CN1864853A - Biological micromolecule or its analogue bonded chiral stationery phase - Google Patents
Biological micromolecule or its analogue bonded chiral stationery phase Download PDFInfo
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- CN1864853A CN1864853A CN 200610011752 CN200610011752A CN1864853A CN 1864853 A CN1864853 A CN 1864853A CN 200610011752 CN200610011752 CN 200610011752 CN 200610011752 A CN200610011752 A CN 200610011752A CN 1864853 A CN1864853 A CN 1864853A
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Abstract
The invention relates to a process of preparing chiral fixed phase bonded by biomicromolecule or its analogues. It is characterized in that the connection arm bonded on carrier comprises ring structure or side chain, which bonds chiral selective agent through conjugated linkage or electrovalent bond. The ring structure or side chain on connection arm is double dentation or single dentation with side chain. The biomicromolecule or its analogues are chiral selective agent, and comprise 2-20 L or D proline, alpha-substituted proline or hydroxyproline and their analogues. The invention is suitable for high efficiency liquid chromatography, preparative chromatography, analog moving bed, microcylinder liquid chromatography and capillary electrochromatography.
Description
Technical field
The present invention relates to fixedly phase and preparation method thereof of biological micromolecule or its analogue bonded chiral.Biological micromolecule or its analogue bonded chiral chromatographic column are fixing to be a class chiral separation material mutually, and the preparation method adopts organic synthesis technology, particularly solid phase synthesis technique.
Background technology
Chiral isolation analysis method and technology are more and more important, especially in chemistry, biology, environmental science, fine chemistry industry, pharmacy and various product quality inspections field.Chromatographic technique is the topmost method of chiral separation, mainly comprises methods such as capillary gas chromatography, high performance liquid chromatography, supercritical fluid chromatography, Capillary Electrophoresis and electrochromatography at present.Directly the chiral separation method is carried out chiral separation with chiral chromatographic column exactly, and this separation can be applied to analyze the preparation with different scales.Chiral chromatographic column is the main tool that realizes chiral separation, and the chiral stationary phase in the chromatographic column is exactly the key of decision chromatogram chiral separation ability.
The high performance liquid chromatography chiral stationary phase mainly contains glycan, brush type, cyclodextrin type, protein type, big cyclohexanol peptide antibiotic type, ligand exchange type, crown ether type, acid amide type and quinine type etc. at present.The carrier material that uses mainly contains silica gel, zirconium glue, titanium glue, polymer and graphitic carbon etc., and most popular is silica gel.Also having another kind of chiral stationary phase in addition, is the fixedly phase of chiral selector exactly with the oligopeptides.Naobumi i etc. has reported that employing L-valine is a chiral selector, s-triazine L-L-valine ester, s-triazine L-valine dipeptides ester and s-triazine L-valine tripeptide ester chiral column have been prepared, find that s-triazine L-valine tripeptide ester chiral column has stronger chiral separation ability (Naobumi i, J.Chromatogr.A, 1996,722,229).Tingyu Li finds that proline oligopeptides chiral stationary phase has very strong chiral separation ability (U.S. Patent Publication No., US2004/0226889 A1, US2005/0092686 A1).Chiral molecules and chiral stationary phase are by temporary transient diastereo-isomerism complexs of formation such as bag and effect, hydrogen bond action, dipole-dipole effect, π-π effects, because two enantiomers are different with the bonded energy between chiral selector, the feasible diastereo-isomerism complexes stability difference that forms, and be achieved chiral separation.The oligopeptides fixedly chiral separation of phase mainly is based on the effect of hydrogen bond, and three-dimensional effect.Pirkle has systematically studied the influence of chiral stationary phase linking arm to chiral separation, and result of study points out that linking arm has important function (William H.Pirkle, J.Chromatogr.A, 1992,589,45) for the function of performance chiral selector.Pirkle has also prepared bidentate silane group chiral stationary phase, the bidentate linking arm can reduce chiral molecules and fixing alternate nonspecific action, strengthen the HYDROGEN BOND INTENSITY between chiral selector and separated chiral molecules, there is the linking arm rigidity of circulus strong, thereby significantly increasing post imitates and chiral separation ability (William H.Pirkle, J.Chromatogr.A, 1994,659,69).Kirkland has prepared fixedly phase of bidentate silane group C18 achirality, can separate alkaline matter (J.J.Kirkland mutually down flowing of pH=11, Anal Chem.1998,70,4344), the bidentate linking arm that liquid-phase chromatographic column is described can reduce the effect of silica gel remained on surface silicon hydroxyl, increases the stability of post.S-triazine L-valine tripeptide ester chiral column is done linking arm with the s-triazine, because the s-triazine is the structure of an electron rich, in the chiral separation process, play a part the π alkali of power supply, can form π-π effect with the π acid in the separated chiral molecules, linking arm has become the part of chiral selector in fact here.Linking arm, chiral selector and carrier are organic wholes, and linking arm can directly influence the performance of chiral selector function on carrier.Therefore having the bonded chiral of extensive with stronger chiral separation ability fixing mutually, is to be connected to carrier surface by chiral selector and the linking arm that adapts with it by covalent bond or ionic bond to form.The proline oligopeptides chiral stationary phase of Tingyu Li preparation adopts the methyl glycoleucine of straight chain as linking arm.The methyl glycoleucine of used Fmoc protection is a kind of thick liquid, synthesizing and purifying difficulty, long preservation conditional request height.The linking arm of this fixedly phase all is the monodentate structure, does not therefore possess the bidentate stability of structure.Should fix is covalent bonding mutually.We know that the chiral stationary phase by the ionic bond bonding also is (Naobumi i etc., J.Chromatogr.A, 1989,462,382) that the chiral separation ability is arranged.And this proline oligopeptides chiral stationary phase does not have the structure of ionic bond bonding.
Summary of the invention
Technical problem to be solved by this invention is: fixedly phase and preparation method thereof of a class biological micromolecule or its analogue bonded chiral chromatographic column is provided.The linking arm that is adopted comprises circulus or side chain, with biological micromolecule and analog thereof as chiral selector, by the linking arm bonding on covalent bond or ionic bond and the carrier.Fixedly phase structure provided by the present invention is stable, and the preparation method is simpler.
The carrier of indication of the present invention can be a silica gel, zirconium glue, titanium glue, polymer and graphitic carbon etc., and specifically the preferred carrier material of the present invention is a silica gel.The carrier particle diameter changes with used pillar size, but is generally 1 μ m to 10mm, preferred 1 μ m to 300 μ m.Carrier can be amorphous or spherical, and is preferred spherical.The most handy porous carrier.When using porous carrier, the average pore size 1nm of carrier～100nm, preferred 5nm～50nm.
Particularly, the invention provides fixedly phase of a kind of bonded chiral, its comprise carrier, with the linking arm of carrier bonding and with the chiral selector of linking arm bonding, the linking arm of bonding comprises circulus or side chain on the wherein said carrier, by covalent bond or ionic bond with the chiral selector bonding.
In one embodiment of the invention, described carrier is a silica gel, and its particle diameter is 1 μ m to 300 μ m, and average pore size is 5nm～50nm.
In another embodiment of the invention, described linking arm has bidentate structure or monodentate structure.
In another embodiment of the invention, described chiral selector is that the proline that replaced by proline, α or hydroxy-proline and their analog combine, and they are L or D spatial configuration, and quantity is 2～20.
Silica gel bonded linking arm provided by the present invention is as follows:
In the formula:
Y
1And Y
2Be 0～3 integer, must make this ring from the three-membered ring to the octatomic ring.Y
3It is 0～10 integer.
Biological micromolecule involved in the present invention and analog thereof are mainly by proline, L or the D type hydroxy-proline of L or D type proline, L or D type α replacement and oligopeptides or the oligopeptides analog that their analog is formed.The spatial configuration of wherein preferred L type.Biological micromolecule specifically involved in the present invention and analog thereof have following architectural feature:
In the formula, as the substituting group on each five-membered ring of construction unit, can be identical, also can be different, thereby different combinations can be arranged, produce numerous species like thing.The silica gel bonded linking arm that the L representative links to each other with ionic bond with covalent bond.R
1It is END CAPPED GROUP.R
1Can be that hydrogen atom, carbon number are that 1～12 alkoxy carbonyl group, carbon number are that 1～12 acyl group, carbon number are that 1～12 substituted aminocarbonyl, carbon number are that 1～18 contain or do not contain heteroatomic aromatic series oxygen carbonyl, carbon number are 1～18 contain or do not contain heteroatomic aromatic series substituted aminocarbonyl, be preferably as follows group especially:
The integer of n=0-20.
Work as X=O, S, CH
2The time, there is not R
5
When X=CH, R
5Be hydroxyl, be that carbon number is that 1～12 carbamate groups, carbon number are that 1～12 urea groups or ghiourea group, carbon number are that 1～12 ester group, carbon number are that 1～18 contain or do not contain heteroatomic aromatic amino ester formate base, carbon number are 1～18 contain or do not contain heteroatomic urea groups or ghiourea group, be preferably as follows group especially:
R
2, R
3, R
4Be that carbon number is that 1～12 alkyl, carbon number are 1～18 aromatic radical, are preferably as follows group especially:
Be the concrete structure of chiral stationary phase of the present invention below.
One, the fixing structure of phase of bidentate structure linking arm chiral chromatogram:
R in the formula
1, R
2, R
3, R
4, R
5, X, n is the same.
Y
1, Y
2=1～2, Y
3=0～10, the integer of m=0～1
R
6＝-CH
3 -CH
2-CH
3 -O-CH
3 -O-CH
2-CH
3
Two, the fixing structure of phase of monodentate structure linking arm chiral chromatogram:
R in the formula
1, R
2, R
3, R
4, R
5, X, n, Y
1, Y
2, Y
3, m is the same.
R in the formula, R
2, R
3, R
4, R
5, L, X, n is the same.
In a preferred embodiment, chiral chromatogram of the present invention is fixed and is had one of following structure mutually:
The present invention also provides a kind of liquid chromatogram chiral separation method, adopts fixedly phase of above-mentioned any bonded chiral.
The present invention also provides a kind of liquid-phase chromatographic column, comprises analytic type post, preparation type post, simulation moving-bed and electric chromatographic column, has wherein filled fixedly phase of above-mentioned any bonded chiral.
The chiral chromatogram of the present invention fixedly preparation method of phase covalent bond mould assembly is: silica-gel carrier is through overpickling, dry, reactions such as 3-aminopropyl silylating reagent with two (trimethoxy silicon propyl group) amine, 3-aminopropyl silylating reagent or nitrogen replacement, obtain amino bonded silica gel, this also is short link arm bonded silica gel simultaneously.This amino bonded silica gel can be further and the cyclic amino acids reaction of amido protecting, behind the amino deprotection, obtains long link arm amino bonded silica gel.One or more five-membered ring structure unit processes of these link arm bonded silica gels and the unprotected chiral selector of amido protecting carboxyl then; behind the amino deprotection; after passing through the coupling of suitable number of times again; last desamidizate protecting group obtains covalent bond mould assembly of the present invention with the capping reagent reaction and fixes mutually.
The chiral chromatogram of the present invention fixedly preparation method of phase ionic bond mould assembly is: above-mentioned amino link arm bonded silica gel is packed into the homogenate method behind the performance liquid chromatographic column.Oxolane (THF) solution with triethylamine is made the mobile pillar that washes mutually with 100% THF, allow band N hold the flowing of THF solution of the chiral selector of END CAPPED GROUP to flow through pillar mutually then, at last with the n-hexane flushing pillar of 10% isopropyl alcohol, up to baseline stability, obtain fixedly phase of ionic bond mould assembly chiral chromatogram of the present invention.
The chiral chromatographic column provided by the present invention fixedly concrete synthesis step of phase is as follows:
1, the preliminary treatment of silica gel: add a certain amount of concentrated sulfuric acid and red fuming nitric acid (RFNA) mixed liquor in the silica gel, the volume (mL) of silica gel weight (g) and acid is than being 1: 10～1: 20, heating is 10-48 hour in 90 ℃～160 ℃ oil baths, after the cooling, filter, silica gel is washed till neutrality with distilled water, and it is inferior to give a baby a bath on the third day after its birth with acetone again, in 110 ℃～180 ℃ drying for standby.
2, preparation link arm bonded silica gel: short link arm bonded silica gel obtains with silica gel and silylating reagent coupling.In pretreated silica gel, add dry toluene and silylating reagent, silica gel (g): coupling agent (mL): the ratio of toluene (mL) is 1: 3～10: 10～40.Under nitrogen protection, heating is 12-36 hour in 90 ℃～150 ℃ oil baths, after the cooling, filters, and solid is washed abundant washing with toluene and acetone, and is standby in 60 ℃～80 ℃ vacuum drying, so just obtains short link arm bonded silica gel.Silylating reagent can be the 3-aminopropyl silylating reagent of two (trimethoxy silicon propyl group) amine, 3-aminopropyl silylating reagent, nitrogen replacement etc.Be the synthetic more amino bonded silica gel of the linking arm of long-chain; at anhydrous N; in the dinethylformamide (DMF); add short link arm bonded silica gel; DIC; N-hydroxy benzo triazole (HOBT) and uncle's fourth oxygen oxygen carbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc) are protected amino cyclic amino acid; ratio DMF (mL): silica gel (g): DIC (N; N '-DIC) (mmol): HOBT (mmol): cyclic amino acid (mmol) is 5～10: 1: 1～8: 1～3: 1～8; stir after 2～24 hours; filter solid DMF; methyl alcohol and washed with dichloromethane.The Boc protecting group was sloughed with the dichloromethane solution reaction of 50% trifluoroacetic acid in 30～120 minutes, filtered, and solid washs with DMF, carrene and 50%N, N-diisopropylethylamine (DIPEA) dichloromethane solution, drying.The Fmoc protecting group was sloughed with the 20%DMF solution reaction of piperidines in 30～60 minutes, filtered solid DMF and washed with dichloromethane.Obtain the amino bonded silica gel of long-chain linking arm.
3, the fixedly preparation of phase of the chiral chromatogram of covalent bond mould assembly of the present invention: at anhydrous N, in the dinethylformamide (DMF), add aminolink bonded silica gel, 2-(7-azo BTA)-N, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester (HATU) and N, N-diisopropylethylamine (DIPEA).Add then Boc or the amino chiral selector unit of Fmoc protection are arranged.DMF (mL): silica gel (g): HATU (mmol): DIPEA (mmol): the ratio of chiral selector unit is 5～10: 1: 1～6: 1～3: 1～5.Stir after 2～24 hours, filter, solid DMF, methyl alcohol and washed with dichloromethane.The Boc protecting group was sloughed with the dichloromethane solution reaction of 50% trifluoroacetic acid in 30～120 minutes, and Fmoc sloughed with the 20%DMF solution reaction of piperidines in 30～60 minutes.Filter and washing back drying.This is a coupling process.Through after the coupling of suitable number of times, acid, acid anhydrides or acyl chloride reaction that silica obtained and end-blocking is used, acid, acid anhydrides or acyl chlorides are wanted excessive 3～6 times at least with respect to amino.Stir 2～24 hours anti-after, filter, solid is with DMF, methyl alcohol and washed with dichloromethane.Obtain the fixedly phase of covalent bond mould assembly of the present invention.
4, prepared the fixing of covalent bond mould assembly of the present invention loads performance liquid chromatographic column by homogenate method high pressure mutually.Carbon tetrachloride: dioxane (2: 1, volume ratio) is a homogenate, and ethanol is displacement fluid.
5, the fixedly preparation of phase of ionic bond mould assembly chiral chromatographic column of the present invention: on the silica-gel carrier bonding behind the linking arm, with homogenate method dress pillar, with the mobile post of washing mutually of the THF that contains the 2mL triethylamine (40mL), allow then and contain the THF solution (40mL) that 2g contains the chiral selector of END CAPPED GROUP and flow through pillar, at last with the n-hexane flushing pillar of 10% isopropyl alcohol, up to baseline stability, obtain the fixedly fixing phase of ionic bond mould assembly of phase of chiral chromatogram of the present invention.
Covalent bond mould assembly post provided by the present invention is applicable to positive, anti-phase mobile mutually with polarity, and the fixing of covalent bond mould assembly bidentate structure can tolerate higher pH mutually.Ionic bond mould assembly column preparation method is simple.Preparation method of the present invention is easy, cost is lower, and method is widely applicable.Prepared silica gel bonded fixedly phase surface bonding rate is higher, has higher stability, is applicable to analytical and preparative high performance liquid chromatography, micro column liquid chromatography, capillary electric chromatogram and separates science, technical field as relevant parting material etc.
Description of drawings
Fig. 1 fixedly is separated 2,2 with embodiment 2, and the high-efficient liquid phase chromatogram of 2-three fluoro-1-(anthryl)-ethanol (TFAE) enantiomer, two peaks, front and back are respectively ((+/-)-2,2,2-three fluoro-1-(anthryl)-ethanol).
Fig. 2 is with the fixing magnificent high-efficient liquid phase chromatogram that makes (Warfarin) enantiomer of phase separation method of embodiment 2, and two peaks, front and back are respectively that ((+/-)-Fa Hua makes).
Fig. 3 is with fixedly the be separated high-efficient liquid phase chromatogram of 1-(2-naphthyl) ethanol (1-(2-naphthyl) ethanol) enantiomer of embodiment 3, and two peaks, front and back are respectively ((+/-)-1-(2-naphthyl) ethanol (1-(2-naphthyl) ethanol)).
Fig. 4 fixedly is separated with embodiment 3 to separate the high-efficient liquid phase chromatogram of Troger ' s Base enantiomer, and two peaks, front and back are respectively ((+/-)-Troger ' s Base).
Fig. 5 is with fixedly the be separated high-efficient liquid phase chromatogram of hydrobenzoin (Hydrobenzoin) enantiomer of embodiment 4, and two peaks, front and back are respectively ((+/-)-hydrobenzoins).
Fig. 6 be with embodiment 4 fixedly be separated dinaphthalene amine (1,1 '-Binaphthyl-2,2 '-diamine) high-efficient liquid phase chromatogram of enantiomer, two peaks, front and back are respectively ((+/-)-dinaphthalene amine).
The specific embodiment
Give further instruction below by embodiment to the present invention.Embodiment only limits to the present invention is described and is not limitation of the invention.
A kind of bidentate structure linking arm silica gel.The chiral chromatogram fixedly preparation method of phase has two kinds, and a kind of is linking arm and chiral selector elder generation and silane coupler reaction, is being coupled to (William H.Pirkle, J.Chromatogr.A, 1992,589,45) on the silica gel then.Another kind is that silane coupler is coupled on the silica gel earlier, and then reacts with linking arm or chiral selector, is fixed phase (U.S. Patent Publication No., US2004/0226889 A1).The preferred a kind of preparation method in back of the present invention.Silica gel and silylating reagent coupling.The pretreated silica gel of 4.0 grams (Kromasil, 5 μ m, 100 , Switzerland) in, add dry toluene 120mL, two (trimethoxy silicon propyl group) amine (Gelest, Inc., Morrisville, PA.USA) 30mL.Under nitrogen protection, heating is 24 hours in 140 ℃ of oil baths, after the cooling, filters, and solid is washed abundant washing with toluene and acetone, in 80 ℃ of vacuum drying, obtains a kind of bidentate structure linking arm silica gel.This is similar in new linking arm structure and the document (William H.Pirkle, J.Chromatogr.A, 1992,589,45), and the latter is a triethoxysilylpropyl, and to adopt be first kind of preparation method.Linking arm elemental analysis bonded amount in the present embodiment, %C, 3.85; %H, 0.90; %N, 1.22; With nitrogen element calculation key resultant is 0.87mol/g.
Embodiment 2
In the 20mL dry DMF; add bonded silica gel 3.8 grams, 4 gram HATU (Sigma-Aldrich Corp.St.Louis among the embodiment 1; MO; USA) and 1.4 gram DIPEA (Sigma-AldrichCorp.St.Louis; MO USA), adds proline (Fmoc-Pro-OH) (the CHEM-IMPEX INTERNATIONAL of fluorenylmethyloxycarbonyl protection then; IL, USA) 4.0 grams.Stir after 12 hours, filter, silica gel DMF, methyl alcohol and washed with dichloromethane.The Fmoc protecting group was sloughed with the 20%DMF solution reaction of 20mL piperidines in 60 minutes.Filter solid DMF and washed with dichloromethane.Dry.This is the coupling process first time.Silica obtained through again through after twice identical coupling, in the silica obtained adding 20mL carrene, add again 1.6 the gram DIPEA and 1.4 the gram trimethyl-aceyl chlorides (Sigma-Aldrich Corp.St.Louis, MO, USA).Stir after 12 hours, filter, solid DMF, methyl alcohol and washed with dichloromethane.Obtain the bidentate structure of a kind of covalent bond mould assembly of the present invention fixedly phase (U.S. Patent Publication No., US2004/0226889A1).Coupling process and proline bonded amount detect the fluorenylmethyloxycarbonyl concentration of deprotection with ultraviolet spectra literature method detects ((Wang, Y.; Li, T.Anal.Chem., 1999; 71 (19); 4178) be 0.31mmol/g, through three times coupling proline bonded amount.Bonded silica gel is packed into by common homogenate method high pressure that (column parameter is 4.6mm * 50mm in the column sleeve.Isolation Technologies, Hopedale, MA USA), obtains the chiral high performance liquid chromatography post.
Common high performance liquid chromatography experiment condition is as follows: column temperature is a room temperature, and ultraviolet 254nm detects, and flow velocity is 1mL/min.Sample is dissolved in the isopropyl alcohol.
Separate 2,2, (Sigma-Aldrich Corp.St.Louis, MO USA), are the phase that flows with n-hexane/isopropyl alcohol (88/12, volume ratio) to 2-three fluoro-1-(anthryl)-ethanol (TFAE) enantiomer.The chiral separation spectrogram, as shown in Figure 1.2,2,2-three fluoro-1-(anthryl)-ethanol (TFAE) enantiomer has obtained good chiral separation.
Partition method China makes (Warfarin) enantiomer that (Sigma-Aldrich Corp.St.Louis, MO USA), are the phase that flows with n-hexane (0.1% trifluoroacetic acid)/isopropyl alcohol (96/4, volume ratio).The chiral separation spectrogram, as shown in Figure 2.Method China makes (Warfarin) enantiomer obtain good chiral separation.
Embodiment 3
Method according to embodiment 1 prepares 3-aminopropyl silica gel.Be the synthetic more amino bonded silica gel of the monodentate linking arm of long-chain, in dry DMF, add 4.0 these bonded silica gels of gram, 750 milligrams of DIC (Sigma-Aldrich Corp.St.Louis, MO, USA), 800 milligrams of HOBT (Sigma-AldrichCorp.St.Louis, MO is USA) with 700 milligrams of 4-(1-Boc piperidines) acetate (Sigma-AldrichCorp.St.Louis, MO, USA), stir after 12 hours, filter, silica gel DMF, methyl alcohol and washed with dichloromethane.The Boc protecting group was sloughed with the dichloromethane solution reaction of 50% trifluoroacetic acid in 60 minutes; solid DMF, carrene and 50%N; N-diisopropylethylamine (DIPEA) (Sigma-Aldrich Corp.St.Louis; MO; USA) dichloromethane solution washing; drying obtains the amino bonded silica gel of the monodentate linking arm of long-chain.Press the method for embodiment 2, use Fmoc-Pro-OH,, obtain the fixedly phase of long linking arm covalent bond mould assembly monodentate structure of the present invention through after three times the coupling and end-blocking.The detection of coupling process and proline bonded amount is 0.28mmol/g with embodiment 2 through three times coupling proline bonded amounts.Bonded silica gel is packed in the column sleeve by homogenate method high pressure, obtains the chiral high performance liquid chromatography post.
The high performance liquid chromatography experiment condition is as follows: column temperature is a room temperature, and ultraviolet 254nm detects, and flow velocity is 1mL/min.Sample is dissolved in the isopropyl alcohol.
(Sigma-AldrichCorp.St.Louis, MO USA), are the phase that flows with n-hexane/isopropyl alcohol (98/2, volume ratio) to separate 1-(2-naphthyl) ethanol (1-(2-Naphthyl) ethanol) enantiomer.The chiral separation spectrogram, as shown in Figure 3.1-(2-naphthyl) ethanol (1-(2-Naphthyl) ethanol) enantiomer has obtained good chiral separation.
(Sigma-Aldrich Corp.St.Louis, MO USA), are the phase that flows with n-hexane to separate Troger ' s Base enantiomer.The chiral separation spectrogram, as shown in Figure 4.Troger ' s Base enantiomer has obtained good chiral separation.
Embodiment 4
Method according to embodiment 1 prepares 3-aminopropyl silica gel, and bonded silica gel is by homogenate method high pressure filling performance liquid chromatographic column.With the mobile post of washing mutually of the THF that contains the 2mL triethylamine (40mL); allow then and contain proline dipeptides (Boc-Pro-Pro-OH) (the CHEM-IMPEX INTERNATIONAL that the 2g pivaloyl group is protected; IL; USA) pillar is flow through in flowing of THF solution (40mL) mutually; at last with the n-hexane flushing pillar of 10% isopropyl alcohol; used flow velocity all is 1mL/min, up to baseline stability, obtains the fixedly phase of ionic bond mould assembly monodentate structure of the present invention.The proline bonded amount is 0.48mmol/g.
The high performance liquid chromatography experiment condition is as follows: column temperature is a room temperature, and ultraviolet 254nm detects, and flow velocity is 1mL/min.Sample is dissolved in the isopropyl alcohol.Be the phase that flows with n-hexane/isopropyl alcohol (95/5, volume ratio).
Separation hydrobenzoin (Hydrobenzoin) enantiomer (Sigma-Aldrich Corp.St.Louis, MO, chiral separation spectrogram USA), as shown in Figure 5.Hydrobenzoin (Hydrobenzoin) enantiomer has obtained good chiral separation.
Separate dinaphthalene amine (1,1 '-Binaphthyl-2,2 '-diamine) enantiomer (Sigma-Aldrich Corp.St.Louis, MO, chiral separation spectrogram USA), as shown in Figure 6.Dinaphthalene amine (1,1 '-Binaphthyl-2,2 '-diamine) the enantiomer enantiomer has obtained good chiral separation.
Claims (10)
1. the fixing phase of a bonded chiral, its comprise carrier, with the linking arm of carrier bonding and with the chiral selector of linking arm bonding, the linking arm of bonding comprises circulus or side chain on the wherein said carrier, by covalent bond or ionic bond with the chiral selector bonding.
2. according to the fixing phase of the bonded chiral of claim 1, wherein said carrier is a silica gel, and its particle diameter is 1 μ m to 300 μ m, and average pore size is 5nm～50nm.
3. according to the fixing phase of the bonded chiral of claim 1, wherein said linking arm has bidentate structure or monodentate structure.
4. according to the fixing phase of the bonded chiral of claim 3, wherein said linking arm has bidentate structure as follows or monodentate structure:
Wherein:
Y
1And Y
2Be 0～3 integer, must make this ring from the three-membered ring to the octatomic ring, Y
3Be 0～10 integer, R
6, R
7Be selected from the group of forming by following: methoxyl group, ethyoxyl, isopropyl, methyl, ethyl, R formula alkyl, thiazolinyl, cycloalkyl, phenyl, 3,5-3,5-dimethylphenyl, 2,4-dinitrophenyl and 3,5-dinitrophenyl.
5. according to the fixing phase of the bonded chiral of claim 1, wherein said chiral selector is that the proline that replaced by proline, α or hydroxy-proline and their analog combine, and they are L or D spatial configuration, and quantity is 2～20.
6. according to the fixing phase of the bonded chiral of claim 1, wherein said chiral selector has following architectural feature:
Wherein, as the substituting group on each five-membered ring of construction unit, can be identical, also can be different, the silica gel bonded linking arm that the L representative links to each other with ionic bond with covalent bond, R
1Being END CAPPED GROUP, can be that hydrogen atom, carbon number are that 1～12 alkoxy carbonyl group, carbon number are that 1～12 acyl group, carbon number are that 1～12 substituted aminocarbonyl, carbon number are that 1～18 contain or do not contain heteroatomic aromatic series oxygen carbonyl, carbon number are 1～18 contain or do not contain heteroatomic aromatic series substituted aminocarbonyl, be preferably as follows group especially:
The integer of n=0-20,
Work as X=O, S, CH
2The time, there is not R
5,
When X=CH, R
5Be hydroxyl, carbon number be 1～12 carbamate groups, carbon number be 1～12 urea groups or ghiourea group, carbon number be 1～12 ester group, carbon number be 1～18 contain or do not contain heteroatomic aromatic amino ester formate base, carbon number be 1～18 contain or do not contain heteroatomic urea groups or ghiourea group, be preferably as follows group especially:
R
2, R
3, R
4Be that carbon number is that 1～12 alkyl, carbon number are 1～18 aromatic radical, be preferably as follows group especially:
-CH
2-CH＝CH
3 
7. according to the fixing phase of the bonded chiral of claim 1, it has one of following two class formations:
(1) the fixing structure of phase of the bonded chiral of bidentate structure linking arm:
Wherein
R
1Being END CAPPED GROUP, can be that hydrogen atom, carbon number are that 1～12 alkoxy carbonyl group, carbon number are that 1～12 acyl group, carbon number are that 1～12 substituted aminocarbonyl, carbon number are that 1～18 contain or do not contain heteroatomic aromatic series oxygen carbonyl, carbon number are 1～18 contain or do not contain heteroatomic aromatic series substituted aminocarbonyl, be preferably as follows group especially:
The integer of n=0-20,
Work as X=O, S, CH
2The time, there is not R
5,
When X=CH, R
5Be hydroxyl, carbon number be 1～12 carbamate groups, carbon number be 1～12 urea groups or ghiourea group, carbon number be 1～12 ester group, carbon number be 1～18 contain or do not contain heteroatomic aromatic amino ester formate base, carbon number be 1～18 contain or do not contain heteroatomic urea groups or ghiourea group, be preferably as follows group especially:
R
2, R
3, R
4Be that carbon number is that 1～12 alkyl, carbon number are 1～18 aromatic radical, be preferably as follows group especially:
-CH
2-CH＝CH
3
Y
1, Y
2=1～2, Y
3=0～10, the integer of m=0～1
R
6=-CH
3-CH
2-CH
3-O-CH
3Or-O-CH
2-CH
3
(2) the fixing structure of phase of the bonded chiral of monodentate structure linking arm:
Wherein
R
1Being END CAPPED GROUP, can be that hydrogen atom, carbon number are that 1～12 alkoxy carbonyl group, carbon number are that 1～12 acyl group, carbon number are that 1～12 substituted aminocarbonyl, carbon number are that 1～18 contain or do not contain heteroatomic aromatic series oxygen carbonyl, carbon number are 1～18 contain or do not contain heteroatomic aromatic series substituted aminocarbonyl, be preferably as follows group especially:
The integer of n=0-20,
Work as X=O, S, CH
2The time, there is not R
5,
When X=CH, R
5Be hydroxyl, carbon number be 1～12 carbamate groups, carbon number be 1～12 urea groups or ghiourea group, carbon number be 1～12 ester group, carbon number be 1～18 contain or do not contain heteroatomic aromatic amino ester formate base, carbon number be 1～18 contain or do not contain heteroatomic urea groups or ghiourea group, be preferably as follows group especially:
R
2, R
3, R
4Be that carbon number is that 1～12 alkyl, carbon number are 1～18 aromatic radical, be preferably as follows group especially:
-CH
2-CH＝CH
3 
Y
1, Y
2=1～2, Y
3=0～10, the integer of m=0～1,
8. according to the fixing phase of the bonded chiral of claim 1, it has following structure:
9. a liquid chromatogram chiral separation method is wherein used according to the bonded chiral of any one among the claim 1-8 and is fixed fixedly phase of conduct mutually.
10. liquid-phase chromatographic column comprises analytic type post, preparation type post, simulation moving-bed and electric chromatographic column, has wherein filled according to any one the fixing phase of bonded chiral among the claim 1-8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113905797A (en) * | 2019-06-14 | 2022-01-07 | 株式会社大赛璐 | Liquid chromatography-based separation method for amines |
| CN115814772A (en) * | 2022-10-19 | 2023-03-21 | 华东理工大学 | Silica gel stationary phase of bond and dipeptide and application of silica gel stationary phase in chitosan oligosaccharide chromatographic separation |
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2006
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113905797A (en) * | 2019-06-14 | 2022-01-07 | 株式会社大赛璐 | Liquid chromatography-based separation method for amines |
| CN113905797B (en) * | 2019-06-14 | 2023-04-18 | 株式会社大赛璐 | Liquid chromatography-based separation method for amines |
| CN115814772A (en) * | 2022-10-19 | 2023-03-21 | 华东理工大学 | Silica gel stationary phase of bond and dipeptide and application of silica gel stationary phase in chitosan oligosaccharide chromatographic separation |
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