Patent Application: US-98723804-A

Abstract:
a general chiral column with a multipleproline - based chiral stationary phase . embodiments include chiral stationary phases of the following formula : wherein n is any integer of 2 or greater , and analogs and isomers thereof .

Description:
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 ( fig1 ). instead of having a primary amino group as in other α - amino acids , it contains a secondary amine . because of the cyclic structure , rotation around the nitrogen - α - carbon bond is restricted . also because of the cyclic structure , proline is not ideally suited for α - helix or β - 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 ( fig1 ), 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 : 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 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 ). 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 . 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 - α - fmoc - l - proline . 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 kromasile silica gel ( particle size 5 μm , pore size 100 å , and surface area 298 m 2 / g ) was purchased from akzo nobel ( eka chemicals , bohus , sweden ). selecto silica gel ( 32 - 63 μm ) 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 ). 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 × 4 . 6 mm hplc column using a standard slurry packing method . 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 × 4 . 6 mm hplc column using the standard slurry packing method . 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 × 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 × 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 × 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 ) 4 -( me ) ahx - o - rink resin . 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 %). 1 h nmr ( cd 2 cl 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 + ). a mixture of fmoc -( pro ) 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 ® silica gel ( 5 μm spherical silica , 100 å , 298 m 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 × 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 × 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 r − t 0 )/ t 0 in which t r is the retention time and t 0 is the dead time . the separation factor ( α ) 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 t 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 . this example compares chromatographic resolution of racemic compounds with chiral columns , including embodiments of the present invention ( pro 2 ( csp2 ), pro 4 csp3 ), pro 6 ( csp4 )). in the following table , k 1 is the retention factor of the least retained enantiomer and the separation factor ( α ) 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 . 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 . 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 . 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 . a . stinson , s . c . chemical & amp ; 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