Resolution Of Racemates

Pasteur devised three methods to resolve paratartaric acid: the first was manual, the second was chemical, and the third could be considered biological or physiological. Because paratartaric acid (also called racemic acid) was the first inactive compound to be resolved into optical isomers (enan-tiomers), an equimolar mixture of two enantiomers is now called a racemate.

A. Manual Separation

As indicated in the first lecture, Pasteur, using a hand lens and pair of tweezers, laboriously separated a quantity of the sodium ammonium salt of paratartaric acid into two piles, one of left-handed crystals and the other of right-handed crystals, and in this way accomplished the first resolution of a racemate. After purifying the free tartaric acids from the separate salt solutions, he found one acid to be identical to the previously characterized ordinary tartaric acid (which was dextrorotatory) and the other acid to be the previously unknown levorotatory isomer. Pasteur was extremely fortunate in this area of his research. The tartrate used by him is one of the very few substances that undergo a spontaneous separation into enantiomeric (hemihedral) crystals, thereby allowing resolution by hand. That is, most enantiomers do not form enantiomeric crystals. Moreover, this separation takes place only below 27°C (7). If Pasteur had been working in southern France during a torrid Mediterranean summer, rather than in Paris, we may have praised another chemist as being the first to resolve a racemate,

B. Chemical Formation of Diastereomers

The physical properties of enantiomers are identical in an achiral environment. However, chemical reactions that add another asymmetric center create a diastereomeric pair, each of which has physical properties that are not completely the same. Therefore, although an enantiomeric pair cannot be separated by ordinary chromatographic means or fractional recrystal-lization, the diastereomeric pair can often be separated easily by these means, as is indicated in the chapter by Joseph Gal. After separation, the pure enantiomers can then be regenerated by chemical means. This is today the most fundamental way of resolving a racemate.

Pasteur, in his second lecture, gives the following account, in which the optically active basic alkaloids quinicine or cinchonicine were used to convert the two enantiomeric tartaric acids into diastereomers:

We have seen that all artificial or natural chemical compounds, whether mineral or organic, must be divided into two great classes: non-asymmetric compounds with superposable image and asymmetric compounds with non-superposable image.

Taking this into account, the identity of properties above described in the case of the two tartaric acids and their similar derivatives, exists constantly, with the unchangeable characters which I have referred to, whenever these substances are placed in contact with any compound of the class with superposable image, such as potash, soda, ammonia, lime, baryta, aniline, alcohol, ethers—in a word, with any compounds whatever which are non-asymmetric, non-hemihedral in form, and without action on polarised light.

If, on the contrary, they are submitted to the action of products of the second class with non-superposable image—asparagine, quinine, strychnine, brucine, albumen, sugar, etc., bodies asymmetric like themselves—all is changed in an instant. The solubility is no longer the same. If combination takes place, the crystalline form, the specific weight, the quantity of water of crystallisation, the more or less easy destruction by heating, all differ as much as in the case of the most distantly related isomers.

Here, then, the molecular asymmetry of a substance obtrudes itself on chemistry as a powerful modifier of chemical affinities. Towards the two tartaric acids, quinine does not behave like potash, simply because it is asymmetric and potash is not. Molecular asymmetry exhibits itself henceforth as a property capable by itself, in virtue of its being asymmetry, of modifying chemical affinities. I do not believe that any discovery has yet made so great a step in the mechanical part of the problem of combination. . . .

Here is a very interesting application of the facts which have just been explained.

Seeing that the right and left tartaric acids formed such dissimilar compounds simply on account of the rotative power of the base, there was ground for hoping that, from this very dissimilarity, chemical forces might result, capable of balancing the mutual affinity of the two acids, and thereby supply a chemical means of separating the two constituents of paratartaric acid. I sought long in vain, but finally succeeded by the aid of two new bases, quinicine and cinchonicine, isomers of quinine and cinchonine, which I obtained very easily from the latter without the least loss.

I prepare the paratartrate of cinchonicine by neutralising the base and then adding as much of the acid as was necessary for the neutralisation, I allow the whole to crystallise, and the first crystallisations consist of perfectly pure left tartrate of cinchonicine. All the right tartrate remains in the mother liquor because it is more soluble. Finally this itself crystallises with an entirely different aspect, since it does not possess the same crystalline form as the left salt. We might also believe that we were dealing with the crystallisation of two distinct salts of unequal solubility.

C. Use of Living Organisms

Pasteur also discovered a method for resolving paratartaric acid while he was deeply involved in the study of fermentation. In essence, it depends on the capacity of certain microorganisms to discriminate between enan-tiomers and selectively to metabolize one instead of the other. This method is obviously less desirable than the chemical method since, at best, only one pure enantiomer can be obtained. The particular example described below by Pasteur in his second lecture grew out of his study of the fermentation of ammonium paratartrate.

Knowing this, I set the ordinary right tartrate of ammonia to ferment in the following manner. I took the very pure crystallised salt, dissolved it, adding to the liquor a clear solution of albumenoid matter. One gram of albumenoid matter was sufficient for one hundred grams of tartrate. Very often it happens that the liquid ferments spontaneously when placed in an oven. I say very often; but it may be added that this will always take place if we take care to mix with the liquid a very small quantity of one of those liquids with which we have succeeded in obtaining spontaneous fermentation.

So far there is nothing peculiar; it is a tartrate fermenting. The fact is well known.

But let us apply this method of fermentation to paratartrate of ammonia, and under the above conditions it ferments. The same yeast is deposited. Everything shows that things are proceeding absolutely as in the case of the right tartrate. Yet if we follow the course of the operation with the help of the polarising apparatus, we soon discover profound differences between the two operations. The originally inactive liquid possesses a sensible rotative power to the left, which increases little by little and reaches a maximum. At this point the fermentation is suspended. There is no longer a trace of the right acid in the liquid. When it is evaporated and mixed with an equal volume of alcohol it gives immediately a beautiful crystallisation of left tartrate of ammonia.

Let us note, in the first place, two distinct things in this phenomenon. As in all fermentation properly so called, there is a substance which is changed chemically, and correlatively there is a development of a body possessing the aspect of a mycodermic growth. On the other hand, and it is this which it is important to note, the yeast which causes the right salt to ferment leaves the left salt untouched, in spite of the absolute identity in physical and chemical properties of the right and left tartrates of ammonia as long as they are not subjected to asymmetric action.

Here, then, the molecular asymmetry proper to organic substances intervenes in a phenomenon of a physiological kind, and it intervenes in the role of a modifier of chemical affinity. It is not at all doubtful that it is the kind of asymmetry proper to the molecular arrangement of left tartaric acid which is the sole and exclusive cause of the difference from the right acid, which it presents in relation to fermentation.

Thus we find introduced into physiological principles and investigations the idea of the influence of the molecular asymmetry of natural organic products, of this great character which establishes perhaps the only well marked line of demarcation that can at present be drawn between the chemistry of dead matter and the chemistry of living matter.

Later qualified, modified, and generalized by others, Pasteur's new method became applicable to the separation of a number of other race-mates (8).

Pasteur then ends his second lecture with the following:

Such, gentlemen, are in co-ordinated form the investigations which I have been asked to present to you.

You have understood, as we proceeded, why I entitled my exposition, "On the Molecular Asymmetry of Natural Organic Products." It is, in fact, the theory of molecular asymmetry that we have just established, one of the most exalted chapters of the science. It was completely unforeseen, and opens to physiology new horizons, distant, but sure.

I hold this opinion of the results of my own work without allowing any of the vanity of the discoverer to mingle in the expression of my thought. May it please God that personal matters may never be possible at this desk. These are like pages in the history of chemistry which we write successively with that feeling of dignity which the true love of science always inspires.

Although popularly known chiefly for his great work in bacteriology and medicine, Pasteur was by training a chemist, and this work in chemistry alone would have earned him a position as an outstanding scientist.

The development of stereochemical ideas entered a new stage in 1858 when August Kekule (Fig 6) introduced the idea of the valence bond and the pictorial representation of molecules as atoms connected by valence bonds. His main thesis was that the carbon atom is tetravalent, and that a carbon atom can form valence bonds with other carbon atoms to form open chains and that sometimes the carbon chains can be closed to form rings (9). This led directly to his proposal for the structure of benzene. On the occasion of celebrations held in his honor, Kekule in 1890 delivered a speech before the German Chemical Society describing the origin of his idea of the linking of atoms (9).

During my stay in London I resided for a considerable time in Clapham Road in the neighborhood of the Common. I frequently, however, spent my evenings with my friend Hugo Muller at Islington, at the opposite end of the giant town. . . . One fine summer evening I was returning by the last omnibus, "outside," as usual, through the deserted streets of the metropolis, which are at other times so fully of life. I fell into a reverie and lo, the atoms were gambolling before my eyes! Whenever, hitherto, these diminutive beings had appeared to me, they had always been in motion; but up to that

Figure 6 Kekule. [From Japp (9),]

time I had never been able to discern the nature of their motion. Now, however, I saw how, frequently, two smaller atoms united to form a pair; how a larger one embraced two smaller ones; how still larger ones kept hold of three or even four of the smaller; whilst the whole kept whirling in a giddy dance. I saw how the larger ones formed a chain, dragging the smaller ones after them, but only at the ends of the chain. . . . The cry of the conductor: "Clapham Road," awakened me from my dreaming; but I spent a part of the night in putting on paper at least sketches of these dream forms. This was the origin of the Structurtheorie.

Then he related a similar experience of how the idea for the structure of benzene occurred to him.

I was sitting writing at my textbook, but the work did not progress; my thoughts were elsewhere. I turned my chair to the fire and dozed. Again the atoms were gambolling before my eyes. This time the smaller groups kept modestly in the background. My mental eye, rendered more acute by repeated visions of this kind, could now distinguish larger structures of manifold conformations; long rows, sometimes more closely fitted together; all twisting and turning in snake-like motion. But look! What was that? One of the snakes had seized hold of its own tail, and the form whirled mockingly before my eyes. As if by a flash of lightning I awoke; and this time also I spent the rest of the night working out the consequences of the hypothesis. Let us leam to dream, gentlemen, and then perhaps we shall find the truth . . .but let us beware of publishing our dreams before they have been put to the proof by the waking understanding.

in speculating on the kind of atomic arrangements that could produce molecular asymmetry, Pasteur, as already indicated, suggested tentatively in 1860 that the atoms of a right-handed compound, for example, might be "arranged in the form of a right-handed spiral, or situated at the corners of an irregular tetrahedron." But he never developed these suggestions. The solution to this problem of what is the cause of molecular asymmetry was presented in the publications of van't Hoff and Le Bel. On September 5,1874, van't Hoff, while he was still a student at the University of Utrecht and only 22 years of age, published a pamphlet entitled "Proposal for the extension of the structural formulae now in use in chemistry into space, together with a related note on the relation between the optical active power and the chemical constitution of organic compounds" (10). An English translation is presented in van't Hoff (11). Starting with the ideas of August Kekule on the tetravalency of carbon, van't Hoff states, at the beginning of his pamphlet: "It appears more and more that the present constitutional formulas are incapable of explaining certain cases of isomerism; the reason for this is perhaps the fact that we need a more definite statement about the actual positions of the atoms." He then proposed that the four valences of a carbon atom are directed toward the corners of a tetrahedron with the carbon atom at the center, based on the concept of the isomer number, which is illustrated below.

For any atom Y, only one substance of formula CH3Y has ever been found. For example, chlorination of methane yields only one compound of formula CH3C1. Indeed, the same holds true if Y represents, not just an atom, but a group of atoms (unless the group is so complicated that in itself it brings about isomerism); there is only one CH3OH, and only one CH3C02H. This suggests that every hydrogen atom in methane is equivalent to every other hydrogen atom, so that replacement of any one of them gives rise to the same product. If the hydrogen atoms of methane were not equivalent, then replacement of one would yield a different compound than replacement of another, and isomeric substitution products would be obtained. In what ways can the atoms of methane be arranged so that the four hydrogen atoms are equivalent? There are three such arrangements (Fig. 7): a planar arrangement (I) in which carbon is at the center of a rectangle (or square) and a hydrogen atom is at each corner; a pyramidal arrangement (II) in which carbon is at the apex of a pyramid and a hydrogen atom is at each comer of a square base; a tetrahedral arrangement (III) in which carbon is at the center of a tetrahedron and a hydrogen atom is at each corner. By then comparing the number of isomers that have been prepared for di-, tri- and tetrasubstituted methanes with the number predicted by the above three spatial arrangements, it is possible to decide which one is correct.

For example, v^ith a disubstituted compound CH2R2 (Fig. 8); (1) if the molecule is planar, then two isomers are possible. This planar configuration can be either square or rectangular; in each case, there are two isomers only. (2) If the molecule is pyramidal, then two isomers are also possible. There are only two isomers, whether the base is square or rectangular. (3) If the molecule is tetrahedral, then only one form is possible. The carbon atom is at the center of the tetrahedron. In actuality, only one disubstituted isomer is known. Therefore, only the tetrahedral model for a disubstituted methane agrees with the evidence of the isomer number.

Figure 7 Spatial models for methane where the four hydrogen atoms are equivalent. I, planar; II, pyramidal; III, tetrahedral.
FIGURE 8 Spatial models for a ¿¡substituted methane. Top, planar; middle, pyramidal; bottom, tetrahedral.

For tetrasubstituted compounds of the type CR1R2R3R4 (Fig. 9); (1) if the molecule is planar, then three isomers are possible. (2) If the molecule is pyramidal, then six isomers are possible. Each of the forms in Fig. 9, top, drawn as a pyramid, is not superimposable on its mirror image. Thus, three pairs of enantiomers are possible (one of which is shown in Fig. 9, middle). (3) If the molecule is tetrahedral, two isomers are possible, related to one another as object to mirror image. In actuality, only two tetrasubstituted isomers of methane are known (pair of enantiomers). This is strong evidence for the tetrahedral model for the carbon atom. Similar reasoning leads to the same conclusion for trisubstituted methanes.

The tetrahedral model for the carbon atom has withstood the test of time very well. Hundreds of thousands of organic compounds have been synthesized since it was first proposed. The number of isomers obtained has always been consistent with the concept of the tetrahedral carbon atom.

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