CN108383850B - A kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC and preparation method thereof - Google Patents
A kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC and preparation method thereof, and structural formula is as follows:CD22 non-natural sialic acid fluorescence probe BPC-FITC of the invention is directed to B cell CD22 target spot, and energy specific recognition simultaneously lights CD22 positive B-cells, is of great significance for monitoring B cell abnormal division and related disease detection.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC and
Preparation method.
Background technique
CD22 is a kind of special antigen differentiation cluster, belongs to one kind point of sialic acid binding domain-immunoglobulin lectin family
Son, in mature bone-marrow-derived lymphocyte surface expression.It can prevent the excessive activation of immune system as regulatory molecule and itself exempt from
The development of epidemic disease disease.CD22 is that sugar combines transmembrane protein, special using immunoglobulin (Ig) structural domain of the end N-
Property bound sialic acid, the presence of Ig structural domain make CD22 become immunoglobulin superfamily member.CD22 is in B-cell receptor
(BCR) play inhibition in signal transduction.
CD22 is B cell specificity glycoprotein, is occurred in the cell in the ontogenetic later period B cell stage first.
Then, CD22 is transferred to the plasma membrane with B cell maturation, until plasma cell differentiation.Thick liquid cell does not express molecule.CD22 has 7
The outer Ig spline structure domain of a cytoplasm, belongs to Ig superfamily.It is used as carbon water on extensive cell surface and shla molecule in vivo
The receptor of compound determinant.With CD19 on the contrary, CD22 may serve as B cell work by enhancing the threshold value of BCR inducement signal
The antagonist of change.After BCR is participated in, the main phosphorylation in the ITIM in its cytoplasmic domains of CD22.Phosphorylation is mainly by Lyn
It mediates, expands ring downstream in the Lyn kinases that CD19 is relied on.If CD22 raises SHP-1 and SHIP phosphatase by Lyn phosphorylation,
Cause CD22 activation/SHP-1/SHIP to adjust access, lowers the signal transduction of CD19 phosphorylation and BCR mediation.Therefore, CD19 and
CD22 together defines the key signal of extension periphery Blymphocyte repertoire.The connection of CD22 and BCR and subsequent SHP-1 activation are logical
It crosses inhibition map kinase ERK2, JNK and p38 and participates in the dephosphorylation molecule of the earliest events for the activation that BCR is mediated to inhibit
B cell activation.These include BCR itself, by the target of tyrosine kinase (such as syk) and these kinases of Ig α β phosphorylation activation
(including adaptin BLNK and PLC).Due to the total combination of CD22 and BCR reduce B cell activation, the interaction of CD22 with
Its ligand may participate in lowering B cell activation.
CD22 is genetically related with autoimmune disease, shows that CD22 defect and subsequent autoimmunity development signal pass
Lead the possibility effect of approach.Have shown that the B1 cell mass of CD22 deficient mice can expand and increase serum IgM.These are small
The B cell of mouse is highly reactive by the stimulation progress to BCR, shows that CD22 is the important inhibition in the activation of BCR dependence B cell
Property receptor.Although these mouse do not develop autoimmune disease, myeloperoxidase and cuorin can be generated high affine
The autoantibody of power.CD22 has been used successfully as patients with non Hodgkin lymphoma and Sjogren syndrome and the life of SLE patient
The target of object therapy, by using the Humanized monoclonal antibodies for being directed to CD22.This monoclonal antibody is induced by exhausting
Apoptosis is still remained to be confirmed by the degree that CD22 inhibits B cell activation to play its effect.
In the prior art, in the cancer caused by all kinds of B cell malignancies, B cell is largely proliferated and disperses to be free on
In blood, it is not easy to monitor.
Summary of the invention
It is an object of the invention to overcome prior art defect, a kind of CD22 non-natural sialic acid fluorescence probe BPC- is provided
FITC。
Another object of the present invention is to provide the preparation sides of above-mentioned CD22 non-natural sialic acid fluorescence probe BPC-FITC
Method.
Technical scheme is as follows:
A kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC, structural formula are as follows:
The preparation method of above-mentioned CD22 non-natural sialic acid fluorescence probe BPC-FITC, reaction equation are as follows:
In a preferred embodiment of the invention, include the following steps:
(1) after mixing by BPC-COOH, EDCHCl, HOBT, triethylamine, N- tertbutyloxycarbonyl ethylenediamine and DMF
It is protected from light 45~50h of stirring, passes through CH after evaporation of solvent2Cl2:: MeOH=20: 1 column chromatographic purifying obtains the first compound,
For white solid；
(2) the first compound, trifluoroacetic acid are uniformly mixed with methylene chloride and are reacted under normal temperature and pressure, supervised by TLC
Survey to evaporation of solvent after fully reacting, then with K2CO3It is uniformly mixed with DMF；
(3) FITC is uniformly mixed with DMF and is added dropwise in step (1) resulting material, be protected from light stirring 10~14, steamed
Hair passes through CH after removing solvent2Cl2:: MeOH=5: 1 column chromatographic purifying obtains the CD22 non-natural sialic acid fluorescence probe
BPC-FITC is yellow solid.
In further preferably, in the step (1), BPC-COOH, EDCHCl, HOBT, triethylamine, N- tertiary butyloxycarbonyl
The ratio of base ethylenediamine and DMF be 8.3~8.7mmol: 10~11mmol: 10~11mmol: 25~26mmol: 8.3~
8.7mmol: 140~160mL.Still more preferably, in the step (1), BPC-COOH, EDCHCl, HOBT, three second
The ratio of amine, N- tertbutyloxycarbonyl ethylenediamine and DMF is 8.6mmol: 10.3mmol: 10.3mmol: 25.8mmol: 8.6mmol:
150mL。
In further preferably, in the step (2): the first compound, trifluoroacetic acid, methylene chloride, K2CO3With DMF's
Ratio is 5.3~5.5mmol: 45~55mL: 45~55mL: 20~22mmol: 90~110mL.Still more preferably, described
In step (2): the first compound, trifluoroacetic acid, methylene chloride, K2CO3Ratio with DMF is 5.35mmol: 50mL: 50mL:
21.7mmol∶100mL。
In further preferably, in the step (3): the molar ratio of FTIC and the first compound for 0.8~1.2: 0.8~
1.2.Still more preferably, in the step (3): the molar ratio of FTIC and the first compound is 1: 1.
The beneficial effects of the present invention are:
CD22 non-natural sialic acid fluorescence probe BPC-FITC of the invention is directed to B cell CD22 target spot, can specific recognition
And CD22 positive B-cells are lighted, it is of great significance for monitoring B cell abnormal division and related disease detection.
Detailed description of the invention
Fig. 1 is the cytotoxicity result figure of the BPC-FITC in the embodiment of the present invention 1.
Fig. 2 is the BPC-FITC labeled in vitro B cell photo in the embodiment of the present invention 1.
Fig. 3 is that the BPC-FITC in the embodiment of the present invention 1 marks one of the streaming figure after cell.
Fig. 4 is that the BPC-FITC in the embodiment of the present invention 1 marks two of the streaming figure after cell.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment combination attached drawing.
Embodiment 1
1, the synthesis of fluorescence probe BPC-FITC
N-boc-ethylenediamine-BPC (1): by BPC-COOH (1.7g, 8.6mmol), EDCHCl
(2.0g, 10.3mmol), HOBT (1.4g, 10.3mmol), triethylamine (3.6mL, 25.8mmol), N- tertbutyloxycarbonyl ethylenediamine
(1.38g, 8.6mmol) is uniformly mixed with DMF (150mL).It is protected from light stirring 48h.Pass through CH after evaporation of solvent2Cl2∶∶MeOH
=20: 1 column chromatographic purifying, obtains the first compound, is white solid (2.54g, 7.48mmol, 87%).
FITC-ethylenediamine-BPC (2): by the first compound (1.8g, 5.35mmol), trifluoroacetic acid
(50mL) is uniformly mixed with methylene chloride (50mL)；It is monitored by TLC to (≈ 1h) when fully reacting.Evaporation of solvent, will
Residue, K2CO3 (3.0g, 21.7mmol) are uniformly mixed with DMF (100mL)；By FITC (2.0g, 5.35mmol) and DMF
(10mL) is uniformly mixed and said mixture is added dropwise.Mixture is protected from light stirring 12h.Pass through after evaporation of solvent
CH2Cl2:: MeOH=5: 1 column chromatographic purifying obtains the CD22 non-natural sialic acid fluorescence probe BPC-FITC, solid for yellow
Body (2.42g, 3.85mmol, 72%).
Above-mentioned reaction route is as follows:
2, the cytotoxicity of BPC-FITC
B cell is isolated from human blood first.Test tube of hepari processing is carried out after taking out whole blood in human body, uses GE company
The Ficoll-Paque-Plus of product isolates PBMCs (peripheral blood mononuclear cells).Then MiltenyiBiotec reagent is used
Box isolates B cell by Solid phase.By the B cell isolated and purified in 37 DEG C of sterile constant-temperatures training containing 5% carbon dioxide
It supports and is cultivated in case, used medium is the RPMI containing 10% fetal calf serum.Cell is passed on 96 orifice plates, every hole is 10,000 thin
Born of the same parents remove original culture medium after cell is adherent, and the RPMI culture medium of the BPC-FITC containing various concentration is added, continues to cultivate
24h.Then cytotoxicity is detected with MTT method.As a result as shown in Figure 1, it can be seen that BPC-FITC is substantially nontoxic to B cell
Property, therefore fluorescent marker B cell is used for feasibility.
3, the labeled in vitro B cell of BPC-FITC
By B cell passage to being copolymerized on burnt special cell culture dish, cultivated in the RPMI culture medium containing 10%FBS to
50% density.Original culture medium is removed, the RPMI culture medium for containing 100 micromole BPC-FITC is added, cultivates 1h.By original training
It supports base to remove, 1 milliliter of PBS rinse is added three times, 10 micromolar Di1 are added, continue culture 10 minutes.Finally use fresh cultured
Base replacement is placed on the burnt lower observation of copolymerization, analyzes the cellular localization of BPC-FITC and Di1.As a result as shown in Fig. 2, BPC-
FITC can be interacted to CD22 sun for the CD22 receptor on CD22 positive cell by the high-affinity between receptor-ligand
Property cell carry out specific marker.It can clearly position cell film location, this is for positioning CD122 positive cell and to it
Function carries out visual research and is of great significance.
4, BPC-FITC promotes airflow classification B cell type after marking B cell in vitro
CD22 positive cell is extracted from hairy cell leukemia patient's peripheral blood, is added and is contained 100 micromole BPC-FITC
RPMI culture medium, be incubated for 1h.Original culture medium is removed later, 1 milliliter of PBS rinse is added three times, it is then thin using streaming
The detection of born of the same parents' instrument.As a result as shown in figure 3, region shown in 1 indicates hairy cell oncocyte, region shown in 2 indicates normal B cells.From
As can be seen that can effectively distinguish canceration B cell and normal B cells after BPC-FITC carries out fluorescent marker in figure.This is
Because in the more CD22 receptors of canceration B cell surface expression, to show stronger fluorescence.
In order to further verify BPC-FITC to the label of CD22 positive cell and the CD22 expression quantity of cell surface in just
Correlation has then isolated CD22 positive cell out of different types of leukaemia human body, after being marked using BPC-FITC
With flow cytometer quantitative detection result as shown in figure 4, what CLL indicated to extract from chronic lymphatic leukemia patient's peripheral blood
B cell, surface C D22 expression are lower.MZL indicates the B cell extracted from the lymphomatosis human peripheral of splenic marginal zone,
Its surface C D22 expression is moderate.HCL indicates the B cell extracted from hairy cell leukemia patient's peripheral blood, surface
CD22 expression is higher.From the graph it is found that BPC-FITC can effectively be marked CD22 positive cell and can basis
The expression difference of CD22 distinguishes the B cell of different illness types in patient body.This illness type for leukemia patient
Diagnosis be of great significance.
Associated antibodies are mainly clinically used for the screening of CD22 positive cell at present, price is more expensive and operation is multiple
Miscellaneous, what the BPC-FITC of the application can be specific carries out fluorescent marker to B cell, convenient for utilizing naked eyes identification, and through BPC-
FITC can distinguish different types of leukaemia after carrying out fluorescent marker, and another evidence can be provided for leukemia diagnosis.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e.,
Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Claims (9)
1. a kind of CD22 non-natural sialic acid fluorescence probe BPC-FITC, it is characterised in that: its structural formula is as follows:
2. the preparation method of CD22 non-natural sialic acid fluorescence probe BPC-FITC described in claim 1 a kind of, feature exist
In: its reaction equation is as follows:
3. preparation method as claimed in claim 2, characterized by the following steps:
(1) BPC-COOH, EDCHCl, HOBT, triethylamine, N- tertbutyloxycarbonyl ethylenediamine and DMF are protected from light after mixing
45~50h is stirred, passes through CH after evaporation of solvent2Cl2: MeOH=20: 1 column chromatographic purifying obtains the first compound, is white
Color solid；
(2) the first compound, trifluoroacetic acid are uniformly mixed with methylene chloride and are reacted under normal temperature and pressure, by TLC monitor to
Evaporation of solvent after fully reacting, then with K2CO3It is uniformly mixed with DMF；
(3) FITC is uniformly mixed with DMF and is added dropwise in step (2) resulting material, be protected from light 10~14h of stirring, evaporated
Pass through CH after removing solvent2Cl2: MeOH=5: 1 column chromatographic purifying obtains the CD22 non-natural sialic acid fluorescence probe BPC-
FITC is yellow solid.
4. preparation method as claimed in claim 3, it is characterised in that: in the step (1), BPC-COOH, EDCHCl,
HOBT, triethylamine, N- tertbutyloxycarbonyl ethylenediamine and DMF ratio be 8.3~8.7mmol: 10~11mmol: 10~11mmol
: 25~26mmol: 8.3~8.7mmol: 140~160mL.
5. preparation method as claimed in claim 3, it is characterised in that: in the step (1), BPC-COOH, EDCHCl,
HOBT, triethylamine, N- tertbutyloxycarbonyl ethylenediamine and DMF ratio be 8.6mmol: 10.3mmol: 10.3mmol: 25.8mmol
∶8.6mmol∶150mL。
6. preparation method as claimed in claim 3, it is characterised in that: in the step (2): the first compound, trifluoroacetic acid,
Methylene chloride, K2CO3With the ratio of DMF be 5.3~5.5mmol: 45~55mL: 45~55mL: 20~22mmol: 90~
110mL。
7. preparation method as claimed in claim 6, it is characterised in that: in the step (2): the first compound, trifluoroacetic acid,
Methylene chloride, K2CO3Ratio with DMF is 5.35mmol: 50mL: 50mL: 21.7mmol: 100mL.
8. preparation method as claimed in claim 3, it is characterised in that: the molar ratio of FTIC and the first compound is 0.8~1.2
: 0.8~1.2.
9. preparation method as claimed in claim 8, it is characterised in that: the molar ratio of FTIC and the first compound is 1: 1.
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Non-Patent Citations (3)
| Title |
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| CD22 Ligands on a Natural N‑Glycan Scaffold Efficiently Deliver Toxins to B‑Lymphoma Cells;Wenjie Peng et al.;《Journal of the American Chemical Society》;20170822;第12450-12458页 * |
| From a Library of MAG Antagonists to Nanomolar CD22 Ligands;Mesch, Stefanie et al.;《ChemMedChem》;20111011;第7卷;第134-143页 * |
| New Human CD22/Siglec-2 Ligands with a Triazole Glycoside;Horst Prescher et al.;《ChemBioChem》;20170519;第18卷;第1216-1225页 * |
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