Chiral supports for resolution of racemates

An inorganic oxide impregnated with poly(aminostyrene) whose amino groups are covalently bonded to carboxyl groups of chiral organic hydroxy acids may be used as the solid stationary phase in the chromatographic separation of racemic mixtures.

BACKGROUND OF THE INVENTION 
Ever since Pasteur discovered the property of optical activity displayed by 
chiral compounds, the resolution of racemic mixtures into their 
enantiomeric components has posed a challenge. Substantial progress in 
separating enantiomeric pairs has been achieved since Pasteur's laborious 
hand separation of the enantiomeric crystals of racemic sodium ammonium 
tartrate, yet methods of resolution, and the materials used therefor, 
remain a formidable obstacle to commercial production of optically active 
organic substances. 
A traditional method of resolution comprises reacting a racemic mixture 
with a second optically active substance to form a pair of diastereomeric 
derivatives. Such derivatives generally have different physical properties 
which permit their separation by conventional means. For example, 
fractional crystallization often permits substantial separation to afford 
at least one of the diastereomers in a pure state, or largely so. An 
appropriate chemical transformation then converts the purified derivative, 
which was formed initially solely to prepare a diastereomeric pair, into 
one enantiomer of the originally racemic compound. This traditional method 
is exemplified by the reaction of naturally occurring optically active 
alkaloids, for example, brucine, with racemic acids to form diastereomeric 
salts, with release of an optically active organic acid from a purified 
diastereomer upon acidification of the latter. 
Such traditional methods suffer from many limitations. Generally, only one 
of the enantiomeric pairs can be obtained, so yields are necessarily less 
than 50%. The separation of the material so obtained usually is 
incomplete, leading to materials with enhanced rather than complete 
optical purity. The optically active materials used to form the 
diastereomers frequently are expensive and quite toxic--the alkaloids as a 
class are good examples--and are only partially recoverable. Regeneration 
of optically active material from its derivative may itself cause 
racemization of the desired compound, leading to diminution of optical 
purity. For example, if optically active benzyl alcohols are prepared 
through their diastereomeric ester derivatives, subsequent acid hydrolysis 
of the latter to regenerate the alcohol may be accompanied by appreciable 
racemization. 
With the advent of chromatography diverse variations on the basic method of 
separating diastereomers became possible. These approaches undeniably 
represent substantial advances in the art, yet fail to surmount the basic 
need, and associated problems, to prepare diastereomeric derivatives of 
the desired compound and to transform such derivatives after separation to 
the optically active compounds of interest. 
Chromatographic methods of separation offer advantages of general 
application, mild conditions which generally preclude chemical or physical 
transformation, efficiency of recovery and separation which are limited 
only by the number of theoretical plates employed, and the capability of 
utilization from a milligram to kilogram scale. Translation from a 
laboratory to industrial scale has proved feasible, and commercial 
processes employing chromatographic separation occupy an important 
position in the arsenal of available industrial methods. For such reasons, 
methods based on chromatographic separation remain under intensive 
exploration. 
To circumvent the disadvantage of separating diastereomeric derivatives of 
a compound while retaining the advantages of chromatographic separation, 
recent advances in the art have employed chiral, optically active 
compounds in association with the chromatographic support. The theory 
underlying this approach is that chiral material will have differential 
weak interactions with enantiomers, for example, hydrogen bonding, or 
acid-base interactions generally. Such weak interactions lead to 
reversible formation of entities which we refer to as complexes, and the 
equilibrium constant characterizing complex formation will differ from 
each member of the enantiomeric pair. The different equilibrium constants 
manifest themselves as a differing partition coefficient among the phases 
in a chromatographic process, leading ultimately to separation of 
enantiomers. 
Thus, enantiomers of some chromium complexes were resolved by 
chromatography on powdered quartz, a naturally occurring chiral material. 
Karagounis and Coumolos, Nature, 142, 162 (1938). Lactose, another 
naturally occurring chiral material, was used to separate 
p-phenylene-bis-iminocamphor. Henderson and Rule, Nature, 141, 917 (1938). 
However, despite this knowledge substantiating theoretical considerations, 
advances in the art have been tortuous at best. 
A major obstacle has been development of a chiral solid phase capable of 
resolving, at least in principle, a broad class of racemic organic 
compounds, with a stability which permits repeated usage, and with 
adequate capacity to make separation feasible on a preparative scale. 
Gil-Av has made a major contribution toward one kind of solution by 
gas-liquid phase chromatographic resolution of enantiomers using columns 
coated with N-trifluoroacetyl derivatives of amino acids, di- and 
tri-peptides. Gil-Av and Nurok, "Advances in Chromatography", Volume 10, 
Marcel Dekker (New York), 1974. However, the advances suffer practical 
limitations originating from the need to have volatile substrates and the 
inability to scale up methods employed. 
Another advance is represented by the work of Baczuk and coworkers, J. 
Chromatogr., 60, 351 (1971), who covalently bonded an optically active 
amino acid through a cyanuric acid linkage to a modified dextran support 
and utilized the resulting material in column chromatography to resolve 
3,4-dihydroxyphenylalanine. A different approach is exemplified by 
polymerization of optically active amides with the resulting polymer used 
as a solid phase in liquid-solid chromatography. Blaschke and Schwanghart, 
Chemische Berichte, 109, 1967 (1976). 
General considerations of the characteristics of a solid phase chiral 
chromatographic medium, including such factors as structural integrity, 
flow characteristics, chemical inertness, reusability, capacity, and 
incorporation into well developed commercial processes, suggest that a 
desirable material will be comprised of (1) a solid, largely inorganic 
support, bearing a (2) pendant functional group sufficiently removed from 
the surface of the support so that it may (3) covalently bond with a 
suitable site of a chiral molecule while enabling the latter to at least 
simulate its homogeneous interactions with racemic compounds it 
encounters. 
SUMMARY 
An object of this invention is to provide material suitable for use as a 
solid phase in the chromatographic separation of racemic mixtures. An 
embodiment of this invention comprises an inorganic oxide, selected from 
the group consisting of alumina and silica, impregnated with 
poly(aminostyrene), a substantial portion of whose amino groups are 
covalently bonded through an amide linkage to a chiral hydroxy acid and 
derivatives thereof. In a more specific embodiment more than 50% of the 
amino groups are bonded to the chiral hydroxy acid. In a still more 
specific embodiment the chiral hydroxy acid is tartaric acid or 
derivatives thereof where both hydroxyl groups are esterified. 
DESCRIPTION OF THE INVENTION 
The inventin herein discloses material which may be suitable for use as a 
solid phase chromatographic medium in the separation of racemic mixtures. 
Such material is comprised of a core support, a layer of 
poly(aminostyrene) deposited on the core support, and a chiral organic 
hydroxy acid covalently bonded to the poly(aminostyrene) via an amide 
linkage formed, if only conceptually, by reaction of the amino group with 
the carboxyl group of the organic acid. 
The core support of this invention consists of metal oxides, such as 
silica, alumina, zirconia, thoria, and combinations thereof. Silica and 
alumina are preferred materials of this invention, and among the aluminas 
gamma-alumina is especially preferred. However, other core supports such 
as glass or ceramic materials also may be employed, although not 
necessarily with equivalent results. It is only necessary that the core 
support have the ability to be impregnated with poly(aminostyrene) in such 
a way as to retain the layer of polymeric material deposited thereon. 
In this invention the core support is impregnated with poly(aminostyrene). 
What is meant by impregnation is that there is deposited on the surface, 
and/or within the pores, of the core support a more or less uniform film 
of poly(aminostyrene). The poly(aminostyrene) of this invention has a 
molecular weight from about 10,000 to about 200,000 units. Although it is 
not necessary to cross link the poly(aminostyrene) so deposited, such 
cross linking may result in the polymer becoming more firmly attached to 
the core support, and consequently may be desirable in some circumstances. 
The poly(aminostyrene) may be deposited per se on the core support, or it 
may be formed on the core support in situ from a suitable precursor. 
A substantial portion of the amino groups of the poly(aminostyrene) is 
present as an amide, with the carboxyl functionality arising from the 
chiral organic acid. Generally, at least 30% of the amino groups present 
are so amidated, with the exact amount depending, inter alia, on the total 
concentration of amino groups per gram of support, the nature of the 
chiral organic acid, and the resolution to be performed. The total 
concentration of chiral hydroxy acids, and derivatives thereof, so bonded 
may be from about 0.1 to about 1.0, or even higher, milliequivalents per 
gram of inorganic oxide. 
The chiral molecules of this invention are hydroxy acids and their 
derivatives. A partial, but not exclusive, list of such acids, cited 
solely for illustrative purposes, include tartaric acid, lactic acid, 
malic acid, mandelic acid, glyceric acid, 3-phenyl-2-hydroxypropionic acid 
and ring substituted derivatives thereof, ascorbic acid, and sugar acids 
such as gluconic and glucaric acids. The use of derivatives of hydroxy 
acids often confer benefits which are advantageous. Such derivatives are 
of the hydroxyl group of the hydroxy acid, and ethers and esters are 
especially important. The ethers may be alkyl, aryl, substituted aryl, or 
aralkyl ethers, examples of which include methyl, ethyl, propyl, butyl, 
phenyl, nitrophenyl, dinitrophenyl, benzyl, hydroxybenzyl, nitrobenzyl, 
dinitrobenzyl, 9-anthryl, 9-anthrylmethyl ethers, etc. 
The esters of the hydroxyl group which may be used in this invention may be 
esters of either aliphatic or aromatic acids. Examples include esters of 
acetic, propionic, butyric, caproic, 2-chloroacetic, 2-bromoacetic, 
hydroxyacetic, aminoacetic, benzoic, hydroxybenzoic, nitrobenzoic, 
dinitrobenzoic, halobenzoic acids, etc. 
Specific examples of derivatives which may be used in this invention, which 
are cited solely for purposes of illustration and should not be construed 
as a limitation in any manner, include monoacetyl tartaric acid, dibenzoyl 
tartaric acid, 2-benzyloxysuccinic acid (the benzyl ether of malic acid), 
2-nitrophenoxysuccinic acid, 2-chlorobenzoyloxypropionic acid (the 
chlorobenzoate ester of the hydroxy group of lactic acid), ethyl 
glycerate, dinitrobenzyl lactate, butyl mandelate, the diamide of malic 
acid, glyceric acid, N-anthrylamide, etc. 
As was mentioned previously, the poly(aminostyrene) impregnated inorganic 
oxide may be formed in situ, or may be formed by depositing prior-formed 
poly(aminostyrene) directly on the inorganic oxide. Where the 
poly(aminostyrene) is formed in situ, the alumina is first impregnated 
with polystyrene of suitable molecular weight, and subsequently the dried 
support is nitrated, as for example with fuming nitric acid, to form a 
poly(nitrostyrene) impregnated inorganic oxide. The nitro groups then can 
be reduced, using for example a stannous chloride-hydrochloric acid 
mixture, to amino groups. 
In an alternate mode of preparation, the poly(aminostyrene) can be prepared 
separately from polystyrene, generally via a nitration-reduction sequence. 
A solution of the poly(aminostyrene) in a suitable solvent is then 
contacted with the inorganic oxide for a time sufficient to ensure 
adequate deposition, after which the inorganic oxide is separated, as by 
filtration, and excess, non-adhering poly(aminostyrene) may be removed by 
washing with a suitable solvent. 
A mixture of the anhydride of the chiral acid and a suitable base, such as 
a trialkylamine, in a solvent such as tetrahydrofuran is then contacted 
with the polymer impregnated support at a temperature and for a time 
sufficient to achieve acylation of the amino group by the anhydride. A 
temperature from about 40.degree. to about 100.degree. C. for a time from 
about 1 to about 10 hours generally suffices. Typically about 10% excess 
anhydride is used based on the amino groups present. Solid is then 
separated, as by filtration, and washed to remove adhering but not 
covalently bound organic material. 
The examples listed hereunder are cited solely for illustrative purposes. 
It is to be understood that this invention is not limited thereto.

EXAMPLE I 
A saturated solution of polystyrene, molecular weight 22,000, in reagent 
grade acetone was contacted with about 70 ml. gamma alumina in vacuo for 
about one-half hour. The solid was collected, air dried, then slowly added 
to 50 ml. fuming nitric acid, held at 0.degree.-5.degree. C., and stirred 
at those temperatures for an additional hour. The solid was removed by 
filtration, thoroughly washed with water until the washings were neutral 
and air dried. The nitrated polymer impregnated oxide was reduced by 
addition, to a boiling solution of stannous chloride (50 g) in 50 ml. 
concentrated hydrochloric acid, and the mixture was maintained at the 
boiling point for about one-half hour. The solid then was removed by 
filtration, and washed thoroughly with copious amounts of water. The 
poly(aminostyrene) impregnated support was then dried prior to subsequent 
use. Analysis showed 0.40 milliequivalents amine per gram. 
Prior to acylation, the poly(aminostyrene) impregnated support (45 g) was 
washed with dilute base, to generate the free amine, followed by a 
thorough water wash to remove excess base. A solution of d-(+)-dibenzoyl 
tartaric anhydride in aqueous 90% tetrahydrofuran containing 10% excess 
anhydride, based on the amine functionality on the support, and an 
equivalent amount of triethylamine was contacted with the 
poly(aminostyrene) support at 50.degree. C. for approximately 5.5 hours 
and mixed continually by slow rotation. Upon completion of the reaction, 
the solid was removed by filtration, and the chiral support was washed 
with water and air dried. 
EXAMPLE II 
This example differs from that above in that the poly(aminostyrene) was 
preformed and deposited on the inorganic oxide from a solution of its 
hydrochloride. 
The preformed poly(aminostyrene) was prepared as follows. Polystyrene (22 
g) of molecular weight about 22,000 was added slowly to 100 ml of fuming 
nitric acid maintained at 0.degree.-8.degree. C. The mixture remained at 
this temperature overnight, then cautiously diluted with a large amount of 
ice water. Nitrated polystyrene was collected by filtration, washed well 
with water, then air dried. 
Poly(nitrostyrene), 25 g., was added to a boiling mixture of stannous 
chloride (55 g) in 75 ml concentrated hydrochloric acid. After 30 minutes 
solid was collected from the cooled solution by filtration and redissolved 
in boiling water with the aid of hydrochloric acid. The hot solution was 
filtered, and filtrate was diluted with an equal volume of acetone, then 
cooled to 4.degree. C. The resulting yellow precipitate of 
poly(aminostyrene hydrochloride) was collected by filtration. 
Typically, 4% solutions of poly(aminostyrene) in 3.6% hydrochloric acid as 
solvent were utilized. To 100 volumes of such solution were added 75 
volumes of inorganic oxide, and the mixture was stirred vigorously for 
several minutes. The mixture was then placed in a vacuum oven for 1.5 
hours, after which liquid was decanted off and the resulting solid was air 
dried. Prior to coupling with the anhydride, the free amine was generated 
by addition of 5% sodium hydroxide. Acylation then was conducted as 
described in Example I.