Proline chiral columns with broad chiral selectivity

FIELD OF THE INVENTION

The present invention relates to the field of chiral chemistry. More particularly, the present invention relates to the separation of enantiomers, i.e., those isomers in which the arrangement of atoms or groups is such that the two molecules are not superimposable.

The present inventor has developed a new class of chiral columns that can resolve a large number of racemic compounds. These columns are stable and can be used with a number of mobile phase solvents.

BACKGROUND OF THE INVENTION

Stereoisomers are those molecules which differ from each other only in the way their atoms are oriented in space. Stereoisomers are generally classified as diastereomers or enantiomers; the latter embracing those which are mirror-images of each other, the former being those which are not. The particular arrangement of atoms that characterize a particular stereoisomer is known as its optical configuration, specified by known sequencing rules as, for example, either + or - (also D or L) and/or R or S.

Though differing only in orientation, the practical effects of stereoisomerism are important. For example, the biological and pharmaceutical activities of many compounds are strongly influenced by the particular configuration involved. Indeed, many compounds are only of widespread utility when employed in a given stereoisomeric form.

Living organisms usually produce only one enantiomer of a pair. Thus only (-)-2-methyl-1-butanol is formed in yeast fermentation of starches; only (+)-lactic acid is formed in the contraction of muscle; fruit juices contain only (-)-malic acid, and only (-)-quinine is obtained from the cinchona tree. In biological systems, stereochemical specificity is the rule rather than the exception, since the catalytic enzymes, which are so important in such systems, are optically active. For example, the sugar (+)-glucose plays an important role in animal metabolism and is the basic raw material in the fermentation industry; however, its optical counterpart, or antipode, (-)-glucose, is neither metabolized by animals nor fermented by yeasts. Other examples in this regard include the mold Penicillium glaucum, which will only consume the (+)-enantiomer of the enantiomeric mixture of tartaric acid, leaving the (-)-enantiomer intact. Also, only one stereoisomer of chloromycetin is an antibiotic; and (+)-ephedrine not only does not have any drug activity, but it interferes with the drug activity of its antipode. Finally, in the world of essences, the enantiomer (-)-carvone provides oil of spearmint with its distinctive odor, while its optical counterpart (+)-carvone provides the essence of caraway.

Thus, as enzymes and other biological receptor molecules possess chiral structures, enantiomers of a racemic compound may be absorbed, activated, and degraded by them in different manners. This phenomenon causes that in many instances, two enantiomers of a racemic drug may have different or even opposite pharmacological activities. In order to acknowledge these differing effects, the biological activity of each enantiomer often needs to be studied separately. This and other factors within the pharmaceutical industry have contributed significantly to the need for enantiomerically pure compounds and thus the need for chiral chromatography.

Accordingly, it is desirable and oftentimes essential to separate stereoisomers in order to obtain the useful version of a compound that is optically active.

Separation in this regard is generally not a problem when diastereomers are involved: diastereomers have different physical properties, such as melting points, boiling points, solubilities in a given solvent, densities, refractive indices etc. Hence, diastereomers are normally separated from one another by conventional methods, such as fractional distillation, fractional crystallization or chromatography.

Enantiomers, on the other hand, present a special problem because their physical properties are identical. Thus they cannot as a rule--and especially so when in the form of a racemic mixture--be separated by ordinary methods: not by fractional distillation, because their boiling points are identical; not by conventional crystallization because (unless the solvent is optically active) their solubilities are identical; not by conventional chromatography because (unless the adsorbent is optically active) they are held equally onto the adsorbent. The problem of separating enantiomers is further exacerbated by the fact that conventional synthetic techniques often produce a mixture of enantiomers. When a mixture comprises equal amounts of enantiomers having opposite optical configurations, it is called a racemate; separation of a racemate into its respective enantiomers is generally known as a resolution, and is a process of considerable importance.

Chiral columns that can resolve a large number of racemic compounds (general chiral columns) are in high demand. They are needed routinely in many laboratories, especially in pharmaceutical industry. Prior to the present invention, Daicel columns, macrocyclic antibiotic columns, and the Whelk-O columns were probably known as the industrial leaders in this type of general chiral columns. The present inventor has developed a new class of general chiral columns based on the use of proline and its analogues.

Furthermore, and importantly, the columns of the present invention have the capability of resolving at least a similar or higher percentage of the compounds tested. Furthermore, the columns of the present invention provide better separation on some of the compounds tested and can resolve certain compounds that cannot be resolved with the commonly used commercial columns listed above.

The columns of the present inventions are stable and can be used with a large number of mobile phase solvents. Therefore, the columns of the present invention should find important applications as general chiral columns.

A large number of chiral columns have been prepared in the past; however, only a few demonstrated broad chiral selectivity. As stated above, the successful examples include the popular Daicel columns, the Chirobiotic columns, and the Whelk-O1/2 columns. The Daicel columns are prepared by coating sugar derivatives onto silica gel. Chirobiotic columns are prepared by immobilizing macrocyclic glycopeptides onto silica gel. Whelk-O 1/2 columns contain both electron rich and electron deficient aromatics. These columns have broad chiral selectivity and have been applied successfully to resolve a fair number of racemic compounds. They have different selectivity and stability profiles. Their selectivities complement each other in some cases, while they duplicate each other in other cases. Some of the columns are more suited for reversed phase conditions and others for normal phase conditions. Each column has its own strengths and weaknesses. Despite these progresses, there are still many compounds that cannot be resolved or resolved well using these commercial available columns. Therefore, there is still a significant need to develop new columns that have relatively broad chiral selectivity.

SUMMARY OF THE INVENTION

The present invention is directed to a chiral selector that represents an improvement in the art of enantiomeric separation. Thus, one embodiment of the present invention is a general chiral column with a multiple proline-based chiral selector.

Another embodiment of the present invention is a chiral stationary phase made of peptides with 2 or more prolines, including chiral selectors with 2, 3, 4, 5, 6, or 10 prolines. Also included within the scope of the present invention are analogs and isomers of prolines, and analogs and isomers of the chiral selector compounds of the present invention.

Another embodiment of the present invention is a chiral stationary phase (or column) of the following formula:

##STR00002## wherein n is any integer of 2 or greater, and analogs and isomers thereof. Another embodiment of the present invention is where n is any integer from 2 10.

The separations achieved for analytes are comparable or superior to those achieved on Daicel AD, Daicel OD, and Whelk O2 columns. The multiple proline-based chiral columns of the present invention show promise as a superior general chiral column.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure for amino acid L-Proline and its associated stationary phases Fmoc-Pro-(Me)Ahx-APS(CSP1), Fmoc-Pro.sub.2-(Me)Ahx-APS(CSP2), Fmoc-Pro.sub.4-(Me)Ahx-APS(CSP3); and Fmoc-Pro.sub.6-(Me)Ahx-APS(CSP4). CSP2-4 are embodiments of compounds of the present invention.

FIG. 2 shows the synthesis of one embodiment of the present invention, Fmoc-Pro.sub.4-(Me)Ahx-APS chiral stationary phase (CSP3): Synthesis of Fmoc-Pro.sub.4-(Me)Ahx-APS chiral stationary phase (CSP3): (a) Fmoc-(Me)Ahx-OH, DIC; (b) (1) Piperidine; (2) Fmoc-Pro-OH, HATU; (c) AcOH; (d) aminopropyl-silica gel, HATU.

DESCRIPTION OF THE INVENTION

The present inventor has developed a new chiral column that has relatively broad chiral selectivity, when compared with Daicel columns and Whelk O2 column, as industry standards or industry models. Additionally, the chiral columns of the present invention are stable in a number of mobile phase conditions.

The success rate of the chiral column of the present invention compares well with the best commercially available general chiral columns developed over the last few decades. For 22 racemic compounds chosen based on their availability (see example 4), our Pro4 column (CSP 3) resolved 17 compounds; our Pro2 column (CSP2) resolved 16 compounds; our Pro6 column (CSP4) resolved 15 compounds. In comparison, Daicel OD column resolved 18, Daicel AD resolved 16, and Whelk-O2 resolved 15 compounds. The monoproline column (CSP1) is much less effective, as it can resolve only 6 out of the 22 compounds tested. The achieved resolutions with the monoproline column are also very modest.

Proline is a unique amino acid in many ways (FIG. 1). Instead of having a primary amino group as in other .alpha.-amino acids, it contains a secondary amine. Because of the cyclic structure, rotation around the nitrogen-.alpha.-carbon bond is restricted. Also because of the cyclic structure, proline is not ideally suited for .alpha.-helix or .beta.-sheet conformation; instead, polyproline forms its own unique helical conformation (Polyproline I and polyproline II). The amide bond in polyproline is sterically hindered compared with other oligopeptides. The distinctly different conformational and structural features of polyprolines suggest that they may behave quite differently from other short oligopeptides that have been studied in chiral chromatography.

The present inventors discovered that proline based chiral selectors, including the embodiment tetraproline based chiral stationary phase 3 (FIG. 1), diproline based chiral stationary phase 2, hexaproline based chiral stationary phase 4 have relatively broad chiral selectivity, while mono-proline stationary phase 1 is largely ineffective.

Immobilization of the chiral selectors of the present invention to silica gel is accomplished through a linker group. One example of a linker group of the present invention is a disubstituted amino group. A second example is a N-methylamino group. Another example is 6-N-methylaminohexanoic acid. The amide bond between these linkers and proline residue is more sterically hindered due to the N-methyl or N-alkyl group. (The particular linker group can be selected by one of ordinary skill in the art depending on the analyte to be tested.) For example, when the selector Fmoc-Pro-Pro is immobilized using 6-N-methylaminohexanoic acid, it may resolve about 16 out of about 22 analytes tested. For the same chiral selector, when immobilized using 6-aminohexanoic acid, it resolved only 4 out of the same group of analytes.

Additionally, the stationary phase compounds of the present invention may comprise various end-capping groups as known in the art.

By use of the term proline with respect to the present invention, it is understood that analogs and isomers of proline are included. For example all stereoisomers are included. Additionally, analogs are included. Examples of the analogs that are included herein are those with the following skeleton structure feature such as in D-proline, hydroxyproline, and pipecolinic acid:

##STR00003## wherein n is an integer (such as 1, 2, 3, 4, 5, etc.), X is a heteroatom such as O, S, or N, and other unspecified atoms can be carbon or heteroatoms. For simplicity, unless otherwise noted, proline includes L-proline in this application.

These covalently bound columns of the present invention are stable in common organic solvents, including CH.sub.2Cl.sub.2 and CHCl.sub.3. Therefore, a wide selection of mobile phase conditions could be applied in method development. For several analytes, the present inventor attempted resolution with CH.sub.2Cl.sub.2/hexane as the mobile phase and effective separation was also achieved (example 6). Wider solvent choices have advantages in that some racemic analytes are soluble in only certain solvents and some compounds can be resolved better in certain solvents.

In terms of potential interaction modes with the analytes, examples of the chiral selectors of the present invention are forming attractive hydrogen bonds with the analyte and they may also have attractive polar interactions with the analyte. In addition, steric interaction between analyte and chiral selector could also be important.

The following examples and experimental section are designed to be purely exemplary in nature. Thus, this section should not be viewed as being limiting of the present invention.

EXAMPLES

Throughout this section, various abbreviations are used, including the following: DIC, diisopropylcarbodiimide; HATU, O-(7-Azabenzotriazol-1-yl)-N,N,N,',N'-tetramethyluronium hexafluorophosphate; DIPEA, N,N-Diisopropylethylamine; DMF, N,N-Dimethylformamide; DCM, Dichloromethane; DMAP, 4-(dimethylaminopyridine); NMM: N-methylmorpholine; Fmoc, 9-Fluorenylmethoxycarbonyl; (Me)Ahx: 6-methylaminohexanoic acid; Fmoc-(Me)Ahx-OH, 6-[(9H-fluoren-9-ylmethoxy)carbonyl]methylamino hexanoic Acid; Fmoc-Ahx-OH, 6-[(9H-fluoren-9-ylmethoxy)carbonyl]aminohexanoic acid; Fmoc-Pro-OH, N-.alpha.-Fmoc-L-proline.

General Supplies and Equipment:

Amino acid derivatives were purchased from NovaBiochem (San Diego, Calif.). All other chemicals and solvents were purchased from Aldrich (Milwaukee, Wis.), Fluka (Ronkonkoma, N.Y.), or Fisher Scientific (Pittsburgh, Pa.). HPLC grade Kromasil.RTM. silica gel (particle size 5 .mu.m, pore size 100 .ANG., and surface area 298 m.sup.2/g) was purchased from Akzo Nobel (EKA Chemicals, Bohus, Sweden). Selecto silica gel (32 63 .mu.m) from Fisher Scientific was used for flash column chromatographic purification of target compounds. Thin-layer chromatography was completed using EM silica gel 60 F-254 TLC plates (0.25 mm; E.Merck, Merck KGaA, 64271 Darmstadt, Germany). Elemental analyses were conducted by Atlantic Microlab, Inc. (Norcross, Ga.). HPLC analyses were completed with a Beckman analytical gradient system (System Gold). UV spectra were obtained with a Shimadzu UV 201 spectrometer (cell volume 3 mL; cell pass length 10 mm).

Example 1

Preparation of Chiral Stationary Phase Fmoc-Pro-(Me)Ahx-APS(CSP1)

To 0.80 g of (Me)Ahx-APS silica (the surface (Me)Ahx concentration is 0.64 mmol/g) are added mixtures of Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20 g) in 8 mL of DMF. After agitating for 6 h, the resulting silica is filtered and washed with DMF, Methanol, and DCM to yield the desired chiral stationary phase. The surface Pro concentration is determined to be 0.57 mmol/g based on the Fmoc cleavage method. The resulting chiral stationary phase is packed into a 50.times.4.6 mm HPLC column using a standard slurry packing method.

Example 2

Preparation of Chiral Stationary Phase Fmoc-Pro.sub.2-(Me)Ahx-APS(CSP2)

To 0.80 g of (Me)Ahx-APS silica (the surface (Me)Ahx concentration was 0.64 mmol/g) were added mixtures of Fmoc-Pro-OH (3 equiv., 0.52 g), HATU (3 equiv., 0.58 g), and DIPEA (3 equiv., 0.20 g) in 8 mL of DMF. After agitating for 6 h, the resulting silica was filtered and washed with DMF, Methanol, and DCM. The surface Pro concentration was determined to be 0.55 mmol/g based on the Fmoc cleavage method. The Fmoc protecting group was then removed by treatment of the silica with 10 mL of 20% (V/V) piperidine in DMF for 1 h. The deprotected silica, Pro-(Me)Ahx-APS, was collected by filtration and washed with DMF, Methanol, and DCM. Then another module, Fmoc-Pro-OH, was coupled to the resulting silica following an identical reaction sequence and yielded the desired chiral selector on the silica gel. The surface Fmoc concentration was determined to be 0.52 mmol/g based on the Fmoc cleavage method. The resulting chiral stationary phase was packed into a 50.times.4.6 mm HPLC column using the standard slurry packing method.

Example 3

Preparation of Chiral Stationary Phase Fmoc-Pro.sub.4-(Me)Ahx-APS(CSP3)

To Rink acid resin (100 200 mesh, 3.0 g, 0.43 mmol/g) preswelled with DCM (20 mL, 30 min) was added the mixture of Fmoc-(Me)Ahx-OH (1.42 g, 3.87 mmol), DMAP (0.16 g, 1.29 mmol), NMM (0.39 g, 3.87 mmol), and DIC (0.49 g, 3.87 mmol) in DCM-DMF (1:1 V/V, 10 mL). After agitating for 6 h, the resin was collected by filtration and washed with DMF, DCM, and Methanol (20 mL.times.3). The Fmoc group was then removed by treatment with 20 mL of 20% (V/V) piperidine in DMF for 30 min. The deprotected (Me)Ahx-O-Rink resin was collected and washed with DMF, DCM, and Methanol (20 mL.times.3).

To (Me)Ahx-O-Rink resin was added the mixture of Fmoc-Pro-OH (1.31 g, 3.87 mmol), HATU (1.47 g, 3.87 mmol), and DIPEA (0.50 g, 3.87 mmol) in 20 mL of anhydrous DMF. After agitating for 3 h, the resin was filtered and washed with DMF, DCM, and Methanol (20 mL.times.3). The Fmoc group was then removed and the second, third and fourth modules, Fmoc-Pro-OH, were coupled by following exactly the same procedures as described above to yield the desired Fmoc-(Pro).sub.4-(Me)Ahx-O-Rink resin.

The resin was then treated with 1% TFA in DCM (20 mL, 10 min) to release Fmoc-(Pro).sub.4-(Me)Ahx-OH from the resin. This cleavage reaction was repeated one more time to ensure complete reaction. The crude product obtained was purified by flash column chromatography on silica gel (mobile phase: 5% Methanol in DCM) to yield the desired Fmoc-(Pro).sub.4-(Me)Ahx-OH as a white solid (0.90 g, 92%). .sup.1H NMR (CD.sub.2Cl.sub.2): d 1.2 1.7 (m, 6H), 1.9 2.4 (m, 18H), 2.80 (s, 3H), 3.2 3.6 (m, 10H), 4.2 4.7 (m, 7H), 7.1 7.6 (m, 8H), 9.6 (br, 1H). ESI-MS: m/z 756.0 (M+H.sup.+).

A mixture of Fmoc-(Pro).sub.4-(Me)Ahx-OH (0.90 g, 1.19 mmol), HATU (0.45 g, 1.19 mmol), and DIPEA (0.15 g, 1.19 mmol) in 8 mL of anhydrous DMF was added to 0.7 g of 3-aminopropyl silica gel (APS). APS was prepared from Kromasil.RTM. silica gel (5 .mu.m spherical silica, 100 .ANG., 298 m.sup.2/g) and 3-aminopropyltriethoxysilane. The surface amino concentration is 0.66 mmol/g, based on elemental analysis data of nitrogen (C, 3.11; H, 0.83; N, 0.93). After agitating the mixture for 4 h, the stationary phase was collected by filtration and washed with DMF, DCM, and Methanol (10 mL.times.3). The surface Fmoc concentration was determined to be 0.27 mmol/g based on Fmoc cleavage method. The resulting chiral stationary phase was packed into a 50.times.4.6 mm HPLC column using the standard slurry packing method.

The following examples set forth various chromatographic measurements. Therein, retention factor (k) equals to (t.sub.r-t.sub.0)/t.sub.0 in which t.sub.r is the retention time and t.sub.0 is the dead time. The separation factor (.alpha.) equals k.sub.2/k.sub.1, ratio of the retention factors of the two enantiomers. Separation factor of 1 indicates no separation. The larger the separation factor, the better the separation is. Dead time t.sub.0 was measured with 1,3,5-tri-t-butylbenzene as the void volume marker. Flow rate at 1 mL/min., UV detection at 254 nm.

Example 4

This example compares chromatographic resolution of racemic compounds with chiral columns, including embodiments of the present invention (Pro.sub.2 (CSP2), Pro.sub.4 CSP3), Pro.sub.6(CSP4)). In the following table, k.sub.1 is the retention factor of the least retained enantiomer and the separation factor (.alpha.) is defined earlier. This example also shows that a mono-proline chiral column does not perform sufficiently.

Furthermore, this example shows embodiments of the present invention in comparison with known commercial columns.

TABLE-US-00001 TABLE 1 Chromatographic resolution of racemic compounds with chiral columns. k.sub.1 is the retention factor of the least retained enantiomer. Mobile phases are solutions of specified percentage of IPA and acetic acid in hexanes. Daicel Daicel Whelk- Analyte name Analyte Structure Pro1 Pro2 Pro4 Pro6 OD AD O2 Benzoin ##STR00004## a: 1k.sub.1: 5.783% IPA a: 1.07k.sub.1: 8.223% IPA a: 1.09k.sub.1: 6.353% IPA a: 1.12k.sub.1: 16.05% IPA a: 1.61k.sub.1: 4.683% IPA a: 1.32k.sub.1: 3.4615% IPA a: 2.12k.sub.1: 1.835% IPA Hydrobenzoin ##STR00005## a: 1k.sub.1: 17.714% IPA a: 1.12k.sub.1: 21.154% IPA a: 1.13k.sub.1: 17.984% IPA a: 1.15k.sub.1: 25.798% IPA a: 1k.sub.1: 7.354% IPA a: 1.08k.sub.1: 5.158% IPA a: 1.33k.sub.1: 4.184% IPA Benzoin oxime ##STR00006## a: 1k.sub.1: 12.2820% IPA a: 1.09k.sub.1: 16.0820% IPA a: 1.13k.sub.1: 15.3620% IPA a: 1.20k.sub.1: 41.4530% IPA a: 1.13k.sub.1: 2.8210% IPA a: 1.24k.sub.1: 4.5515% IPA a: 1.31k.sub.1: 1.4010% IPA 2,2,2-Trifluoro-1-(9-anthryl)ethanol ##STR00007## a: 1k.sub.1: 16.4010% IPA a: 1.28k.sub.1: 23.4410% IPA a: 1.56k.sub.1: 18.4810% IPA a: 1.78k.sub.1: 22.5825% IPA a: 1.13k.sub.1: 1.2615% IPA a: 1.47k.sub.1: 1.9910% IPA a: 1.13k.sub.1: 0.6210% IPA .alpha.-(pentafluoroethyl)-.alpha.-(trifluoromethyl)-Benzenemethanol ##STR00008## a: 1k.sub.1: 19.313% IPA a: 1.06k.sub.1: 16.083% IPA a: 1.10k.sub.1: 8.913% IPA a: 1.10k.sub.1: 8.625% IPA a: 1.16k.sub.1: 0.901% IPA a: 1.11k.sub.1: 0.793% IPA a: 1k.sub.1: 0.703% IPA Warfarin ##STR00009## a: 1k.sub.1: 13.9110% IPA &1% AcOH a: 1.11k.sub.1: 10.5710% IPA &1% AcOH a: 1.08k.sub.1: 11.1910% IPA &1% AcOH a: 1.18k.sub.1: 17.5010% IPA &1% AcOH a: 2.49k.sub.1: 6.4015% IPA a:3.94k.sub.1: 5.0220% IPA a: 1.97k.sub.1: 10.0620% IPA Sec-Phenethylalcohol ##STR00010## a: 1k.sub.1: 6.671% IPAin hexane a: 1.02k.sub.1: 11.31% IPAin hexane a: 1.02k.sub.1: 8.071% IPAin hexane a: 1.04k.sub.1: 19.421% IPAin hexane a: 1.37k.sub.1: 8.301% IPAin hexane a: 1k.sub.1: 4.122% IPAin hexane a: 1.03k.sub.1: 3.642% IPAin hexane a-Methyl-2-Naphthalene-methanol ##STR00011## a:1k.sub.1: 13.361% IPA a: 1k.sub.1: 22.361% IPA a: 1.04k.sub.1: 17.621% IPA a: 1k.sub.1: 18.813% IPA a: 1k.sub.1: 6.253% IPA a: 1.05k.sub.1: 1.9810% IPA a: 1.02k.sub.1: 4.393% IPA 1-Acenaphthenol ##STR00012## a: 1k.sub.1: 7.833% IPA a: 1k.sub.1: 13.363% IPA a: 1k.sub.1: 10.283% IPA a: 1k.sub.1: 28.063% IPA a: 1.16k.sub.1: 5.463% IPA a: 1.08k.sub.1: 6.583% IPA a: 1.28k.sub.1: 4.963% IPA 3-Phenyl-Glycidol ##STR00013## a: 1k.sub.1: 5.233% IPA a: 1k.sub.1: 5.643% IPA a: 1k.sub.1: 6.773% IPA a: 1k.sub.1: 10.385% IPA a: 1.15k.sub.1: 16.8710% IPA a: 1k.sub.1: 8.018% IPA a: 1.37k.sub.1: 8.7410% IPA 1,1'-Bi-2-naphthol ##STR00014## a: 1.04k.sub.1: 11.4875% IPA a: 1.16k.sub.1: 32.8075% IPA a: 1.29k.sub.1: 23.8375% IPA a: 1.42k.sub.1: 9.9490% IPA a: 1.16k.sub.1: 4.498% IPA a: 1.13k.sub.1: 3.5825% IPA a: 1k.sub.1: 1.265% IPA 2,2'-Diol-5,5',6,6',7,7',8,8'-octahydro-1,1'-binaphthalene ##STR00015## a: 1.14k.sub.1: 12.5810% IPA a: 1.17k.sub.1: 11.1010% IPA a: 1.32k.sub.1: 10.9510% IPA a: 1.67k.sub.1: 19.7525% IPA a: 1.32k.sub.1: 3.985% IPA a: 1k.sub.1: 8.5310% IPA a: 1k.sub.1: 4.475% IPA 1,2,3,4-Tetrahydro-4-(4-methoxyphenyl)-6-methyl-2-thioxo-5-pyrimidinecarbo- xylicacid ethylester ##STR00016## a: 1.05k.sub.1: 14.8115% IPA a: 1.18k.sub.1: 18.6815% IPA a: 1.24k.sub.1: 12.6015% IPA a: 1.20k.sub.1: 23.5315% IPA a: 1.15k.sub.1: 2.1315% IPA a: 1.40k.sub.1: 3.4415% IPA a: 1.16k.sub.1: 2.2715% IPA 1,2,3,4-Tetrahydro-4-(4-hydroxyphenyl)-6-methyl-2-thioxo-5-Pyrimidine-carb- oxylic acidethyl ester ##STR00017## a: 1.12k.sub.1: 44.6630% IPA a: 1.20k.sub.1: 27.8050% IPA a: 1.41k.sub.1: 25.0730% IPA a: 1.32k.sub.1: 30.1370% IPA a: 1.30k.sub.1: 2.8715% IPA a: 1.36k.sub.1: 4.6215% IPA a: 1k.sub.1: 2.2315% IPA 1-[1,2,3,4-Tetrahydro-4-(4-methoxyphenyl)-6-methyl-2-thioxo-5-pyrimidinyl]- ethanone ##STR00018## a: 1k.sub.1: 27.0315% IPA a: 1.20k.sub.1: 40.6015% IPA a: 1.21k.sub.1: 25.7215% IPA a: 1.34k.sub.1: 33.9025% IPA a: 1.18k.sub.1: 2.6215% IPA a: 1.70k.sub.1: 3.6215% IPA a: 1k.sub.1: 1.8315% IPA Hexobarbital ##STR00019## a: 1k.sub.1: 28.861% IPA a: 1k.sub.1: 22.281% IPA a: 1k.sub.1: 16.981% IPA a: 1k.sub.1: 11.263% IPA a: 1.12k.sub.1: 6.265% IPA a: 1.46k.sub.1: 2.428% IPA a: 1k.sub.1: 1.955% IPA Temazepam ##STR00020## a: 1.09k.sub.1: 22.032% IPA a: 1k.sub.1: 25.542% IPA a: 1k.sub.1: 20.242% IPA a: 1k.sub.1: 25.525% IPA a: 1k.sub.1: 3.3925% IPA a: 1k.sub.1: 4.1225% IPA a: 1.19k.sub.1: 3.4025% IPA 5-Methyl-5-(2,5-dichloro-phenyl)hydantoin ##STR00021## a: 1k.sub.1: 11.2115% IPA a: 1.16k.sub.1: 14.615% IPA a: 1.17k.sub.1: 8.5515% IPA a: 1.34k.sub.1: 11.6425% IPA a: 1.08k.sub.1: 4.298% IPA a: 1k.sub.1: 4.808% IPA a: 1.11k.sub.1: 1.9610% IPA 5-Methyl-5-phenylhydantoin ##STR00022## a: 1k.sub.1: 16.248% IPA a: 1.10k.sub.1: 25.008% IPA a: 1.15k.sub.1: 15.68% IPA a: 1.32k.sub.1: 10.720% IPA a: 1.09k.sub.1: 4.068% IPA a: 1k.sub.1: 3.248% IPA a: 1.46k.sub.1: 1.6710% IPA Mephenytoin ##STR00023## a: 1k.sub.1: 5.862% IPA a: 1.14k.sub.1: 7.862% IPA a: 1.14k.sub.1: 6.932% IPA a: 1.27k.sub.1: 9.853% IPA a: 1.10k.sub.1: 5.204% IPA a: 1.37k.sub.1: 3.564% IPA a: 1k.sub.1: 3.354% IPA sec-Butylcarbanilate ##STR00024## a: 1k.sub.1: 3.621% IPA a: 1k.sub.1: 7.301% IPA a: 1k.sub.1: 7.641% IPA a: 1k.sub.1: 15.343% IPA a: 1k.sub.1: 6.182% IPA a: 1.04k.sub.1: 3.352% IPA a: 1.05k.sub.1: 3.361% IPA MethylMandelate ##STR00025## a: 1.02k.sub.1: 7.971% IPA a: 1.10k.sub.1: 10.81% IPA a: 1.17k.sub.1: 8.311% IPA a: 1k.sub.1: 17.493% IPA a: 1.25k.sub.1: 2.823% IPA a: 1.08k.sub.1: 1.8510% IPA a: 1.07k.sub.1: 1.1210% IPA

Example 5

Specific Embodiments, for Exemplary Purposes, of the Stationary Phase Compounds of the Present Invention and Silica Supports

This example sets forth poly-proline compounds of the present invention, including embodiments with different end-capping groups. The end-capping groups are bonded to the nitrogen atom that is further away from the support. As is noted in the example, some end-capping groups such as pivaloyl (PIV) (CSP-6) are more effective for some analytes than others, such as TAPA. Overall, several different end-capping groups useable with the present invention such as Piv, Fmoc, Boc, Cbz, Aca, Dmb, Tpa all work well. CSP-5, which has no end-capping group, did not perform as well with respect to some analytes.

TABLE-US-00002 TABLE 2 Impact of end-capping groups. Pro-Pro-N(Me)-Ahx-APS: CSP-5 ##STR00026## Piv-Pro-Pro-N(Me)-Ahx-APS: CSP-6 ##STR00027## Fmoc-Pro-Pro-N(Me)-Ahx-APS: CSP-2 ##STR00028## Boc-Pro-Pro-N(Me)-Ahx-APS: CSP-7 ##STR00029## Cbz-Pro-Pro-N(Me)-Ahx-APS: CSP-8 ##STR00030## Aca-Pro-Pro-N(Me)-Ahx-APS: CSP-9 ##STR00031## Tapa-Pro-Pro-N(Me)-Ahx-APS: CSP-10 ##STR00032## Dmb-Pro-Pro-N(Me)-Ahx-APS: CSP-11 ##STR00033## Tpa-Pro-Pro-N(Me)-Ahx-APS: CSP-12 ##STR00034## Analyte name CSP-5 CSP-6 CSP-2 CSP-7 CSP-8 CSP-9 CSP-10 CSP-11 CSP-12 Benzoin a: 1 a: 1.12 a: 1.07 a: 1.07 a: 1.08 a: 1 a: 1 a: 1.10 a: 1.07 k.sub.1: 5.84 k.sub.1: 6.34 k.sub.1: 8.22 k.sub.1: 4.45 k.sub.1: 4.63 k.sub.1: 7.71 k.sub.1: 19.02 k.sub.1: 7.76 k.sub.1: 6.00 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA Hydrobenzoin a: 1.07 a: 1.22 a: 1.12 a: 1.11 a: 1.11 a: 1.10 a: 1.14 a: 1.13 a: 1.16 k.sub.1: 16.71 k.sub.1: 17.00 k.sub.1: 21.15 k.sub.1: 10.71 k.sub.1: 13.33 k.sub.1: 16.81 k.sub.1: 26.41 k.sub.1: 18.09 k.sub.1: 15.17 4% IPA 4% IPA 4% IPA 4% IPA 4% IPA 4% IPA 4% IPA 4% IPA 4% IPA Benzoin oxime a: 1 a: 1.12 a: 1.09 a: 1 a: 1 a: 1 a: 1 a: 1.10 a: 1.08 k.sub.1: 11.44 k.sub.1: 14.65 k.sub.1: 16.08 k.sub.1: 10.09 k.sub.1: 12.23 k.sub.1: 13.44 k.sub.1: 16.03 k.sub.1: 15.00 k.sub.1: 11.71 20% IPA 20% IPA 20% IPA 20% IPA 20% IPA 20% IPA 20% IPA 20% IPA 20% IPA 2,2,2-Trifluoro-1- a: 1 a: 1.58 a: 1.28 a: 1.28 a: 1.33 a: 1.16 a: 1.28 a: 1.30 a: 1.40 (9-anthryl) k.sub.1: 17.23 k.sub.1: 22.4 k.sub.1: 23.44 k.sub.1: 15.08 k.sub.1: 15.82 k.sub.1: 20.02 k.sub.1: 31.59 k.sub.1: 20.47 k.sub.1: 16.03 ethanol 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA .alpha.- a: 1 a: 1.14 a: 1.06 a: 1.11 a: 1.10 a: 1.06 a: 1.10 a: 1.09 a: 1.09 (pentafluoroethyl)- k.sub.1: 9.72 k.sub.1: 8.89 k.sub.1: 16.08 k.sub.1: 7.55 k.sub.1: 5.76 k.sub.1: 6.18 k.sub.1: 8.13 k.sub.1: 13.80 k.sub.1: 5.75 .alpha.-(trifluoromethyl)- 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA Benzenemethanol Warfarin a: 1 a: 1.20 a: 1.11 a: 1 a: 1.16 a: 1.12 a: 1 a: 1.16 a: 1.14 k.sub.1: 41.10 k.sub.1: 12.41 k.sub.1: 10.57 k.sub.1: 19.53 k.sub.1: 11.55 k.sub.1: 14.34 k.sub.1: 28.61 k.sub.1: 13.41 k.sub.1: 12.68 90% IPA 10% IPA 10% IPA 25% IPA 10% IPA 10% IPA 10% IPA 25% IPA 10% IPA & 1% & 1% & 1% & 1% & 1% & 1% AcOH AcOH AcOH AcOH AcOH AcOH Sec-Phenethyl a: 1 a: 1.08 a: 1.02 a: 1.03 a: 1 a:1 a: 1 a: 1 a: 1 alcohol k.sub.1: 6.47 k.sub.1: 8.42 k.sub.1: 11.3 k.sub.1: 6.30 k.sub.1: 6.02 k.sub.1: 6.84 k.sub.1: 9.37 k.sub.1: 19.34 k.sub.1: 1.90 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA a-Methyl-2- a: 1 a: 1 a: 1 a: 1 a: 1.02 a: 1.10 a: 1 a: 1.04 a: 1 Naphthalene- k.sub.1: 15.30 k.sub.1: 17.31 k.sub.1: 22.36 k.sub.1: 13.07 k.sub.1: 12.96 k.sub.1: 14.89 k.sub.1: 17.15 k.sub.1: 7.65 k.sub.1: 1.19 methanol 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 5% IPA 1% IPA 1% IPA 1-Acenaphthenol a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 k.sub.1: 8.12 k.sub.1: 10.78 k.sub.1: 13.36 k.sub.1: 7.34 k.sub.1: 8.45 k.sub.1: 11.04 k.sub.1: 21.41 k.sub.1: 13.90 k.sub.1: 8.95 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 7% IPA 3% IPA 3% IPA 3-Phenyl- a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 Glycidol k.sub.1: 9.54 k.sub.1: 11.13 k.sub.1: 5.64 k.sub.1: 10.75 k.sub.1: 9.12 k.sub.1: 11.29 k.sub.1: 20.85 k.sub.1: 15.49 k.sub.1: 9.78 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 3% IPA 1,1'-Bi-2- a: 1.05 a: 1.34 a: 1.16 a: 1.17 a: 1.17 a: 1.35 a: 1 a: 1.18 a: 1.30 naphthol k.sub.1: 13.31 k.sub.1: 20.90 k.sub.1: 32.80 k.sub.1: 13.21 k.sub.1: 17.07 k.sub.1: 15.73 k.sub.1: 28.63 k.sub.1: 21.89 k.sub.1: 19.51 75% IPA 75% IPA 75% IPA 75% IPA 75% IPA 75% IPA 75% IPA 75% IPA 75% IPA 2,2'-Diol- a: 1 a: 1.08 a: 1.17 a: 1.06 a: 1.14 a: 1.33 a: 1.34 a: 1.24 a: 1 5,5,6,6',7,7',8,8- k.sub.1: 13.03 k.sub.1: 17.92 k.sub.1: 11.10 k.sub.1: 8.80 k.sub.1: 12.16 k.sub.1: 12.33 k.sub.1: 7.83 k.sub.1: 13.54 k.sub.1: 12.73 octahydro-1,1'- 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA 10% IPA binaphthalene 1,2,3,4- a: 1 a: 1.30 a: 1.18 a: 1.19 a: 1.15 a: 1.22 a: 1.48 a: 1.21 a: 1.26 Tetrahydro-4-(4- k.sub.1: 11.52 k.sub.1: 11.34 k.sub.1: 18.68 k.sub.1: 7.91 k.sub.1: 12.93 k.sub.1: 13.36 k.sub.1: 23.07 k.sub.1: 12.98 k.sub.1: 9.61 methoxyphenyl)- 10% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 6-methyl-2-thioxo-5- pyrimidinecarboxylic acid ethyl ester 1,2,3,4- a: 1.07 a: 1.49 a: 1.20 a: 1.40 a: 1.36 a: 1.37 a: 1.26 a: 1.40 a: 1.36 Tetrahydro-4-(4- k.sub.1: 36.72 k.sub.1: 39.00 k.sub.1: 27.80 k.sub.1: 23.11 k.sub.1: 31.14 k.sub.1: 27.18 k.sub.1: 28.56 k.sub.1: 24.64 k.sub.1: 16.80 hydroxyphenyl)- 70% IPA 30% IPA 50% IPA 30% IPA 30% IPA 30% IPA 40% IPA 40% IPA 30% IPA 6-methyl-2- thioxo-5- Pyrimidine carboxylic acid ethyl ester 1-[1,2,3,4- a: 1 a: 1.29 a: 1.20 a: 1.24 a: 1.11 a: 1.34 a: 1 a: 1.34 a: 1.23 Tetrahydro-4-(4- k.sub.1: 26.54 k.sub.1: 26.27 k.sub.1: 40.60 k.sub.1: 19.00 k.sub.1: 32.93 k.sub.1: 31.72 k.sub.1: 50.85 k.sub.1: 34.45 k.sub.1: 17.30 methoxyphenyl)- 10% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 6-methyl-2- thioxo-5- pyrimidinyl]ethanone Hexobarbital a: 1 a: 1 a: 1 a: 1.22 a: 1.10 a: 1 a: 1 a: 1 a: 1. k.sub.1: 29.92 k.sub.1: 11.51 k.sub.1: 22.28 k.sub.1: 7.21 k.sub.1: 13.11 k.sub.1: 15.02 k.sub.1: 9.74 k.sub.1: 14.94 k.sub.1: 15.27 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 2% IPA 1% IPA 1% IPA Temazepam a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 a: 1 k.sub.1: 20.77 k.sub.1: 17.73 k.sub.1: 25.54 k.sub.1: 12.52 k.sub.1: 17.34 k.sub.1: 27.75 k.sub.1: 25.44 k.sub.1: 20.24 k.sub.1: 27.30 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 10% IPA 2% IPA 2% IPA 5-Methyl- a: 1 a: 1.30 a: 1.16 a: 1 a: 1 a: 1 a: 1 a: 1.22 a: 1.16 5-(2,5-dichloro k.sub.1: 9.35 k.sub.1: 10.00 k.sub.1: 14.6 k.sub.1: 12.39 k.sub.1: 15.78 k.sub.1: 8.27 k.sub.1: 7.52 k.sub.1: 9.91 k.sub.1: 7.30 phenyl)hydantoin 10% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 15% IPA 5-Methyl-5- a: 1 a: 1.18 a: 1.10 a: 1.08 a: 1.16 a: 1 a: 1.16 a: 1.10 a: 1 phenyl k.sub.1: 18.12 k.sub.1: 17.91 k.sub.1: 25.00 k.sub.1: 12.93 k.sub.1: 14.71 k.sub.1: 13.54 k.sub.1: 13.44 k.sub.1: 19.00 k.sub.1: 14.25 hydantoin 8% IPA 8% IPA 8% IPA 8% IPA 8% IPA 8% IPA 8% IPA 8% IPA 8% IPA Mephenytoin a: 1 a: 1 a: 1.14 a: 1 a: 1 a: 1 a: 1.10 a: 1 a: 1.07 k.sub.1: 7.42 k.sub.1: 5.73 k.sub.1: 7.86 k.sub.1: 5.43 k.sub.1: 5.43 k.sub.1: 6.27 k.sub.1: 9.74 k.sub.1: 8.09 k.sub.1: 9.00 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA 2% IPA sec-Butyl a: 1.47 a: 1 a: 1 a: 1.04 a: 1 a: 1 a: 1 a: 1 a: 1 carbanilate k.sub.1: 4.09 k.sub.1: 9.04 k.sub.1: 7.30 k.sub.1: 5.91 k.sub.1: 5.91 k.sub.1: 7.74 k.sub.1: 11.89 k.sub.1: 9.64 k.sub.1: 9.61 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA Methyl a: 1 a: 1.23 a: 1.10 a: 1 a: 1 a: 1.10 a: 1 a: 1 a: 1 Mandelate k.sub.1: 9.24 k.sub.1: 9.00 k.sub.1: 10.8 k.sub.1: 6.50 k.sub.1: 8.46 k.sub.1: 6.27 k.sub.1: 19.37 k.sub.1: 19.00 k.sub.1: 17.64 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA 1% IPA

Example 6

This example compares chromatographic resolution of racemic compounds with Fmoc-Pro-Pro-Pro-Pro-N(Me)Ahx-APS (CSP-3) which is an embodiment of the present invention, in two mobile phase systems. Accordingly, this example helps demonstrate the flexibility of chiral stationary phases of the present invention in different mobile phase systems.

TABLE-US-00003 TABLE 3 Chromatographic resolution of racemic compounds with Fmoc-Pro- Pro-Pro-Pro-N(Me)Ahx-APS (CSP-3) in two mobile phase systems Analyte name IPA/Hex DCM/Hex/MeOH Benzoin a: 1.09 a: 1.07 k.sub.1: 6.35 k.sub.1: 11.61 3% IPA 5% DCM in Hexane Hydrobenzoin a: 1.13 a: 1.12 k.sub.1: 17.98 k.sub.1: 12.93 4% IPA 40% DCM in Hexane Benzoin oxime a: 1.13 a: 1.08 k.sub.1: 15.36 k.sub.1: 15.85 20% IPA 100% DCM 2,2,2-Trifluoro-1- a: 1.56 a: 1.20 (9-anthryl) k.sub.1: 18.48 k.sub.1: 9.54 ethanol 10% IPA 100% DCM .alpha.-(pentafluoroethyl)- a: 1.10 a: 1.06 .alpha.-(trifluoromethyl)- k.sub.1: 8.91 k.sub.1: 28.23 Benzenemethanol 3% IPA 30% DCM in Hexane Warfarin a: 1.08 a: 1 k.sub.1: 11.19 k.sub.1: 5.84 10% IPA & 30% DCM 1% in Hexane & AcOH 1% AcOH Sec-Phenethyl a: 1.02 a: 1.02 alcohol k.sub.1: 8.07 k.sub.1: 13.08 1% IPA 10% DCM in Hexane a-Methyl-2- a: 1.04 a: 1 Naphthalenemethanol k.sub.1: 17.62 k.sub.1: 23.66 1% IPA 10% DCM in Hexane 1-Acenaphthenol a: 1 a: 1 k.sub.1: 10.28 k.sub.1: 7.31 3% IPA 30% DCM in Hexane 3-Phenyl-Glycidol a: 1 a: 1 k.sub.1: 6.77 k.sub.1: 7.39 3% IPA 30% DCM in Hexane 1,1'-Bi-2-naphthol a: 1.29 a: 1.06 k.sub.1: 23.83 k.sub.1: 12.21 75% IPA 1% MeOH in Hexane 2,2'-Diol- a: 1.32 a: 1 5,5',6,6',7,7',8,8'- k.sub.1: 10.95 k.sub.1: 12.08 octahydro-1,1'-binaphthalene 10% IPA 50% DCM in Hexane 1,2,3,4-Tetrahydro-4- a: 1.24 a: 1.18 (4-methoxyphenyl)- k.sub.1: 12.60 k.sub.1: 6.26 6-methyl-2-thioxo- 15% IPA 60% DCM 5-pyrimidinecarboxylic acid in Hexane ethyl ester 1,2,3,4-Tetrahydro-4- a: 1.41 a: 1.19 (4-hydroxyphenyl)- k.sub.1: 25.07 k.sub.1: 12.72 6-methyl-2-thioxo-5- 30% IPA 3% MeOH Pyrimidinecarboxylic acid in Hexane ethyl ester 1-[1,2,3,4-Tetrahydro-4- a: 1.21 a: 1.22 (4-methoxyphenyl)- k.sub.1: 25.72 k.sub.1: 12.20 6-methyl-2-thioxo- 15% IPA 60% DCM 5-pyrimidinyl]ethanone in Hexane Hexobarbital a: 1 a: 1 k.sub.1: 16.98 k.sub.1: 8.08 1% IPA 30% DCM in Hexane Temazepam a: 1 a: 1 k.sub.1: 20.24 k.sub.1: 4.62 2% IPA 10% DCM in Hexane 5-Methyl- a: 1.17 a: 1.12 5-(2,5-dichloro k.sub.1: 8.55 k.sub.1: 13.56 phenyl)hydantoin 15% IPA 100% DCM 5-Methyl-5-phenyl a: 1.15 a: 1.11 hydantoin k.sub.1: 15.6 k.sub.1: 18.51 8% IPA\ 100% DCM Mephenytoin a: 1.14 a: 1.17 k.sub.1: 6.93 k.sub.1: 17.10 2% IPA 20% DCM in Hexane sec-Butyl carbanilate a: 1 a: 1.12 k.sub.1: 7.64 k.sub.1: 4.16 1% IPA 10% DCM in Hexane Methyl Mandelate a: 1.17 a: 1 k.sub.1: 8.31 k.sub.1: 8.18 1% IPA 10% DCM in Hexane

The invention being described, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the Attachments be considered as exemplary only, and not intended to limit the scope and spirit of the invention.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, experimental results, and so forth used in the Specification and Attachments are to be understood as being modified by the term "about." Accordingly, unless specifically indicated to the contrary, are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

REFERENCES

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