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The Stereochemistry of Biochemical Molecules: A Subject To Revisit Josep J. Centelles* and Santiago Imperial Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Martí i Franquès, 1, 08028-Barcelona, Spain; *
[email protected] The discovery of the existence of many naturally occurring compounds in enantiomeric forms, and the fact that one enantiomer is predominant in living cells (for example, the L-amino acids in proteins), was an important advance in early vitamin B12 chemistry. The poor facilities for showing threedimensional structures at that time hindered the communication of data concerning newly described spatial conformation of chemical structures. The original contribution of Emil H. Fischer (Nobel Prize for Chemistry, 1902) with his popular projection method improved this situation for chiral compounds. Nevertheless, it is impossible to represent a complex molecule by a simple Fischer projection. In biochemistry, there are many examples of stereoisomerism, as most biochemical compounds have chiral carbons. Carbohydrates, such as glucose and fructose, polyhydroxyaldehydes or polyhydroxyketones have many chiral carbons. Natural amino acids, except glycine, have at least one chiral carbon, while others (isoleucine and threonine) have two chiral carbons. Some lipids, such as fats and steroids, also have interesting stereochemistry. Most fatty acids have cis double bonds and many isoprenoids have trans double bonds. Diacylglycerides and phosphatidic acids have at least one chiral carbon; the β-carbon from glycerol. Sphyngosine has two chiral carbons and a trans double bond. Finally, nucleosides and nucleotides have chiral carbons as a result of their ribose stereoisomerism. Owing to the importance of stereoisomerism in biochemistry textbooks, books should correctly indicate the stereoisomerism of biochemical molecules.
to the corrin ring system. The fifth atom (the axial α-ligand) is N1 of the 5,6-dimethylbenzimidazole moiety. This base forms a nucleotide by binding N3 to the C1 of an α-D-ribofuranose-3´-phosphate moiety. It should be noted that this nucleotide forms a loop, as it is bound through the phosphate
R CH2–CO–NH2 H
H
2
N
Co3+ N
H
C
α
H2N –OC–H2C
12
H 13
D H3C
18
CH3 CH3
14
19
CH2–CH2 –CO–NH2
15
16
CH3 17
H
CH2 CH2 C
O
HN H2C
H C
CH3
O O
An Example of a Stereochemical Molecule
P
N
CH3
N
CH3
OH HO
H H
To show how poorly stereochemistry is presented, a review of the structure of vitamin B12 was performed on 25 of the most common textbooks. The structure of this molecule (Figure 1) was established between 1950 and the early 1960s by X-ray crystallographic techniques in the laboratory of Dorothy Hodgkin (Nobel Prize for Chemistry, 1964). The molecule contains a cobalt ion, a corrin ring, the 5,6dimethylbenzimidazole moiety, the 1-α-D-ribofuranoside-3´phosphate moiety, a cyanide ion and several amide groups. The general formula is C63H88O14N14PCo. The corrin ring system is not flat as in a porphyrin ring system (Figure 2). The direct link between rings A and D (between C1 and C19) is formed by two sp3 carbon atoms. As the rest of the structure is a conjugated system of double bonds, the result is a kind of wave (see Figure 2). The cobalt atom is octahedrally coordinated to six atoms or groups in all the B12 derivatives that contain cobalt(III). The coordination is distorted by the asymmetry of the corrin ring system. Five of the six coordination atoms of vitamin B12 are nitrogen atoms. Four atoms (the equatorial ligands) belong •
N
1
H3C
10
N 11
A
H2N –OC–H2C H3C
β
4
3
H2N –OC–H2C –H2C
CH2–CH2 –CO–NH2
CH3 B 9
6
5
O
www.JCE.DivCHED.org
8
7
H 3C
HOH2C
H O
H
OH
OH
H
H
H
R=
R=
CN
H O
CH2
cyanocobalamin (vitamin B12)
N N
R= N
CH3
methylcobalamin
N NH2
5⬘-deoxyadenosylcobalamin (coenzyme B12)
Figure 1. Structure of vitamin B12 and the active forms of vitamin B12. The oxidation state of cobalt is +3 and the β-ligand is a cyano group (vitamin B 12 or cyanocobalamin), a methyl group (methylcobalamin) or a 5´-deoxyadenosyl radical (coenzyme B12 or adenosylcobalamin).
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In the Classroom
group to the substituent of C17 in the corrin ring system. Finally, the axial β-ligand depends on the cobalamin under consideration. Nomenclature of cobalamins depends on the axial βligand (the upper axial ligand in Figure 1). Thus, for aquacobalamin (or vitamin B 12a), the β-ligand is water; for cyanocobalamin (or vitamin B12), it is a cyano group; for methylcobalamin, it is a methyl group; for adenosylcobalamin (coenzyme B12), it is a 5´-deoxyadenosyl radical; for hydroxocobalamin (or vitamin B12b), it is a hydroxyl group; and for nitritocobalamin (vitamin B12c), it is a nitrite group. The oxidation state of cobalt (+1, +2, or +3) depends on the metabolism of cobalamin. For aquacobalamin, all oxidation states are possible, their respective names being: vitamin B 12s (or aquacob(I)alamin), vitamin B 12r (or aquacob(II)alamin), vitamin B12a (or aquacob(III)alamin). Vitamin B12 (cyanocobalamin) is converted in the body into its biologically active forms: methylcobalamin and adenosylcobalamin (coenzyme B12), that act in a number of enzymes catalyzing methyl group transfer and intramolecular rearrangements.
17
15 16
13
HN
N
15 16
12
18 19
17
14
12
18
11
20
13 14
19
N
N
11
NH
N
9
10
NH
1
N
2 4 3
2
8
6 5
10 1
9
4
7
3
+ 0.70
+ 0.60 + 0.20
+ 0.42
D
+ 0.25
− 0.37
7
corrin ring
porphyrin ring + 0.09
8
6 5
− 0.07
C
+0.05 N
− 0.07 N
− 0.05
0.00 M
+ 0.29
N + 0.05
− 0.13 N − 0.06
A
− 0.06
B
− 0.18
+ 0.14 − 0.14
+ 0.05 + 0.22
− 0.01
− 0.37
Figure 2. Differences between the porphyrin and the corrin ring system. The porphyrin ring contains 20 carbons, whereas the corrin ring contains only 19. Structure of the corrin ring is not planar owing to the nonaromaticity of the four dihydropyrrole rings (A, B, C, and D). The level of the carbons are shown in terms of the level of the cobalt atom: positive values indicate above the cobalt atom and negative values indicate below the cobalt atom. Values adapted from ref 26.
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Results Despite the importance of vitamin B12 in metabolism, some biochemical textbooks omit the structure (1–5). In many textbooks (6–25) the structure of the corrin ring system is depicted (Table 1). However, the corrin ring is usually not labeled, which can be a problem for the student. A student rapidly looking at the nonlabeled corrin ring structure might confuse it with a porphyrin ring system. The porphyrin ring is flat and contains 20 carbons, whereas the corrin ring is not flat and contains 19 carbons (Figure 2). Although the Rawn textbook names the rings I, II, III, and IV (22); the accepted labeling system is A, B, C, and D (6– 8, 21). To clearly indicate substituents in the corrin ring one should be aware that one of the N atoms is bound to a H atom (see Figure 2). This atom can be located either in ring A or in ring D. The latter was preferred in the following textbooks (9–12, 20, 22–24). In the textbook by Garret and Grisham (19) a double bond in the corrin ring is missing and in (21) positions of all six double bonds are wrong. All textbooks show cobalt in the center of the corrin ring, octahedrally coordinated to six atoms or groups, 4 of them being the nitrogens from the 4 tetrahydropyrrole rings. The α-ligand is N1 of the 5,6-dimethylbenzimidazole moiety, depicted at the bottom of the corrin system in all the books except Armstrong (24). The β-ligand for vitamin B12 is a cyano group (cyanocobalamin). In some textbooks, instead of vitamin B12 (cyanocobalamin), coenzyme B12 (5´-deoxyadenosylcobalamin) is shown (6–10, 13–16, 18). Other books indicate vitamin B12 in the figure legend, although X (11, 12), R (21–23), or methyl (20) is depicted. It should be noted that methylcobalamin and 5´deoxyadenosylcobalamin (coenzyme B12) are the active vitamin B12 derivatives, but only cyanocobalamin is known as vitamin B12. For the metal ion of the corrin ring, some authors do not show the charge of cobalt (11–16, 25) and others show Co+ (9, 10, 17, 20–24). It should be noted that the molecule with Co+ is very reactive and is rapidly oxidized to Co3+. Although Co3+ from cyanocobalamin can be reduced by the actions of flavoprotein reductases to a supernucleophile (Co+), which can attack ATP to form the active coenzyme 5´deoxyadenosylcobalamin, this active form contains Co3+ and is also valid for cyanocobalamin. Therefore, the molecule with Co3+ is the one that should be depicted. If we look through the substituents in the corrin ring system, we find that some authors do not include the stereoisomerism of substituents. In the corrin ring system, carbons 1, 2, 3, 7, 8, 13, 17, 18, and 19 are chiral carbons. In some books, the enantiomer of corrin ring is depicted (6, 7, 18) and the α- and β-ligands from cobalt are also exchanged. In other cases, the stereoisomerism of all corrin ring carbons is missing (9, 10, 20–23). Some books show the stereoisomerism of some carbons but not of all the chiral carbons of the corrin ring (6–8, 11–16, 18, 19, 24, 25). The substituents of the corrin ring usually agree with the real structure of vitamin B12. Zubay’s textbooks present a methyl group on C8 that should not be there (13–16). Other methyl groups on C3 and C13 are incorrectly shown in the Zubay’s first edition and in Champe’s book (25), but
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In the Classroom
Table I. Mistakes Observed in the Vitamin B12 Structurea Depicted in Different Books Re f
C harge on C o
C orrin Ring N ome nclat ure (A , B , C , D)
C orrin Ring A t oms w it h St e re oche mis t ry N ot Show n (or N ot C le arly Show n)
L at e ral C hain St e re oche mis t ry
Ribos e St e re oche mis t ry
6
+3
Ye s
1,3,19, Enant iome r
No
Ye s
C 12 of corrin: t w o ⫺C H 2 ins t e ad of t w o ⫺C H 3
7
+3
Ye s
1,3,19, Enant iome r
No
Ye s
C 12 of corrin: t w o ⫺C H 2 ins t e ad of t w o ⫺C H 3
8
+3
Ye s
1, 3, 19
No
Ye s
9
+1
No
1, 2, 3, 7, 8, 13, 17, 18, 19
No
NCb
10
+1
No
1, 2, 3, 7, 8, 13, 17, 18, 19
No
NC
11
---
No
17, 18, 19
No
NC
12
---
No
17, 18, 19
No
NC
Ot he r Errors
N ins t e ad of O in ribos e
13
---
No
3, 13, 18, 19
No
No
C 8 of corrin: ⫺C H 3 s hould be ⫺H
14
---
No
3, 13, 18, 19
No
No
C 8 of corrin: ⫺C H 3 s hould be ⫺H
15
---
No
3, 13, 18, 19
No
No
C 8 of corrin: ⫺C H 3 s hould be ⫺H
No
C 3 of corrin: ⫺C H 3 s hould be ⫺H C 8 of corrin: ⫺C H 3 s hould be ⫺H C 13 of corrin: ⫺C H 3 s hould be ⫺H
16
---
No
18, 19
No
17
+1
No
Ye s
Ye s
Ye s
18
+3
No
1,3,19, Enant iome r
No
Ye s
19
+3
No
1
Ye s
Ye s
Double bond mis s ing in D ring of corrin (C 16 of corrin)
20
+1
No
1, 2, 3, 7, 8, 13, 17, 18, 19
No
NC
Double bonds in corrine ring not in t he corre ct pos it ions Double bonds are not locat e d w e ll C 5 of corrin: has f iv e bonds , as one ⫺C H 3 s hould be de le t e d C 11 of corrin has f iv e bonds (t w o double bonds and a s ingle bond)
21
+1
Ye s
1, 2, 3, 7, 8, 13, 17, 18, 19
No
NC
22
+1
N o : I, II, III, IV (I ⬅A )
1, 2, 3, 7, 8, 13, 17, 18, 19
No
NC
23
+1
No
1, 2, 3, 7, 8, 13, 17, 18, 19
No
Ye s
24
+1
No
1, 3, 19
No
Ye s
25
---
No
18, 19
No
No
C 3 of corrin: ⫺C H 3 s hould be ⫺H C 8 of corrin: ⫺C H 3 s hould be ⫺H C 13 of corrin: ⫺C H 3 s hould be ⫺H
a N ot all re f e re nce s de pict v it amin B 12 (cy anocobalamin): in re f s 6–10,13–16,18 coe nzy me B 12 (ade nos y lcobalamin) is de pict e d, w he re as ot he r t e x t book s indicat e v it amin B 12 in t he le ge nd and de pict X (11-–12), R (21–23), or me t hy l (20). b
St ruct ure is not comple t e .
are corrected in later editions of Zubay’s books (13–15). Such differences in structure for different editions are also observed in Voet’s textbooks (6–8). On C12, instead of a methyl group, two –CH2 groups are depicted in the first and second editions (6, 7) but not in the third edition (8). Harper shows two methyl groups on C5, giving it five bonds although only one methyl should be present (21). Stereoisomeric problems are not restricted to the corrin ring. The dimethylbenzimidazole group is well depicted in all textbooks, as it does not contain stereoisomerism. But in some textbooks, the α-D-ribofuranose-3´-phosphate stereoisomerism is either not shown (13–16, 25) or not completely shown (9–12, 20–22). Devlin’s textbook shows a tetrahydropyrrole ring instead of a tetrahydrofuran ring in ribofuranose (11) that was correct in a previous edition (12). In the cases www.JCE.DivCHED.org
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where coenzyme B12 is depicted, stereoisomerism of adenosine is also absent in some books (9, 10, 13–16). Finally, only two textbooks show the stereoisomerism of the chiral carbon from the nucleotide loop of cobalamins (17, 19). The textbook by Mathews (17) is the only one out of the 20 books studied that shows the complete stereoisomerism of all chiral carbons in vitamin B12. Unfortunately, in this book Co+ is shown and the corrin ring nomenclature (A, B, C, D) is not used. Conclusions Textbooks are a primary source of information for many students learning biochemistry. These are excellent tools prepared by the collaborative effort of professional teachers and
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researchers. Textbooks should be a reliable source of information. When mistakes are unadvertedly present, they can be transmitted to the next generation of academics and researchers. On comparison of 25 of the best-known biochemistry textbooks, some discrepancies and mistakes concerning the structure of vitamin B12 were found. Five textbooks do not examine the structure of the vitamin; certain other books do not show a three-dimensional structure of the vitamin; others have problems with the corrin ring system or with the oxidation state of cobalt. We advise biochemistry and molecular biology textbook authors to look closely at stereoisomerism and, if necessary, include graphics showing stereoscopic models for complicated structures. The stereochemistry of chiral carbons from biochemical molecules must be clearly indicated in textbooks. Both biochemistry textbook writers and teachers must be fully aware of this situation so that confusion among students is avoided. Literature Cited 1. Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of the Cell, 2nd ed.; Garland Publishing, Inc.: New York, 1989. 2. Boyer, R. F. Modern Experimental Biochemistry; The Benjamin/ Cummings Publishing Company, Inc.: Menlo Park, CA, 1986. 3. Blackstock, J. C. Guide to Biochemistry; Wright: London, 1989. 4. Stephenson, W. K. Concepts in Biochemistry, 3rd ed.; Wiley and Sons: New York, 1988. 5. Elliot, W. H.; Elliot, D. C. Biochemistry and Molecular Biology, 2nd ed.; Oxford University Press: New York, 2001. 6. Voet, D.; Voet, J. G. Biochemistry, 1st ed.; Wiley and Sons: New York, 1990. 7. Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley and Sons: New York, 1995. 8. Voet, D.; Voet, J. G; Pratt, C. W. Fundamentals of Biochemistry; Wiley and Sons: New York, 1999.
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9. Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Company: New York, 1995. 10. Stryer, L. Biochemistry, 3rd ed.; W. H. Freeman and Company: New York, 1988. 11. Devlin, T. M. Textbook of Biochemistry with Clinical Correlations, 4th ed.; Wiley and Sons: New York, 1997. 12. Devlin, T. M. Textbook of Biochemistry with Clinical Correlations, 3rd ed.; Wiley and Sons: New York, 1992. 13. Zubay, G. L. Biochemistry, 4th ed.; Wm. C. Brown Publishers: Dubuque, IA, 1998. 14. Zubay, G. L. Biochemistry, 3rd ed.; Wm. C. Brown Publishers: Dubuque, IA, 1993. 15. Zubay, G. L. Biochemistry, 2nd ed.; MacMillan Publishing Company: New York, 1988. 16. Zubay, G. L. Biochemistry, 1st ed.; Addison-Wesley Publishing Company: Reading, MA, 1984. 17. Mathews, C. K.; van Holde, K. E. Biochemistry, 2nd ed.; Benjamin/Cummings: Menlo Park., CA, 1996. 18. Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed.; Worth, NY, 1993. 19. Garret, R. H.; Grisham, C. M. Biochemistry; Saunders College Publishing: Orlando, FL, 1995. 20. Bohinski, R. C. Modern Concepts in Biochemistry, 5th ed.; Allyn and Bacon, Inc.: Boston, 1987. 21. Murray, R. K.; Granner, D. K.; Mayers, P. A.; Rodwell, V. W. Harper’s Biochemistry, 24th ed.; Prentice-Hall: Stamford, CT, 1996. 22. Rawn, J. D. Biochemistry; McGraw Hill Interamericana: Madrid, Spain, 1989. 23. Horton, H. R.; Moran, L. A.; Ochs, R. S.; Rawn, J. D.; Scimgeour, K. G. Principles of Biochemistry, 2nd ed.; Prentice Hall, Inc.: Upper Saddle River, NJ, 1986. 24. Armstrong, F. B. Biochemistry, 3rd ed.; Oxford University Press: New York, 1989. 25. Champe, P. C.; Harvey, R. A. Biochemistry, 2nd ed.; J. B. Lippincott Company: Philadelphia, PA, 1994. 26. Dolphin, D. B12 Volume 1: Chemistry; Wiley and Sons, New York. 1982.
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