Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof

Definitions

• 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.
• 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.
• 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.
• diastereomers have different physical properties, such as melting points, boiling points, solubilities in a given solvent, densities, refractive indices etc.
• diastereomers are normally separated from one another by conventional methods, such as fractional distillation, fractional crystallization or chromatography.
• Enantiomers present a special problem because their physical properties are identical.
• Chiral columns that can resolve a large number of racemic compounds are in high demand. They are needed routinely in many laboratories, especially in pharmaceutical industry.
• Daicel columns, macrocyclic antibiotic columns, and the Whelk-0 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.
• the columns of the present invention have the capability of resolving at least a similar or higher percentage of the compounds tested.
• 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.
• the successful examples include the popular Daicel columns, the Chirobiotic columns, and the Whelk-Ol/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-0 1/2 columns contain both electron rich and electron deficient aromatics.
• the present invention is directed to a chiral selector that represents an improvement in the art of enantiomeric separation.
• 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.
• Another embodiment of the present invention is a chiral stationary phase (or column) of the following formula:
• 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 02 columns.
• the multiple proline-based chiral columns of the present invention show promise as a superior general chiral column.
• Figure 1 shows the structure for amino acid L-Proline and its associated stationary phases Fmoc-Pro-(Me)Ahx-APS (CSPl), Fmoc-Pro 2 -(Me)Ahx-APS (CSP2), Fmoc-Pro 4 -(Me)Ahx-APS (CSP3); and Fmoc-Pro 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 4 - (Me)AhX-APS chiral stationary phase (CSP3): Synthesis of Fmoc-Pro 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.
• CSP3 Fmoc-Pro 4 - (Me)AhX-APS chiral stationary phase
• the present inventor has developed a new chiral column that has relatively broad chiral selectivity, when compared with Daicel columns and Whelk 02 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-02 resolved 15 compounds.
• the monoproline column (CSPl) 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 ( Figure 1). Instead of having a primary amino group as in other ⁇ -amino acids, it contains a secondary amine. Because of the cyclic structure, rotation around the nitrogen- ⁇ -carbon bond is restricted. Also because of the cyclic structure, proline is not ideally suited for ⁇ -helix or ⁇ -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.
• proline based chiral selectors including the embodiment tetraproline based chiral stationary phase 3 ( Figure 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.
• a linker group of the present invention is a N-alkylamino 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-methylaniinohexanoic 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.
• the stationary phase compounds of the present invention may comprise various end-capping groups as known in the art.
• proline 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:
• n is an integer (such as 1, 2, 3, 4, 5, etc.) and X is a heteroatom such as O 5 S 5 N; and other unspecified atoms can be carbon or heteroatoms.
• n is an integer (such as 1, 2, 3, 4, 5, etc.) and X is a heteroatom such as O 5 S 5 N; and other unspecified atoms can be carbon or heteroatoms.
• These covalently bound columns of the present invention are stable in common organic solvents, including CH 2 Cl 2 and CHCl 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 2 Cl 2 /hexane as the mobile phase and effective separation was also achieved (example 6).
• 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.
• steric interaction between analyte and chiral selector could also be important.
• DIC diisopropylcarbodiimide
• HATU O-(7-Azabenzotriazol- 1 -y ⁇ )-N,N,N',N -tetramethyluronium hexafluorophosphate
• DIPEA N,N-Diisopropylethylamine
• DMF N,N-Dimethylformamide
• DCM Dichloromethane
• DMAP 4-(dimethylaminopyridine
• ⁇ MM ⁇ -methylraorpholine
• 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)carbon
• Amino acid derivatives were purchased from ⁇ ovaBiochem (San Diego, CA). All other chemicals and solvents were purchased from Aldrich (Milwaukee, WI), Fluka (Ronkonkoma, NY), or Fisher Scientific (Pittsburgh, PA). HPLC grade Kromasil ® silica gel (particle size 5 ⁇ m, pore size 100 A, and surface area 298 m 2 /g) was purchased from Akzo Nobel (EKA Chemicals, Bohus, Sweden). Selecto silica gel (32-63 ⁇ m) from Fisher Scientific was used for flash column chromatographic purification of target compounds.
• the Fmoc protecting group was then removed by treatment of the silica with 10 mL of 20% (WV) piperidine in DMF for 1 h.
• the deprotected silica, Pro-(Me)Ahx-APS was collected by filtration and washed with DMF, Methanol, and DCM.
• 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 x 4.6 mm HPLC column using the standard slurry packing method.
• Example 3 Preparation ofchiral stationary phase Fmoc-Pr ⁇ 4-(Me)Ahx-APS (CSP 3) 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).
• CSP 3 Preparation ofchiral stationary phase Fmoc-Pr ⁇ 4-(Me)Ahx-APS
• the resin was then treated with 1% TFA in DCM (20 mL, 10 min) to release Fmoc-(Pro) 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)4-(Me)Ahx-OH as a white solid (0.90 g, 92%).
• the stationary phase was collected by filtration and washed with DMF, DCM, and Methanol (10 mL x 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 x 4.6 mm HPLC column using the standard slurry packing method.
• retention factor (k) equals to (t r -to)/to in which t r is the retention time and to is the dead time.
• the separation factor ( ⁇ ) equals k 2 /k 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 to was measured with 1,3,5-tri-t-butylbenzene as the void volume marker. Flow rate at 1 niL/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 2 (CSP2), Pro 4 CSP3), Pr ⁇ 6 (CSP4)).
• CSP2 Pro 2
• Pro 4 CSP3 Pro 4 CSP3
• Pr ⁇ 6 CSP4
• ki is the retention factor of the least retained enantiomer and the separation factor ( ⁇ ) is defined earlier
• ⁇ is defined earlier
• This example also shows that a mono-proline chiral column does not perform sufficiently.
• this example shows embodiments of the present invention in comparison with known commercial columns.
• Table 1 Chromatographic resolution of racemic compounds with chiral columns, ki is the retention factor of the least retained enantiomer.
• Mobile phases are solutions of specified percentage of IPA and acetic acid in hexanes.
• 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.
• some end- capping groups such as pivaloyl (PIV) (CSP-6) are more effective for some analytes than others, such as TAPA.
• PAV pivaloyl
• CSP-6 are more effective for some analytes than others, such as TAPA.
• TAPA Triavaloyl
• CSP-5 which has no end-capping group, did not perform as well with respect to some analytes.
• Pro-Pro-N(Me)- Ahx- APS CSP-5 Piv-Pro-Pro-N(Me)-Ahx-APS: CSP-6 Fmo Boc-Pro-Pro-N(Me)-Ahx-APS: CSP-7 Cbz-Pro-Pro-N(Me)-Ahx-APS: CSP-8 Aca-Pro-Pro-N(Me)-Ahx-APS: CSP-9 Tapa-Pro-Pro-N(Me)-Ahx-APS: CSP-IQ Dmb-Pro-Pro-N(Me)-Ahx-APS: CSP-Il Table 2. Impact of end-capping groups.
• 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 3. Chromatographic resolution of racemic compounds with Fmoc-Pro-Pro-Pro-Pro-N(Me)Ahx-APS (CSP-3) in two mobile phase systems

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• 2004
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