the so-called "Black Friday" (July 9, 1971). [55] : 39-40,107-118 [9] : 97-99 [3] : 168-169 [5] ( 0:07:54-0:09:33 ) [18] : 1568-1569

This introduction of methyl groups could draw on exploratory studies on model corrins [7] : 13-14 [8] : 375-377 [80] [18] : 1528,1530-1532 as well as on exploratory experiments carried out at ETH on cobester [note 9] and its (c→C-8)-lactone derivative. [55] : 27-43 Chloromethyl benzyl ether alkylated the meso position C-10 of cobester, but not that of the corresponding lactone, the difference in behavior reflecting the difference in steric hindrance exerted on the meso position C-10 by its neighboring substituents. [55] : 37-39 This finding was decisive for the choice of the substrate to be used for introducing methyl groups at meso positions C-5 and C-10 of E-37/HE-44. [9] : 96-99 [55] : 19 [3] : 167 [18] : 1567-1568 In this final phase of the synthesis, HPLC again turned out to be absolutely indispensable for separation, isolation, characterization and, above all, identification of pure isomers of dicyano-cobalt(III)-complexes of totally as well as partially synthetic origin. [9] : 96-102 [3] : 165 [55] : 61-63 [5] ( 0:21:13-0:25:28 ) [18] : 1566-1567

The first step was to convert the c-N,N-dimethylcarboxamide group of E-37/HE-44 into the (c→C-8)-lactone derivative E-38/HE-45 by treatment with iodine/AcOH effecting iodination at C-8, followed by intramolecular O-alkylation of the carboxamide group to an iminium salt that hydrolyses to the lactone. [63] : 23,90-108 [3] : 166-167 [4] ( 2:02:18-2:09:02 ) This lactonization leads to cis-fused rings. [55] : 19 [5] ( 0:09:34-0:10:43 ) Reaction of (c→C-8)-lactone E-38/HE-45 with chloromethyl benzyl ether in acetonitrile in the presence of LiCl gave, besides mono-adduct, the bis-benzyloxy adduct E-39/HE-46. When treated with thiophenol, this produced the bis-phenylthio-derivative E-40/HE-47. Treatment with Raney nickel in MeOH not only set free the two methyl groups at the meso positions, but also reductively opened the lactone ring to the free c-carboxyl group at ring B, producing the correct α-configuration at C-8. Esterification of c-carboxyl with diazomethane afforded hexamethylester-f-nitrile E-41/HE-48. [55] : 19-21,39-43,146-205 [3] : 167-169 For steric reasons, only the predominant [55] : 19 [63] : 24 [4] ( 2:08:20-2:09:02 ) C-3 α-epimer (with the C-3 side chain below the plane of the corrin ring) reacted to a 5,15-disubstituted product E-38/H-45, the reaction thus amounting to a chemical separation of the C-3 epimers. [55] : 40 [5] ( 0:12:51-0:14:33,0:15:56-0:16:24 )

In improved procedures developed at Harvard later in 1972, [18] : 1569 footnote 62 the reagent chloromethyl benzyl ether was replaced by formaldehyde/sulfolane/HCl in acetonitrile for the alkylation step, and Raney nickel in the reduction step was replaced by zinc/acetic acid to give E-41/HE-48. [5] ( 0:00:32-0:21:12 )

Concentrated H2SO4 at room temperature converted the nitrile function of pure (3α,8α,13α)-E-41/HE-48 into the primary f-amide group of E-42/HE-49, besides partial epimerization at C-13 [9] : 100-103 [55] : 21,134-136 [3] : 150-151,169-170 an alternative procedure for the selective f-nitrile→f-amide conversion (BF3 in CH3COOH) later developed at Harvard proceeded without epimerization at C-13. [18] : 1569 footnote 62 [5] ( 0:46:40-0:49:45 ) [55] : 21 A crystalline sample of the 3α,8α,13α-epimer of dicyano-cobalt (III)-a,b,c,d,e,g-hexamethyl-cobyrinate-f-amide E-42/HE-49, isolated by HPLC, was the first totally synthetic intermediate to be chromatographically and spectroscopically identified with a relay sample made from vitamin B12. [55] : 136-141 [3] : 170

In the remaining steps of the synthesis, only epimerization at C-13 played an important role, [55] : 19-21 with 13α being the configuration of the natural corrinoids, and 13β known as neo-epimers of vitamin B12 and its derivatives [3] : 169-170 [81] these are readily separable by HPLC. [5] ( 0:19:30-0:20:21 ) [55] : 135,208-209

In the course of 1972, comprehensive identifications (HPLC, UV/VIS, IR, NMR, CD, mass spectra) of crystalline samples of totally synthetic intermediates with the corresponding compounds derived from vitamin B12 were carried out in both laboratories: individually compared and identified were the 3α,8α,13α and 3α,8α,13β neo-epimer of f-amide E-42/HE-49, as well as the corresponding pair of C-13-epimeric nitriles E-41/HE-48. [55] : 206-221 [57] : 46-47 [5] ( 0:27:28-0:46:32 ) All these dicyano-cobalt(III)-complexes are soluble in organic solvents [56] : 11 in which the separation power of HPLC by far exceeds that of analytical methods operating in water, [55] : 44-45 the solvent in which cobyric acid was to be identified, and where it exists as two easily equilibrating aquo-cyano complexes, epimeric regarding the position of the two non-identical axial Co ligands. [63] : 196-197 [57] : 49-60

These thorough identifications of the totally synthetic with partially synthetic materials mark the accomplishment of the two syntheses. They also reciprocally provided structure proof for a specific constitutional isomer isolated from a mixture of isomeric mono-amides formed in the partial ammonolysis of the B12-derived cobester, [note 9] tentatively assigned to be the 3α,8α,13α-f-amide E-42/HE-49 (see fig. 30). [56] : 9-18,67-70 [55] : 226-239 [59]

The final task of reaching cobyric acid from f-amide E-42/HE-49 required the critical step of hydrolysing the singular amide function into a free carboxyl function without touching any of the six methoxycarbonyl groups around the molecule's periphery. Since exploratory attempts by the conventional method of amide hydrolysis via nitrosation led to detrimental side reactions at the chromophore, a novel way of "hydrolysing" the f-amide group without touching the six methylester groups was conceived and explored at ETH: treatment of f-amide E-42/HE-49 (B12-derived relay material) with the unusual reagent α-chloro-propyl-(N-cyclohexyl)-nitrone [82] and AgBF4 in CH2Cl2, then with HCl in H2O/dioxane, and finally with dimethylamine in isopropanol afforded the f-acid E-43/HE-50 in 57% yield. [63] : 24-25,159-172 [3] : 170-172 [5] ( 0:53:17-0:58:30 ) Sustained experimentations at Harvard eventually showed the nitrosation method to be successful (N2O4, CCl4, NaOAc) and to produce the f-carboxyl group even more effectively. [3] : 172-173 [5] ( 0:58:19-0:59:15 )

It was also at Harvard that conditions for the last step were explored, conversion of all remaining ester groups into primary amide groups by ammonolysis. Liquid ammonia in ethylene glycol, in the presence of NH4Cl and the absence of oxygen, converted f-carboxy-hexamethylester E-43/HE-50 into f-carboxy-hexa-amide E-44/HE-51 (= cobyric acid). [3] : 173-175 [55] : 24 This was crystallised and shown both as the α-cyano-β-aquo and the α-aquo-β-cyano form to be chromatographically and spectroscopically identical with the corresponding forms of natural cobyric acid. [5] ( 0:59:53-1:09:58 ) [3] : 175-176 [63] : 26-27,196-221 At Harvard, the transformation E-43/HE-50E-44/HE-51 was eventually carried out starting with f-amide that had been obtained by total synthesis via the A/B approach. [57] : 47-61 The ETH group contented itself with a corresponding f-amide → cobyric acid conversion and subsequent cobyric acid identification where the actual starting material f-amide was derived from vitamin B12. [55] : 22 [63] : 15 [12] : footnote 45 [18] : 1570-1571


Best Forms of Vitamin B12: Methylcobalamin vs Cyanocobalamin vs Hydroxocobalamin vs Adenosylcobalamin

Let’s explore all forms of vitamin B12, one by one.

Cyanocobalamin

(Also CN-Cbl, or Cyano B12)

Cyano B12 is a cheap, synthetic, slightly-toxic, inactive form of B12 that is made with a cyanide donor and is used commercially. It is the most stable form, because the cyanide molecule has the greatest attraction to the cobalamin, so it protects it from conditions like very high temperatures. Cyano B12, however, doesn’t absorb well and requires a methyl group to detoxify it before it can ever convert to a useful form.

When cyano B12 does absorb, it converts to hydroxo B12 (hopefully discarding of the cyanide in the process). Then, it converts to methyl B12 and adenosyl B12. Taken orally, cyano B12 absorption is drastically reduced if you have any gastric acid issues.

We can’t recommend this form to nobody.

Sure, it’s cheap, but it comes with a price. Again, our body must use a methylation reaction to cleave the cyanide out of this form. Only then it can be converted to an active form that you can use and absorb. This is a demanding process:

Although the amount of cyanide is considered toxicologically insignificant, humans must remove and detoxify the cyanide molecule, reduce the cobalamin to its usable +1 oxidation state, and then enzymatically convert the cobalamin into one of two metabolically active coenzyme forms. Nutritional inadequacies, enzyme defects, and pathological changes to tissues can all contribute to a reduced ability of the body to accomplish the synthesis of the active forms of vitamin B12 from CN-Cbl.

The Coenzyme Forms of Vitamin B12

Methylation is one way through which your body detoxifies. But it requires methyl molecules, which are often in low supply (modern life is full of toxins), especially in folks with a B12 deficiency. This is why people with methylation issues (like autistic children) can get worse on CN-Cbl but not on other forms of B12.

Then why does cyano B12 even exist?

Commercial cyano B12 exists because, when making hydroxo B12 from bacteria, some of the cobalamin binds to cyanide during the charcoal filtration process.

If the indiscriminate dumping of industrial cyanide waste continues unchecked with the inherent risk of pollution of food and water supplies there may well come a time when more widespread chronic cyanide neurotoxicity occurs in the Western hemisphere from a dietary source in persons with a genetic or acquired error of cyanide or vitamin B12 metabolism.

We all get cyanide into our systems – pollution, barbecue, bonfire, second hand smoke, even almonds. The body always has to detoxify it. By taking cyano B12, you’re further depriving your body of methyl groups, a natural antidote, an antagonist for toxins.

Also, some people may have conditions which inhibit them from converting this form of B12 into the active forms that you can absorb. In these cases, serum B12 levels will shoot up (the blood test counts inactive B12 too), but there will be a deficiency of adenosyl B12 and methyl B12 in the cells, tissues and body fluids.

It takes more than 48 hours for cyano B12 to convert to usable methyl B12. Even then, only a small amount is converted. And remember, when it does convert, it requires the interaction (possibly the depletion) of glutathione and other agents. For that reason, it can make Leber’s optic atrophy worse, and nobody should ever use it in that case.

Why can you even buy cyano B12 then?

Despite all that, cyano B12 is the most prescribed form. Reason? It’s cheap. But it’s also the least safe, least effective, and most demanding type of B12.

Not natural to mammals whatsoever.

Why would you try to improve your health with something that requires the depletion of other crucial substances? It makes no sense, especially when there are other, better forms of vitamin B12 that don’t cost much at all.

Methylcobalamin

(also Mecobalamin, MeCbl, MetCbl, MetB12, MeB12, or Methyl B12)

Methylcobalamin as an endogenous coenzyme plays as important a role in transmethylation of methionine synthetase in the synthesis of methionine from homocysteine. It is transported to nerve cell organelles, and promotes nucleic acid and protein synthesis. Transportation of methylcobalamin to nerve cell organelles is better than both hydroxocobalamin and cyanocobalamin. It is involved in the synthesis of thymidine from deoxyuridine, promotion of deposited folate utilisation and metabolism of nucleic acid. It promotes nucleic acid and protein synthesis more than adenosylcobalamin does.

Methylcobalamin promotes axonal transport and axonal regeneration. Methylcobalamin normalises axonal skeletal protein transport in nerve cells in animal models of diabetes mellitus. It exhibits neuropathological and electrophysiological protective effect on nerve degeneration in animal models of axonal degeneration (adriamycin, acrylamide, and vincristine-induced neuropathies) with spontaneous diabetes mellitus. It promotes myelination (phospholipid synthesis). Methylcobalamin promotes the synthesis of lecithin, the main constituent of medullary sheath lipids, and increases myelination of neurons in tissue culture, more than adenosylcobalamin does. It restores delayed synaptic transmission and diminished neurotransmitters to normal.

Oxford Biosciences.

Methylcobalamin is one of the two active forms of vitamin B12. It reduces homocysteine and generates SAMe (S-adenosyl methionine), the most crucial methyl donor we have. In other words, it supplies methyl groups for the crucial chemical reactions we discussed.

Where methyl B12 shows its greatest utility is with people suffering from degenerative neurological symptoms, where it is often the only promising treatment. It bypasses several potential issues in the absorption cycle and helps relieve or reverse symptoms. It is the best form to help regenerate nerves and treat peripheral neuropathies.

High doses of methyl B12 have been used to treat amytropic lateral sclerosis and Parkinson’s. It also improves visual and auditory symptoms in multiple sclerosis, and it improves memory and intellectual function in Alzheimer’s patients.

Is methyl B12 really so effective?

In fact, so much that Japan uses it almost exclusively to treat B12 deficiency. Of all vitamin B12 forms, the science on methyl B12 is the most breathtaking:

When hemolytic hyperchromic anemia and impairment of hematopoiesis in the bone marrow were induced in rabbits, a decrease in methylcobalamin in the blood serum was observed. Methyl B12 administration completely normalized some hematopoiesis and blood patterns, improved the ratio between the B12 forms, and completely regenerated total B12 content. Adenosyl B12, the other active form, showed a much lower effect.

As you see, methyl B12’s applications are endless

Under experimental conditions, methyl B12 (adenosyl and hydroxo B12 as well, actually) inhibited HIV-1 infection of normal human blood lymphocytes and monocytes.

In one study, methyl B12 at daily doses of 6,000mcg for four months improved sperm count by 37.5%. In another one, at doses of 1,500mcg a day for 4-24 weeks, it increased sperm concentrations in 38% of cases, total sperm count in 54% of cases, and sperm motility in 50% of cases. This is incredible.

One study reported a case of a 48 year old woman with motor weakness, dementia, sensory disturbances, and widespread coarse hair. Classic B12 deficiency symptoms. In response to methyl B12 shots (500mcg every other day), her weird sensations resolved, dementia was reduced, hand grip strengthened, hair texture normalized, and she was now able to walk on tiptoe. Results like this are not by any means rare.

Also, remember we talked about B12 converting homocysteine to methionine?

This is specfically the job of methylcobalamin, and it’s why high levels of homocysteine can be a sign of low methyl B12 levels. In one study, high homocysteine levels went from 14.7 down to 10.2 nmol/ml following methyl B12 shots. Because of this effect, methyl B12 is useful in children with autism, and in reducing cognitive decline and cardiovascular outcomes in older patients.

On methyl B12 and sleep

The science on methyl B12 and sleep is really promising. We don’t fully understand how, but it seems like this type of B12 could modulate the synthesis of melatonin, a hormone involved in regulating your sleep-wake cycle:

Eight young males were subjected to a single blind cross-over test to see the effects of vitamin B12 (methylcobalamin) on the phase-response of the circadian melatonin rhythm to a single bright light exposure. VB12 (0.5 mg/day) or vehicle was injected intravenously at 12:30 h for 11 days, which was followed by oral administration (2 mg x 3/day) for 7 days. A serial blood sampling was performed under dim light condition (less than 200 lx) and plasma melatonin rhythm was determined before and after a single bright light exposure (2500 lx for 3 h) at 07:00 h. The melatonin rhythm before the light exposure showed a smaller amplitude in the VB12 trial than in the placebo. The light exposure phase-advanced the melatonin rhythm significantly in the VB12 trail, but not in the placebo. These findings indicate that VB12 enhances the light-induced phase-shift in the human circadian rhythm.

Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans

It makes sense, because the formation of melatonin requires a methyl group. Another study reported that methyl B12 shots improved alertness and increased rectal temp in later daytime hours. This suggests that B12 indeed affects the circadian clock.

One study tested the effects of methyl and cyano B12 on circadian rhythms, well-being, alertness, and concentration in healthy subjects. Sleep time was significantly lower in the methyl B12 group, who reported better sleep quality, focus and a refreshed feel. The authors concluded that “only methylcobalamin has a positive psychotropic alerting effect with a distribution of the sleep-wake cycle toward sleep reduction.”

Some personal cases

Here’s a case of a 13 year old boy with adrenoleukodystrophy who had developed a sleep-wake disorder after a complete loss of vision. His sleep-wake cycle had been 25 hours, but normalized after taking methyl B12, which caused his plasma melatonin and beta-endorphin levels to more or less match those of healthy volunteers. His peak cortisol time shifted backwards. We also saw a case where methyl B12 successfully treated a 32 year old man suffering from recurrent hypersomnia for 12 years.

Here is another interesting case:

Two adolescent patients suffering from persistent sleep-wake schedule disorders appear to have responded to treatment with vitamin B12 (methylcobalamin). A 15-year-old girl with delayed sleep phase syndrome (DSPS) and a 17-year-old boy with hypernychthemeral syndrome complained of not being able to attend school despite many trials of medication. The improvement of the sleep-wake rhythm disorders appeared immediately after the administration of high doses (3,000 micrograms/day) of methylcobalamin. Neither patient showed any laboratory or clinical evidence of vitamin B12 deficiency or hypothyroidism (which can cause B12 deficiency). Serum concentrations of vitamin B12 during treatment were in the high range of normal or above normal.

Treatment of persistent sleep-wake schedule disorders in adolescents with methylcobalamin (vitamin B12)

So, are you having issues with your sleep-wake schedule?

Then methyl B12 may help modulate your melatonin secretion, enhance your light sensitivity, and normalize your circadian and sleep-wake rhythm.

Any other benefits to methyl B12?

In a randomized study, 67 stroke patients received daily doses of 1500mcg methyl B12, while 68 remained untreated. After two years, sensory nerve in the treated group improved significantly compared to the untreated group.

Some Chinese studies found methyl B12 helpful in lumbar disc herniation, thalamic pain, glaucoma, cervical spondylosis, and cubital tunnel syndrome. They also found methyl B12 shots with acupuncture to help against intractable facial paralysis.

Methyl B12 vs Cyano B12

Now, how does methylcobalamin compare to cyanocobalamin, the previous form we examined, when it comes to absorption rate and bio-availability?

Because of the effort needed to reduce cyano B12 to an active form, its absorption rate varies greatly between people. In contrast, methyl B12 absorbs and retains in the body much better. It is so potent even orally it may help treating pernicious anemia:

A 73-year-old Japanese man with Hashimoto’s disease and diabetes mellitus received regular medical checkups for type 2 diabetes care. Blood tests indicated macrocytic anemia. The laboratory data demonstrated a normal folic acid level with a low vitamin B12 level. An endoscopic examination indicated no signs of gastric or intestinal bleeding. Positive results for anti-intrinsic factor antibodies were strongly suggestive of pernicious anemia. The patient refused cobalamin injections to treat the anemia. However, the oral administration of mecobalamin for the treatment of diabetic neuropathy was simultaneously initiated. Subsequently, the anemia gradually improved. Oral mecobalamin was presumably effective for pernicious anemia management.

Mecobalamin improved pernicious anemia in an elderly individual with Hashimoto’s disease and diabetes mellitus

Of course, we’re not suggesting you treat PA with oral tablets. Injections should always be the treatment (their absorption is far superior, and with PA you can’t take risks). But it still goes to show you the power of methyl B12 among all vitamin B12 forms.

Hydroxocobalamin

(Also Hydroxycobalamin, Hydroxo B12, OH-Cbl, or B12a)

Hydroxocobalamin, a predominant form in B12 rich foods, is an inactive type of B12. It has the benefit over cyano B12 in that it doesn’t have a toxic cyanide donor, so you bypass the need for detoxification and get to preserve the glutathione source.

Compared to cyano B12, it has a higher affinity to plasma protein and a longer half life, retaining longer in the blood. This may help reduce injection frequency.

Like cyano B12, hydroxo B12 has to convert in the body to either methyl B12 or adenosyl B12. But it converts much easier. Cyano B12 doesn’t react easily to anything (the cyanide makes it very stable), so the body must expend energy for it to convert.

Actually, when you take methyl B12, a good amount converts to hydroxo B12 as soon as it donates its methyl group. Then, it has to receive another methyl group if your body ever wants to convert and use it as active vitamin B12.

Hydroxo B12 reacts chemically with cyanide, nitric oxide, and nitrous oxide. In fact, this form is common as an antidote for cyanide toxicity. Therefore, anybody can use it safely in tobacco amblyopia and in pernicious anemia with optic neuropathy. It’s also good for patients with cobalamin metabolic diseases. But careful:

Suppressing nitric oxide could have adverse affects like elevated blood pressure, digestive disturbances, impotence, susceptibility to infection, even increased risk of cancer. This is especially true during pregnancy, where NO helps control the feto-lacental circulation. Therefore, you’d be better off with methyl or adenosyl B12.

Adenosylcobalamin

(Also AdeCbl, cobamamide, cobinamide, dibencozide, or AdoB12)

Adenosylcobalamin is the mitochondrial form of B12. The enzyme methylmalonyl-CoA mutase uses it to convert methylmalonyl-CoA to succinylcholine CoA (in the synthesis of porphyrin). This is why methylmalonic acid (MMA) levels get high when you’re low on adenosyl B12. It also acts as an intermediate in the pathway for valine, threonine, methionine, thymine, isoleucine, cholesterol and odd-chain fatty acids.

Actuallly, our body stores most of our B12 reserves in the liver as adenosyl B12, and converts it to methyl B12 whenever needed.

But what about supplementing with adenosyl B12?

In one study, carnitine and adenosylcobalamin promoted cerebral mass growth, pyramidal neuron volume, neocortical layer thickness, and fully restored normal structure of the neocortex in a model of anorexia nervosa. In the patients, carnitine and adenosyl B12 accelerated body weight gain and gastrointestinal function normalization. Latent fatigue was gone and mental performance sharply increased.

Speaking of anorexia, another study found that the combined use of carnitine and adenosyl B12 eliminated fluctuations in work rate and normalized the scope and productivity of intellectual work in patients with anorexia nervosa in the stage of cachexia. In this one, latent fatigue wasn’t fully gone though.

An Italian study treated 37 persons suffering from viral hepatitis with either adenosyl B12 or cyano B12. The authors found adenosyl B12 to be significantly better than cyano B12 in normalizing total bilirubin, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase values.

Overall, 13/18 of subjects receiving the adenosyl B12 had their total bilirubin normalized, 15/18 had their SGOT normalized, 10/18 had their SGPT normalized, and 18/18 (all) had their alkaline phosphatase normalized.

Adenosyl B12 vs methyl B12

So, which one is better for you?

Remember, the two inter-convert in the body.

The main benefit of methyl B12 over adenosyl B12 is that it comes with a very beneficial methyl donor, further enhancing your health in a myriad of ways. Also, adenosyl B12 isn’t available in shots, and some people can’t absorb B12 in any other way. However, if you can absorb B12 through the stomach, adenosyl B12 tablets can be a great option.

If your chronic fatigue seems to get better only with adenosyl B12 (and not with methyl B12), it is possible that you have some rare condition preventing your body from inter-converting them. In this case, use a mixture of both methyl B12 and adenosyl B12.


1.1: Biological Significance of Iron, Zinc, Copper, Molybdenum, Cobalt, Chromium, Vanadium, and Nickel

The transition metals and zinc are among the least abundant metal ions in the sea water from which contemporary organisms are thought to have evolved (Table 1.1). 1-5 For many of the metals, the concentration in human blood plasma greatly exceeds that in sea water. Such data indicate the importance of mechanisms for accumulation, storage, and transport of transition metals and zinc in living organisms.

Table 1.1: Concentrations of transition metals and zinc in sea water and human plasma.a*Data from References 1 - 5 and 12
Element Sea Water (M) x 10 8 Human Plasma (M) x 10 8
Fe 0.005 - 2 2230
Zn 8.0 1720
Cu 1.0 1650
Mo 10.0 1000
Co 0.7 0.0025
Cr 0.4 5.5
V 4.0 17.7
Mn 0.7 10.9
Ni 0.5 4.4

The metals are generally found either bound directly to proteins or in cofactors such as porphyrins or cobalamins, or in clusters that are in turn bound by the protein the ligands are usually O, N, S, or C. Proteins with which transition metals and zinc are most commonly associated catalyze the intramolecular or intermolecular rearrangement of electrons. Although the redox properties of the metals are important in many of the reactions, in others the metal appears to contribute to the structure of the active state, e.g., zinc in the Cu-Zn dismutases and some of the iron in the photosynthetic reaction center. Sometimes equivalent reactions are catalyzed by proteins with different metal centers the metal binding sites and proteins have evolved separately for each type of metal center.

Iron is the most common transition metal in biology. 6,7 Its use has created a dependence that has survived the appearance of dioxygen in the atmosphere ca. 2.5 billion years ago, and the concomitant conversion of ferrous ion to ferric ion and insoluble rust (Figure 1.1 See color plate section, page C-1.). All plants, animals, and bacteria use iron, except for a lactobacillus that appears to maintain high concentrations of manganese instead of iron. The processes and reactions in which iron participates are crucial to the survival of terrestrial organisms, and include ribonucleotide reduction (DNA synthesis), energy production (respiration), energy conversion (photosynthesis), nitrogen reduction, oxygen transport (respiration, muscle contraction), and oxygenation (e.g., steroid synthesis, solubilization and detoxification of aromatic compounds). Among the transition metals used in living organisms, iron is the most abundant in the environment. Whether this fact alone explains the biological predominance of iron or whether specific features of iron chemistry contribute is not clear.

Many of the other transition metals participate in reactions equivalent to those involving iron, and can sometimes substitute for iron, albeit less effectively, in natural Fe-proteins. Additional biological reactions are unique to nonferrous transition metals.

Zinc is relatively abundant in biological materials. 8,9 The major location of zinc in the body is metallothionein, which also binds copper, chromium, mercury, and other metals. Among the other well-characterized zinc proteins are the Cu-Zn superoxide dismutases (other forms have Fe or Mn), carbonic anhydrase (an abundant protein in red blood cells responsible for maintaining the pH of the blood), alcohol dehydrogenase, and a variety of hydrolases involved in the metabolism of sugars, proteins, and nucleic acids. Zinc is a common element in nucleic-acid polymerases and transcription factors, where its role is considered to be structural rather than catalytic. Interestingly, zinc enhances the stereoselectivity of the polymerization of nucleotides under reaction conditions designed to simulate the environment for prebiotic reactions. Recently a group of nucleic-acid binding proteins, with a repeated sequence containing the amino acids cysteine and histidine, were shown to bind as many as eleven zinc atoms necessary for protein function (transcribing DNA to RNA). 10 Zinc plays a structural role, forming the peptide into multiple domains or "zinc fingers" by means of coordination to cysteine and histidine (Figure 1.2A See color plate section, page C-l.). A survey of the sequences of many nucleic-acid binding proteins shows that many of them have the common motif required to form zinc fingers. Other zinc-finger proteins called steroid receptors bind both steroids such as progesterone and the progesterone gene DNA (Chapter 8). Much of the zinc in animals and plants has no known function, but it may be maintaining the structures of proteins that activate and deactivate genes. 11

Copper and iron proteins participate in many of the same biological reactions:

  1. reversible binding of dioxygen, e.g., hemocyanin (Cu), hemerythrin (Fe), and hemoglobin (Fe)
  2. activation of dioxygen, e.g., dopamine hydroxylase (Cu) (important in the synthesis of the hormone epinephrine), tyrosinases (Cu), and catechol dioxygenases (Fe)
  3. electron transfer, e.g., plastocyanins (Cu), ferredoxins, and c-type cytochromes (Fe)
  4. dismutation of superoxide by Cu or Fe as the redox-active metal (superoxide dismutases).

The two metal ions also function in concert in proteins such as cytochrome oxidase, which catalyzes the transfer of four electrons to dioxygen to form water during respiration. Whether any types of biological reactions are unique to copper proteins is not clear. However, use of stored iron is reduced by copper deficiency, which suggests that iron metabolism may depend on copper proteins, such as the serum protein ceruloplasmin, which can function as a ferroxidase, and the cellular protein ascorbic acid oxidase, which also is a ferrireductase.

Cobalt is found in vitamin B12 , its only apparent biological site. 12 The vitamin is a cyano complex, but a methyl or methylene group replaces CN in native enzymes. Vitamin-B12 deficiency causes the severe disease of pernicious anemia in humans, which indicates the critical role of cobalt. The most common type of reaction in which cobalamin enzymes participate results in the reciprocal exchange of hydrogen atoms if they are on adjacent carbon atoms, yet not with hydrogen in solvent water:

(An important exception is the ribonucleotide reductase from some bacteria and lower plants, which converts ribonucleotides to the DNA precursors, deoxyribonucleotides, a reaction in which a sugar -OH is replaced by -H. Note that ribonucleotide reductases catalyzing the same reaction in higher organisms and viruses are proteins with an oxo-bridged dimeric iron center.) The cobalt in vitamin B12 is coordinated to five N atoms, four contributed by a tetrapyrrole (corrin) the sixth ligand is C, provided either by C5 of deoxyadenosine in enzymes such as methylmalonyl-CoA mutase (fatty acid metabolism) or by a methyl group in the enzyme that synthesizes the amino acid methionine in bacteria.

Nickel is a component of a hydrolase (urease), of hydrogenase, of CO dehydrogenase, and of S-methyl CoM reductase, which catalyzes the terminal step in methane production by methanogenic bacteria. All the Ni-proteins known to date are from plants or bacteria. 13,14 However, about 50 years elapsed between the crystallization of jack-bean urease in 1925 and the identification of the nickel component in the plant protein. Thus it is premature to exclude the possibility of Ni-proteins in animals. Despite the small number of characterized Ni-proteins, it is clear that many different environments exist, from apparently direct coordination to protein ligands (urease) to the tetrapyrrole F430 in methylreductase and the multiple metal sites of Ni and Fe-S in a hydrogenase from the bacterium Desulfovibrio gigas. Specific environments for nickel are also indicated for nucleic acids (or nucleic acid-binding proteins), since nickel activates the gene for hydrogenase. 15

Manganese plays a critical role in oxygen evolution catalyzed by the proteins of the photosynthetic reaction center. The superoxide dismutase of bacteria and mitochondria, as well as pyruvate carboxylase in mammals, are also manganese proteins. 16,17 How the multiple manganese atoms of the photosynthetic reaction center participate in the removal of four electrons and protons from water is the subject of intense investigation by spectroscopists, synthetic inorganic chemists, and molecular biologists. 17

Vanadium and chromium have several features in common, from a bioinorganic viewpoint. 18a First, both metals are present in only small amounts in most organisms. Second, the biological roles of each remain largely unknown. 18 Finally, each has served as a probe to characterize the sites of other metals, such as iron and zinc. Vanadium is required for normal health, and could act in vivo either as a metal cation or as a phosphate analogue, depending on the oxidation state, V(lV) or V(V), respectively. Vanadium in a sea squirt (tunicate), a primitive vertebrate (Figure 1.2B), is concentrated in blood cells, apparently as the major cellular transition metal, but whether it participates in the transport of dioxygen (as iron and copper do) is not known. In proteins, vanadium is a cofactor in an algal bromoperoxidase and in certain prokaryotic nitrogenases. Chromium imbalance affects sugar metabolism and has been associated with the glucose tolerance factor in animals. But little is known about the structure of the factor or of any other specific chromium complexes from plants, animals, or bacteria.



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