Stereotopicity and Liver Alcohol Dehydrogenase

At one level the question "Are protons A and B equivalent" seems silly, since all protons are obviously the same.

However just as obviously protons in different environments can behave differently. For example protons in different acids are more or less inclined to dissociate from the atom to which they are attached (HCl is more acidic than CH3COOH, which is a lot more acidic than CH4). We often say protons are inequivalent, when we really mean their environments are inequivalent.

Thus when comparing two protons we should consider their surroundings within the molecule to which they are attached. If we are feeling ambitious we should also include surrounding molecules of the solvent or of other reagents with which the molecule might be reacting.

No one will be surprised to know that protons that differ in Constitution (in the nature and sequence of the bond network to which they are attached) should behave differently. For example the proton attached to O in CH3CH2OH is more acidic (more likely to dissociate) than the ones attached to C. One would expect the protons of the CH2 group to behave somewhat differently from those of the CH3 group, because of their different environments.

Questions about the Configuration or Conformation of the environment are more subtle than those about its Constitution, and require us to think about the topic of Topicity (which we could call "Stereotopicity" to exclude the obvious case of constitutionally different environments).

What is the definition of topicity?
-ity is a suffix "expressing state or condition" (Oxford English Dictionary) and is more or less equivalent to the suffix -ness.
Thus chirality and handedness are two different ways of saying the same thing - the condition of being like a hand (not superimposable on its mirror image, see below).

Cheir- (Χειρ-) is the Greek root for hand - from which we derive, with the soft sound for the ch in chirurgeon, "Surgeon, one whose profession it is to cure bodily diseases and injuries by manual operation."

Topicity (which you won't find in the O.E.D.)was coined fairly recently by analogy to chirality. It means literally the condition of having a place (Greek topos (topos) for place). I suppose one could call it "placeness", but no one does.

The idea is than an atom, or group, occupies a place or environment, that includes everything else in the molecule (If you are taking the broad view you might include surrounding molecules as well, as in a solvent, or a crystal. A particularly important neighboring molecule would be a reagent with which the group might react).

The environment of one atom might be superimposable on that of another atom (they would be homotopic), or they might be different (heterotopic). In the last category it is useful to discriminate diastereotopic (completely different) from enantiotopic (different in the way enantiomeric objects are, as non-superposable mirror images).

The distinction betwen diastereotopic and enantiotopic is useful because diastereotopic atoms, or groups, may behave completely differently (for example in the position of their signal in a particular spectrum, or in their reactivity with some reagent). On the other hand enantiotopic groups will have identical properties unless they interact with something else that is itself chiral. In this circumstance, if one takes the broad view, including both the molecule in question AND the chiral molecule with which it is interacting, the enantiotopic groups have become diastereotopic, because the environment includes the chiral molecule, which by definition is not its own mirror image. (see example of liver alcohol dehydrogenase below)

Note that homotopic, heterotopic, diastereotopic,and enantiotopic are best regarded as terms of COMPARISON. Thus the environment of one group might be the mirror image of that of a second group, just plain different from that of a third group, and identical with that of a fourth group, even though all of the groups have the same constitution.

Probably the best way to become familiar with organic stereochemistry is by manipulating molecular models.  An increasingly strong second best is to use a computer program, like Jmol, to visualize the spatial arrangement of atoms and their surroundings.  A previous course TA prepared a Wiki giving instructions for implementing Jmol on your computer.

The following models use Jmol to show a particular conformation of cyclooctane (not the lowest-energy one; soon you may be able to guess how to change the conformation to give a lower energy.)  If the display doesn't work on your computer, you can click here for a single view of the molecule.

Some of the hydrogens in the models are colored to keep track of which is which, but there is of course no intrinsic difference in the hydrogens of different color. Consider the Black hydrogen and the variously colored hydrogens (Green, White, Red, Dark Blue, Pink, Yellow). Try rotating one model to make one of its colored hydrogens and its environment look just like the Black hydrogen and its environment in the other model (neglecting other color differences). If it is impossible to make them look the same, see if you can rotate the molecule to make its colored hydrogen look like the mirror image of the black hydrogen in the reference model (e.g. reflected top to bottom). You can also look for a plane of symmetry in a single molecule that would relate the two environments.
Describe the relationship of the Black hydrogen to each of the colored hydrogens using the topicity terms above.

If one could change the conformation freely so as to consider only time-averaged environments
(i.e. consider only configurational stereotopicity), what would the relationships between the various pairs of hydrogens be?

(Check your answer here)

Topicity and Yeast Alcohol Dehydrogenase

Consider the topochemical relationship among the methyl (CH3) protons of ethanol. The gauche proton whose site (i.e. environment) is shown in the middle structure is diastereotopic with the anti proton (on the left) and enantiotopic with the gauche' proton (on the right). That is, the sites are diastereomeric and enantiomeric, respectively. However, it is a little silly to make very much out of these differences because, as shown below, 120 rotation of the methyl group interconverts these sites, and it will occur VERY rapidly [barrier <4 kcal/mole means that the rate will be greater than 1013/sec x 10-3/4 x 4 = 1010 per second]. Pairs of methyl protons are only conformationally heterotopic (diastereotopic or enantiotopic).

[Note: Of course we should ignore the absence of symmetry created by the way the OH group is drawn. Its H would actually be pointing back into the page rather than to the right and it would wag right and left equally often.]

Now consider the methylene (CH2) protons. Like the gauche and gauche' protons above they are enantiotopic with one another, BUT their sites are NOT interconverted by rotation of the back carbon about the C-C bond. The methylene proton sites are mirror images and the protons are configurationally enantiotopic. Note that in naming the methylene protons, we promote the site whose proton is to be named to higher priority than its enantiotopic mate.

Why do we care?

The enantiotopic pro-R and pro-S methylene protons, being mirror images, should share all properties except for interaction with a single enantiomer of a chiral substance. If such a chiral substance interacts with the molecule, the sites (which now include the new substance) cannot be mirror images of one another, even when time averaged, because the new reagent located next to the pro-R hydrogen is NOT the mirror image of the new reagent located next to the pro-S hydrogen (because the new reagent cannot be its own mirror image if it is chiral). With the new chiral molecule present, the methylene hydrogens become diastereotopic rather than enantiotopic, and they will behave differently, e.g. one could react more readily than the other. Note in the figure below that the mirror image (equal energy) of a right hand reacting with the pro-R hydrogen, is not the right hand reacting with the pro-S hydrogen, but the pro-S hydrogen reacting with the left hand. If the chiral substance is resolved and contains only right hands, the enantiotopic hydrogens should behave differently.

Natural enzymes are chiral and come as a single enantiomer (like right hands), so they should be able to discriminate between the enantiotopic hydrogens. For example Liver Alcohol Dehydrogenase (LAD) removes one methylene hydrogen and one OH hydrogen from ethanol during its metabolism (it doesn't really give H2, but more of that next semester):

Might LAD specifically remove one of the methylene hydrogens preferentially (as shown)? Probably, but how could you prove it?

How Do You Know?

You could prove that LAD removes only the pro-R hydrogen by starting with ethanol whose pro-S hydrogen is replaced by deuterium and showing that the aldehyde product contains only deuterium, which would have been lost from the aldehyde to the extent that the pro-S hydrogen was abstracted. (You could use nmr spectroscopy to see how much deuterium is in the aldehyde.)

But where in the world would you obtain resolved mono-deutero ethanol?

If the enzyme catalyst does indeed operate specifically, you could get it by running the dehydrogenation backward using the Law of Mass Action! You could start from aldehyde containing deuterium in the presence of a big excess of what we are calling "H2" to generate the "deuterium-labeled" ethanol, then react the labeled ethanol with LAD in the absence of "H2" to make the reaction run in the normal direction. If the enzyme is not specific, it will introduce H randomly in the pro-R and pro-S methylene positions, and them remove H and D at random, so that only about half the ultimate aldehyde product will still contain the original deuterium. If the enzyme is specific, it will put on H in the pro-R position, and then remove it again, leaving all of the original deuterium with the final aldehyde.

This works!

If you run the second reaction in the presence of a lot of "H2" then the first reaction in the absence of "H2" you in fact find that all of the original deuterium is retained by the aldehyde. So the enzyme is perfectly specific in selecting between the enantiotopic hydrogens.

However the result would have been exactly the same if the enzyme had specifically donated H to, and then removed it from, the pro-S position. It was more work to show which position it works with.

One neat trick is to use one enzyme to add the H2 and another one to remove H2 (or HD). This approach is able to divide enzymes into two classes, those that work on the pro-R hydrogen and those that work on the pro-S. There are a number of examples of each.

Consider the two hands shown below.

One could clearly consider them Constitutional Homomers ("Finger bone connected to the hand bone; hand bone connected to the wrist bone," etc.).

How about from the point of view of Configuration and Conformation?


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copyright 2001 J.M.McBride