CN109504652A - Promote the method and application of organism interaction - Google Patents
Promote the method and application of organism interaction Download PDFInfo
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- CN109504652A CN109504652A CN201811450846.XA CN201811450846A CN109504652A CN 109504652 A CN109504652 A CN 109504652A CN 201811450846 A CN201811450846 A CN 201811450846A CN 109504652 A CN109504652 A CN 109504652A
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Abstract
The present invention provides the methods and application of a kind of promotion organism interaction, it is related to technological field of biochemistry, organism including making needs interact is metabolized the basic metabolism substance for being modified with the click chemistry group for capableing of match reaction respectively, obtain the organism with click chemistry group and the organism with the click chemistry group for capableing of match reaction with the click chemistry group, by the match reaction between click chemistry group, promote the interaction of organism.This method is not on the basis of changing its basic activity characteristic, so that the tolerance of organism is more preferable；It is quickly furthered the distance between organism by the bio-orthogonal reaction between organism simultaneously, realizes the specific surfaces identification of two kinds of organisms, and then promote the interaction between organism.Method provided by the invention is more direct, easy to operate；And reaction condition is mild, small toxicity；This method also has good reproducibility simultaneously, is suitable for popularization and application.
Description
Technical field
The present invention relates to technological field of biochemistry, more particularly, to a kind of method that promotion organism interacts and answer
With.
Background technique
" cell recognition " is a kind of biological cell to the understanding and identification of another biological cell, is related to many great diseases
The occurrence and development of disease, such as identification of the pathogen cell to host cell, can only identify just to can be carried out and infect, cause a disease.Simultaneously
Cell recognition is also related to the treatment of many serious diseases, such as identification of the immunocyte in immunization therapy to tumour cell
Deng, thus enhancing cell recognition has important role and research significance to the treatment of many diseases.
Intercellular identification includes the selection by cell surface receptor or ligand and other cell surface ligands or receptor
Property interaction, so as to cause a series of biochemical reactions signal transmit.Either any identifying system, there is one
A common fundamental characteristics is exactly to have selectivity, or that have specificity.
But the natural identification of cell is easy the interference by complicated microenvironment or other each side's factors in life entity, example
Such as, tumour cell itself face body immune system pressure, gradually selectively lose cell membrane surface antigen presentation,
MHC-1 molecule, p microglobulin etc. not only can reduce antigen presentation in the immune initial stage, but also can weaken the spy in immunological effect stage
Specific cell toxic action.In addition, tumour cell also can be gradually to interference in progression due to the presence that fastidious mechanism is immunized
The cell factor of the immune effector cells such as element secretion becomes insensitive.These phenomenons explanation, while immune system fight tumour,
There is plastotype effect to tumour again, i.e., Immune Selection pressure is implemented to tumour cell, weak immunogene tumour cell is enable to escape
Ease and further growth.
Click chemistry base group modification monosaccharide or cholinomimetic at present, and pass through metabolic pathway of the substance in cell
Although realization is widely used the transformation of cell surface, with this method in the surface modification clickization of tumour in more documents
Group is learned, the cancer target of the chemical reagent such as nanometer diagnosis and treatment integration reagent, molecular probe is realized using the modification of pairing group
Effect.But not by the technology practical application in the identification of two kinds of cells, especially immune cell therapy.Seok Hyun
The combination of two kinds of cells may be implemented in the bridge joint key for the click group that Yun seminar proposes, but simultaneously due to bridge joint key length
It cannot be guaranteed that the distance of two kinds of cells is suitble to identify (as shown in FIG. 1A and 1B), while this method is comparatively laborious.Dimension, exploitation
One kind identifies between can be improved organism, and then promotes the method for organism interaction particularly important.
Summary of the invention
The first purpose of this invention is to provide a kind of method of promotion organism interaction, existing at least to alleviate
One of technical problem present in technology.
Second object of the present invention is that the method for providing above-mentioned promotion organism interaction is exempted from preparation enhancing
Application in the drug of epidemic disease treatment.
The present invention provides a kind of methods of promotion organism interaction, which comprises
Make that the organism to interact is needed to be metabolized the basis for being modified with the click chemistry group for capableing of match reaction respectively
Metabolite obtains the organism with click chemistry group and with the point for capableing of match reaction with the click chemistry group
The organism for hitting chemical group promotes the interaction of organism by the match reaction between click chemistry group.
Further, the organism includes one of cell, bacterium or virus or a variety of；
Preferably, the basic metabolism substance includes in monosaccharide, monosaccharide derivatives, choline, cholinomimetic or amino acid
It is one or more；
Preferably, the monosaccharide derivatives include mannosamine hydrochloride, glucosamine HCL or galactosamine hydrochloric acid
Salt；
The cholinomimetic includes dimethylethanolamine.
Further, the click chemistry group includes nitrine, alkynes, octyne, diphenyl cyclooctyne or tetrazine；
Preferably, the click chemistry group includes DBCO, DIBO, DIFO, BARAC or BCN.
Further, it by carrying out click chemistry base group modification on the amino or hydroxyl to the basic metabolism substance, obtains
To basic metabolism substance-click chemistry analog；
Wherein, the basic metabolism substance is monosaccharide or monosaccharide derivatives；
Preferably, by carrying out click chemistry base group modification on the amino or hydroxyl to the basic metabolism substance, to hydroxyl
After base is closed, basic metabolism substance-click chemistry analog is obtained；
Preferably, it is closed by hydroxyl of the acetic anhydride to the basic metabolism substance-click chemistry analog；
Preferably, the click chemistry group is BCN, and the basic metabolism substance-click chemistry analog has such as formula
(I) structural formula shown in:
Wherein, in formula (I), R1 is monosaccharide groups or monosaccharide derivatives base；
It is highly preferred that the general formula structure of the basic metabolism substance-click chemistry analog is as follows:
It is further preferred that the structure of the basic metabolism substance-click chemistry analog is in following (ii)-(iv)
It is at least one:
Wherein, as in above formula (i)-(iv), R2-R5、R6-R9、R10-R13It is independently hydrogen or acetyl group.
Further, the Bromo-intermediates of click chemistry group are provided, by the Bromo-intermediates of the click chemistry group
Basic metabolism substance-click chemistry analog is generated with basic metabolism substance reaction；
Wherein, the basic metabolism substance is choline or cholinomimetic.
Further, the basic metabolism substance for being modified with the click chemistry group for capableing of match reaction is trained altogether with organism
It supports, after the metabolism of the organism, obtains the organism with click chemistry group and have and the click chemistry
Group is capable of the organism of the click chemistry group of match reaction.
Further, the organism for needing to interact is cell；
Preferably, the method for promoting organism interaction includes the following steps:
(a) the first cell is incubated in cell culture fluid altogether with the basic metabolism substance for being modified with click chemistry group,
Obtain first cell with click chemistry group；
(b) by the second cell and be modified with can be with the base of the click chemistry group of the click chemistry group match reaction
Plinth metabolite is incubated for altogether in cell culture fluid, is obtained with the click for capableing of match reaction with the click chemistry group
Learn the second cell of group；
(c) by the first cell that step (a) obtains and the second mixing with cells that step (b) obtains, pass through click chemistry base
Match reaction between group promotes the interaction of the first cell and the second cell；
Preferably, the basic metabolism substance for being modified with click chemistry group is the monosaccharide analog of BCN modification, described
It is modified with the list that can be modified with the basic metabolism substance of the click chemistry group of the click chemistry group match reaction for nitrogen
Sugar analogue.
Further, in total incubation, the concentration of the basic metabolism substance for being modified with click chemistry group is 5-
50umol/L, preferably 10-40umol/L；Described be modified with can be with the click chemistry of the click chemistry group match reaction
The concentration of the basic metabolism substance of group is 5-50umol/L, preferably 10-40umol/L；
Preferably, the time being incubated for altogether is 48-96 hours, preferably 48-84 hours, more preferably 48-72 hours；
Preferably, the first cell and the cell quantity of the second mixing with cells ratio are 1:0.2-5, preferably 1:0.5-2.
Further, step (a) further includes removing impurity after total incubation, obtains having the first of click chemistry group thin
The step of born of the same parents；
Preferably, step (b) further includes removing impurity after total incubation, obtain have can with the click chemistry group
The step of second cell of the click chemistry group of match reaction；
Preferably, step (a) and step (b) remove impurity independently by the mode of cleaning cell and/or centrifugation.
In addition, the present invention also provides above-mentioned to promote the method for organism interaction in preparation enhancing immunization therapy
Application in drug.
The method provided by the invention for promoting organism interaction, including making to need the organism that interacts generation respectively
It thanks to the basic metabolism substance for being modified with the click chemistry group for capableing of match reaction, obtains the organism with click chemistry group
With the organism with the click chemistry group for capableing of with the click chemistry group match reaction, by between click chemistry group
Match reaction, promote the interaction of organism.This method is with the different click chemistry base group modifications for capableing of match reaction
Basic metabolism substance realizes that click chemistry group passes through expression of the metabolism in different organism surfaces of organism itself, not
On the basis of changing its basic activity characteristic, so that the tolerance of organism is more preferable；Recycle the quick of click chemistry group
Match reaction is quickly furthered the distance between organism by the bio-orthogonal reaction between organism, realizes two kinds of organisms
Specific surfaces identification, and then promote organism between interaction.Also, due to being typically free of cycloalkyne in biosystem
Base and azido, therefore, the biological precursor reactant of entire the reaction system only organism containing cycloalkynyl radical and azido, to protect
Specificity is demonstrate,proved.Compared with the connection by bridge joint key reaction, method provided by the invention is more direct, easy, saves centre
It is the step of reaction, easy to operate；And reaction condition is mild, and toxicity is smaller；This method also has good reproducibility simultaneously, fits
In popularization and application.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Figure 1A is in the prior art by the illustraton of model of bridge joint two kinds of cells of key connection；
Figure 1B is the illustraton of model for bridging two kinds of cells of key connection by secondary modification in the prior art；
Fig. 2 is cell provided by the invention with click chemistry group and with can match with the click chemistry group
The illustraton of model that click chemistry group progress match reaction is passed through to the cell of the click chemistry group of reaction；
Fig. 3 is 1 reaction process schematic diagram of the embodiment of the present invention；
Fig. 4 A is the laser scanning figure for the T cell that the surface metabolism that the embodiment of the present invention 1 provides has azido group；
Fig. 4 B is the laser scanning figure for the Raji cell that the surface metabolism that the embodiment of the present invention 1 provides has BCN group；
Fig. 4 C is that the surface metabolism that the embodiment of the present invention 1 provides has the T cell of azido group and surface metabolism to have BCN group
Raji cell be incubated for altogether after laser scanning figure；
Fig. 4 D is that the surface metabolism that the embodiment of the present invention 1 provides has the T cell of azido group and surface metabolism to have BCN group
Raji cell be incubated for altogether after micrograph results figure；
Fig. 5 A is the laser scanning figure for T cell of the surface without azido group that the embodiment of the present invention 1 provides；
Fig. 5 B is the laser scanning figure for Raji cell of the surface without BCN group that the embodiment of the present invention 1 provides；
Fig. 5 C is that T cell and surface of the surface without azido group that the embodiment of the present invention 1 provides are free of BCN group
Raji cell be incubated for altogether after laser scanning figure；
Fig. 5 D is that T cell and surface of the surface without azido group that the embodiment of the present invention 1 provides are free of BCN group
Raji cell be incubated for altogether after micrograph results figure.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with embodiment, it is clear that described reality
Applying example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
The present invention provides a kind of methods of promotion organism interaction, which comprises
Make that the organism to interact is needed to be metabolized the basis for being modified with the click chemistry group for capableing of match reaction respectively
Metabolite obtains the organism with click chemistry group and with the point for capableing of match reaction with the click chemistry group
The organism for hitting chemical group promotes the interaction of organism by the match reaction between click chemistry group.
Wherein, " click chemistry " also translations click chemistry, quick-acting close combined chemical, dynamic combinatorial chemistry, purport
It is the splicing by junior unit, completes the chemical synthesis of varied molecule fast and reliablely.In certain embodiments, point
It hits chemistry and is presented as cycloaddition reaction general between azide and alkynes.
" click chemistry group " is used to indicate that click chemistry can be participated in together with the second reactive functionality appropriate sometimes
First reactive functionality of reaction, wherein the second reactive functionality is also click chemistry group.First and second click chemistry bases
Group, or the entity (such as molecule) comprising such group, can be with complementary pairing.In certain embodiments, clickization
Group is can be with the molecule with azide of complementary pairing and the alkynes or alkynes of biomolecule progress cycloaddition reaction
Hydrocarbon derivative；In another embodiment, click chemistry group is can to divide with complementary pairing with alkynes molecule and biology
Son carries out the azide or azide derivatives of cycloaddition reaction.
" organism " all contains protein and nucleic acid in basic component, to need to absorb nourishment from the external world, and
Microcosmic life entity with metabolism.
" basic metabolism substance " is that can be digested, be absorbed in vivo, operated, decomposed etc. and physiology relatedization
The substance of process, without limitation to the type of basic metabolism substance, the substance that all organisms can be metabolized is the present invention
The basic metabolism substance of definition.
" interaction of organism " for example can be, but be not limited between organism recognition reaction, targeting or kill
Wound effect.
The method provided by the invention for promoting organism interaction is with the different click chemistry bases for capableing of match reaction
Group's modification basic metabolism substance realizes that click chemistry group passes through table of the metabolism in different organism surfaces of organism itself
It reaches, on the basis of not changing its basic activity characteristic, so that the tolerance of organism is more preferable；Recycle click chemistry group
Quick match reaction, quickly furthered the distance between organism by the bio-orthogonal reaction between organism, realize two kinds
The specific surfaces of organism identify, and then promote the interaction between organism.Also, due to being typically free of in biosystem
There are cycloalkynyl radical and azido, therefore, the biological precursor reactant of entire the reaction system only organism containing cycloalkynyl radical and azido,
To ensure that specificity.Compared with the connection by bridge joint key reaction, method provided by the invention is more direct, easy, saves
It is the step of intermediate reaction, easy to operate；And reaction condition is mild, and toxicity is smaller；This method also has good heavy simultaneously
Existing property, is suitable for popularization and application.
In some preferred embodiments, the organism includes one of cell, bacterium or virus or a variety of.
Preferably, the basic metabolism substance includes in monosaccharide, monosaccharide derivatives, choline, cholinomimetic or amino acid
It is one or more.When basic metabolism substance is monosaccharide, monosaccharide derivatives, choline, cholinomimetic or amino acid, it is easier to
Metabolism is carried out in organism, to click chemical group in organism surface exposure.
Wherein, monosaccharide refers to the sugar containing 3-6 carbon atom in molecular structure, such as the glyceraldehyde of triose；Tetrose
Erythrose, Su Li sugar；Arabinose, ribose, xylose, the lyxose of pentose；Glucose, mannose, the fructose, half of hexose
Lactose；Choline (α-hydroxyl-IV, IV-front three ethylethanolamine) is positively charged tetravalence base, is the constituent of all biomembranes
With the precursor of the acetylcholine in cholinergic neuron；Amino acid is the change after the hydrogen atom on carboxylic acid carbon atom is replaced by amino
Object is closed, contains amino and carboxyl Liang Zhong functional group in amino acid molecular, they are the basic units for constituting protein.
Preferably, the monosaccharide derivatives include mannosamine hydrochloride, glucosamine HCL or galactosamine hydrochloric acid
Salt；
The cholinomimetic includes dimethylethanolamine.
In some preferred embodiments, the click chemistry group includes nitrine, alkynes, octyne, diphenyl cyclooctyne
Or tetrazine.
Preferably, the click chemistry group includes DBCO, DIBO, DIFO (bifluoride cyclooctyne), BARAC
(biarylazacyclooctynone) or BCN ((1R, 8S, 9r)-bicyclic [6.1.0] nonyl- 4- alkynyl -9- base methanol).
In some preferred embodiments, by carrying out click on the amino or hydroxyl to the basic metabolism substance
Base group modification is learned, obtains basic metabolism substance-click chemistry analog, that is, be modified with the basic metabolism of click chemistry group
Substance；
Wherein, the basic metabolism substance is monosaccharide or monosaccharide derivatives.
Preferably, by carrying out click chemistry base group modification on the amino or hydroxyl to the basic metabolism substance, to hydroxyl
After base is closed, basic metabolism substance-click chemistry analog is obtained.
Basic metabolism substance-click can be made by closing to basic metabolism substance-click chemistry analog hydroxyl
It learns analog and realizes better endocytosis effect, improve the recognition efficiency between organism, and then promote organism interaction.
Preferably, it is closed by hydroxyl of the acetic anhydride to the basic metabolism substance-click chemistry analog.
In a specific embodiment, the click chemistry group is BCN, the basic metabolism substance-click
Learning analog has the structural formula as shown in formula (I):
Wherein, in formula (I), R1 is monosaccharide groups or monosaccharide derivatives base；
It is highly preferred that the general formula structure of the basic metabolism substance-click chemistry analog is as follows:
It is further preferred that the structure of the basic metabolism substance-click chemistry analog is in following (ii)-(iv)
It is at least one:
Wherein, as in above formula (i) ,-OAc a substituent group or-(C1-C4) are at least contained on monosaccharide ring (oxygen-containing hexatomic ring)
Alkylidene-OAc substituent group (is directly connected with carbon atom on ring), and each carbon atom is at most connected with an above-mentioned substituent group
It connects；Remaining carbon atom (in addition to being connected carbon atom with amino) is connected with hydroxyl.
In formula (ii)-(iv), R2-R5、R6-R9、R10-R13It is independently hydrogen or acetyl group.
And more preferably due to acetylation monosaccharide analog cell compatibility, thus, as above in logical formula (II), R2-R5In at least
One R base is acetyl group；It is furthermore preferred that R2-R5It is acetyl group；
In logical formula (III), R6-R9In at least one R base be acetyl group；It is furthermore preferred that R6-R9It is acetyl group；
In logical formula (IV), R10-R13In at least one R base be acetyl group；It is furthermore preferred that R10-R13It is acetyl group.
In a specific embodiment, monosaccharide is mannose, is that basic metabolite synthesizes four acetyl with mannose
The synthetic route of the BCN monosaccharide analog (Ac4ManN-BCN) of change is as follows:
In some preferred embodiments, the Bromo-intermediates of click chemistry group are provided, by the click chemistry base
The Bromo-intermediates and basic metabolism substance reaction of group generate basic metabolism substance-click chemistry analog, that is, are modified with
The basic metabolism substance of click chemistry group；
Wherein, the basic metabolism substance is choline or cholinomimetic.
In a specific embodiment, it is carried out by the straight chain amine for replacing the click chemistry group and halogen anti-
It answers, obtains the Bromo-intermediates of click chemistry group.The straight chain amine that typical halogen replaces can be 2- bromine ethamine, 2- bromine propylamine
Or 2- bromine butylamine.
In some preferred embodiments, the basic metabolism object for the click chemistry group for capableing of match reaction will be modified with
Matter and organism co-culture, and after the metabolism of the organism, obtain the organism with click chemistry group and have
It is capable of the organism of the click chemistry group of match reaction with the click chemistry group.
It is co-cultured by the way that the basic metabolism substance for capableing of the click chemistry group of match reaction and organism will be modified with, energy
Enough realize will click on chemical group metabolism in the surface of organism, be capable of the click of match reaction in different organism surface exposures
Chemical group, to realize the interaction of two kinds of life entities by the bio-orthogonal reaction clicked between group.
In some preferred embodiments, the organism for needing to interact is cell.Its model such as Fig. 2 institute
Show.
Preferably, the method for promoting organism interaction includes the following steps:
(a) the first cell is incubated in cell culture fluid altogether with the basic metabolism substance for being modified with click chemistry group,
Obtain first cell with click chemistry group；
(b) by the second cell and be modified with can be with the base of the click chemistry group of the click chemistry group match reaction
Plinth metabolite is incubated for altogether in cell culture fluid, is obtained with the click for capableing of match reaction with the click chemistry group
Learn the second cell of group；
(c) by the first cell that step (a) obtains and the second mixing with cells that step (b) obtains, pass through click chemistry base
Match reaction between group promotes the interaction of the first cell and the second cell.
Typical cell culture fluid for example can be, but be not limited to DMEM culture solution or 1640 culture medium.
In actual operation, first cell and the second cell can be through cultures after a period of time again and with click
The basic metabolism substance of chemical group is incubated for altogether.For example, using the DMEM culture medium for containing 10% fetal calf serum, in 37 DEG C, 5%
CO2, 95% humidity condition of culture under cultivate, replace culture solution every other day, passage in every 4-6 days is primary.Before being incubated for altogether, first with buffering
Liquid cleans cell, and replaces fresh culture.
Preferably, the basic metabolism substance for being modified with click chemistry group is the monosaccharide analog of BCN modification, described
It is modified with the list that can be modified with the basic metabolism substance of the click chemistry group of the click chemistry group match reaction for nitrogen
Sugar analogue.
In some preferred embodiments, in total incubation, the basic metabolism object for being modified with click chemistry group
The concentration of matter is 5-50umol/L, such as can be, but be not limited to 5umol/L, 10umol/L, 20umol/L, 30umol/L,
40umol/L or 50umol/L, preferably 10-40umol/L；When the concentration of the basic metabolism substance with click chemistry group exists
When in above range, organism metabolic efficiency is higher, can be improved the recognition efficiency between organism, and then promote organism mutual
Effect.
Described be modified with can be with the basic metabolism substance of the click chemistry group of the click chemistry group match reaction
Concentration be 5-50umol/L, such as can be, but be not limited to 5umol/L, 10umol/L, 20umol/L, 30umol/L,
40umol/L or 50umol/L, preferably 10-40umol/L；When with the click with the click chemistry group match reaction
When learning the concentration of the basic metabolism substance of group within the above range, organism metabolic efficiency is higher, can be improved between organism
Recognition efficiency, and then promote organism interaction.
Preferably, the time being incubated for altogether is 48-96 hours, such as can be, but be not limited to 48 hours, 60 hours,
72 hours, 84 hours or 96 hours, preferably 48-84 hours, more preferably 48-72 hours；When total incubation time is in above-mentioned model
When enclosing interior, organism metabolic efficiency is higher, can be improved the recognition efficiency between organism, and then promotes organism interaction.
Preferably, the first cell and the cell quantity of the second mixing with cells ratio are 1:0.2-5, such as can be, but unlimited
In 1:0.5,1:1,1:1.5,1:2,1:3,1:4 or 1:5, preferably 1:0.5-2.When the mixing ratio of the first cell and the second cell
Example within the above range when, organism metabolic efficiency is higher, can be improved the recognition efficiency between organism, and then promote organism
Interaction.
In some preferred embodiments, step (a) further includes removing impurity after total incubation, obtains having clickization
The step of learning the first cell of group；It is preferred that washed with the buffer that pH is 7.2-7.6, can remove extra has clickization
The basic metabolism substance of group is learned, reaction efficiency is improved；And/or the organism after total incubation is collected by centrifugation.Typical buffer
It can be PBS buffer solution or HEPES buffer solution.
Preferably, step (b) further includes removing impurity after total incubation, obtain have can with the click chemistry group
The step of second cell of the click chemistry group of match reaction；It is preferred that being washed with the buffer that pH is 7.2-7.6, can remove
The extra basic metabolism substance with click chemistry group improves reaction efficiency；And/or the biology after total incubation is collected by centrifugation
Body.Typical buffer can be PBS buffer solution or HEPES buffer solution.
In addition, the present invention also provides above-mentioned to promote the method for organism interaction in preparation enhancing immunization therapy
Application in drug.
By promoting organism interaction, can be realized the targets identification between promotion immunocyte and target cell and exempting from
Epidemic disease identification, such as targets identification and Immune discrimination between T cell and cancer cell, the targets identification between macrophage and bacterium
Targets identification and Immune discrimination between Immune discrimination, natural killer cells and virus, and then promote organism interaction,
Enhance immunization therapy.
In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.
Embodiment 1
(a) metabolism of the BCN sweet dew sugar analogue (Ac4ManN-BCN) in B lymphoma cell (Raji cell)
(1) activation of BCN
The BCN of 5mmol is dissolved in 100mL anhydrous methylene chloride, and the pyridine of 10mmol is added, and is added after several minutes of stirring
Enter the p-nitrophenyl chloro-formate (pNC) to 5mmol, reacts 30min under room temperature；
After saturated ammonium chloride solution is added, extract, organic phase is dry with anhydrous sodium sulfate, is evaporated, silica gel column chromatography
It isolates and purifies, obtains white solid BCN-pNC, yield 70%.
(2) synthesis of BCN-Man:
By the mannosamine hydrochloride salt of 4.4mmol in the n,N-Dimethylformamide of 10mL, it is added 10mmol's
The pyridine of BCN-pNC and 10mmol reacts half an hour under room temperature；
After reaction, it is evaporated, silica gel column chromatography separating purification, obtains white solid BCN-Man, yield 72%；
The synthesis of the BCN- sweet dew sugar analogue (Ac4ManN-BCN) of (3) four acetylations
5mmol BCN-Man is dissolved in the n,N-Dimethylformamide (DMF) of 20mL, the acetic anhydride of 10mmol is added
And 10mmol pyridine, after being sufficiently stirred, normal-temperature reaction is overnight；
Reaction solution concentration, silica gel column chromatography separating purification arrive white solid Ac4ManN-BCN, yield 65%.
Its reaction process is referring to Fig. 3.
(4) by 2 × 105A Raji cell culture in 24 orifice plates, with contain 10% fetal calf serum DMEM culture medium, 37
DEG C, 5%CO2, 95% humidity condition of culture under be incubated overnight, by certain density Ac4ManN-BCN dimethyl sulphoxide solution
It is added in the culture solution of Raji cell, makes its final concentration in 25umol/L, mixed liquor is incubated for 72 hours in the incubator, makes Raji
Cell surface is sufficiently metabolized BCN group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes cell twice with PBS, from
The heart collects cell, obtains the Raji cell (BCN-Raji cell) of surface modification BCN group.
(b) metabolism of the sweet dew sugar analogue (AC4ManAz) of nitrine modification in immunocyte (T cell)
By 2 × 105A T cell is cultivated in 24 orifice plates, with the DMEM culture medium for containing 10% fetal calf serum, in 37 DEG C, 5%
CO2, 95% humidity condition of culture under be incubated overnight, by certain density AC4ManAz dimethyl sulphoxide solution be added T cell
Culture solution in, make its final concentration in 25umol/L, mixed liquor is incubated for 72 hours in the incubator.Make T cell surface abundant generation
Thank to nitrine (N3) group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes cell twice with PBS, is centrifuged, is collected thin
Born of the same parents obtain the T cell (N3-T cell) of surface modification azido group.
(c) interaction of two kinds of cells
BCN-Raji cell is mixed according to a certain percentage (1:1) with N3-T cell, is analyzed and is modified by laser co-focusing
Since bio-orthogonal reaction is situated between after the total incubation of Raji cell (BCN-Raji cell) after T cell (N3-T cell) and modification afterwards
The interaction between two kinds of cells led, as shown in Fig. 4 A-4D and Fig. 5 A-5D.With no Raji for carrying BCN and N3 group
Cell and T cell can quickly be gathered in BCN-Raji cell peripheral as control, discovery N3-T cell, relative to control group,
It combines speed to increase 8 times or more, improves 3 times or more to BCN-Raji cell killing rate.
Embodiment 2
(a) metabolism of the DBCO glucalogue in liver cancer cells (HepG2 cell)
(1) synthesis of DBCO- glucose (DBCO-Glc):
By 4.4mmol mannosamine hydrochloride salt in 10mL n,N-Dimethylformamide (DMF), 20mmol is added
The EDCHCl of the NHS and 20mmol of DBCO-COOH and 20mmol, normal-temperature reaction are overnight；After reaction, it is evaporated, silicagel column
Chromatography purifying, obtains white solid DBCO-Glc, yield 78%.
The synthesis of the DBCO- glucose (Ac4DBCO-Glc) of (2) four acetylations
5mmol DBCO-Glc is dissolved in 20mL n,N-Dimethylformamide (DMF), be added 15mmol acetic anhydride and
15mmol pyridine, after being sufficiently stirred, normal-temperature reaction is overnight.Reaction solution concentration, silica gel column chromatography separating purification arrive white solid
Ac4DBCO-Glc, yield 70%.
(3) by 1 × 105A HepG2 cell culture in 24 orifice plates, with contain 10% fetal calf serum DMEM culture medium,
In 37 DEG C, 5%CO2, 95% humidity condition of culture under be incubated overnight, by certain density DBCO glucalogue dimethyl
Formamide solution is added in the culture solution of HepG2 cell, makes its final concentration in 5umol/L, mixed liquor is incubated for 96 in the incubator
Hour, so that HepG2 cell surface is sufficiently metabolized DBCO group.After being incubated for altogether, mixed liquor is collected, is centrifuged, is washed with PBS
Cell is collected in cell twice, centrifugation, obtains the HepG2 cell (DBCO-HepG2cell) of surface modification DBCO group.
(b) metabolism of the glucalogue of nitrine modification in immunocyte (T cell)
By 4 × 105A T cell is cultivated in 24 orifice plates, with the DMEM culture medium for containing 10% fetal calf serum, 37 DEG C,
5%CO2, 95% humidity condition of culture under be incubated overnight, the glucalogue dimethyl that certain density nitrine is modified
Formamide solution is added in the culture solution of T cell, makes its final concentration in 50umol/L, it is small that mixed liquor is incubated for 48 in the incubator
When.T cell surface is set sufficiently to be metabolized nitrine (N3) group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes two with PBS
Cell is collected in secondary cell, centrifugation, obtains the T cell (N3-T cell) of surface modification azido group.
(c) interaction of two kinds of cells
DBCO-HepG2 cell is mixed according to a certain percentage (1:0.2) with N3-T cell, is swashed by what embodiment 1 provided
Light is total to the interaction between focus analysis experiment two kinds of cells of detection, with no HepG2 cell for carrying DBCO and N3 group
With T cell as compareing, discovery N3-T cell can quickly be gathered in DBCO-HepG2 cell peripheral, relative to control group,
7 times or more are increased in conjunction with speed, 2 times or more is improved to DBCO-HepG2 cell killing rate.
Embodiment 3
(a) metabolism of the BCN- cholinomimetic in cervical cancer cell (HeLa cell)
By 4 × 105A HeLa cell culture in 24 orifice plates, with contain 10% fetal calf serum 1640 culture mediums, 37
DEG C, 5%CO2, 95% humidity condition of culture under be incubated overnight, certain density BCN- cholinomimetic dimethyl sulfoxide is molten
Liquid is added in the culture solution of HeLa cell, makes its final concentration in 50umol/L, mixed liquor is incubated for 48 hours in the incubator, is made
HeLa cell surface is sufficiently metabolized BCN group.After being incubated for altogether, mixed liquor is collected, is centrifuged, is washed with HEPES thin twice
Cell is collected in born of the same parents, centrifugation, obtains the HeLa cell (BCN-HeLa cell) of surface modification BCN group.
(b) metabolism of the cholinomimetic of nitrine modification in immunocyte (T cell)
By 1 × 105A T cell is cultivated in 24 orifice plates, with 1640 culture mediums for containing 10% fetal calf serum, 37 DEG C,
5%CO2, 95% humidity condition of culture under be incubated overnight, the cholinomimetic dimethyl that certain density nitrine is modified is sub-
Sulfolane solution is added in the culture solution of T cell, makes its final concentration in 5umol/L, mixed liquor is incubated for 48 hours in the incubator.Make T
Cell surface is sufficiently metabolized nitrine (N3) group.After being incubated for altogether, mixed liquor is collected, is centrifuged, is washed with HEPES thin twice
Cell is collected in born of the same parents, centrifugation, obtains the T cell (N3-T cell) of surface modification azido group.
(c) interaction of two kinds of cells
BCN-HeLa cell is mixed according to a certain percentage (1:5) with N3-T cell, the laser provided by embodiment 1 is total
Interaction between focus analysis experiment two kinds of cells of detection, with no HeLa cell and T cell for carrying BCN and N3 group
As control, it is found that N3-T cell can quickly be gathered in BCN-HeLa cell peripheral, relative to control group, increases in conjunction with speed
8 times or more are added, 2 times or more are improved to BCN-HeLa cell killing rate.
Embodiment 4
(a) metabolism (1) Ac of BCN sweet dew sugar analogue (Ac4ManN-BCN) in (Raji cell)4The conjunction of ManN-BCN
At step referring to embodiment 1.
(2) by 2 × 105A Raji cell culture in 24 orifice plates, with contain 10% fetal calf serum DMEM culture medium, 37
DEG C, 5%CO2, 95% humidity condition of culture under be incubated overnight, by certain density Ac4ManN-BCN dimethyl sulphoxide solution
It is added in the culture solution of Raji cell, makes its final concentration in 25umol/L, mixed liquor is incubated for 72 hours in the incubator, makes Raji
Cell surface is sufficiently metabolized BCN group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes cell twice with PBS, from
The heart collects cell, obtains the Raji cell (BCN-Raji cell) of surface modification BCN group.
(b) the sweet dew sugar analogue (AC of nitrine modification4ManAz) the metabolism in anaerobic bacteria YB1
By 2 × 105A YB1 is cultivated in strain test tube, and certain density AC4ManAz dimethyl sulphoxide solution is added
In the culture solution of YB1, make its final concentration in 50umol/L, mixed liquor is incubated for 72 hours in the incubator.Make the surface YB1 abundant generation
Thank to nitrine (N3) group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes cell twice with PBS, is centrifuged, is collected thin
Born of the same parents obtain the T cell (N of surface modification azido group3-YB1)。
(c) interaction of bacterium and cell
BCN-Raji cell is mixed according to a certain percentage (1:1) with N3-YB1 cell, is repaired by laser co-focusing analysis
Anaerobic bacteria (N after decorations3- YB1) and modification after Raji cell (BCN-Raji cell) altogether be incubated for after due to bio-orthogonal it is anti-
The interaction of the bacterium and cell that should mediate.Relative to the bacterium and tumour cell not handled, N3-YB1 bacterium can be quickly
Be gathered in BCN-Raji near tumor cells, enhance its targeting to tumour.
Embodiment 5
(a) metabolism of the BCN sweet dew sugar analogue (Ac4ManN-BCN) in (Raji cell)
(1) synthesis step of Ac4ManN-BCN is referring to embodiment 1.
(2) by 2 × 105A A549 cell culture in 24 orifice plates, with contain 10% fetal calf serum DMEM culture medium, 37
DEG C, 5%CO2, 95% humidity condition of culture under be incubated overnight, by certain density Ac4ManN-BCN dimethyl sulphoxide solution
It is added in the culture solution of A549 cell, makes its final concentration in 25umol/L, mixed liquor is incubated for 72 hours in the incubator, makes A549
Cell surface is sufficiently metabolized BCN group.After being incubated for altogether, mixed liquor is collected, is centrifuged, washes cell twice with PBS, from
The heart collects cell, obtains the A549 cell (BCN-A549cell) of surface modification BCN group.
(b) modification of the sweet dew sugar analogue (AC4ManAz) of nitrine modification to the surface slow virus latin-virus
By 5 × 105A HEK293T cell culture in 6 orifice plates, with contain 10% fetal calf serum DMEM culture medium, 37
DEG C, be incubated overnight under the condition of culture of 5%CO2,95% humidity, by certain density AC4ManAz dimethyl sulphoxide solution is added
In the culture solution of HEK293T cell, make its final concentration in 25umol/L, mixed liquor is incubated for 24 hours in the incubator, is made
HEK293T cell surface is sufficiently metabolized nitrine (N3) group.After being incubated for altogether, mixed liquor is collected, is centrifuged, is washed with PBS
Cell is collected in cell twice, centrifugation, obtains the HEK293T cell (N3-HEK293T cell) of surface modification N3 group.Then
Transfection calcium phosphate transfection kit (Promega), which is wrapped, with virus transfects slow virus latin-virus.After 6-8 hours, trained in mixing
The AC4ManAz for supporting addition same concentrations in base continues to cultivate 48h.Include N3The supernatant of-latin-virus passes through 25000
Rev/min, the centrifugation for continuing 2h obtains.
(c) interaction of virus and cell
By BCN-A549 cell and N3- latin-virus engineering virus mixes according to a certain percentage (1:1), passes through laser
Engineering virus (N after focus analysis modification altogether3- latin-virus) and modification after Raji cell (BCN-A549 cell) altogether
The interaction of the virus and cell that are mediated after incubation due to bio-orthogonal reaction.Relative to the virus and cell not handled,
N3- latin-virus bacterium can quickly be gathered in BCN-A549 near tumor cells, enhance its infection ability to cell.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. a kind of method for promoting organism interaction, which is characterized in that the described method includes:
Make that the organism to interact is needed to be metabolized the basic metabolism for being modified with the click chemistry group for capableing of match reaction respectively
Substance obtains the organism with click chemistry group and with the click for capableing of match reaction with the click chemistry group
The organism for learning group promotes the interaction of organism by the match reaction between click chemistry group.
2. the method according to claim 1 for promoting organism interaction, which is characterized in that the organism includes thin
One of born of the same parents, bacterium or virus are a variety of；
Preferably, the basic metabolism substance includes one in monosaccharide, monosaccharide derivatives, choline, cholinomimetic or amino acid
Kind is a variety of；
Preferably, the monosaccharide derivatives include mannosamine hydrochloride, glucosamine HCL or galactosamine hydrochloride；
The cholinomimetic includes dimethylethanolamine.
3. the method according to claim 1 for promoting organism interaction, which is characterized in that the click chemistry group
Including nitrine, alkynes, octyne, diphenyl cyclooctyne or tetrazine；
Preferably, the click chemistry group includes DBCO, DIBO, DIFO, BARAC or BCN.
4. the method according to claim 1 for promoting organism interaction, which is characterized in that by the basic generation
It thanks to progress click chemistry base group modification on the amino or hydroxyl of substance, obtains basic metabolism substance-click chemistry analog；
Wherein, the basic metabolism substance is monosaccharide or monosaccharide derivatives；
Preferably, by the amino or hydroxyl to the basic metabolism substance carry out click chemistry base group modification, to hydroxyl into
After row closing, basic metabolism substance-click chemistry analog is obtained；
Preferably, it is closed by hydroxyl of the acetic anhydride to the basic metabolism substance-click chemistry analog；
Preferably, the click chemistry group is BCN, and the basic metabolism substance-click chemistry analog has such as formula (I) institute
The structural formula shown:
Wherein, in formula (I), R1 is monosaccharide groups or monosaccharide derivatives base；
It is highly preferred that the general formula structure of the basic metabolism substance-click chemistry analog is as follows:
It is further preferred that the structure of the basic metabolism substance-click chemistry analog be in following (ii)-(iv) at least
It is a kind of:
Wherein, as in above formula (ii)-(iv), R2-R5、R6-R9、R10-R13It is independently hydrogen or acetyl group.
5. the method according to claim 1 for promoting organism interaction, which is characterized in that provide click chemistry group
Bromo-intermediates, the Bromo-intermediates of the click chemistry group and basic metabolism substance reaction are generated into basic metabolism object
Matter-click chemistry analog；
Wherein, the basic metabolism substance is choline or cholinomimetic.
6. the method according to claim 1 for promoting organism interaction, which is characterized in that will be modified with can match
The basic metabolism substance of the click chemistry group of reaction and organism co-culture, and after the metabolism of the organism, obtain
Organism with click chemistry group and with the click chemistry group for capableing of match reaction with the click chemistry group
Organism.
7. the method according to claim 1-6 for promoting organism interaction, which is characterized in that the needs
The organism of interaction is cell；
Preferably, the method for promoting organism interaction includes the following steps:
(a) the first cell is incubated in cell culture fluid altogether with the basic metabolism substance for being modified with click chemistry group, is obtained
The first cell with click chemistry group；
(b) by the second cell and be modified with can be with the basic generation of the click chemistry group of the click chemistry group match reaction
It thanks to substance to be incubated for altogether in cell culture fluid, obtain with the click chemistry base for capableing of match reaction with the click chemistry group
Second cell of group；
(c) by the first cell that step (a) obtains and the second mixing with cells that step (b) obtains, by between click chemistry group
Match reaction, promote the first cell and the second cell interaction；
Preferably, the basic metabolism substance for being modified with click chemistry group is the monosaccharide analog of BCN modification, the modification
There is the monosaccharide that can be modified with the basic metabolism substance of the click chemistry group of the click chemistry group match reaction for nitrogen
Like object.
8. the method according to claim 7 for promoting organism interaction, which is characterized in that described in total incubation
The concentration for being modified with the basic metabolism substance of click chemistry group is 5-50umol/L, preferably 10-40umol/L；The modification
Having can be 5-50umol/ with the concentration of the basic metabolism substance of the click chemistry group of the click chemistry group match reaction
L, preferably 10-40umol/L；
Preferably, the time being incubated for altogether is 48-96 hours, preferably 48-84 hours, more preferably 48-72 hours；
Preferably, the first cell and the cell quantity of the second mixing with cells ratio are 1:0.2-5, preferably 1:0.5-2.
9. it is according to claim 7 promote organism interaction method, which is characterized in that step (a) further include
Impurity is removed after being incubated for altogether, obtains that there is the step of the first cell of click chemistry group；
Preferably, step (b) further includes removing impurity after total incubation, and obtaining having can match with the click chemistry group
The step of second cell of the click chemistry group of reaction；
Preferably, step (a) and step (b) remove impurity independently by the mode of cleaning cell and/or centrifugation.
10. the method for promoting organism to interact as claim 1-9 is described in any item is preparation enhances immunization therapy
Application in drug.
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| CN111718904A (en) * | 2020-06-30 | 2020-09-29 | 深圳先进技术研究院 | Click chemistry mediated targeting method |
| CN114621925A (en) * | 2020-12-10 | 2022-06-14 | 深圳先进技术研究院 | Click chemistry mediated targeting method |
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