Patent Application: US-83360204-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 present inventor found from the same library a chiral selector [ fmoc - hyp ( tbu )- pro ] that has broad chiral selectivity when compared to chiral selectors such as those mentioned above , which may lead to a general chiral column . although this high throughput approach is designed to identify effective chiral selectors for a given analyte , newly prepared columns could be evaluated quickly for general chiral separations . in certain embodiments , this column contains only hydrogen bonding acceptor groups but no hydrogen bonding donor groups . it was subsequently evaluated using racemic compounds that contain one or more hydrogen bonding donor groups ( ho or nh group ). of the thirteen compounds chosen based on their availability , it resolved nine . since many compounds contain one or more hydrogen bonding donor groups , the column of the present invention could prove to be a useful general chiral column . 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 . according to the online catalogue of chiral technologies , distributor of the most successful chiral columns , their five best columns could resolve 80 % of the racemic compounds submitted to them . 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 1 ( fig1 ) and diproline based chiral stationary phase 2 have relatively broad chiral selectivity , while mono - pro stationary phase 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 n - alkylamino group . a second example is a n - methylamino group . another example is 6 - n - methylaminohexanoic acid . the particular linker group can be selected by on 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 - amonihexanoic acid , it resolved about 4 out of the same group of analytes . preferred linkers of the present invention do not introduce a hydrogen - bonding donor nh group next to the pro tetramer . the amide bond between this linker and pro residue is also more sterically hindered due to the n - methyl group . a preferred linker of the present invention is shown in connection with the synthesis shown in fig2 . 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 , analog are included . examples of the analogs that are included herein are those with the following structure feature : wherein n is an integer ( such as 1 , 2 , 3 , 4 , 5 , etc .) and x is o , s , n . the columns of the present invention were evaluated using 22 racemic mixtures chosen based on their availability . the separation factors along with the capacity factors for the fast eluting enantiomer are shown below . for comparison , resolution of these 22 compounds was also studied with daicel ad , daicel od , and whelk o2 columns , the three most popular chiral columns used in normal phase mode . their separation factors along with the capacity factors for the fast eluting enantiomer are also shown in an example , below . as seen in the examples , the di - proline and tetra - proline columns of the present invention are superior . 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 , finding column efficiency . 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 . 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 ; fmoc -( me ) ahx - oh , 6 -[( 9h - fluoren - 9 - ylmethoxy ) carbonyl ] methylamino hexanoic 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 kromasil ® 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 prepared previously ( 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 prepared previously ( 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 was packed into a 50 × 4 . 6 mm hplc column using the standard slurry packing method . to 0 . 80 g of ( me ) ahx - aps silica prepared previously ( the surface ( me ) ahx concentration is 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 is filtered and washed with dmf , methanol , and dcm to yield the desired chiral stationary phase . the surface pro concentration was determined to be 0 . 57 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 . 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 . 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 ( pro2 , pro4 , pro6 ). k 1 is the retention factor of the least retained enantiomer . this example also shows that a mono - proline chiral column does not perform suffieiently . furthermore , this example shows embodiments of the present invention in comparison with known columns . 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 - 1 ) 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 - 11 , which has not 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 , 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 . welch , c . j . ; pollard , s . d . ; mathre , d . j . ; reider , p . j . improved method for rapid evaluation of chiral stationary phase libraries , 2001 . murer , p . ; lewandowski , k . ; svec , f . ; frechet , j . m . j . chemical communications ( cambridge ) 1998 , 2559 – 2560 . weingarten , m . d . ; sekanina , k . ; still , w . c . journal of the american chemical society 1998 , 120 , 9112 – 9113 . wang , y . ; bluhm , l . h . ; li , t . analytical chemistry 2000 , 72 , 5459 – 5465 . bluhm , l . h . ; wang , y . ; li , t . analytical chemistry 2000 , 72 , 5201 – 5205 . maclean , d . ; baldwin , j . j . ; ivanov , v . t . ; kato , y . ; shaw , a . ; schneider , p . ; gordon , e . m . pure and applied chemistry 1999 , 71 , 2349 – 2365 . blodgett , j . ; wang , y . ; li , t . ; polavarapu , p . l . ; drabowicz , j . ; pietrusiewicz , k . m . ; zygo , k . analytical chemistry 2002 , 74 , 5212 – 5216 . periasamy , m . aldrichimica acta 2002 , 35 , 89 – 101 . stinson , s . c . chemical & amp ; engineering news 1995 , 73 , 44 – 74 . okamoto , y . ; kawashima , m . ; hatada , k . journal of the american chemical society 1984 , 106 , 5357 – 5359 . yashima , e . ; yamamoto , c . ; okamoto , y . journal of the american chemical society 1996 , 118 , 4036 – 4048 . berthod , a . ; chen , x . ; kullman , j . p . ; armstrong , d . w . ; gasparrini , f . ; d &# 39 ; acquarica , i . ; villani , c . ; carotti , a . analytical chemistry 2000 , 72 , 1767 – 1780 . ekborg - 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