Chemistry 185H
Laboratory #8: Synthesis and Spectroscopy of a Vitamin B12 Model Coordination Complex
Introduction
A cobalt(III) coordination complex having a number of structural similarities with vitamin B12
will be synthesized in this laboratory exercise. The synthesis exploits the expectation that strong-field ligands displace weaker-strong-field ligands in transition-metal complexes; the absorption spectra
of the starting CoCl2 material and the end product can be compared to see the increasing energy
gap between the d orbitals that occurs owing to the substitution of the stronger field
dimethylglyoximato and pyridine ligands.
Background Reading
Please read about the crystal-field/ligand-field theory in chapter 16 of Atkins/Jones. Especially
important is the concept of the spectrochemical series.
Vitamin B12
In 1926, Minot and Murphy showed that pernicious anemia, a serious illness characterized by a marked and progressive decrease in the number of red blood cells and increase in their size as well as by paleness, weakness, and gastrointestinal and nervous disturbances, could be cured by introducing large amounts of liver to the diet of the afflicted patient. Their work was stimulated by Whipple’s observation that anemic dogs could be cured by feeding them raw liver. Whipple, Minot, and Murphy received the Nobel Prize in Medicine and Physiology for this work in 1934. The substance responsible for curing pernicious anemia was isolated from liver tissue in 1948 by Folkers and his co-workers at Merck Laboratories and by Smith and Parker at Glaxo Laboratories. The anti-anemia factor,
now known as cyanocobalamin or vitamin B12, was
determined by elemental analysis of the isolated material to have the approximate empirical formula
C61-64H84-90N14O13-14PCo. Crystals of the complex were obtained for the first time in 1948, but the molecule’s size and complexity were such that methods suitable for solving its structure from X-ray diffraction data were not available until a few years later. The structure (see
Figure 1) was finally determined by Dorothy Hodgkin and her crystallography group at Oxford in 1956. Hodgkin
received the Nobel Prize in Chemistry for this work in 1964. The molecule contains a corrin (a cobalt(III) ion complexed in a porphyrin-like macrocycle) and a pendant dimethylbenzimidazole moiety. The cobalt ion is coordinated in an octahedral environment composed of four N ligands from the corrin ring, a N in the benzimidazole unit, and an axial cyanide ligand. Note that while a
porphyrin or chlorophyll macrocycle contains mostly sp2 hybridized carbons and extensive
conjugation, the corrin contains mostly sp3 hybridized carbons around the outer ring.
N N N N H2N O NH2 O NH2 NH2 O O O Co O H H2N O NH O P O O -O O N N HO H H NH2 H H OH R
Figure 1. Structure of vitamin B12 or
cobalamin; R= CN- for vitamin B12, R= adenosyl for coenzyme B12, and R= CH3 for methylcobalamin. The central cobalt ion is in the +3 oxidation state.
Robert Burns Woodward (Harvard University) and Albert Eschenmoser (Swiss Federal
Institute of Technology, Zurich) announced the synthesis of vitamin B12 in 1959. Ninety separate
reactions, 100 co-workers, and eleven years were need to synthesize this “complex” molecule. Woodward, one of the most important chemists in history, is also recognized for the synthesis of other natural product molecules, such as cholesterol and chlorophyll. He received the Nobel Prize in Chemistry in 1965, and, had he lived long enough, it is fairly certain that he would have received a second prize in Chemistry with Roald Hoffmann in 1981 for his work in developing the Woodward-Hoffman rules for electrocyclic transformations (such as in the interconversion of cyclobutene to butadiene)
Methylcobalamin (R = CH3 in the structure shown in Figure 1) is synthesized by a variety of microorganisms, but not by plants and animals. Methylcobalamin has been implicated in the methylation of heavy metals to their toxic methyl derivatives. An example of such a conversion is the transformation of inorganic mercury to methylmercury halides and dimethyl mercury.
Therefore, the reactions of methylcobalamin with inorganic compounds are important for an understanding of the cycling of heavy metals in the environment. These reactions are studied in
many laboratories. Because methylcobalamin and other vitamin B12 derivatives are difficult to
synthesize, not easily isolated from organisms, and expensive, model compounds that are easily
prepared in the laboratory and that behave in a manner similar to the natural complex molecules
are frequently used. Such a model compound is chloro(pyridine)bis
(dimethylglyoximato)-cobalt(III), the compound that you will prepare in this experiment (see Figure 2). The tetradentate
corrin macrocycle in vitamin B12 is modeled by a pair of bidentate dimethylglyoximato ligands;
the axial benzimidazole ligand in vitamin B12 is modeled by a pyridine ligand. The resulting
complex exhibits ligand field properties that are similar to those exhibited by vitamin B12.
Synthetic Approach
The starting materials–cobalt dichloride hexahydrate (CoCl2•6H2O), pyridine (C5H5N), and
2,3-dioxobutane dioxime (C4H8N2O2), also known as dimethylglyoxime, are routinely available
chemicals. The reaction (Figure 2) is carried out in ethanolic solution in which the
2 CoCl2 HO N C C N HO CH3 CH3 C2H5OH OH N N H3C H3C O Co2+ N N CH3 CH3 O OH 4 HCl + 4 2 + N N H3C H3C O OH Co3+ Cl N N N CH3 CH3 O OH N + 4 , + 1/2 O2 NH+Cl -H2O + 2 2 +
dimethylglyoxime is more soluble than in water. In the first step bis
(dimethylglyoximato)-cobalt(II) is formed by replacement of H2O, Cl-, and ethanol bound to Co(II) by the bidentate
anion of dimethylglyoxime that is derived from a tautomeric form of dimethylglyoxime (Figure 3). A small excess (10%) of dimethylglyoxime will improve the yield of the complex. When pyridine is added and air is bubbled through the solution, cobalt(II) is oxidized to
cobalt(III). The final product is formed by coordination of one pyridine molecule and one chloride anion to the cobalt(III) center. This complex is insoluble in ethanol at room temperature.
Consequently, it precipitates and is easily isolated by filtration. Formally, the cobalt carries three
positive charges. We start with Co2+ in CoCl2 and then remove one electron from the cobalt
during the oxidation with air. The charge of the resulting Co3+ is neutralized by the negative
charges on the chloride anion and the two dimethylglyoximato anions. Thus, the complex is electrically neutral.
Safety
Be very careful with open flames and hot plates to avoid igniting the ethanol used. In case of contact with any of the chemicals used, wash skin immediately and thoroughly with soap and water. Pyridine should be handled in the fume hood.
Procedure-Week 1
Read this procedure thoroughly prior to beginning work, and note that some calculations are required to determine the proper amount of reactants to use in the synthesis. Do these calculations
in your laboratory notebook prior to arrival at lab in order to save time.
1. Weigh a clean, dry watch glass to the nearest 0.01 g on the top-loading balance. Add to the
watch glass the approximate amount of CoCl2•6H2O needed for the preparation of 0.60 g of
the complex. Assume a 70% yield; consider the starting cobalt compound as the limiting reagent. Record the mass used (to the nearest 0.01 g) in your notebook. Transfer the
CoCl2•6H2O quantitatively into a clean 100-mL beaker.
2. Using a clean, dry watch glass and the analytical balance, weigh out the mass of dimethylglyoxime approximately corresponding to 110 percent of the mass required to
convert the amount of CoCl2•6H2O that you are using to the complex. Record the mass used,
and completely transfer the compound into the beaker containing the CoCl2•6H2O.
3. Slowly add 30 mL of 95% ethanol to the mixture of cobalt chloride and dimethylglyoxime. Record in your notebook the color changes observed during the initial stages of this
procedure.
4. Place the 100-mL beaker containing the ethanolic reaction mixture on a hot plate. Bring the mixture to a very gentle boil. Some of the solid material in the mixture may not dissolve. Discontinue boiling after 5 minutes.
HO N C C N HO CH3 CH3 O N C C N HO CH3 CH3 O N C C N HO CH3 CH3 H + H+
5. While the solution is still reasonably hot, filter it into a clean flask using vacuum filtration to remove any undissolved material. Rinse the beaker with 3–4 mL of ethanol, and run the rinse through the filter collecting it in the flask containing the solution. Then, allow the solution in the flask to cool to within 10 degrees of room temperature.
6. Take the flask containing the filtered ethanolic solution to the fume hood. Keep the pyridine container and your flask inside the fume hood while you add a stoichiometric quantity of pyridine dropwise. Assume that one drop of pyridine weighs approximately 0.04 g. Swirl the flask gently to mix the contents. Record any observation of color change and other
indications of a reaction.
7. With the flask in the hood, bubble air through the solution for 20 minutes. The bubbling should not be too vigorous; the TA will help you adjust the flow of air to maintain just a steady bubbling (a few bubbles per second is sufficient). Record any observation of color change and crystal formation.
8. Collect the crystals on a piece of filter paper using vacuum filtration. (Gravity filtration could be used to collect the crystals; however, vacuum filtration speeds the removal of solvent.) Wash the crystals with 10 mL of distilled water and then with 10 mL of 95% ethanol. Carefully remove the filter paper from the funnel. Put the wet filter paper and crystals on a stack of two or three filter paper circles or paper towels. Allow the crystals and paper to dry on the bench top. Record observations of the color and nature (size, shape, etc.) of the crystals.
9. After the paper has dried for a while, carefully transfer the crystals to a glass sample bottle provided by your TA. Weigh the sample bottle before transferring the crystals so that you can determine the mass of the crystals by computing the difference.
10. Give the bottle containing your product crystals to your TA for storage in a desiccator until your next lab period. This will ensure that the crystals are dry before you measure the final mass in order to avoid overestimating the yield.
Procedure-Week 2
1. Weigh the sample bottle containing your product crystals. Compute the percent yield using the reaction stoichiometry, the amount of starting material, and the amount of product. 2. Using some of the product crystals, prepare a solution in heptane and/or acetone and obtain
the absorption spectrum with the Ocean Optics spectrograph/CCD setup. Also, obtain spectra
of the starting material CoCl2 dissolved in water and in 95% ethanol.
Introduction
• This section should describe the objectives of the experiment and provide an overview of the synthesis of the model compound.
Experimental
• In this section of your laboratory report, specify the quantities of reagents used in the synthesis, their manufacturer, and grade or purity. Show the calculations used to determine
Results
• The results section should include a description of the chloro(pyridine)bis
(dimethylglyox-imato)cobalt(III) crystals, the percent yield of the product, and the absorption spectra
collected for the CoCl2 starting material and the product. The percent yield calculation
should be included with the other calculations in an appendix.
Discussion
• In your laboratory report, discuss the appearance of the spectra in terms of the position of the
lowest-energy band (which is assignable to the d-d or ligand-field transition) and the structure
(ligands) of the complex. Can you come up with assignments for any of the other transitions you observe?
• Use the PyMOL program to study the structure (available on the course web site) of your
model complex and that of vitamin B12. Comment on the differences and similarities that you
observe. For instance, how do the metal-ligand bond lengths compare? Does the bis
-dimethylglyoximato ligand setup look the same [especially in the CPK (van der Waals radii
or space-filling) representation] as the corrin macrocycle of vitamin B12 with respect to the
size of the equatorial disk-like structure that surrounds the cobalt ion? How do the axial
ligands of the model complex compare in bulk with those of vitamin B12? Based on your
comparison of the structures of the model complex and vitamin B12, do you think that the
cobalt(III) complex that you synthesized is a reasonable model for vitamin B12? Justify your
response.
Conclusions
• Briefly summarize the results of your synthesis and what you learned from the UV/Visible
spectra collected and examination of the structures of the model complex and vitamin B12.
Acknowledgments
This laboratory is based on an exercise described in the following text:
Peck, L.; Irgolic, Kurt J. Measurement and Synthesis in the Chemistry Laboratory;
Macmillan Publishing Company: New York, 1992.
A discussion of vitamin B12 and its reactions can be found in the following introductory text:
Lippard, Stephen J.; Berg, Jeremy M. Principles of Bioinorganic Chemistry; University
Science Books: Mill Valley, California, 1994.
