CN101065185A - Methods for purifying corrin-containing molecules - Google Patents

Methods for purifying corrin-containing molecules Download PDF

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CN101065185A
CN101065185A CNA2005800372446A CN200580037244A CN101065185A CN 101065185 A CN101065185 A CN 101065185A CN A2005800372446 A CNA2005800372446 A CN A2005800372446A CN 200580037244 A CN200580037244 A CN 200580037244A CN 101065185 A CN101065185 A CN 101065185A
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cbi
sorbent material
compound
corrin
solution
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瑟吉·N·费多索夫
托本·E·彼得森
埃巴·奈克索
拉斯·E·伯格伦德
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Cobento AS
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to affinity adsorbent materials that contain high-specificity functional groups, in particular tetrazole, capable of binding corrin-containing compounds, such as cobalamin (Cbl, or vitamin B<SUB>12</SUB>).<SUB/>The invention also relates to methods for making such adsorbent materials, and their use in purifying corrincontaining molecules, such as cobalamin. Compositions containing cobalamin purified by the method are also provided.

Description

The method that is used for purifying corrin-containing molecules
All patents and the non-patent literature quoted in the present invention all are incorporated herein by reference in full at this.
Invention field
The present invention relates to a kind of compatibility sorbent material (affinity adsorbent materials), its comprise can with contain for example cobalamin (CbI, or Cobastab of corrin compound 12) height-specificity functional group of combination.The invention still further relates to the method for this sorbent material of preparation, and the purposes in purifying corrin-containing molecules such as cobalamin.The composition that comprises cobalamin by this method purifying also is provided.
Background of invention
Cobastab 12(or cobalamin CbI) is the biology confactor that relates in mammiferous two significant process: be methionine with the homocysteine reparation and propionic acid is converted into succinate (1).All mammals all depend on the B of external source 12, this is because it only synthesizes (2) by microorganism.Herbivore obtains CbI from the various a large amount of bacteriums that reside in its intestines system.Other animal comprises the mankind, obtains vitamin (3,4) from animal sources such as meat, milk and egg.Lack B 12To cause serious illness, comprise neurological unusual and anaemia (4,5).CbI is a molecule (Fig. 1) the most complicated in the confactor, and comprises three parts:
1) have the corrinoid ring of center cobalt atom,
2) at the nucleotides ring of lower shaft position ([α]-position) by its base and cobalt coordination,
3) at higher shaft position ([β]-position) [β] group with the cobalt coordination.
Produce B 12Synthetic simultaneously cobalamin of microorganism and analog thereof, this analog on its structure with B 12Different and in mammalian cell, do not have a catalysis (2,6).Most typical analog is the corrinoid (for example false B12, the A factor) with wrong nucleotide base or has lacked part molecule (for example cobinamide).The synthetic various intermediate products of cobalamin belong to the latter.All cobalamins and most of analog are the coloring matters with characteristic absorption spectrum, and this has simplified the mensuration of its concentration and different corrin rings (6) is distinguished from each other.
Bioactive CbI with correct skeleton can comprise the upper axis group of different and cobalt atom coordinations last in [β]-position (Fig. 1).Modal [β]-group is: other organic and inorganic compound (6) of methyl, desoxyadenossine, cyanide, water and some.Form of ownership (no matter character of its [β]-group) with CbI of correct skeleton can be confactor methyl-or desoxyadenossine-cobalamin (CbI-Met, CbI-Ado) (3) of tool catalytic activity by the enzymatic conversion in the cell.The intermediate forms of CbI is moisture-CbI (aquo-CbI) (CbI-OH in this conversion 2, Cobastab 12a).Therefore, CbI-OH 2, CbI-Ado and CbI-Met be the ubiquitous cobalamin (3,4,7) from any natural origin.The cyano group form (CbI-CN) of CbI is commercially pure typical products, and is also referred to as Cobastab 12
The preparation of CbI is the existing good method of setting up, and it comprises a series of active material separation steps (8,9) from bacterial suspension that make.Though this accurate details is not obtained by the public, its main step can be assumed that: 1) fermentation; 2) pyrolysis of the cell of interpolation cyanide; 3) non-specific adsorption of the little organic molecule (comprising CbI) on matrix such as charcoal; 4) wash-out and the absorption on other resin such as hydrophobic adsorbent XAD; 5) with phenol from the aqueous phase wash-out and the extraction CbI; 6) water-acetone mixture extracts CbI from phenol; 7) drying; 8) be dissolved in the hot water and in room temperature crystallization from acetone-water solution; 9) last drying.The product of purifying is a CbI-CN (Cobastab 12), but CbI-OH 2(Cobastab 12a) preparation need other chemical treatment and repurity.
Said process has adopted a series of non-specific steps that are used for separating.On the contrary, also certainly utilize the characteristic feature of CbI, that is, and the ability in [β]-position with some compound coordination.For example, the matrix with [β]-specificity group of coupling can be used to extract CbI from solution.This means the technical staff can be only in a step from thick extract purifying vitamin.This method of successful implementation is to be worth expectation, and this is that this aglucon can be replaced by the group that combines with adsorbent because the CbI in the medium of source has the aglucon on the axle top of weak combination.
As mentioned above, the principal mode of biogenic obtainable CbI is CbI-Met, CbI-Ado and CbI-OH 2But, even the carbon of CbI-Met and CbI-Ado-cobalt key also is (6) of sensitiveness to light in scattered beam illumination.Consequently, if do not take special prevention to handle, these two kinds of forms all can take place to CbI-OH in the aqueous solution 2Spontaneous conversion.The CbI-OH that obtains 2Has the weak molecule (H that connects in its [β]-position 2O), this molecule can easily be replaced (6,10) by a series of chemical agent.Some known compounds can be with different compatibilities and CbI-OH 2In conjunction with, this has caused the characteristic of CbI absorption spectrum to change.This has simplified the mensuration (6,10) to the dissociation constant of the compound of preparation: CN -(K d＜10 -12M), SO 3 -2＞(K d=10 -7M), the SH-base (K of cysteine d=10 -6M), NO 2 -(K d=10 -5M), N 3-(K d=10 -5M), imidazoles and histidine (K d=10 -4M), different amino (K d=10 -3And acetate (K M), d=1M).But, these simple inorganic ions with the matrix coupling after lose the character that CbI-combines easily.In addition, some in them (as containing the SH chemical agent) can cause beginning minimizing and destroying (6) of CbI.
Early stage amino-agarose resin being used for and the CbI-OH that low compatibility is arranged 2In conjunction with (Nexo1975, Biochim Biophys Acta 379:189-192).But this combination only just takes place when the CbI high concentration and very slowly (16-20 hour).This method thereby can not be used to purifying vitamin from lower biogenic of vitamine concentration.
Therefore, exist to simply and promptly from biological and other source separation of C bI and other contain the needs of the method for corrin compound.
Summary of the invention
First aspect the invention provides a kind of sorbent material that is suitable for the purifying corrin-containing compound, wherein said sorbent material:
Comprise tetrazolium as functional group,
And/or
Obtain by the method that comprises following step:
I) provide the activated substrate that comprises cyanide and
Ii) use the described matrix of solution-treated that comprises triazo-compound,
And/or
Have [β]-specificity functional group, wherein said functional group in pH3～7.5,5 ℃～60 ℃ down and CbI-OH 2In conjunction with, its K dLess than 100 μ M, and wherein said sorbent material does not cause described minimizing and the destruction that contains the corrin compound.
First, sorbent material is developed into and can combine with containing the corrin compound in [β]-position with high-affinity, and do not cause described minimizing or the destruction that contains the corrin compound.
In yet another aspect, the invention provides the method that a kind of preparation is suitable for the sorbent material of purifying corrin-containing compound, this method comprises following step:
I) provide the activated substrate that comprises cyanide, and
Ii) with the described matrix of solution-treated that comprises triazo-compound.
Do not fettered by a kind of particular theory, the method that chemistry causes and the feature of functional group point out that the tetrazolium residue is the functional group in the sorbent material, i.e. active material.
In yet another aspect, the present invention relates to be used to prepare the method for sorbent material, it comprises makes tetrazol group link to each other with matrix, or causes the formation of tetrazol group on matrix.
The present invention also provides a kind of and has been used for from the method for solution purifying corrin-containing compound, and described method comprises described solution is exposed to sorbent material of the present invention.
Therefore, in yet another aspect, the present invention relates to be used for the method from solution purifying corrin-containing compound, it comprises following step:
I) provide and comprise the solution that contains the corrin compound,
The sorbent material of the present invention that ii) provides this paper to define,
Iii) described solution is exposed in the described sorbent material,
Iv) never be adsorbed to and separate the compound that contains the corrin compound that combines with described sorbent material in the composition of the solution on the sorbent material,
V) randomly wash, and
The vi) described corrin compound that contains of wash-out from described sorbent material.
In yet another aspect, the invention provides cobalamin and composition by method purifying of the present invention, as pharmaceutical composition or food compositions, it comprises the cobalamin of described purifying.
Description of drawings
Fig. 1. the structure of cobalamin.Cobalamin (CbI, Cobastab 12) molecule comprises three parts: 1) have the corrinoid ring of center cobalt atom, it is according to the state of coordination and in difference aspect its valence; 2) at the lower axle position (α) and the nucleotides of base coordination; 3) at the axle position (β) on top and the group of cobalt coordination.Different groups can be with the β-position combination of different compatibilities at CbI.
Fig. 2. the formation of tetrazolium with and character.Last figure is the agarose of CNBr-activation and the reaction between the triazo-compound.The scheme of suggestion comprises two steps, wherein at triazo-compound after the initial connection of cyanide, the conversion that encircles along with the appearance of tetrazolium.In document 11, provided this reaction.Figure below: tetrazolium and group thereof are electronegative in neutral pH, and at pK a4-5 and pK b(3)-(2) have the ability that links two protons.
Fig. 3 .CbI-OH 2And the reaction between amino-tetrazolium.A) do not have tetrazolium (solid line) and have 2mM amino-tetrazolium (chain-dotted line) in the presence of, in 0.2M phosphate buffer, pH7.5,50 μ M CbI-OH 37 ℃ the time 2Spectrum.B) under described condition as A, with after the amino-tetrazolium (25-250 μ M) of variable concentrations mixes, CbI-OH 2The variation of absorbance.The curve of inferring demonstrates and meets bimolecular reaction CbI+ATZ  CbI-ATZ, wherein k +=306 ± 20M -1s -1And k_=(1.2 ± 0.3) 10 -3s -1
Fig. 4 .A) time dependent CbI-OH 2Absorption at the affinity adsorbent material.With CbI-OH 2Solution mixes with the CbI-specific adsorption agent material of different volumes.Final concentration is: 50 μ M CbI, 25-250 μ M and the functional group matrix coupling.This is reflected at pH7.5,37 ℃ and to carrying out under the mixture appropriateness stirring condition.At certain at interval, get 0.5ml sample and filtration rapidly.Measurement is CbI-OH in solution 2Amount.The curve demonstration of inferring meets bimolecular reaction CbI+ sorbent material  CbI-sorbent material, wherein k +=55 ± 6M -1s -1And k_=(4.1 ± 0.3) 10 -3s -1B) make 20 μ MCbI-OH 2The balance that combines with the variable concentrations sorbent material.The time of cultivating is 5 hours and 22 hours (pH6.0,20 ℃).The match of curve has provided following equilibrium dissociation constant: K d=2.8 μ M (5 hours), and K d=1.1 μ M (22 hours).
The adsorbing pH optimum condition of Fig. 5 .CbI.Will be on concrete sorbent material CbI-OH 2The absorption initial velocity be depicted as the function (20 ℃) of pH.Final concentration is 50 μ M CbI and 400 μ M conjugated groups.The pH optimum condition is in the zone of pH3-7.Has pK 12.0 and pK 27.8 the protonated-deprotonation of two groups will influence combination.Be not subjected to concrete theoretical restriction, the pK value that interacting molecule is known compares, can advise the condition that is used for effective combination as described below: 1) tetrazol group of adsorbent needs deprotonation (pH＞2), 2) CbI need be moisture form (aquo-form) (CbI-OH 2, pH＜7.5) rather than OH-form (hydroxo-form) (CbI-OH, pH＞7.5).
Fig. 6. make CbI-OH 2The mixture of+CbI-Ado combines with adsorbent.The combination of CbI is at pH7.5, carries out under 20 ℃ and the different illumination condition, and wherein said CbI is the typical case in the natural origin.The mixture of 50 μ M CbI, it comprises 20% CbI-OH 2(the CbI-CN of weak [β]-yl), 70% CbI-Ado (strong [β]-Ji is to photaesthesia) and ≈ 10% (strong [β]-Ji has resistance to the illumination of appropriateness).The condition of absorption is: 1) in the dark, 2) under scattered light, 3) with the preliminary illumination of scattered light after 30 minutes.At the remaining absorbance of the t → ∞ CbI-CN corresponding to 4 μ M, it is present in the preparation of CbI-Ado as pollutant.
Fig. 7 .CbI is from the desorption of concrete sorbent material.The wash-out of CbI from adsorbent (0.1ml) be in 20 ℃, carry out with the following solution of 1ml: 1) 10mM KCN, pH5.5 (open circles); 2) 10mM KCN, pH7.5 (triangle), pH9.5 (square), pH12 (rhombus); 3) 1M HCl (filled circles).
Fig. 8. the spectrum of the CbI of wash-out.In that (product is CbI-OH with KCN (product is CbI-CN (Fig. 8 A)) or 1MHCl 2(Fig. 8 B)) after the wash-out CbI, measure the spectrum of the prepared product that obtains and compare with standard (solid line).The prepared product (pH7.5) that dash line obtains after corresponding to wash-out at once.The short spectrum that dotted line shown the final prepared product of CbI (desalination, freeze-drying and dissolving) again of drawing.
The thin-layer chromatography of Fig. 9 .CbI sample.A) sample behind absorption and the wash-out: the CbI-CN of 1-standard; 2-in 37 ℃ with the CbI-CN behind the 1M HCl hydrolysis 3h; 3-from the compatibility material with the CbI-CN of 10mMKCN wash-out, desalination with freeze-drying; 4-is by CbI-OH 2The CbI-CN of preparation, it carries out wash-out with HCl, desalination with freeze-drying; The carboxyl analog of 5-CbI-CN.B) standard purification of CbI: the CbI-CN of 1-standard; The CbI-CN of the 2-purifying (CbI-OH that from blood plasma, adsorbs 2, carry out wash-out with HCl, be converted into CbI-CN, desalination with freeze-drying); The carboxyl analog of 3-CbI-CN.
Figure 10. the derivative of tetrazolium, amino-tetrazolium passes through its amine and following three kinds of different materials conjugation: epoxy agarose 6B, CM agarose and carboxyl-Amberlite (Amberlite) IRC76.
Detailed Description Of The Invention
Definition
Term used herein " functional group " refer to according to the separated interactional chemical group of molecule (here for containing the corrin molecule), the separate mode of this isolated molecule be for separating of condition under make separated molecule still be attached to sorbent material. " functional group " should not be interpreted as described group is to be attached to the unique chemical group on the matrix or to be interpreted as being attached on the matrix in a certain concrete mode. For example, Fig. 2 has shown to have tetrazolium as an example of the sorbent material of functional group, but can adopt other analog material, and in described material, for example tetrazolium is connected with matrix by being different from chemical part shown among Fig. 2, referring to Figure 10.
Term used herein " matrix " refers to be suitable for connecting the material of (conjugation) or initiation functional group, preferred polymeric material. This matrix can for example be the form of resin, piller, solid carrier or other surface of solids (solid surface). Matrix is agar glycosyl resin in a preferred embodiment. Preferred actual substrate is agarose (TM), epoxy agarose 6B, CM agarose and the hydroxyl-Amberlite IRC76 of CNBr-activation.
Sorbent material of the present invention and preparation method
In one aspect, the invention provides a kind of sorbent material that is suitable for the purifying corrin-containing compound, wherein said sorbent material:
Comprise tetrazolium as functional group,
And/or
Obtained by the method that comprises following step:
I) provide a kind of activated substrate that comprises cyanide, and
Ii) with the described matrix of solution-treated that comprises triazo-compound,
And/or
Have [β]-specificity functional group, wherein said functional group in pH3～7.5,5 ℃～60 ℃ of lower and CbI-OH2In conjunction with, its KdLess than 100 μ M, and wherein said sorbent material does not cause described minimizing and the destruction that contains the corrin compound.
In preferred embodiments, described contain the corrin compound be contain the corrin compound moisture form or with the form that contains corrin compound balance of moisture form. In the preferred embodiment of another kind, the described corrin compound that contains is cobalamin or its analog, preferably moisture-cobalamin.
Sorbent material with high-affinity with contain the corrin compound and be combined. In preferred embodiments, described sorbent material in pH3～7.5,5 ℃～60 ℃ of lower and CbI-OH2In conjunction with, its KdBe 0.01～100 μ M, for example KdBe 0.1～50 μ M, for example KdBe 0.5～20 μ M, for example KdBe 1～7 μ M. KdPreferably according to detecting described in embodiment 1.
In another preferred embodiment, when being filled in the post by gravity, sorbent material has maximum Sa, namely in every liter sorbent material, have 0.1～500mmol, for example 0.5～100mmol, for example 1～50mmol, for example 4～8mmol's contains the corrin compound.
In yet another embodiment, sorbent material is not combined with CbI-CN or CbI-Ado.
Can use many activated substrate as known in the art as activated substrate. Preferred activated substrate is the agarose of CNBr-activation or the Agar Gel sugar of CNBR-activation.
In yet another aspect, the invention provides the method for the preparation of the sorbent material that comprises at least a functional group, this sorbent material is suitable for the purifying corrin-containing compound, and described method comprises following step:
I) provide a kind of activated substrate that comprises cyanide, and
Ii) with the described matrix of solution-treated that comprises triazo-compound,
In yet another aspect, the present invention relates to the method for the preparation of sorbent material, it comprises the formation that tetrazol group is connected with matrix or causes tetrazol group in matrix. This tetrazol group can be connected in any suitable manner with matrix, for example by being described in any suitable substituted radical on the carbon atom of tetrazolium among embodiment 4 and Figure 10.
The method of purifying corrin-containing compound
The sorbent material of the present invention definition can be used for from various sources comprise biogenic the purifying corrin-containing compound.
Therefore, another main aspect, the present invention relates to be used for method from solution purifying corrin-containing compound, it comprises following step:
I) provide and comprise the solution that contains the corrin compound,
Here the sorbent material of definition ii) is provided,
Iii) described solution is exposed to described sorbent material,
Iv) never be adsorbed to and separate the compound that contains the corrin compound that combines with described sorbent material in the composition of the solution on the sorbent material,
V) randomly carry out washing step, and
The vi) described corrin compound that contains of wash-out from described sorbent material.
Contain the corrin compound and know in the art, it comprises for example corrinoid, synthetic intermediate, cobinamide, the false B of CbI 12, the A factor etc.In preferred embodiments, described contain the corrin compound be contain the corrin compound moisture form or with the form that contains corrin compound balance of moisture form.In another kind of embodiment preferred, the described corrin compound that contains is cobalamin or its analog, and is preferably moisture-cobalamin.
In some embodiments of above-mentioned purification process, described solution step I ii) before and/or among be exposed under the light.This can for example be converted into the form that combines with described material, for example CbI-OH with the CbI form that does not combine with absorber material 2
And in some embodiments, the method for purifying is included in the solution preliminary treatment of step I before ii), to contain the corrin compound from for example discharging the compound of protein with other molecule.This preliminary treatment preferably by preferably low pH heat as pH3 as described in solution carry out.
In preferred embodiments, the exposure of the step I of purification process in ii) is to carry out between pH3～7.
Purification process can contain corrin solution and undertaken by the post that has loaded sorbent material of the present invention by making.Therefore, in some embodiments of said method, step I ii) and iv) comprises makes described solution by comprising the post of described sorbent material.
The wash-out that contains the corrin molecule for absorption can carry out according to many modes.
In some embodiments, elution step vi) comprises use cyanide (CN -).Preferably carry out at pH7-12, preferably carry out after following step, described step is converted into single cyano group form by for example acidifying with the dicyano form.
In other embodiment, in step wash-out vi), comprise and use for example HCl of acid, preferably then neutralization procedure is converted into the step of cyano group form with another by the interpolation cyanide.
Purification process preferably includes described another step that contains the corrin compound of purifying in the salt from be contained in sample.This can be according to comprising dialysis and/or using many modes of the absorption of active carbon to carry out.
When using active carbon, described purifying preferably by will describedly contain the corrin compound be adsorbed on back on the active carbon, never separated in the solution composition of charcoal absorption combine with active carbon contain the corrin compound and wash-out adsorbs from the activated carbon contains the corrin compound.
Cobalamin and composition of the present invention
In yet another aspect, the present invention relates to the cobalamin that obtains by purification process of the present invention.
In yet another aspect, the present invention relates to composition, it comprises cobalamin that obtained by purification process of the present invention or available.
In yet another aspect, the present invention relates to pharmaceutical composition, it comprises cobalamin that obtained by purification process of the present invention or available and acceptable diluents, carrier or excipient.
Composition of the present invention is applicable to any suitable approach such as per os (comprise and sucking or the hypogloeeis), local (comprise suck, hypogloeeis or transdermal) or the stomach and intestine administration of outer (comprising subcutaneous, intramuscular, intravenous, intracutaneous).Such preparation can be prepared according to any method known in the drug world, for example by active component is mixed with carrier or excipient.
Be applicable to that oral pharmaceutical preparation can be the form of dispersal unit, for example capsule or tablet, powder or particle, the solution in water-based or non-aqueous liquid or suspension, edible foams or chewable tablets (whips) or oil-in-water liquid emulsion or Water-In-Oil liquid emulsion.
The pharmaceutical preparation that is applicable to topical can be prepared as ointment, creme, supensoid agent, lotion, powder, solution, paste, gel, spray, aerosol or finish.Be applicable to that the pharmaceutical preparation of topical comprises lozenge (lozenges), lozenge (pastilles) and mouth wass in the oral cavity.
The pharmaceutical preparation that is applicable to parenteral comprises aseptic injectable solution water-based or nonaqueous, and it can comprise antioxidant, buffer solution, bacteriostatic agent and make preparation and solute that intended recipient's blood etc. oozes; Sterile suspensions water-based or nonaqueous, it can comprise suspending agent and thickener.Preparation can be ampoule or the bottle that the form of the form of dosage unit or multi-dose container for example seals, and also can store under the condition of freeze-drying, and its need add sterile liquid carrier before use such as water for injection gets final product.Interim injection solution and suspension can be by aseptic powdery, particle and preparation tablets.
Preferred dosage unit preparation comprises daily dose or sub-doses or its suitable part of active component.
Should be understood that except above mentioned concrete composition, preparation can also comprise the conventional additives of other this area that is used for related preparation type, for example be applicable to oral can comprise flavouring.
In yet another aspect, the invention provides food supplement, it comprises the cobalamin by method purifying of the present invention.
Embodiment
Embodiment 1
1.1 the preparation of absorber material
The sepharose 4B (Pharmacia) of CNBr-activation is loaded in the post, washs widely with 1mM HCl, afterwards with 0.02M NaN 3At 0.1M NaH 2PO 4Buffer solution (P i-buffer solution) in pH7.5, room temperature, reacts.Within an hour, the medium pump that makes two bed volumes is through post.
As the matrix of using acid elution of 1 volume and the 0.02M NaN of 2～3 volumes 3At 0.2M P iSolution among-buffer solution, the pH7.5 was cultivated 1 hour under regularly stirring, and the reaction between agarose and the triazo-compound also can be carried out in batches.After the coupling step, sorbent materials 0.1M P i-buffer solution, the pH6.0-7.0 washing washes with water afterwards.The material of preparation is according to the absorber material that suspends: the ratio of water (1: 1) is preserved at room temperature.
1.2 the chemical action of reaction
The agarose of CNBr-activation and the definite reaction mechanism between the triazo-compound are unknown.According to the related data in the document (11), the scheme of Fig. 2 has proposed possible reaction mechanism.This reaction may comprise two steps, wherein triazo-compound for the first time attached to agarose on, be converted into by ring then that tetrazol group carries out.In order to test this hypothesis, analyzed water soluble amino-tetrazolium to CbI-OH 2Combination on (pH7.5,37 ℃).Be intended to comparison amino-tetrazolium to NaN 3With to the preparation sorbent material on speed constant.
Mixing the back with amino-tetrazolium at CbI-OH as passing through 2In the change of detected spectrum indicated, amino-tetrazolium can be coordinated to CbI [β]-(Fig. 3 a) in the position.The spectrum that obtains is suitable typical C bI-N-R (6).The binding ability that contains [β]-position of tetrazole compound and CbI is strange.By timely monitoring CbI-OH 2Spectrum to the CbI-tetrazolium changes, and has studied the dynamics of this reaction.Relevant speed general knowledge obtains (Fig. 3 b) by the curve calculation that shows.The present invention compares triazo-compound, amino-tetrazolium and sorbent material in the table 1 that the results are shown in of mensuration.It has supported that the functional group to agarose imports is this theory of tetrazolium-group.
Table 1. detects the CbI-OH that is coordinated in that different compounds obtain 2The speed constant of [β]-position
Compound of reaction k +(M -1 s -1) k_(s -1) K d(μM) Condition
NaN 3 2500 0.13 50 37℃，pH7.5
Amino-tetrazolium 306 12×10 -4 3.9 37℃，pH7.5
Amino-tetrazolium 75 3.4×10 -4 4.5 20℃，pH7.5
Sorbent material 55 4.1×10 -4 7.4 37℃，pH7.5
Sorbent material 17 1.1×10 -4 6.4 20℃，pH7.5
Sorbent material 25 1.0×10 -4 4.1 20℃，pH6.0
Sorbent material The K of balance combination d＝2.8(5h)；1.1(22h) 20℃，pH6.0
Embodiment 2.CbI-OH 2The character of particular adsorbent
2.1. with the matrix of CbI with batch (-type) saturated (Batch saturation) triazo-compound-activation of maximum possible degree
Tested CbI-OH 2With combining of the sorbent material for preparing.When the sorbent material that 0.05ml is suspended: the 1mM CbI-OH of water (1: 1) and 0.5ml 2At the 0.1M of pH6 P i-buffer solution mixes and cultivated 5 minutes, observes the abundant saturated sorbent material with CbI.With sample centrifugal (10,000rpm, 30s), with the 0.1M P of 2ml pH6 iThe coloured supernatant of-buffer solution, flush away 1 minute.Sorbent material is by centrifugal and precipitated, and according to as 2.2 joints description from bead the CbI of elution of bound.Detect the concentration of wash-out CbI according to its absorption spectrum.The concentration of CbI-conjugated group is estimated as 4-8mM in the adsorbent that loads.The amount of active group depends on original agarose in resin.The agarose of fresh CNBr-activation has shown the high-caliber group that is caused of being handled by nitrine.
2.2. in the assay determination method from sorbent material wash-out CbI
The sorbent material that CbI-is saturated (0.02-0.1ml) was cultivated 5 minutes under pH7-9 and 95 ℃ at the 2mM of 1ml KCN, caused the CbI of absorption all to discharge, promptly had whole releases with the CbI (CN-CbI-CN) of the CN-group of cobalt coordination at higher and low shaft position with two-cyano group form.The CbI of wash-out by its absorbance quantitatively.Than being easier to detect two-cyano group-CbI, this is to have the highest absorptivity (6) because other CbI of this form and all of part compares.
2.3. sorbent material does not have the post of leakage saturated
When the post of the 0.2ml with adsorbent in pH6 with 100 μ M CbI-OH 2Flow handle, before any visual leakage, can reach the concentration of the 2mM in the compatibility material.At the saturated level greater than 2mM, the eluent from post begins to comprise the CbI-OH of the amount of increasing gradually 2(5-10 μ M or higher).On the other hand, the solution when application does not comprise any free CbI-OH 2The time, there is not any significant leakage in the sorbent material that is saturated to par.The CbI of absorption is as the CbI-OH to the outside 2Be responsive, wherein the latter attacks the part in the sorbent material and cause slight outflow when height is saturated.
2.4. adsorbent and CbI-OH 2Between the dynamics that reacts to each other
Reaction between adsorbent and the CbI is assumed that bimolecular reversible reaction A+B  AB, and it is considered to correct at least when the sorbent material appropriateness is saturated.In order to detect the kinetic parameter of this reaction, with the sorbent material (corresponding in final suspension, being the functional group of 25-250 μ M concentration) and the 50 μ M CbI-OH of difference amount 2Mix.This reaction can be carried out pH7.5,37 ℃, 20 ℃ or under pH6.0,20 ℃ condition.After cultivation, the suspension of 0.5ml part is filtered rapidly by the sponge that is loaded in the pasteur pipet with gentle agitation.The concentration of detection CbI in filtered sample, and be used to draw through the time absorption curve (Fig. 4 a).Describe according to the speed constant shown in the table 1, experimental point is gratifying.Combining of CbI and material can be carried out 5 ℃ or 60 ℃ or at 20-37 ℃, but the proportional deceleration/acceleration (proportional deceleration/acceleration) with adsorption process.In another experiment, analyzed the balance of combination.The sorbent materials of different amounts in gentle agitation with the 20 μ M CbI-OH of 1ml 2Cultivated 5-22 hour.With solubility CbI-OH 2Minimizing to the concentration of functional group in suspension mapping (Fig. 4 b).Calculated equilibrium dissociation constant (table 1) afterwards.Its value is than the K that is calculated by speed constant in kinetic determination dIt is lower slightly that (Fig. 4 a).Compare CbI-OH 2Adsorption dynamics adsorption kinetics clearly shows: compare with the CbI-specific adsorption agent material with triazo-compound, the CbI-specific adsorption agent material with amino-tetrazolium has higher similitude.This has supported following hypothesis: the functional group in the agarose of triazo-compound-activation is the triazo-compound of tetrazol group rather than coupling.
Above-mentioned dynamic experiment can be predicted the state about adsorbent in loading post.Therefore, make and comprise CbI-OH 2Medium filter (in sorbent material, being the supposition tetrazol group of 4mM) by being loaded into sorbent material in the post, 95% of the CbI that being adsorbed as in the time of will causing being the per minute two bed volumes with the flow velocity applies, 99.8% (pH6, the room temperature) of the CbI that being adsorbed as when perhaps with the flow velocity being per minute one bed volume applies.Aforementioned calculation has been confirmed in measuring, and be presented at flow velocity for 2ml/min time filter has the absorption (pH6, room temperature) of 95-96% through 2.5 μ mol CbI of 1ml post.(1 volume of adsorbent material mixes with the CbI solution that contains of 99 volumes in the suspension of dilution, suppose the tetrazol group of 40 μ M, final dilute strength is 1 μ M CbI, pH6.0,20 ℃) batch (-type) absorption (batchadsorption), after cultivating in 30 minutes, demonstrate about CbI-OH 280% absorption.
2.5. absorption is for the dependence of pH
Consider that the many groups that exist have soda acid character, can expect CbI-OH in reaction molecular 2With combining of tetrazol group is the pH-sensitivity: i) pK of second proton on the tetrazolium is (2)-(1), and ii) the pK of first proton on the tetrazolium is 2-4, and iii) the nucleotide base of CbI is because protonated and pK is 0, iiii) CbI-OH+H + CbI-OH 2The pK of acid-base balance be 7.6.The solution that will have relative broad range pH0-12 is as being used for CbI-OH on sorbent material 2Absorption medium and detect.When mixing, the medium that contains 60 μ M CbI as the suspension of 0.2ml sorbent material and 1ml combines with batch (-type).Between 1-30 minute, followed the tracks of removing of from supernatant CbI by optics test.With the initial velocity of combination to pH mapping (Fig. 5).This figure shows that optimal conditions are between pH3～pH7.The approximate pK that influences the group of combination is pK 2.0 and pK 7.8.
As shown in Figure 5, do not detect absorption at pH 0 (1M HCl).In low pH value is not because of due to the deterioration of sorbent material in conjunction with this not, in case this is because pH is adjusted to 4-7, the sorbent material that cleans with HCl has just recovered it and catch CbI-OH from solution 2Ability.As known for tetrazol group, this is effective similarly to be by protonated cause (11) of sorbent material when hanging down pH.
PH is elevated to the deceleration that also causes combination greater than 7, this may be since moisture-CbI due to the conversion of its hydroxyl-form (wherein the OH-base has higher compatibility than water to [β]-position of CbI).But, shown in the data in table 1, compare with pH6, combination is not difficult when pH7.5.
2.6. biologically Xiang Guan CbI and TZ-agarose combines
The principal mode of obtainable CbI is CbI-Ado, CbI-Met and CbI-OH in the natural source 2, wherein the above two are photosensitivities.Therefore, 20%CbI-OH 2Detect under the following conditions with the characterization of adsorption of 80%CbI-Ado mixture: 0.1ml sorbent material, 0.9ml buffer solution, 50 μ MCbI, pH6,20 ℃.Sample differently shines: 1) preserve in the dark, 2) in the illumination of the chamber of scattering, cultivate 3 in the cohesive process) before combination, carry out scattered light illumination in 30 minutes.The result describes (Fig. 6) with optical density that keeps in the solution and incubation time.Positive according to expectation, in all samples, 20% CbI-OH 2Promptly from medium, removed.On the contrary, under study for action, the CbI-Ado that three kinds of samples are kept is difference as a result.Therefore, when preserving sample in the dark, what carry out after the phase I of absorption fast is the combination of CbI-Ado very slowly.The appropriate illumination that suspension is carried out has caused the remarkable acceleration of absorption, and makes that for the pre-illumination that solution carries out the speed of combination almost can not be from pure CbI-OH 2In distinguish.
Can derive following conclusion, that is, by CbI-Ado to CbI-OH 2Photochemical transformations be highly effective, even and under the irradiation of appropriateness after 1 hour cultivation the initial CbI-Ado in fact whole pond (wholepool) be accumulated in the sorbent material.As being found that from the spectrum of record absorbance residual in the supernatant is neither relevant with CbI-Ado, also not with CbI-OH 2Relevant, and the CbI-CN (not shown) of automatic pollution.
2.7. the desorption of CbI from sorbent material under different condition
CbI can carry out according to the purpose of work in many ways from the release of compatibility sorbent material.In 2-10mM KCN in pH7.5,95 ℃ down rapidly quantitatively the method for wash-outs be applicable to the purpose of analysis and be described in the 2.2nd joint.Two kinds of other methods that more are applicable to purifying CbI in enormous quantities are described below.
First method also adopts 10mM KCN, but desorption is to carry out under the condition that relaxes.Therefore, if the time long enough of cultivating, can be under the condition of room temperature and pH7-12 wash-out CbI (Fig. 7).Be enough to from sorbent material, to remove the CbI of 99% absorption in about 10 hours.Because this reaction is irreversible, therefore in fact do not need excessive eluant, eluent from the post that loads, to discharge whole CbI.Obtain part, and release from sorbent material has been quickened in the existence of cyanide on two coordination positions as CN-CbI-CN.Medium is acidified to pH2-3 have been caused in 20 minutes to list-cyano group form (CbI-CN, Cobastab 12) change.Obtain the absorbance spectrum of material and do not have different (Fig. 8) of CbI-CN standard liquid.The advantage of KCN wash-out is the conversion of CbI to cyano group-form and dicyano-form, and described two kinds of forms are very stable for different chemical reagent.But the shortcoming of this method is to have used hazardous compound KCN.
Consistent with the pH dependence of CbI combination, the wash-out of the CbI of absorption can also carry out (Fig. 5) in 1M HCl.This method is rapidly, and sorbent material therein: the ratio of acid is the wash-out (Fig. 7) that batch culture (batch incubation) reaches 90-95% in 1: 10 the acid after 1 minute.The release of the CbI of residue 5-10% needs fresh HCl, is reversible with the opposite absorption-desorption process in acid of wash-out that above-mentioned irreversible KCN-induces promptly.In the desorption of post, the 1M HCl that pumps into two bed volumes is enough by sorbent material (each volume 5 minutes) with the part that discharges 95% combination.If necessary, adsorbent recovers fully and can realize by cleaning with the acid of two volumes again.Do not recommend CbI is kept among the 1M HCl for a long time, this is because it will slowly be hydrolyzed to carboxyl-analog.Therefore, the sample of wash-out neutralizes with 5M NaOH, and adds the level (pH5 or pH7.5) of buffer solution pH is remained on requirement.Spectroscopic assay demonstrates, and what obtain comprises CbI-OH 2Sample and spectrum do not have different (Fig. 8) with standard items.By adding the KCN more excessive a little than CbI, CbI's moisture-form can easily be converted into stable cyano group-form when pH5.Because the protective nature of cyanide, it can be preferred changing CbI-CN into.
2.8. the stability of the special sorbent material of CbI-in different medium
The stability of sorbent material is important for the prolonged application of material, and the adsorbent of preparation is proved to be and can resists degraded.For example, sorbent material can be in room temperature at water or 1-10mM NaN 3Preserved 1 year in (antiseptic) and any visible deterioration do not take place.Not only preserve, and the condition of CbI desorption all may influence the character of sorbent material.Therefore, we have measured the influence of wash-out medium for sorbent material.
Measure to show that adsorbent can be in room temperature at 10mM KCN, 0.1M Pi, pH7.5 preserved at least two weeks and do not have any loss of CbI-binding ability.Maximum saturation degree and adsorption rate be identical with fresh material all.
On the other hand, it also is quite necessary measuring for the sorbent material after cultivating in 1M HCl, this be since the pH of this solution outside the stability range (pH2-11) that the agarose of report is used.This mensuration is following carries out.The sorbent material that to cultivate in acid after 30-50 hour places CbI-OH 2In solution (0.1M Pi, pH6.0, the 200 μ M CbI) stream.Detect the absorbance of eluting liquid, and in a single day this experiment reaches the 10% just termination that 20 μ M promptly reach used medium in the leakage of CbI.Measure the amount of ligand of absorption then, data are represented (table 2) with the concentration of CbI in sorbent material.
The CbI-of table 2. sorbent material after cultivating different time with 1M HCl is in conjunction with character
Time in HCl The degree of saturation that CbI-leaks does not take place in flowing:
Two bed volumes/minute The attached column bed volume/minute
0h 2.5mM 3.1mM
30h 2.2mM 2.4mM
50h 1.2mM 1.9mM
Cultivation in 1M HCl has caused the part deterioration of resin, and follows early leaking and lower degree of saturation of CbI.Should effect when the absorption of flowing fast (two bed volumes/minute) more obvious, but and it is represented obvious like that when being unlike in flowing of reducing (attached column bed volume/minute).In a word, even in 1M HCl, cultivate after 50 hours, also can 0.5 volume/minute flow velocity under post is saturated to the level of 2mmol CbI in every liter of sorbent material.This result shows that the CbI-specific materials may preserve the attached circulations of 300 absorption-desorptions (keeping 10 minutes) and seriously damage in each HCl wash-out.
2.9. the desalination of CbI-sample behind wash-out from sorbent material
Prepare CbI by the compatibility sorbent material, owing to the NaCI (carry out behind the HCl wash-out NaOH neutralization) of high concentration is arranged or have dissociate KCN (KCN wash-out) and can not use at once behind wash-out of 7-8mM.Two kinds of following methods are to be suitable for removing from solution low-molecular-weight material most.
Separation of C bI can utilize the difference of its quality to carry out (CbI is 1300, and salt is＜100) from salt.This can be undertaken by the dialysis of water, its adopt have bore dia be equivalent to 500-1000 by the size film.It also is possible adopting some dilution-concentration circulations of the film of same characteristic features.Method based on dialysis is effectively for CbI-CN, and this is to change because of comparing with standard items not introduce in UV and visible spectrum.But, to final CbI-OH 2The analysis of preparation has shown some distortion in spectral shape, this may be owing to the reaction of [β]-position with impurity.Thin-layer chromatographic analysis shows exist (10-20%) of yellow CbI, and but it drifts about soon slightly than principal component has still covered its (Fig. 9 A, swimming lane 3).If the dialysis and freeze drying before with CbI-OH 2Be converted into CbI-CN, then do not find this material (Fig. 9 A, swimming lane 4), at the concentrated CbI-OH of preparation 2In exist small amount of impurities suggestion by conventional method recrystallization sample.
Desalination to the CbI sample also can be used active carbon method.This step can not be applied to CbI-OH 2, this is because its [β]-position is responsive (the end-product flavescence of CbI also comprises [β]-group rather than water) to some composition in the active carbon.On the other hand, the CbI-CN with [the β]-group of combining closely is applicable to such processing.In experiment, having obtained the maximum saturation degree is every 1g active carbon 20-25 μ mol CbI.Suction-operated takes place immediately, and solid constituent can separate from solution by gravitational settling (2-3h), of short duration centrifugal (1 fen 3000g) or filtration.The ball water is cleaned, and with the CbI of absorption with 96% ethanol with two parts (per step cultivation 30 minutes) wash-out.CbI freeze drying with wash-out.
The model purifying of embodiment 3.CbI
3.1.CbI the source
Employing CbI-OH 2Saturated CbI-is in conjunction with albumen intrinsic factor (IF) (5,12), and joins in the human plasma and set up model system, and described human plasma is 1: 5 with 0.1M citrate buffer pH3.0 dilution.The final concentration of IF-CbI is 10 μ M (CbI of ≈ 0.01mg/ml), and its cumulative volume is 5ml.The concentration of contaminating protein is about 15mg/ml.
3.2.CbI extraction
The sample that will contain CbI is 95 ℃ of heating 30 minutes, to discharge CbI-OH from albumen composition 2Be accompanied by the degraded of about 30% part in the processing of pH3.As a comparison, the similar processing at pH7 has caused CbI-OH 2Degraded fully, this may be because the minimizing that contains the SH compound that exists in blood plasma.Be neutralized to pH5.0 and by the centrifugal chip of removing with the sample cooling of heating and with NaOH.Clean the ball that is equivalent to cumulative volume 1/3 with the citrate buffer solution pH5.0 of 0.1M then, centrifugal and collected two kinds of supernatants.The final volume of sample is 6ml.Also can adopt other extracting process.
3.3. the absorption on the TZ-agarose
With the flow velocity of 0.2ml/ branch (two bed volumes per minute), part is adsorbed on the CbI-specific adsorption agent material (0.1ml) that is loaded in the pasteur pipet.To be combined with the 1MNaCI of the adsorbent of CbI with 1ml, 0.1M Tris, the water washing of 1ml is used in the pH7.5 washing afterwards.
3.4.CbI wash-out
The part of absorption is with 0.3ml 1M HCl wash-out from post, wherein with 5M NaOH neutralization solution and interpolation citrate buffer to the concentration of 0.1M pH is kept ≈ 3.0.By adding 1mMKCN and sample being heated 5 minutes at 95 ℃, with the CbI-OH of preparation 2Be converted into CbI-CN.
3.5. desalination and freeze drying
It is 500 the film salting liquid to water dialysis CbI that use has by molecular weight (cut off molecular), afterwards with the sample freeze-drying.The analysis of preparation to dissolving shows that it is at UV and both absorption spectrums of visible light part and (Fig. 8) in full accord of standard items.Thin-layer chromatography (Fig. 9 B) has demonstrated a colored component (swimming lane 2), and it has the mobility that is equivalent to standard items (swimming lane 1).The amount of purifying CbI is equivalent to add to the 60-65% in the model sample.The main loss of 25-30% occurs in and extract CbI-OH from blood plasma 2Process in.
The conjugated of embodiment 4. amino-tetrazolium and different substrates
The active character of tetrazol group is identified in independent experiment.Therefore, the derivative Aminotetrazole of tetrazolium passes through its amino and three kinds of different materials conjugated: epoxy agarose 6B, CM agarose and carboxyl-Amberlite IRC76 (Figure 10).Consequently, need the CbI binding ability of 2mM and 6mM to the inert material (inert) of CbI material (epoxy-and CM-agarose), and consistent in all others with the behavior of early stage described matrix.CbI is to have quickened 10 times (factor 10) after linking tetrazolium to the slow and nonspecific combination of Amberlite.
4.1 experimental detail
The matrix that 10ml is measured is with excessive water washing, and the slurry of preparation feedback mixture: i) 20ml 0.1M amino-tetrazolium pH12.3 is mixed with the epoxy agarose 6B of 10mL; Or ii) 0.1M amino-tetrazolium, 0.2M N-(3-the dimethylamino-propyl)-N '-ethyl carbodiimides pH5.5 of 20mL mix with 10mL CM agarose or carboxyl-Amberlite IRC76.The pH of latter's mixture is monitored and maintain pH5.0-5.5 by adding 5M NaOH in first hour of reaction.Continue at room temperature to cultivate and leniently stirred two days.Afterwards, the matrix of activation is with excessive water washing and detect its CbI binding ability.
The conclusion of embodiment
By using sodium azide (NaN 3) handle the agarose that CNBr-activates, successfully on insoluble material, cause CbI-specificity group.Be not subjected to the restriction of particular theory, dynamic analysis points out that forcefully the active material in the sorbent material that obtains is a tetrazol group, rather than azido group.Solution and with the tetrazol group of carrier band material coupling, because of combining with CbI effectively in its [β]-position with the cobalt coordination.The character of tetrazolium is earlier strange.The material that obtains shows CbI-OH 2High-affinity, it is Kd=1-7 μ M (table 1) when pH6-7.5, and maximum degree of saturation is the CbI of every liter of adsorbent 4-8mmol.When minimum leakage, can obtain the degree of saturation of 2-3mM.
CbI-OH 2Combination to sorbent material can be observed in the relative broad range of pH3～pH7.From solution, capture CbI-OH 2In two processes that post filters and produces in batches all is effective.Sorbent material does not combine with CbI by [the β]-Ji that combines closely, for example CbI-CN or biology form A bI-Ado.But the light scattering of CbI latter's form causes being converted into the CbI-OH that adsorbs with effectively 2
Carried out the method for two kinds of wash-outs from the saturated compatibility material of CbI-.First kind is adopted 10mM KCN and obtains end-product with pure CbI-CN.The advantage of this method is to have protected the reaction of CbI [β]-position to prevent that itself and pollutant from may carry out.Second kind of technology adopts 1M HCl as eluant, eluent and to make the product that will obtain be CbI-OH 2, it comprises 10-20% and has CbI rather than the water of [β]-Ji.This is owing to causing with some pollutant reactions in concentrated CbI sample process.
The compatibility sorbing material is durable and the reagent of all application is had fabulous patience.The analysis of carrying out has shown that this material has after 300 absorption of experience the potential ability with the circulation of 1M HCl wash-out.
Carried out typical purifying.Use CbI-OH 2Be attached to the specific proteins intrinsic factor of mixing with plasma protein.Main purification phase is: 1) in pH3 and 95 ℃ of extractions, 2) be adsorbed on the compatibility material 3) with 1M HCl wash-out, neutralization and with CbI-OH 2Be converted into CbI-CN, 4) with solution desalination and freeze-drying.According to absorption spectrum and the mobility on thin-layer chromatography, the product that obtains does not have different with standard items.
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Claims (26)

1. the sorbent material that is used for the purifying corrin-containing compound, wherein said sorbent material:
Comprise tetrazolium as functional group,
And/or
Obtain by the method that comprises following step:
I) provide the activated substrate that comprises cyanide and
Ii) use the described matrix of solution-treated that comprises triazo-compound,
And/or
Have [β]-specificity functional group, wherein said functional group is at 3～7.5,5 ℃～60 ℃ of pH and CbI-OH 2In conjunction with, its K dLess than 100 μ M, and wherein said sorbent material does not cause described minimizing or the destruction that contains the corrin compound.
2. the sorbent material of claim 1, the wherein said corrin compound that contains is cobalamin or its analog.
3. the sorbent material of aforementioned each claim, wherein said contain the corrin molecule be contain the moisture form of corrin compound preferably moisture-cobalamin, the perhaps form that contains the corrin compound that balances each other with the moisture-form that contains the corrin compound.
4. the sorbent material of aforementioned each claim, wherein said sorbent material is at 3～7.5,5 ℃～60 ℃ of pH and CbI-OH 2In conjunction with, its K dBe 0.01～100 μ M, for example Kd is 0.1～50 μ M, is 0.5～20 μ M as Kd, is 1～7 μ M as Kd.
5. the sorbent material of aforementioned each claim, wherein when by gravity loading in post the time, described sorbent material has maximum Sa, promptly in every liter sorbent material, have 0.1～500mmol, for example 0.5～100mmol, for example 1～50mmol, for example 4～8mmol's contains the corrin compound.
6. the sorbent material of aforementioned each claim, wherein said activated substrate are the agarose of CNBr-activation.
7. be used for preparing the method for the sorbent material that defines in above-mentioned each claim, it comprises makes tetrazol group link to each other with matrix, or causes the formation of tetrazol group on matrix.
8. the method for claim 7, wherein said matrix is epoxy agarose 6B, CM agarose or carboxyl-Amberlite (Amberlite) IRC76.
9. be used for preparing the method at the sorbent material of each definition of aforesaid right requirement 1-6, it comprises the steps:
I) provide the activated substrate that comprises cyanide and
Ii) with the described matrix of solution-treated that comprises triazo-compound.
10. be used for from the method for solution purifying corrin-containing compound, it comprises the steps:
I) provide and comprise the solution that contains the corrin compound,
The sorbent material of each definition among the claim 1-6 ii) is provided,
Iii) described solution is exposed in the described sorbent material,
Iv) never be adsorbed to and separate the compound that contains the corrin compound that combines with described sorbent material in the composition of the solution on the sorbent material,
V) randomly wash, and
The vi) described corrin compound that contains of wash-out from described sorbent material.
11. the method for claim 10, the wherein said corrin compound that contains is cobalamin or its analog.
12. the method for claim 10 or 11, wherein said contain the corrin molecule be contain the moisture form of corrin compound preferably moisture-cobalamin, the perhaps form that contains the corrin compound that balances each other with the moisture-form that contains the corrin compound.
13. each method in the claim 10～12, its be included in step I ii) before and/or among described solution is exposed in the light.
14. each method in the claim 10～13, its be included in step I ii) before to described solution carry out preliminary treatment with will contain the corrin compound from the compound of other molecule such as protein discharge.
15. the method for claim 14, wherein said preliminary treatment is undertaken by heating described solution.
16. each method in the claim 10～15 is to carry out between pH 3～7 in the exposure of step I in ii) wherein.
17. each method in the claim 10～16, wherein step I ii) and iv) comprises and makes described solution by comprising the post of described sorbent material.
18. each method in the claim 10～17, wherein step vi) comprises use cyanide (CN -) solution.
19. the method for claim 18, wherein step is vi) carried out in pH 7-12.
20. the method for claim 18 or 19, the step that wherein is converted into single cyano group form are for example to be undertaken by acidifying after vi) in step.
21. each method in the claim 10～17, wherein step vi) comprises for example HCl of use acid.
22. the method for claim 21, wherein neutralization procedure carries out after vi) in step.
23. claim 21 or 22, another step that wherein is converted into the cyano group form are to be undertaken by adding cyanide.
24. each method in the claim 10～23, it comprises described another step that contains the corrin compound of purifying in the salt from be included in sample.
25. the method for claim 24, wherein said purifying is finished by dialysis.
26. the method for claim 24, wherein said purifying be by with described contain the corrin compound be adsorbed on the active carbon back, never separated in the solution composition of charcoal absorption combine with active carbon contain the corrin compound and wash-out adsorbs from activated carbon the corrin compound that contains is finished.
CNA2005800372446A 2004-09-01 2005-09-01 Methods for purifying corrin-containing molecules Pending CN101065185A (en)

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