CN101203497A - Triazines as protein binding ligands - Google Patents

Abstract

Compounds of the general formula (I): in which inter alia Q<1> represents -NR<1>R<3>, -OR<1> or -SR<1> and Q<2> represents -NR<2>R<4>, -OR<2> or -SR<2>, and A represents the point of attachment to a support matrix, are useful as protein binding ligands when (a) at least one of R<1>, R<2>, R<3> and R<4> includes an alkyl group -CnH2n+1 in which n is greater than or equal to 7; (b) at least two of R<1> , R<2>, R<3> and R<4> independently include an alkyl group -CnH2n+1 or a cycloalkyl group -CnH2n-1 in which n is greater than or equal to 4; or (c) at least three of R<1>, R<2>, R<3> and R<4> independently include a C1-12 alkyl group substituted by -NR<5>R<6> or aryl.

Description

Triazines as protein binding ligands

Technical field

The present invention relates to new compound and as the application of triazines as protein binding ligands.

Background technology

Characteristics of numerous protein are all to exist the surface can reach hydrophobic pocket (pocket) or hydrophobic patch (patch).Utilize these hydrophobic regions, can isolated protein, and can be used as different hydrophobic solutes are arranged.Hydrophobic interaction chromatography (HIC) is existing outside chromatogram support particle combination and the method for purifying proteins that is covered with hydrophobic grouping that utilize.Polarity of solvent around regulating for example adds water structure salt (water-structuring salt), by hydrophobic interaction, protein is combined with the hydrophobic surface of chromatography matrix.Remove water structure salt and/or add polarity and reduce solvent, can will elute by hydrophobic interaction bonded protein.The used hydrophobic grouping of HIC often comprises straight chained alkyl or the phenyl that directly links to each other with supported matrix by ehter bond or amino key.The HIC stromal surface contains the hydrophobic grouping of high density usually, in order that change surface polarity, forms general hydrophobic surface.Because the ubiquity of face-face binding interactions, the selectivity of these materials is compared with protein bound, and is often all lower, so its purification degrees is also lower, often needs other purification step, reaches required purification degrees.If can develop hydrophobic ligand, hydrophobic grouping is embedded in a kind of compound ligand structure with these group particular spaces and orientation, then these parts just might interact with specific hydrophobic site on the protein, hydrophobic site characteristic on relative protein bound and the protein, its selectivity is improved.

Use at present and carry out the protein affinity purification by the chemically derived compound of triazine.For example, set forth Cynuric Chloride among WO97/10887, WO 2000/067900 and the WO 2004/035199 and be substituted the ligand structure of deriving and obtaining after alkylamine and the arylamines replacement.WO 97/10887 lectures Cynuric Chloride and make part after two substituted aryl compounds or a substituted aryl amine compound and a substituted alkyl amine compound replacement.WO 2000/067900 has put down in writing the ligand compound that contains with the triazine ring derivatives of various cation groups.WO 2004/035199 put down in writing be substituted with aminophenyl and carboxyl-, hydroxyl-or the application of Cynuric Chloride aspect the antibody fragment purifying of amino-propyl group.

We find, different with these structures of putting down in writing in the prior art is, have linearity, branch or cycloalkyl, not with any other hydrophilic or the triazine of charged group and protein purification that related compound can be used in particular for having hydrophobic pocket or hydrophobic surface.In addition, these new ligand structures are more excellent than the phenyl or the alkyl chain effect that directly link to each other with supported matrix.

Summary of the invention

So first aspect of the present invention provides the compound shown in a kind of general formula (I)

Wherein,

Among the symbol X, an X represents nitrogen-atoms, and another X represents nitrogen-atoms or has the carbon atom of chlorine atom or cyano group;

Q ¹Expression-NR ¹R ³,-OR ¹Or-SR ¹And

Q ²Expression-NR ²R ⁴,-OR ²Or-SR ²

R ¹, R ², R ³And R ⁴Can be identical or different, they are independently expression separately:

(i) hydrogen; Perhaps

(ii) can in the middle of be inserted with O or S and can by one or more OH of being selected from, halogen ,-NR ⁵R ⁶The C that substituting group replaced with aryl _1-12Alkyl, wherein, R ⁵And R ⁶Independent separately expression hydrogen or C _1-8Alkyl; Perhaps

(iii) can by one or more be selected from OH, halogen ,-NH ₂The C that substituting group replaced with aryl _4-12Cycloalkyl; Perhaps

(iv) can be by one or more C that are selected from _1-8Alkyl, C _1-8Alkoxyl group, C _1-8Acyloxy, C _1-8Amido ,-OH ,-NH ₂, carboxyl, carbamyl, aminosulfonyl (sulphamoyl) and-SO ₃R ⁷The aryl that substituting group replaced; Wherein, R ⁷Expression hydrogen or C _1-8Alkyl;

But, have following restricted condition:

(a) R ¹, R ², R ³And R ⁴In have at least one to comprise alkyl-C _nH _2n+1, wherein n is more than or equal to 7;

(b) R ¹, R ², R ³And R ⁴In have at least two independently to comprise alkyl-C separately _nH _2n+1Or cycloalkyl-C _nH _2n-1, wherein n is more than or equal to 4; Perhaps

(c) R ¹, R ², R ³And R ⁴In have at least three independently to comprise separately and being substituted with-NR ⁵R ⁶Or the C of aryl _1-12Alkyl;

And

A represents can be by inserting the tie point that space key (spacerlinkage) links to each other with supported matrix between the compound shown in matrix and the general formula (I).

Preferred compound of the present invention be shown in the general formula (I), symbol X all represents the compound of nitrogen-atoms, i.e. triaizine compounds.

Preferred compound of the present invention be shown in the general formula (I), Q ¹Expression-NR ¹R ³, Q ²Expression-NR ²R ⁴Compound.

In one group of preferred compound of the present invention, R ¹And R ²Can be the same or different, all expression can in the middle of be inserted with O or S and can by one or more OH of being selected from, halogen ,-NR ⁵R ⁶The C that substituting group replaced with aryl _4-12Alkyl.

In another group preferred compound, R ¹And R ²Can be the same or different, all represent C _7-12Alkyl.

In another group preferred compound of the present invention, R ¹And R ²In have at least the expression can be by one or more C of being selected from _1-8Alkyl, C _1-8Alkoxyl group, C _1-8Acyloxy, C _1-8Amido ,-OH ,-NH ₂, carboxyl, carbamyl, aminosulfonyl and-SO ₃R ⁷The aryl that substituting group replaced, R wherein ⁷Expression hydrogen or C _1-8Alkyl.In the particularly preferred subbase of these compounds is rolled into a ball, R ¹And R ²In have an expression at least by C _6-12Alkyl or C _6-12The aryl that alkoxyl group replaced.This aryl preferentially is selected from phenyl, naphthyl, 1-phenylpyrazole, indazole, benzothiazole, benzoxazole and benzoglyoxaline, and override is selected from phenyl.

In another group preferred compound of the present invention, all R ¹, R ², R ³And R ⁴All represent C _1-12Alkyl.

Further preferred compound subgroup is those wherein R ³And R ⁴The compound subgroup and the R that all represent hydrogen ³And R ⁴The compound subgroup of all representing methyl.

In the preferred compound of another group shown in general formula (I), R ¹And R ²All represent C _4-6Alkyl, R ³And R ⁴Independent separately expression hydrogen or can in the middle of be inserted with O or S and can by one or more OH of being selected from, halogen and-NR ⁵R ⁶The C that substituting group replaced _1-12Alkyl.

Should be appreciated that the chemical formula-C that mentions herein _nH _2n+1Shown alkyl comprises straight chain and branched-chain alkyl simultaneously.Equally, mentioned chemical formula C _nH _2n-1Shown cycloalkyl comprises the group that all carbon atom arrangement circularize, and as cyclohexyl, also comprises the group of part carbon atom looping, as the cyclopropyl ethyl.

At least the alkyl that contains four carbon atom for example includes but not limited to butyl, isobutyl-, 1-methyl-propyl, 2-methyl-propyl, phenyl, 1-methyl butyl, 2-methyl butyl, 3-methyl butyl, 1,1-dimethyl propyl, 1,2-dimethyl propyl, hexyl, 1-aminomethyl phenyl, 2-aminomethyl phenyl, 3-aminomethyl phenyl, 4-aminomethyl phenyl, 2-ethyl-butyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, heptyl, 1,4-dimethyl amyl group, 2,4-dimethyl amyl group, octyl group, nonyl, decyl, undecyl and dodecyl.

At least the alkyl that contains 7 carbon atoms for example includes but not limited to: heptyl, octyl group, nonyl, decyl, undecyl and dodecyl.

At least the cycloalkyl that contains 4 carbon atoms for example comprises cyclopentyl, cyclohexyl and norcamphane.

The compound that the present invention is confined to meet the following conditions:

(a) R ¹, R ², R ³And R ⁴In have at least one to comprise alkyl-C _nH _2n+1, wherein n is more than or equal to 7;

(b) R ¹, R ², R ³And R ⁴In have at least two to comprise alkyl-C _nH _2n+1Or cycloalkyl-C _nH _2n-1, wherein n is more than or equal to 4; Perhaps

(c) R ¹, R ², R ³And R ⁴In have at least three independently to comprise separately and being substituted with-NR ⁵R ⁶Or the C of aryl _1-12Alkyl;

But the important point is to notice that mentioned alkyl or cycloalkyl can be by each R ¹, R ², R ³And R ⁴Whole groups in the group constitute, also can be only by each R ¹, R ², R ³And R ⁴Part in the group constitutes.So, R ¹, R ², R ³And R ⁴Can represent alkyl or cycloalkyl, perhaps can represent to comprise the part of such group.

R ⁵, R ⁶And R ⁷The concrete group that can represent comprises straight chain and side chain C _1-4Alkyl is as methyl, ethyl and sec.-propyl.

Following structure shown in (iii)-(x) is a concrete preferred compound of the present invention:

It is apparent to those skilled in the art by tie point A the compound shown in the general formula (I) to be connected to method used on the supported matrix.Suitable key comprises amino key, ehter bond or thioether bond.In addition, can also be conspicuous for a person skilled in the art as the material behavior of supported matrix.Supported matrix for example includes but not limited to polysaccharide, agarose, Mierocrystalline cellulose, chitin, dextran, methacrylic ester, hydroxyalkyl methacrylate, SDEB styrene diethylenebenzene, polyvinyl alcohol, glass, silica, metal oxide, aluminum oxide and zirconium white.For avoiding doubt, any material can be used as supported matrix, as long as use this material easily part to be separated from the solute of contact solution.As the preferred material of supported matrix comprise can the activatory polysaccharide, agarose, Mierocrystalline cellulose, chitin, dextran, methacrylic ester, hydroxyalkyl methacrylate, SDEB styrene diethylenebenzene or polyvinyl alcohol.

Part of the present invention can also pass through spacer groups (space group) and link to each other with supported matrix.If you are using, described spacer groups can comprise the spacer groups shown in the following general formula

-TLV-

Wherein, T be amino or-NHR ⁸-, ether or thioether group, L contains 1-20 carbon atom, the dispensable linearity of substituting group or branch's alkyl, V be amino or-NHR ⁹-Ji, ether or thioether group, wherein, R ⁸And R ⁹Independent separately expression contains linearity or branch's alkyl of 1-20 carbon atom.In addition, according to circumstances, linking group L for example can be substituted with ether or hydroxyl.

A kind of method for preparing compound shown in the general formula (I) is by compound shown in the general formula (II) and the supported matrix reaction that contains amine,

Wherein, Q ¹, Q ²With symbol X all with compound shown in the general formula (I) in defined identical.Compound shown in this method and the general formula (II) all belongs to another aspect of the present invention.

The method that another kind can prepare compound shown in the general formula (I) comprises compound shown in the general formula (XI) and the supported matrix reaction that contains amine, generates the compound shown in the general formula (XII), wherein, in the general formula (XI), Q ¹With symbol X all with compound shown in the general formula (I) in defined identical, in the general formula (XII), Q ¹With A all with compound shown in the general formula (I) in defined identical;

Then with compound and formula H-Q shown in the general formula (XII) ²Shown in compound reaction.Compound shown in this method and the general formula (XI) all constitutes another aspect of the present invention.

Ligand structure of the present invention can be used for proteinic extraction, purifying, quantitatively, characterize or reclaim.Preferred material of the present invention is to use in the column chromatography operation, during operation, material is loaded chromatographic column, forms the post bed that supplies sample and washing lotion to flow through.Select the condition that combines that can promote hydrophobic grouping absorption on protein and the part for use.Add buffering salt and metal cation salt, particularly add the water structure salt (water structuringsalt) that includes but not limited to ammonium sulfate, sodium sulfate, sodium-chlor and Repone K, can promote protein adsorption.Regulate pH or ionic strength, can be with bonded protein wash-out.Especially, remove buffering salt and metal ion, and/or add polarity and reduce solvent, can quicken the wash-out of institute's conjugated protein, described polarity reduces solvent and includes but not limited to ethanol, propyl alcohol, Virahol, butanols, ethylene glycol, glycerol, butyleneglycol and hexylene glycol.In addition, can also in the post washing lotion, add the chaotropic agent (chaotropic agent) that includes but not limited to urea, Guanidinium hydrochloride, Sodium Thiocyanate 99 and potassiumiodide, be used for promoting the protein desorption.During practical application, can utilize ligand structure screening library to select only material of the present invention for use.Working method is very simple, material of the present invention can be installed in the microfluidic chromatography post, perhaps makes up the part array at slide or chip surface, and uses together in conjunction with the biological analyser that detects the protein-ligand effect.These class methods can filter out many different ligand structures simultaneously, and can assess the keying action of selected target protein.Can also use this triage techniques, the application (comprising the selection and the optimization of required combination of column chromatography and elution requirement) of part is optimized.

So, according to a further aspect in the invention, the invention provides the library of related compound shown in the above-mentioned general formula (I), described compound is connected with the supported matrix of using always by tie point A.

Of the present inventionly on the one hand provide application of compound shown in (i) a kind of general formula (I) again, as mentioned above, be used for peptide and proteinicly separate, extraction, purifying, sign, evaluation, quantitatively or reclaim; And

The method of (ii) a kind of separation, purifying or recovery proteinaceous materials, this method comprises that the sample that will contain this material contacts with compound shown in the above-mentioned general formula (I).

The protein of using relevant a kind of particular type with the present invention is interleukin-18 binding proteins or IL-18BP.Referring now to following examples,, the present invention is done detailed description further only in the elaboration mode.

Embodiment

Embodiment 1

Affinity ligand sorbent material solid phase synthesis

1.1 sepharose 4B epoxy activation

Beaded agarose (6% crosslinked, 100mm (Purabead 6XL), 1kg) suspendible with after 640ml water and 80ml 10M NaOH and the washing adds the 72ml Epicholorohydrin, and 40 ℃ were stirred 3 hours.With 10 premium on currency washing gained epoxy activated agarose, and the gravity discharge opeing.

1.2 sepharose 4B amination

Epoxy activated agarose pearl suspendible with preparation in 800ml water and above-mentioned 1.1 adds 200ml ammoniacal liquor (proportion 0.88) then, and 40 ℃ were stirred 16 hours, and gained amination agarose washs with 10 premium on currency.

1.3 amination agarose triazine activation

The amination agarose of preparation in above 1.2 is mixed with 1 liter of 1M potassium phosphate buffer (pH7.0), and the gravity discharge opeing.Again the amination agarose is acutely mixed with 0.5 liter of 0.5M potassium phosphate buffer (pH7.0) and 0.5 liter of acetone, and be cooled to 0-5 ℃.Add the freshly prepd acetone soln that contains the 25g Cynuric Chloride of 250ml, 0-5 ℃ was stirred 1 hour.Mixture is forwarded in the filter funnel, successively with acetone and the washing of 10 premium on currency of the acetone of 5 liters of 50%v/v, 5 premium on currency, 5 liters of 50%v/v.Before in making up ligand library, using, make the discharge opeing of gained dichlorotriazine-activated sepharose 4B gravity.

1.4 triazine activated agarose and amine replace molecular reaction

The dichlorotriazine-activated agarose of preparation in above 1.3 is divided into every part of 30g, is cooled to 5 ℃.With 30ml 50%v/v DMF dissolving 3mmol amine (shown in table 1 the 2nd hurdle), first amine aqueous solution so that preparation will be coupled is adjusted to pH9-10 with 10M NaOH then, is cooled to 5 ℃.Add amine aqueous solution in each part dichlorotriazine-activated agarose, 5 ℃ were mixed 1 hour.After the end, the gained list is replaced intermediate product successively use 150ml 50%v/v DMF and 300ml water washing, gravity discharge opeing then.With 30ml 50%v/v DMF dissolving 6mmol amine (shown in table 1 the 3rd hurdle), second amine aqueous solution so that preparation will be coupled is adjusted to pH9-10 with 10M NaOH then.This amine aqueous solution is added to above-mentioned single the replacement in the intermediate product, and 60 ℃ were mixed 24 hours.After the end, the affinity adsorbent that obtains is successively used 150ml50%DMF, 150ml water, 150ml 0.1M hydrochloric acid, 150ml 30% Virahol/0.2M sodium hydroxide, 300ml water washing, forward at last in 20% ethanol, store.Supernatant liquor is answered in negate, detects whether contain the above-mentioned chlorion that discharges in the reaction process that is coupled, to monitor the reaction of above first and second amine.By above reactions steps, make affinitive material of the present invention, shown in table 1 the 1st hurdle.

Table 1

Part	First amine	Second amine
			III	The N-tuaminoheptane	The N-tuaminoheptane
IV	Hexylamine	Hexylamine
			V	Octylame	Octylame
VI	1-amino-4-hexyloxy benzene	1-amino-4-hexyloxy benzene
			VII	2-amino-5-methyl hexane	S-benzyl cysteamine
VIII	1-amino-2-methyl propane	1-amino-4-hexyloxy benzene
			IX	2-amino-4-phenyl butane	N '-benzyl-N, the N-dimethyl-ethylenediamine
X	The amino norcamphane of 2-	1-amino-4-hexyloxy benzene

Embodiment 2

The protein bound of affinity ligand detects

With embodiment 1 described material filling micro-column (0.25ml) of the present invention,, carry out the micro-column balance with 3ml phosphate-buffered salt (PBS) solution (pH7.0) flushing.25 ℃, the Chinese hamster ovary celI substratum that 0.5ml is contained 0.5mg/ml interleukin-18 binding proteins (IL-18BP) is loaded in each post after the balance, continues 10 minutes.Afterwards, laden each post is used 3 * 0.5ml level pad (PBS, pH7.0) washing respectively, conjugated protein uses 2 * 0.5ml to contain the PBS wash-out of 50% ethylene glycol (pH7.0) and 0.5M NaCl respectively.Afterwards, elder generation washes with the solution that 1ml contains the pure and mild 0.2MNaOH of 30%v/v propane-2-with post, with the 2.0ml washing, makes its regeneration again.Adopt the enzyme linked immunosorbent assay (ELISA) and the Bradford total protein assay method of SDS-polyacrylamide gel electrophoresis, employing IL-18BP monoclonal antibody specific, the post stream sample of collecting in loading and the elution process is analyzed.SDS-PAGE and total protein analytical results show that all parts all filter out in conjunction with IL-18BP, and show very high level of purification.Under all situations, SDS PAGE all shows, contains IL-18BP in the elution fraction.

Embodiment 3

2,4-two chloro-6-[N-tuaminoheptanes]-triazine is synthetic

Cynuric Chloride (5.0g) is dissolved in the acetone (35ml), is cooled to-2 ℃.N-tuaminoheptane (3.12g) is dissolved in the acetone (20ml), dropwise is added to then in the top cold soln, temperature maintenance is below 2 ℃.Dropwise add 10M sodium hydroxide (2.71ml) water (20ml) solution again, by cooling off temperature maintenance below 0 ℃.Use ethyl acetate extraction, separate obtaining yellow oil product (6.78g, 95%).Behind the HPLC purifying, purity is greater than 95%.

Embodiment 4

Compound III is synthetic

4.1 preparation epoxy agarose

In RO water (800ml), stir Purabead 6HF (1200g is treatment gel), add 10M sodium hydroxide (108ml).Add Epicholorohydrin (154ml) again, 20 ℃ were stirred 17 hours.Continue to add Epicholorohydrin (38.5ml) and 10M sodium hydroxide (27ml), then stirred again 2 hours.After the epoxy activation Purabead filtration that obtains, water (12 * 1 liters/equal portions) washing.

4.2 amination

Add entry (960ml) and proportion among the epoxy activation Purabead of preparation in above 4.1 and be 0.88 ammoniacal liquor (240ml), be heated to 40 ℃, stirred 17 hours.Purabead after the amination is filtered, wash (13 * 1.2 liters/equal portions) again with water

4.3 2,4-two chloro-6-[N-tuaminoheptanes]-triazine and amination Purabead reaction

The amination Purabead suspendible to 1.2 of preparation in above 4.2 is risen among the DMF, and the gravity discharge opeing.Add DMF (900ml), add again be dissolved in 2 in 295ml DMF and the 13.2ml diisopropylethylamine, 4-two chloro-6-[N-tuaminoheptanes]-(20.66g) solution of triazine (press embodiment 3 described methods prepares).20 ℃ were stirred 2 hours, afterwards product is filtered, and successively with the 75%v/v DMF aqueous solution (2 * 1.2 liters), the 50%v/v DMF aqueous solution (2 * 1.2 liters) and water (10 * 1 liters) washing.

4.4 react with the N-tuaminoheptane

With products therefrom suspendible in above 4.3 steps in 1145ml DMF.Add 26.1ml N-tuaminoheptane again,, stirred 17 hours mixture heating up to 60 ℃.Afterwards products therefrom (III) is filtered,, place preservative solution 20% aqueous ethanolic solution to store then with 50%v/v DMF (4 * 1.2 liters) and water (10 * 1.2 liters) washing.

Embodiment 5

With The compounds of this invention purifying IL-18BP

Detect affinity adsorbent (the column volume 10ml that is filled with preparation among the embodiment 1 below; Post bed height 13cm) the purifying ability of IL-18 conjugated protein in the post pair cell culture medium.With the PBS damping fluid (pH7.0) of 5 column volumes, the post of filling filler with the flow velocity flushing of 3ml/min makes its balance.With purified IL-18BP material (Switzerland Serono laboratory; 15.5mg) be loaded in each post with the 1.5ml/min flow velocity, use the PBS damping fluid (pH7.0) of 5 column volumes to wash then with same flow velocity.Wash with the 3.0ml/min flow velocity with the PMS damping fluid (pH7.0) that contains 0.5M NaCl and 50%v/v ethylene glycol, with the conjugated protein wash-out.After the wash-out, contain the Virahol cleaning post of 0.2M NaOH again with 30%v/v.By SDS-PAGE, Western hybridization, total protein is measured and rp-hplc method to loading component, not analyzing in conjunction with component and elution fraction.Purification result is as shown in table 2.

Table 2

Sorbent material	Frac n	[protein] (μ g/ml)	Volume (ml)	Gross protein (μ g)	[IL18BP] (μg/ml)	Total IL-18BP (μ g)	% purity	% reclaims	Gross protein E+ET (μ g)	IL-18BP E+ET (μg)	(E+ ET) purity %	(E+ ET) reclaims %
													X	Load NB E ET	612 109 994 260	80 118 10 5	48943 12893 9938 1301	180 33 793 240	14438 3945 7927 1201	29.5 30.6 79.8 92.3	100 27.3 54.9 8.3	11238	9128	81.2	63.2
III	Load NB E ET	612 74 968 279	80 116 10 5	48943 8603 9679 1395	180 8 732 358	14438 964 7317 1792	29.5 11.2 75.6 128.5	100 6.7 50.7 12.4	11074	9109	82.3	63.1
													VII	Load NB E ET	594 85 1448 374	80 100 10 5	47558 8457 14485 1870	181 12 1042 354	14497 1193 10422 1770	30.5 14.1 72 94.7	100 8.2 71.9 12.2	16354	12193	74.6	84.1
VII	Load NB E ET	650 55 874 287	80 110 10 10	51975 6018 8737 2873	192 9 737 294	15382 994 7372 2940	29.6 16.5 84.4 102.3	100 6.5 47.9 19.1	11611	10312	88.8	67

NB=is not in conjunction with component

The E=elution fraction

ET=wash-out afterbody component (tail fraction)

Embodiment 6

The combination of rnase

Take by weighing among a certain amount of embodiment 4 sorbent material (50mg) after the washing of preparation, install in the clean pipe, add the phosphate buffered saline(PBS) (pH7.0) that 0.95ml contains 5mg/ml ribonuclease A solution.

Pipe is closed the lid, the mixing of turning upside down, room temperature, 1 hour, centrifugal then, make the sorbent material precipitation.To wash the back sorbent material and change 0.5ml phosphate-buffered salt (pH7.0) into, prepare control sample with quadrat method by above.Supernatant liquor in the sample is carefully poured in the clean pipe, measured the absorbancy at UV 280nm place.

Deduct the control sample adsorptive capacity with the sample adsorptive capacity, the binding ability that calculates ribonuclease A is that every milliliter of sorbent material is in conjunction with 15.5mg protein.

Embodiment 7

The combination of polyclone human IgG and wash-out

Will be according to the chromatographic column of the washing sorbent material filling 1ml volume of embodiment 4 preparation, and with 12ml phosphate-buffered salt (pH7.4) balance, contain the phosphoric acid salt of 5mg/ml people's polyclone IgG solution to wherein adding 2ml then.

Subsequently with 5ml level pad washing chromatographic column.The Ig that contains phosphate-buffered salt (pH7.4) elution of bound of 50% ethylene glycol and 0.5M sodium-chlor again with 4ml measures conjugated protein quality in this component with Bradford gross protein quantitative method.After measured, every milliliter of sorbent material can be in conjunction with 8.5mg people's polyclone IgG.

Claims (29)

1. the compound shown in the general formula (I)

Wherein,

Among the symbol X, an X represents nitrogen-atoms, and another X represents nitrogen-atoms or has the carbon atom of chlorine atom or cyano group;

Q ¹Expression-NR ¹R ³,-OR ¹Or-SR ¹And

Q ²Expression-NR ²R ⁴,-OR ²Or-SR ²

R ¹, R ², R ³And R ⁴Can be identical or different, they are independently expression separately:

(i) hydrogen; Perhaps

Be inserted with in the middle of (ii) optional O or S and optional by one or more OH of being selected from, halogen ,-NR ⁵R ⁶The C that substituting group replaced with aryl _1-12Alkyl, wherein, R ⁵And R ⁶Independent separately expression hydrogen or C _1-8Alkyl; Perhaps

(iii) optional by one or more be selected from OH, halogen ,-NH ₂The C that substituting group replaced with aryl _4-12Cycloalkyl; Perhaps

(iv) optional by one or more C that are selected from _1-8Alkyl, C _1-8Alkoxyl group, C _1-8Acyloxy, C _1-8Amido ,-OH ,-NH ₂, carboxyl, carbamyl, aminosulfonyl and-SO ₃R ⁷The aryl that substituting group replaced; Wherein, R ⁷Expression hydrogen or C _1-8Alkyl;

But, have following restricted condition:

(a) R ¹, R ², R ³And R ⁴In have at least one to comprise alkyl-C _nH _2n+1, wherein n is more than or equal to 7;

(b) R ¹, R ², R ³And R ⁴In have at least two independently to comprise alkyl-C separately _nH _2n+1Or cycloalkyl-C _nH _2n-1, wherein n is more than or equal to 4; Perhaps

(c) R ¹, R ², R ³And R ⁴In have at least three independently to comprise separately by-NR ⁵R ⁶Or the C of aryl replacement _1-12Alkyl;

And

A represents optional by inserting the tie point that space key links to each other with supported matrix shown in matrix and the general formula (I) between the compound.

2. compound according to claim 1, wherein, symbol X all represents nitrogen-atoms.

3. compound according to claim 1 and 2, wherein, Q ¹Expression-NR ¹R ³, Q ²Expression-NR ²R ⁴

4. according to the described compound of the arbitrary claim in front, wherein, R ¹And R ²Identical or different, they represent separately to be inserted with in the middle of optional O or S and optional by one or more OH of being selected from, halogen ,-NR ⁵R ⁶The C that substituting group replaced with aryl _4-12Alkyl.

5. according to each described compound among the claim 1-3, wherein, R ¹And R ²Identical or different, each represents C _7-12Alkyl.

6. according to each described compound among the claim 1-3, wherein, R ¹And R ²In have at least an expression optional by one or more C of being selected from _1-8Alkyl, C _1-8Alkoxyl group, C _1-8Acyloxy, C _1-8Amido ,-OH ,-NH ₂, carboxyl, carbamyl, aminosulfonyl and-SO ₃R ⁷The aryl that substituting group replaced, R wherein ⁷Expression hydrogen or C _1-8Alkyl.

7. compound according to claim 6, wherein, R ¹And R ²In have an expression at least by C _6-12Alkyl or C _6-12The aryl that alkoxyl group replaced.

8. according to claim 6 or 7 described compounds, wherein, described aryl is selected from phenyl, naphthyl, 1-phenylpyrazole, indazole, benzothiazole, benzoxazole and benzoglyoxaline.

9. according to claim 6 or 7 described compounds, wherein, described aryl is selected from phenyl.

10. compound according to claim 3, wherein, R ¹, R ², R ³And R ⁴All represent C _1-12Alkyl.

11. compound according to claim 3, wherein, R ³And R ⁴All represent hydrogen.

12. compound according to claim 3, wherein, R ³And R ⁴All represent methyl.

13. compound according to claim 3, wherein, R ¹And R ²Expression C _4-6During alkyl, R ³And R ⁴Be inserted with in the middle of then independent separately expression is optional O or S and optional by one or more OH of being selected from, halogen and-NR ⁵R ⁶The C that substituting group replaced _1-12Alkyl.

14. compound according to claim 1, it has following structure:

15. compound according to claim 1, it has following structure:

16. compound according to claim 1, it has following structure:

17. compound according to claim 1, it has following structure:

18. compound according to claim 1, it has following structure:

19. compound according to claim 1, it has following structure:

20. compound according to claim 1, it has following structure:

21. compound according to claim 1, it has following structure:

22. according to the described compound of the arbitrary claim in front, wherein, this compound is connected with the supported matrix of polysaccharide, agarose, Mierocrystalline cellulose, chitin, dextran, methacrylic ester, hydroxyalkyl methacrylate, SDEB styrene diethylenebenzene or the polyvinyl alcohol form of optional activation at tie point A place.

23. the compound shown in the general formula (II)

Wherein, Q ¹, Q ²With symbol X all with claim 1 in defined identical.

24. the method for compound shown in the described general formula of synthetic claim 1 (I), this method comprise compound shown in the described general formula of claim 23 (II) and the supported matrix that contains amine are reacted.

25. the compound shown in the general formula (XI)

Wherein, Q ¹With symbol X all with claim 1 in defined identical.

26. the method for compound shown in the described general formula of synthetic claim 1 (I), this method comprise compound shown in the described general formula of claim 25 (XI) and the supported matrix reaction that contains amine, generate the compound shown in the general formula (XII)

Wherein, Q ¹With A all with claim 1 in defined identical; Then with compound and formula H-Q shown in the general formula (XII) ²Shown in compound reaction.

27. the library of related compound shown in the described general formula of claim 1 (I), described compound is connected with the supported matrix of using always by tie point A.

28. among the claim 1-21 each described compound in separation, extraction, purifying, sign, evaluation, quantitatively or the application aspect recovering peptide and the protein.

29. the method for a separation, purifying or recovery proteinaceous materials, this method comprises that the sample that will contain this material contacts with compound shown in the described general formula of claim 1 (I).