CN107501142B - Restore chemical linkers and its preparation and purposes of the response type containing double disulfide bond - Google Patents

Restore chemical linkers and its preparation and purposes of the response type containing double disulfide bond Download PDF

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CN107501142B
CN107501142B CN201710756890.2A CN201710756890A CN107501142B CN 107501142 B CN107501142 B CN 107501142B CN 201710756890 A CN201710756890 A CN 201710756890A CN 107501142 B CN107501142 B CN 107501142B
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disulfide bond
double
response type
containing double
chemical linkers
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CN107501142A (en
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吴川六
李卓儒
周顺清
郑艺武
赵一兵
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Xiamen University
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Abstract

Restore chemical linkers and its preparation and purposes of the response type containing double disulfide bond, it is related to the formed chemical linkers with double disulfide bond of the compound with double sulfydryls, it constructs the dynamic covalent linkage with unique redox responsiveness based on the synergistic effect between mutually incoherent double disulfide bond.The covalent linkage has the redox responsiveness completely different with traditional single disulfide bond；Under low-reductant concentration or extracellular conditions, with the stability of superelevation, and quick reduction fracture can occurs under the conditions of strong reducing properties in the cell etc. in it.It is expected to be applied to drug release and bio-sensing field, constructs redox response type drug delivery system, such as monoclonal antibody-drug coupling body.

Description

Restore chemical linkers and its preparation and purposes of the response type containing double disulfide bond
Technical field
The present invention relates to the formed chemical linkers with double disulfide bond of the compound with double sulfydryls, especially It is related to restoring chemical linkers and its preparation and purposes of the response type containing double disulfide bond.
Background technique
Most of drug, the effective therapeutic agent especially in cancer chemotherapy often show internal urgency in the prior art Property toxicity, has that toxic side effect is larger during practical application, which has limited their applications.Therefore new drug Focus on that exploitation more needs to have tumour cell with bigger with safer specific treatment agent, especially antitumor agent Effect property, but the quantity and seriousness of these side effects of pharmaceutical drugs are reduced simultaneously.In addition, the another kind of existing drug the disadvantage is that they Stability in vivo in circulation is insufficient, after most drugs enter in vivo, with blood circulation reach human body each organ and Tissue, wherein the part for eventually arriving at destination organization is less, most of drug decomposes in the circulating cycle, and accumulates in human health group In knitting, therapeutic effect is not achieved.
Part researchs and develops the form that new drug is pro-drug.Pro-drug, both prodrug was active medicine chemistry Be converted to itself inactive or low activity derivative, using chemistry or enzyme effect before or after reaching action site It is converted to parent drug in vivo.To reduce toxicity, this transformation is normally limited to site of action or target tissue rather than recycles System or non-target tissue.
Prodrug is prepared to change the pharmacokinetics of drug, improves dissolubility, stability, reduces toxicity, increases specificity With the duration for the pharmacological effect for increasing drug.Increase drug absorption, distribution, bioconversion by changing pharmacokinetics And excretion, to increase the bioavilability of drug.In prodrug design, it is important to consider that following factor: carrier attachment Selection；Prodrug is necessary for reversible or bio-reversible medicaments derivative and inactive or specific activity ingredient activity is low；Target The specificity of point and compatibility etc..About prodrug reference can be made to such as Rautio, J.et al., Nature Reviews DrugDiscovery 7.3 (2008): 255-70 and Chari, Ravi V.J.et al., Angewandte Chemie International Edition 53.15(2014):3796-827。
Currently, the connecting key that attachment (Linker) uses in prodrug mainly has ester bond, amido bond, acylhydrazone key, thioether bond With the covalent bonds such as disulfide bond.Wherein, disulfide bond is a kind of covalent linkage being widely used.Disulfide bond is important in life entity Chemical bond, under the reducing conditions, it may occur that fracture；Under the action of other thiol compounds, it may occur that bimolecular nucleophilic takes Generation reaction generates new disulfide bond and thiol molecule.Disulfide bond is the most common dynamic covalent bond in protein structure, not only Can effectively regulation protein folding process, and can be made a response for redox environments different in organism. The responsiveness of disulfide bond is mainly reflected in it can generate new disulfide bond between thiol molecule by bimolecular nucleophilic subsititution And thiol molecule.
Due to usually containing sulfydryl in biomolecule, such as the cysteine residues on polypeptide and protein, it is especially advantageous for repairing Decorations, construct the Biofunctional materials of redox response type, such as drug delivery system and activated form fluorescence imaging in which can be convenient Probe etc..In the system for being mostly based on disulfide bond building, disulfide bond is straight mainly as the connecting key of redox response type Body and loading molecule (drug or fluorescence probe) are carried in succession, see, for example, Santra, Santimuk μ L et al., Journal of the American Chemical Society 133.41(2011):16680–16688.These systems are in biotic environment In responsiveness depend primarily on the breaking kinetics of disulfide bond in the environment.It is rung to improve the redox based on disulfide bond The biological property of type system is answered, the stability of disulfide bond, which must pass through, subtly to be regulated and controled, and reaches target position to avoid disulfide bond It is broken in advance (such as in blood circulation and extracellular environment) before point；Also to guarantee it after reaching target site simultaneously (as intracellular) can be broken rapidly, and discharge loading molecule.
Currently, there are mainly two types of the stability that method can be applied to regulation disulfide bond: introducing steric hindrance and improve disulfide bond Stability；Change its stability of disulfide bond study on microenvironment regulation, referring to Kellogg, Brenda A.et al., Bioconjugate Chemistry 22.4(2011):717-27.Although these methods can be existed with the stability of accuracy controlling disulfide bond, disulfide bond The difference of stability but is difficult to be regulated and controled in different redox environments.For traditional single disulfide bond, in redox Response mode in environment is based on disulfide bonds rate and reducing agent (thiol molecule, as between glutathione GSH) concentration Linear relationship.The stability for improving disulfide bond, although reducing the Drug Conjugates degree that cycle interruption is split in vivo, Also it affects it and reaches the efficiency discharged after destination organization.The chemical formula of traditional single disulfide bond is as follows:
Therefore, in practical applications, nonlinear response mode is even more important, i.e., disulfide bond under low-reductant concentration or Need that there is higher stability in the environment of week reduction, and can more rapidly in high reductant concentration or the environment of strong reducing property Generation reduction fracture.This nonlinear response type can not be realized always in traditional single disulfide bond, significantly limit The application potential of Biofunctional materials based on disulfide bond building.Wu et al. in Zhai, LX, et al.Chem.Eur.J., 20.52 (2014): proposing a kind of connecting key form based on two disulfide bond in 17507-14, can be mutual by two The disulfide bond of synergistic effect, which constitutes, a kind of has the characteristics that the connecting key of nonlinear response.However, two acyl of naphthalene used in it is sub- Amine molecule (NDI) parent has some disadvantages: poor in polarity and the dissolubility in nonpolar solvent；Volume is larger, connects to it There may be other effects for the load connect；Responsiveness cannot in further regulation, can not further satisfaction novel prodrugs etc. answer Demand.
Need to construct above-mentioned double disulfide bond attachment systems, basis is the structure for constituting the monomer molecule of its attachment In there are double mercapto functional groups.In nature and current research, more double thiol molecules are had existed.Wherein, natural Molecule present in boundary has lipoic acid, dithiothreitol (DTT), 2,3- dimercaprol dimercaptopropanol and 1,2- dimercaptoethane etc.；Furthermore this kind of knot Structure is also widely present in polypeptide, protein, and Cys-Xaa-Cys and Cys-Xaa-Xaa- is normally behaved as in polypeptide sequence The form of Cys (wherein Cys is cysteine, and Xaa is a kind of any other natural amino acid).Under study for action, there are double sulfydryls The molecule of structure is typically based on the dynamic property of disulfide bond, for constructing in dynamic combinatorial chemistry library.Point being more commonly used Son is carboxyl diphenyl disulfide phenol, bis- (thiopurine methyltransferase) benzoic acid of 3,5-, referring to West, K.R., et al., Organic Letters 7.13 (2005): 2615-8 and Otto, S, et al., Science 297.5581 (2002): 590-3.
Although the above molecule meets the basic demand for constituting double disulfide bond attachments, wherein most can not be formed Double disulfide bond attachments of synergistic effect.The concertedness of sulfydryl is directly influenced by the space structure near sulfydryl, including mercapto Distance between base between the rigidity and sulfydryl of molecular skeleton.Inappropriate structure, which will lead to, can not form synergistic effect, generate too low Or excessively high stability: too low stability causes attachment to be directly broken in the circulating cycle；Crossing high stability causes attachment to reach It can not be broken after target site.Therefore, the molecular skeleton for connecting two mercapto groups need to have certain rigidity, and two sulfydryls Between spacing require more than the bond distance of disulfide bond.
Some double sulfhydryl compounds, the carboxyl diphenyl disulfide phenol and 3 such as before told, bis- (thiopurine methyltransferase) benzoic acid of 5-, autoxidation When thermodynamically stable product be tripolymer or polymer, and some double sulfhydryl compounds directly tend to form the two of intramolecular Sulfide linkage inner ring, the yield that may eventually lead to the formation of the dimer with double disulfide bond are lower.And many common double sulfhydryl compounds, Also lack the site that can further react with modification, make it that can not be used to constitute attachment.
It is polysubstituted from common sense and literature cited it is found that phenyl ring is a kind of suitably with the rigid parent of multidigit point Raw material is easy to get.Phenyl ring is connected to for sulfydryl and the case where sulfydryl is connected to benzyl position, there is suitable synthetic method.But work as mercapto With more than one, phenyl ring parent interval carbon atom and when situation that the carbon atom band that is connected with sulfydryl is substituted, general synthesis makes base Halogen substitution technique is simultaneously not suitable for.Because this kind of site can not replace progress halogenated by benzyl position free radical, and contain conjunction The polysubstituted phenyl ring parent stock of suitable modification group is also prohibitively expensive or can not directly buy, the space limited its application.
In addition, also needing to consider the hydrophilic and hydrophobic of molecule itself, dissolubility, toxicity, stabilization when constructing this kind of attachment Property and structure, these properties all affect whole system.The most ideal situation is that the double disulfide bond attachments introduced only have also The property of original response is all smaller to the other influences of loaded article and composition.
The delivering and release of prodrug are still the major issue for needing to solve, and wherein the optimization of attachment is a key, Although this field has been achieved for centainly being in progress, remain on and need to develop improved attachment, regulate and control attachment stability and Responsiveness, to meet the demand of different drug delivery and release.
Summary of the invention
It is an object of the invention to for existing single insufficient and rigid naphthalimide molecule of disulfide bond medicine-carried system stability The water-soluble poor defect that can not further apply of double disulfide bond systems of building, provides can be used for sensing and delivering, volume Smaller, water-soluble and responsiveness is good, with realize can the reduction response type of reduction response release of accuracy controlling contain double disulfide bond Chemical linkers and preparation method thereof and purposes.
A kind of double sulfhydryl compounds can be connected by double sulfydryls with itself or other substances with double sulfydryls, and formation has The compound of double disulfide bond.
A method of disulfide bond redox responsiveness being regulated and controled based on synergistic effect between key, the method includes providing one Chemical linkers and preparation method thereof of the class containing double disulfide bond, the method can solve traditional single disulfide bond in biotic environment In stability and redox responsiveness (such as blood circulation, extracellular and intracellular environment etc.) be difficult to both with defect, institute State double disulfide bond compounds in method as attachment controlled drug delivering, release and bio-sensing purposes.
The chemical general formula of a kind of double sulfhydryl compounds is as follows:
Wherein Ar indicates aryl or heteroaryl；R1Indicate singly-bound, alkylidene, substituted alkylene；Y indicate carboxyl, amino, Hydroxyl, azido, sulfonic group, halogen；R2Independently selected from hydrogen, alkyl, alkoxy, aryl, heteroaryl, halogen, nitro, cyanogen Base, can have all identical chemical structures or all different chemical structure or in which any several there is phase Same chemical structure, and a is an integer from 0 to 3；R3、R4It is each independently selected from singly-bound, alkylidene, replaces alkylene Base；R5Indicate hydrogen or acetyl group；Work as R3When for singly-bound, R4It cannot be singly-bound；Work as R3When for methylene, R4It cannot be methylene；Institute Straight chain, branch or cricoid saturated hydrocarbyl that term " alkyl " refers to 1~8 carbon atom are stated, the alkyl can be selected from methyl, second One of base, propyl, isopropyl, butyl, isobutyl group, tert-butyl etc., straight chain, the branched alkyl of preferably 1~4 carbon atom； The term " alkylidene " refers to the alkylidene of 1~3 carbon atom of straight chain, the alkylidene of preferably 1~2 carbon atom；The art Language " substituted alkylene " refers to the alkylidene that at least one hydrogen atom is replaced by groups such as alkyl, alkoxy, aryl；It is described Term " alkoxy " refers to the alkoxy of the straight chain of 1~6 carbon atom, branch；The term " aryl " refers to 6~15 carbon originals The monocycle or polynary polycyclic aromatic hydrocarbon of son, the aromatic hydrocarbon of preferably 6~10 carbon atoms, the aromatic hydrocarbon are selected from phenyl or naphthyl, Preferably phenyl；The term " heteroaryl " refers to the aromatic rings comprising 3~15 carbon and containing at least one N, O or S atom, The aromatic rings can be selected from one of pyrrole radicals, pyridyl group, pyrimidine radicals, similar group etc.；The term " halogen " include fluorine, One of chlorine, bromine, iodine etc..
The chemical general formula of a kind of double sulfhydryl compounds may be expressed as:
The preparation methods of a kind of double sulfhydryl compounds the following steps are included:
Method (I): when sulfydryl is directly connected in aryl, using aryl, the heteroaryl compound replaced containing hydroxyl；The One step, by its in n,N-Dimethylformamide with N, the thio formyl chloride of N- dimethylamino alkali effect under be condensed；Second Step, in diphenyl ether, is heated to 200 DEG C of progress rearrangement reactions；Third step is hydrolyzed in ethylene glycol and water by potassium hydroxide； 4th step protects sulfydryl by acetic anhydride and alkali.The reaction route is schematically as follows:
Method (II): when carbon atom is connected with aryl when one, sulfydryl interval, using alkyl-substituted aryl, heteraryl Close object, and at least one hydrogen atom on the carbon atom that is connected with aryl of the alkyl；The first step passes through bromo in carbon tetrachloride Succimide and benzoyl peroxide carry out benzyl position free radical bromo-reaction；Second step is replaced by thioacetic acid potassium Reaction.The reaction route is schematically as follows:
Method (III): when carbon atom is connected with aryl when two, sulfydryl interval, using alkyl-substituted aryl, heteroaryl Compound, and at least one hydrogen atom on the carbon atom that is connected with aryl of the alkyl；The first step passes through in carbon tetrachloride NBS and benzoyl peroxide carry out benzyl position free radical bromo-reaction；Second step obtains triphenyl phosphorus by reacting with triphenyl phosphorus Salt occurs Wei Tixi (wittig) with the compound containing aldehydes or ketones later and reacts.Third step, by drawing with thioacetic acid in light It sends out progress addition reaction under the catalysis and action of ultraviolet radiation of agent or progress addition is anti-under heating by azodiisobutyronitrile catalysis It answers.The reaction route is schematically as follows:
Method (IV): when two, sulfydryl interval, carbon atom is connected with aryl, the aryl, the heteraryl that are replaced using halogen Close object；The first step, by carrying out Suzuki coupling reaction under alkali, the effect of four triphenyl phosphorus palladiums with ene boric acid；Second step passes through Addition reaction is carried out under the catalysis and action of ultraviolet radiation of photoinitiator with thioacetic acid or is existed by azodiisobutyronitrile catalysis Heating is lower to carry out addition reaction.The reaction route is schematically as follows:
The sulfhydryl protected base used in method (I), method (II), method (III) and method (IV) is acetyl group, can Leniently removed at room temperature by alkali, wherein alkali is one in sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide etc. Kind；Solvent is one of water, ethyl alcohol, methanol, acetonitrile, tetrahydrofuran etc..Due to sulfydryl with acetyl group protect so that into When the subsequent synthesis of row, sulfhydryl oxidase will not be led to the problem of and form by-product or sulfydryl the smell is awful.
In the first step of method (II) and method (III), bromo-succinimide and former with each carbon that can react Sub- site inventory molar ratio is that (1~2.5) ︰ 1, benzoyl peroxide rub with the carbon atom site inventory that can each react You are than for (0.05~0.2) ︰ 1, reaction temperature are 60~80 DEG C, and the reaction time is 2~12h.
In the second step of method (II), thioacetic acid potassium and with each bromine atom site inventory mole that can be reacted Than for (1~2.5) ︰ 1, reaction temperature be 20~60 DEG C, the reaction time be 2~for 24 hours.The solvent is acetone, acetonitrile, N, N- bis- One of methylformamide, tetrahydrofuran, 1,4- dioxane, methanol, ethyl alcohol etc..
In the second step of method (III), triphenylphosphine and with each bromine atom inventory molar ratio that can be reacted it is (1.2~2.5) ︰ 1, reaction temperature are 50~80 DEG C, and the reaction time is 8~12h.Compound containing aldehydes or ketones and each can The inventory molar ratio in the triphenylphosphine hydrobromate site of reaction is 1.2~2 ︰ 1, alkali and and the triphenyl that can each react The inventory molar ratio in phosphine hydrobromate site is that (1.2~2) ︰ 1, reaction time are 1~8h.Solvent is water, methylene chloride, four Hydrogen furans etc.；Alkali is one of sodium hydroxide, sodium ethoxide, sodium methoxide, potassium tert-butoxide etc..
In the third step of method (III) and the second step of method (IV), thioacetic acid and the alkene that can each react The inventory molar ratio in site is that (the inventory molar ratio in 2~8) ︰ 1, photoinitiator and the alkene site that can each react is (0.1~0.5) ︰ 1, reaction time are 12~48h.Solvent is one of tetrahydrofuran, 1,4- dioxane, acetonitrile etc.；Light Initiator is one of dimethoxybenzoin, styrax, diethoxy acetophenone etc.；The ultraviolet light light wave a length of 254 used ~365nm.
In the third step of method (III) and the second step of method (IV), thioacetic acid and the alkene that can each react The inventory molar ratio in site is (2~8) ︰ 1, the inventory mole of azodiisobutyronitrile and the alkene site that can each react Than for (0.1~0.5) ︰ 1, reaction temperature are 60~80 DEG C, and the reaction time is 12~48h.Solvent is tetrahydrofuran, 1,4- dioxy One of six rings, carbon tetrachloride, benzene etc..
In the first step of method (IV), the inventory molar ratio in ene boric acid and the halogen site that can each react is (the inventory molar ratio in 1~2) ︰ 1, alkali and the halogen site that can each react is (2~6) ︰ 1, four triphenyl phosphorus palladiums and every The inventory molar ratio in a halogen site that can be reacted is that (0.1~0.5) ︰ 1, reaction temperature are 80~120 DEG C, the reaction time For 6~for 24 hours.Solvent is one of n,N-Dimethylformamide, Isosorbide-5-Nitrae-dioxane, water, toluene etc.；Alkali is sodium carbonate, carbon Sour potassium, it is a kind of in potassium phosphate etc..
In one of method (I), method (II), method (III) and method (IV), corresponding raw material, Ke Yihe are used The equal double thiol molecules of carbon atom number at interval, such as application method (III) can synthesize bis- (2- mercapto ethyl) the benzene first of 3,5- Acid；Application method (IV) can synthesize bis- (2- mercapto the propyl)-benzoic acid of 3,5-.
Combination in method (I), method (II), method (III) and method (IV), uses corresponding raw material, Ke Yihe The unequal double thiol molecules of carbon atom number at interval, such as application method (III) and method (II) combine and 3- (2- can be obtained (acetyl mercapto) ethyl) -5- (2- (acetyl mercapto) methyl) benzoic acid and 3- (2- mercapto ethyl) -5- (thiopurine methyltransferase) benzoic acid.
The chemical general formula of the reduction chemical linkers of the response type containing double disulfide bond are as follows:
Wherein M1And M2It is the compound containing double sulfydryls, forms double disulfide bond by respective double sulfydryls, constitute the dimerization bodily form Formula, S are sulphur atom；M1Selected from one of double sulfhydryl compounds, carboxyl diphenyl disulfide phenol, bis- (thiopurine methyltransferase) benzoic acid of 3,5- etc.；M2 Selected from double sulfhydryl compounds, lipoic acid, 2,3- dimercaprol dimercaptopropanol, 2,3- dimercapto propionic acid, 1,2- dimercapto propylamine, carboxyl benzene two It is bis- (thiopurine methyltransferase) benzoic acid of thiophenol, 3,5-, residual by 3-30 amino acid with Cys-Xaa-Cys, Cys-Xaa-Xaa-Cys sequence One of polypeptide of base composition etc.；Work as M1When for carboxyl diphenyl disulfide phenol, M2It cannot be carboxyl diphenyl disulfide phenol；It is residual in the polypeptide Base Xaa be selected from amino acids Glycine (Gly, G), alanine (Ala, a), valine (Val, V), leucine (Leu, L), different bright ammonia Acid (Ile, I), phenylalanine (Phe, P), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagus fern acyl Amine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), glutamine (Gln, Q), methionine (Met, M), serine It is a kind of in (Ser, S), threonine (Thr, T), proline (Pro, P), histidine (His, H), arginine (Arg, R) etc..
The chemical linkers of the reduction response type containing double disulfide bond can be M1And M2Represent double sulfhydryl compounds, M1 Represent double sulfhydryl compounds and M2Represent the polypeptide.
The preparation method of the reduction chemical linkers of the response type containing double disulfide bond, is original with the compound containing double sulfydryls Material is prepared by the method for the activation of two sulphur, two pyridine or dimethylsulfoxide oxidation, and the solvent that dimethylsulfoxide oxidation uses is dimethyl The volume ratio of sulfoxide and 7.4 phosphate buffer mixed solution of pH, the dimethyl sulfoxide and 7.4 phosphate buffer of pH is 20 ︰ 80, oxidation sulfhydryl compound molar concentration control is about 1mM；Two sulphur, two pyridine and the Sulfhydryl Groups inventory that can each react Molar ratio is that (1~10) ︰ 1, the solvent that two sulphur, two pyridine uses are methanol, ethyl alcohol, acetonitrile, water, dimethyl sulfoxide, N, N- diformazan One of base formamide etc..
Beneficial effects of the present invention:
(1) a kind of double sulfhydryl compounds are compared to common double sulfhydryl compounds with can be with the reactive official of functionalization It can roll into a ball, for building there is multi-functional biomaterial to provide basis.The preparation method is simple, low in cost, is easy to industry Change.Further, it is also possible to easily synthesize different substituted double thiol molecules.Synthesized thiol molecule is protected with acetyl group, Side reaction when subsequent reactions can be reduced.
(2) a kind of double sulfhydryl compounds are by preferably, will not or be hardly formed intramolecular disulfide bond, main composition point Disulfide bond between son forms more disulfide bond macrocycle molecules, primarily forms double disulfide bond molecules.Using described containing double disulfide bond The preparation method of chemical linkers can obtain product with high productivity, reduce by-product common in more sulfhydryl compound reactions.
(3) a kind of chemical linkers containing double disulfide bond, since it forms based on sulfydryl the reaction of disulfide bond, A series of attachments with different reduction response properties can simply be synthesized.In addition, constituting described one kind of the attachment Double sulfhydryl compounds, the carbon atom being connected with sulfydryl can have different substituent groups by the preparation method, so that Chemical environment around sulfydryl is different, also will affect the rate of fracture.It is available to adapt to different biographies by the two methods The attachment of sense and delivering release demand.In addition, such attachment small volume, good water solubility, so that its shadow to load Sound further decreases.
(4) fluorescence probe is constructed using a kind of chemical linkers containing double disulfide bond, which has good Stability, lower side effect, can be efficiently applied to the sensing of bioluminescence imaging and bioreductive environment.
Detailed description of the invention
Fig. 1 is that the release dynamics of 1mM and 10mM GSH of the preferred double disulfide bond attachments in simulation physiological condition are bent Line；
Fig. 2 is cell imaging figure (the fluorescence probe concentration that synthesized double disulfide bond attachments are applied to bio-sensing 2uM), light field, fluorescence superimposed image (a), fluorescent image (b)；
Fig. 3 is cell imaging figure (the fluorescence probe concentration that synthesized double disulfide bond attachments are applied to bio-sensing 5uM), light field, fluorescence superimposed image (a), fluorescent image (b)；
Fig. 4 is the fluorescence spectrum that 10mM GSH restores fluorescence probe F2.
Specific embodiment
The compound of the present invention and method will further be shown by the following example.
In-vitro simulated release experiment is carried out to following preferred chemical linkers and its single disulfide bond object of reference:
When simulating blood circulation, single disulfide bond attachment all quick release in 1mM and 10mM, release half-life period and GSH concentration is in a linear relationship.Unlike, under conditions of 1mM GSH, double disulfide bond attachments only occur seldom when 720min Release；And when simulation intracellular environment, under conditions of 10mM GSH, when 240min, double disulfide bond attachments almost discharged. These results suggest that such double disulfide bond attachment has good nonlinear reduction response property.
Shown in the principle of this nonlinear response: first, traditional single disulfide bond feelings existing for thiol molecule reducing agent Under condition, a step sulfydryl-disulfide bond exchange reaction can occur, directly so as to cause disulfide bonds.It is constant in reductant concentration In the case of, the fracture of disulfide bond follows first-order kinetics, service life (τ1/2) in a linear relationship with the concentration of reducing agent.Its Two, the redox fracture process of double disulfide bond is different from single disulfide bond, in the lower situation of reductant concentration, generating unit Double disulfide bond (i.e. one of disulfide bond is broken) intermediate that disjunction is split due to the synergisticing stable effect between double disulfide bond, Sulfydryl-disulfide bond exchange reaction of intramolecular can occur, to re-form double disulfide bond；But it is higher in reductant concentration In the case where, double disulfide bond that portion fractures occur further can occur sulfydryl-disulfide bond with the reducing agent in environment and exchange instead It answers, is broken in succession so as to cause two disulfide bond.Therefore, double disulfide bond are steady with superelevation under conditions of week reduction It is qualitative, and it is then similar with single disulfide bond under conditions of strong reducing property, it can occur quickly to be broken.Proposed by the present invention double two Sulfide linkage is as follows:
In certain embodiments of the present invention, the fluorescence probe with flowering structure has been synthesized:
And cell imaging experiment, result and the extracorporeal releasing experiment result phase of cell imaging have been carried out using fluorescence probe F2 Meet, such double disulfide bond attachment has non-linear reduction responsiveness.
Specific embodiment is given below.
Embodiment 1
The preparation of bis- (the 2- mercapto ethyl) benzoic acid (1) of 3,5-:
The synthetic route of bis- (the 2- mercapto ethyl) benzoic acid (1) of 3,5- is as follows:
The synthesis of 3,5- divinyl benzoic acid (1d).(1) 3,5- mesitylenic acid (1a) (1.0g, 6.6mmol) is molten In 10mL carbon tetrachloride, the dibenzoyl peroxide of N-bromosuccinimide (2.37g, 13.3mmol) and catalytic amount is added (73mg).It is heated to reflux 3h, cold filtration collects filtrate.Vacuum rotation goes filtrate to obtain bis- (bromomethyl) benzene of white solid 3,5- Formic acid (1b).(2) above-mentioned product (1b) is dissolved in 25mL acetone.It is slowly added to triphenylphosphine (3.49g, 13.3mmol) and flows back Overnight.It is cooling, white solid is collected by filtration, and wash with hexane and ether, obtains intermediate (1c).(3) it is added to salt (3c) 30mL formalin (37% formalin of 240mmol, 20mL, 10mL water).5N hydroxide is slowly dropped into the mixture Sodium water solution (7.5mL, 37.5mmol).Solution is gradually clarified, and generates white precipitate later.React at room temperature 3h.It is heavy to filter out white It forms sediment, pH=2 is neutralized to concentrated hydrochloric acid later.Solid is precipitated, filters and be dried to obtain white solid 0.31g, yield 26.5%.1H NMR(500MHz,CDCl3) δ 8.07 (d, J=1.6Hz, 2H), 7.67 (d, J=1.8Hz, 1H), 6.79 (dd, J=17.5, 10.9Hz, 2H), 5.92-5.85 (m, 2H), 5.39 (d, J=10.9Hz, 2H)13C NMR(125MHz,CDCl3)δ138.30, 135.70,129.95,129.03,127.09,115.57.
Bis- (2- (the synthesis of acetyl mercapto (ethyl) benzoic acid (1e) of 3,5-.3d (50mg, 0.29mmol), thioacetic acid (0.42mL, 5.96mmol) and catalytic amount dimethoxybenzoin (5mg) are dissolved in 3mL tetrahydrofuran.Solution is in 365nm UV- The lower reaction of light (8W) irradiation is for 24 hours.Solvent is removed in rotation, obtains white solid 29mg, yield 31% with column chromatography for separation.1H NMR (400MHz,CDCl3) δ 7.85 (d, J=1.7Hz, 2H), 7.28 (s, 1H), 3.15 (dd, J=8.5,6.5Hz, 4H), 2.94 (dd, J=8.6,6.5Hz, 4H), 2.36 (s, 6H)13C NMR(100MHz,CDCl3)δ195.62,171.73,140.67, 135.70,134.44,129.75,129.49,128.59,126.27,77.36,77.24,77.04,76.72,35.49, 35.44,30.68,30.27,30.21,21.20.ESI MS(m/z):348.8[M+Na]+.
Bis- (2- (the synthesis of acetyl mercapto (ethyl) benzoic acid (1e) of 3,5-.3d (50mg, 0.29mmol), thioacetic acid (0.42mL, 5.96mmol) and catalytic amount azodiisobutyronitrile (10mg) are dissolved in 10mL carbon tetrachloride, and flow back 6h, and cooling back spin is gone Solvent, column chromatography for separation obtain white solid, yield 22%.
The synthesis of bis- (the 2- mercapto ethyl) benzoic acid (1) of 3,5-.3e (20mg, 0.06mmol) is dissolved in 1mL methanol, and 3N hydrogen is added Sodium hydroxide solution (1mL), is stirred at room temperature 1h under nitrogen protection.1N hydrochloric acid is neutralized to pH=2.White solid is precipitated, with centrifugation Separation, vacuum drying obtain white solid 14mg.1H NMR(500MHz,CDCl3) δ 7.83 (d, J=1.7Hz, 2H), 7.64 (d, J=1.7Hz, 1H), 2.99~2.95 (m, 4H), 2.83~2.79 (m, 4H), 1.42~1.39 (m, 2H)
Embodiment 2
The preparation of bis- (the 2- mercapto ethyl) benzoic acid (2) of 2,5-:
The synthetic route of bis- (the 2- mercapto ethyl) benzoic acid (2) of 2,5- is as follows:
The synthesis of 2,5- divinyl benzoic acid (2d).(1) 2,5- mesitylenic acid (2a) (1.0g, 6.6mmol) is molten The dibenzoyl peroxide of N-bromosuccinimide (2.37g, 13.3mmol) and catalytic amount is added in 10mL carbon tetrachloride (73mg).It is heated to reflux 3h, filtrate is collected in cooling filtering.Vacuum rotation goes filtrate to obtain bis- (bromomethyl) benzene of white solid 2,5- Formic acid (2b).(2) above-mentioned product (2b) is dissolved in 25mL acetone.It is slowly added to triphenylphosphine (3.49g, 13.3mmol) and flows back Overnight.It is cooling, white solid is collected by filtration, and wash with hexane and ether, obtains intermediate (2c).(3) it is added to salt (4c) 30mL formalin (37% formalin of 240mmol, 20mL, 10mL water).5N hydroxide is slowly dropped into the mixture Sodium water solution (7.5mL, 37.5mmol).Solution is gradually clarified, and generates white precipitate later.React at room temperature 3h.It is heavy to filter out white It forms sediment, pH=2 is neutralized to concentrated hydrochloric acid later.Solid is precipitated, filters and be dried to obtain white solid 0.4020g, yield 35%.1H NMR(500MHz,CDCl3) δ 8.08 (s, 1H), 7.62~7.60 (m, 2H), 7.60~7.55 (m, 1H), 6.76 (dd, J= 17.6,10.9Hz, 1H), 5.90~5.82 (m, 1H), 5.71 (dd, J=17.4,1.3Hz, 1H), 5.38 (ddd, J=22.3, 11.0,0.8Hz,2H).13C NMR(125MHz,CDCl3)δ171.93,139.54,136.94,135.48,132.13, 130.18,129.14,128.54,127.61,116.62,115.09。
The synthesis of bis- (2- (acetyl mercapto) ethyl) benzoic acid (2e) of 2,5-.2d (50mg, 0.29mmol), thioacetic acid (0.42mL, 5.96mmol) and catalytic amount dimethoxybenzoin (5mg) are dissolved in 3mL tetrahydrofuran.Solution is in 365nm UV- The lower reaction of light (8W) irradiation is for 24 hours.Solvent is removed in rotation, obtains white solid 21mg, yield 22% with column chromatography for separation.1H NMR (500MHz,CDCl3) δ 7.92 (d, J=1.9Hz, 1H), 7.39 (dd, J=7.8,2.0Hz, 1H), 7.31 (d, J=7.8Hz, 1H), 3.27 (t, J=7.7Hz, 2H), 3.19~3.15 (m, 4H), 2.93 (t, J=8.0Hz, 2H), 2.36 (s, 3H), 2.35 (s,3H).13C NMR(125MHz,CDCl3)δ195.85,195.51,171.67,140.77,138.62,133.24,132.07, 131.84,128.28,35.13,34.21,30.64,30.61,30.22,30.17.ESI MS(m/z):348.8[M+Na]+。
The synthesis of bis- (the 2- mercapto ethyl) benzoic acid (2) of 2,5-.2e (20mg, 0.06mmol) is dissolved in 1mL methanol, and 3N hydrogen is added Sodium hydroxide solution (1mL), is stirred at room temperature 1h under nitrogen protection.1N hydrochloric acid is neutralized to pH=2.White solid is precipitated, with centrifugation Separation, vacuum drying obtain white solid 13mg.1H NMR(500MHz,CDCl3) δ 7.93 (d, J=2.0Hz, 1H), 7.38 (dd, J=7.8,2.1Hz, 1H), 7.30 (s, 1H), 3.34~3.30 (m, 2H), 2.99~2.96 (m, 2H), 2.86~2.81 (m, 4H), 1.45 (t, J=8.1Hz, 1H), 1.41 (t, J=7.9Hz, 1H).
Embodiment 3
The preparation of 3- (2- mercapto ethyl) -5- (thiopurine methyltransferase) benzoic acid (3):
The synthetic route of 3- (2- mercapto ethyl) -5- (thiopurine methyltransferase) benzoic acid (3) is as follows:
The synthesis of 3- methyl -5- vinyl benzoic acid (3d).(1) 3,5- mesitylenic acid (3a) (1.0g, 6.6mmol) It is dissolved in the dibenzoyl peroxide that N-bromosuccinimide (1.18g, 6.7mmol) and catalytic amount is added in 10mL carbon tetrachloride (37mg).It is heated to reflux 3h, filtrate is collected in cooling filtering.Vacuum rotation goes filtrate to obtain white solid.(2) by above-mentioned product (5b) is dissolved in 25mL acetone.It is slowly added to triphenylphosphine (1.75g, 6.7mmol) and is refluxed overnight.It is cooling, white is collected by filtration Solid, and washed with hexane and ether, obtain intermediate (3c).(3) to salt (3c) be added 20mL formalin (120mmol, 10mL37% formalin, 10mL water).To the mixture be slowly dropped into 5N sodium hydrate aqueous solution (3.75mL, 18.8mmol).Solution is gradually clarified, and generates white precipitate later.React at room temperature 3h.White precipitate is filtered out, later with concentrated hydrochloric acid It is neutralized to pH=2.Solid is precipitated, filters and be dried to obtain white solid 0.4360g, yield 40.8%.1H NMR(500MHz, CDCl3) δ 7.98 (s, 1H), 7.84 (s, 1H), 7.48 (s, 1H), 6.75 (dd, J=17.5,10.9Hz, 1H), 5.85 (d, J= 17.6Hz, 1H), 5.34 (d, J=10.9Hz, 1H), 2.44 (s, 3H)13C NMR(125MHz,CDCl3)δ170.99, 138.52,137.96,135.92,132.04,129.98,129.54,125.25,115.05,21.19.
The synthesis of 3- (2- (acetyl mercapto) ethyl)-methyl benzoic acid (3e).3d (150mg, 0.92mmol), thioacetic acid (0.50mL, 7.3mmol) and catalytic amount dimethoxybenzoin (30mg) are dissolved in 3mL tetrahydrofuran.Solution is in 365nm UV- The lower reaction of light (8W) irradiation is for 24 hours.Solvent is removed in rotation, obtains white solid 75.9mg, yield 34.7% with column chromatography for separation.1H NMR(500MHz,CDCl3) δ 7.80 (s, 1H), 7.76 (s, 1H), 7.29 (s, 1H), 3.18~3.10 (m, 2H), 2.92~ 2.87(m,2H),2.40(s,3H),2.34(s,3H).13C NMR(125MHz,CDCl3)δ195.64,172.07,140.31, 138.57,134.92,129.05,128.26,127.44,35.45,30.66,30.27,21.19。
The synthesis of 3- (2- (acetyl mercapto) ethyl) -5- (bromomethyl) benzoic acid (3f).3e (70mg, 0.29mmol) is dissolved in N-bromosuccinimide (56mg, 0.32mmol), catalytic amount dibenzoyl peroxide is added in 10mL carbon tetrachloride.It heats back 3h is flowed, cold filtration is spin-dried for filtrate.It is recrystallized with ethyl acetate and hexane, obtains white solid 81mg, yield 89.1%.1H NMR(500MHz,CDCl3) δ 7.98 (s, 1H), 7.86 (s, 1H), 7.39 (s, 1H), 4.51 (s, 2H), 3.22~3.12 (m, 2H), 2.90~2.85 (m, 2H), 2.33 (s, 3H).
The synthesis of 3- (2- (acetyl mercapto) ethyl) -5- (2- (acetyl mercapto) methyl) benzoic acid (3g).3f(81mg, It 0.26mmol) is dissolved in 10mL acetone, thioacetic acid potassium (58mg, 0.52mmol) is added, 3h is stirred at room temperature.Be added ethyl acetate and Water stratification, with ethyl acetate extraction 3 times, solvent is removed in rotation, is recrystallized with ethyl acetate and hexane, obtains white solid 77mg, produces Rate 95.0%.1H NMR(500MHz,CDCl3)δ7.82(s,1H),7.78(s,1H),7.35(s,1H),3.87(s,2H),3.12 ~2.80 (m, 4H), 2.33 (m, 6H).
The synthesis of 3- (2- mercapto ethyl) -5- (thiopurine methyltransferase) benzoic acid (3).3g (20mg, 0.06mmol) is dissolved in 1mL methanol, It is added 3N sodium hydroxide solution (1mL), 1h is stirred at room temperature under nitrogen protection.1N hydrochloric acid is neutralized to pH=2.White solid analysis Out, with centrifuge separation, vacuum drying obtains white solid 12.1mg.1H NMR(500MHz,CDCl3)δ7.80(s,1H),7.76 (s, 1H), 7.30 (s, 1H), 3.80 (s, 2H), 2.95~2.81 (m, 4H), 2.30 (s, 1H), 1.40 (s, 1H)
Embodiment 4
The preparation of bis- (2- mercapto the propyl)-benzoic acid of 3,5-:
The synthetic route of bis- (2- mercapto the propyl)-benzoic acid of 3,5- is as follows:
The synthesis of bis- (the acrylic)-benzoic acid (4b) of 3,5-.Under nitrogen atmosphere, 4a (280mg, 1mmol) is dissolved in 6mL N, Dinethylformamide is added four triphenyl phosphorus palladiums (230mg, 0.2mmol), is stirred at room temperature 10min, be added sodium carbonate (1.1g, 10.5mmol), 4mL water and 1- propylene -1- boric acid (179mg, 2.1mmol), flow back 12h.After cooling, 30mL water, 1N salt is added Acid is neutralized to pH=2, obtains gray precipitate, filtering.Column chromatography for separation white solid 145.4mg, yield 72%.
The synthesis of bis- (2- mercapto the propyl)-benzoic acid (4) of 3,5-.4b (100mg, 0.5mmol), thioacetic acid (0.27mL, 4mmol) and catalytic amount dimethoxybenzoin (30mg) is dissolved in 3mL acetonitrile.Solution is anti-under 365nm UV-light (8W) irradiation It should for 24 hours.Rotary Evaporators concentration, with column chromatography for separation.It is dissolved in 1mL methanol later, is added 3N sodium hydroxide solution (1mL), in 1h is stirred at room temperature under nitrogen protection.1N hydrochloric acid is neutralized to pH=2.White solid is precipitated, and with centrifuge separation, vacuum drying obtains white Color solid 24.2mg, yield 17.9%.1H NMR(500MHz,CDCl3) δ 7.85 (d, J=1.6Hz, 2H), 7.62 (d, J= 1.6Hz, 1H), 3.01~2.72 (m, 6H), 1.47 (d, 6H), 1.41~1.38 (m, 2H) .ESI MS (m/z): 268.9 [M- H]-。
Embodiment 5
The preparation of double disulfide bond attachments of carboxyl diphenyl disulfide phenol and bis- (thiopurine methyltransferase) the benzoic acid compositions of 3,5-:
The synthesized reference of carboxyl diphenyl disulfide phenol and bis- (thiopurine methyltransferase) benzoic acid of 3,5- from West, K.R., et al., Organic Letters7.13(2005):2615-8.By carboxyl diphenyl disulfide phenol (3.7mg, 0.02mmol) and bis- (the mercapto first of 3,5- Base) benzoic acid (4.2mg, 0.02mmol) is dissolved in (20 ︰ in 5mL dimethyl sulfoxide and 7.4 phosphate buffer mixed solution of pH 80), sulfhydryl compound concentration control is about 1mM.Room temperature reaction 2 days uses preparation after Rotary Evaporators concentration after reaction Chromatographic isolation obtains 5.1mg product, yield about 65%.ESI MS(m/z):395.0[M-H]-。
Embodiment 6
The preparation of double disulfide bond attachments of bis- (thiopurine methyltransferase) the benzoic acid compositions of 3,5-:
2,2'-, bis- sulphur, two pyridine (220mg, 1mmol) is dissolved in 20mL methanol.By bis- (thiopurine methyltransferase) benzoic acid of 3,5- (20mg, 0.09mmol) is dissolved in 5mL methanol, and is instilled in the methanol solution of two sulphur, two pyridine, is reacted at room temperature 8h, is rotated later Evaporimeter concentration, prepares chromatographic isolation.The intermediate that preparation chromatographic isolation obtains is dissolved in 5mL methanol, and the 3,5- of equivalent are added Bis- (thiopurine methyltransferase) benzoic acid react at room temperature 1h, prepare chromatogram purification after concentration, obtain product 32.2mg, yield about 81%.ESI MS(m/z):422.3[M-H]-。
Embodiment 7
The preparation of double disulfide bond attachments of bis- (2- mercapto ethyl) the benzoic acid compositions of 3,5-:
Bis- (the 2- mercapto ethyl) benzoic acid (4.8mg, 0.02mmol) of 3,5- are dissolved in 5mL dimethyl sulfoxide and 7.4 phosphoric acid of pH is slow In fliud flushing mixed solution (20 ︰ 80), the control of sulfhydryl compound concentration is about 1mM.Room temperature reaction 2 days, after reaction, rotation are steamed Preparation chromatographic isolation is used after sending out instrument concentration, obtains 3.8mg product, yield about 79%.ESI MS(m/z):479.0[M-H]-。
Embodiment 8
The preparation of double disulfide bond attachments of bis- (2- mercapto ethyl) the benzoic acid compositions of 3,5-:
2,2'-, bis- sulphur, two pyridine (220mg, 1mmol) is dissolved in 20mL methanol.By bis- (the 2- mercapto ethyl) benzoic acid of 3,5- (24mg, 0.1mmol) is dissolved in 5mL methanol, and is instilled in the methanol solution of two sulphur, two pyridine, is reacted at room temperature 8h, is rotated later Evaporimeter concentration, prepares chromatographic isolation.The intermediate that preparation chromatographic isolation obtains is dissolved in 5mL methanol, and the 3,5- of equivalent are added Bis- (2- mercapto ethyl) benzoic acid react at room temperature 1h, prepare chromatogram purification after concentration, obtain product 41.7mg, yield about 87%. ESI MS(m/z):479.0[M-H]-。
Embodiment 9
The preparation of reduction response type fluorescence probe based on double disulfide bond attachments:
3,5- bis- (2- mercapto ethyl) benzoic acid (9a) (3.26mg, 13.5umol), are dissolved in 2mL n,N-Dimethylformamide In, 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester (20mg, 52.6umol), N, N- is added Diisopropylethylamine (13mg, 100umol).1h is reacted at room temperature, 4,7,10- tri- oxygen -1,13- tridecane diamines are added dropwise later (22mg, 100umol) reacts at room temperature 4h, and three (2- carboxyethyl) phosphines are added later and restore 30min.It is concentrated, prepare chromatographically pure Change, white solid (9b) 2.2mg, yield 36% are obtained after freeze-drying.
Intermediate (9b) (2.2mg, 4.9umol) is dissolved in the phosphate buffer of 1mLpH7.4, and be quickly adding into containing In the phosphate buffer of the 1mL pH7.4 of bis- sulphur of 2,2'-, two pyridine (22mg, 98umol).Reaction 3h is stirred at room temperature, later with height Effect liquid phase chromatogram purifying, obtains white solid (9c) 2.3mg, yield 70% after freeze-drying.
Intermediate (9c) and fluoro fluorescein N-hydroxy-succinamide ester are dissolved in anhydrous dimethyl sulphoxide respectively, with for 2mM solution.100 μ L solution are respectively taken, with the mixing of 1 ︰, 1 ratio, and a small amount of n,N-diisopropylethylamine is added and adjusts pH to 8~9, room Warm shaking table reaction 8h obtains yellow solid (9d), is dissolved in anhydrous dimethyl sulphoxide later with high-efficient liquid phase chromatogram purification after freeze-drying In, and, yield 91% quantitative with ultraviolet-visible absorption spectroscopy.
3,5- bis- (2- mercapto ethyl) benzoic acid (9a) (30mg, 0.14mmol), are dissolved in 1mL n,N-Dimethylformamide, add Enter RRRRRK-Dabcyl (52.9mg, 0.045mmol), 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluoro Phosphate (38.5mg, 0.1mmol) and n,N-diisopropylethylamine (35mg, 0.27mmol) react at room temperature 12h.Later plus Enter three (2- carboxyethyl) phosphines reduction 30min.Chromatogram purification is prepared, orange solids (9e) 51.2mg, yield 82% are obtained after freeze-drying.
Intermediate (9d) and intermediate (9e) are dissolved in anhydrous dimethyl sulphoxide respectively, matching is 2mM solution.Respectively take 100 μ L solution is put by 1 ︰, 1 equivalent in the phosphate buffer solution of 2mL pH7.4, reacts 30min, and high-efficient liquid phase chromatogram purification is simultaneously lyophilized Fluorescence probe (F1) is obtained, is dissolved in anhydrous dimethyl sulphoxide, and is quantitative with ultraviolet-visible absorption spectroscopy, yield 91%. MALDI-TOF(m/z):2236.2[M+H]+。
Embodiment 10
The preparation of reduction response type fluorescence probe based on double disulfide bond attachments:
Using the similar approach in embodiment 9, intermediate (10d) is obtained by raw material of ethylenediamine.By itself and polypeptide A c- WGCGGCGRKKRRQRRR-NH2It is dissolved in anhydrous dimethyl sulphoxide respectively, matching is 2mM solution.100 μ L solution are respectively taken, by 1 ︰ 1 Equivalent is put into the phosphate buffer solution of 2mL pH7.4, reacts 30min, and high-efficient liquid phase chromatogram purification and freeze-drying obtain fluorescence spy Needle (F2), is dissolved in anhydrous dimethyl sulphoxide, and quantitative with ultraviolet-visible absorption spectroscopy, yield 92%.MALDI-TOF MS(m/ z):2674.4[M-H]-。
Embodiment 11
In-vitro simulated release experiment:
Double two sulphur attachments and traditional single disulfide bond in the present invention are in glutathione (GSH) solution of various concentration Reduction discharge comparison, environment when blood circulation, ring when 10mM GSH discharges into the cell are simulated with 1mM GSH in test Border.Compound for release experiment is as follows.Wherein, used single disulfide bond attachment on corresponding monomer by shielding Or it after reducing sulfydryl, is obtained using oxidation.
Solution used, which is placed in the round-bottomed flask with rubber stopper before use, is bubbled deoxygenation with nitrogen.In order to avoid The influence of oxygen, all processes carry out in anaerobic culture box.Detailed process is as follows: take 0.2mL phosphate buffer (100mM, PH7.4) in the low protein adsorption centrifuge tube of 1.5mL, being separately added into bis-/mono- disulfide bond compound solutions of 50 μ L, (ultimate density is 5uM) and the glutathione solution of 0.25mL various concentration.At regular intervals, take 40 μ L reaction solutions rapid in empty centrifuge tube It is quenched and is reacted with liquid nitrogen frozen.After to be sampled, each sample is subjected to high performance liquid chromatography (HPLC) and analyzes (sampling volume 30 μ L) it is quantitative, it draws kinetic curve fittings and calculates half-life period.Different single, double disulfide bond attachments are at 1mM and 10mM GSH Release half-life period result referring to table 1, it is providing statistics indicate that double disulfide bond attachments are compared to the single disulfide bond attachment of tradition, tool There is controllable non-linear reduction responsiveness, can be used as the attachment in prodrug, can reach and hardly be discharged in blood circulation, And enter the effectiveness of quick release after target site.For different double disulfide bond attachments, such as D2 and D4, with different Responsiveness is restored, solves the demand discharged under different reducing conditions.Wherein, the release of preferred double disulfide bond attachment D4 Kinetic curve is shown in Fig. 1.
Table 1
Note*: half-life period is extremely short, and the sampling time has influenced last result.
Note**: half-life period is extremely long, and 720min is only broken 7%, can not be fitted.
Note***: referring to Zhai, LX, et al.Chem.Eur.J., 20.52 (2014): 17507-14.
Embodiment 12
The cell imaging experiment of fluorescence probe F2 uses Hela cell in embodiment 10, in 37 DEG C, 5%CO2Under ring In border, cultivated in the DMEM culture medium of serum-free.Before experiment, cell inoculation is in culture plate, every hole concentration 105It is a, upper After being incubated for for 24 hours under the conditions of stating, remove matrix, and cleaned with PBS.The fluorescence probe F2 of respective concentration is added, and is incubated for corresponding Time.Later, it is imaged using fluorescence microscope and carries out cell imaging experiment (488nm laser excitation), experimental result is shown in Fig. 2 and figures 3。
Polypeptide in fluorescence probe F2 has fluorescent quenching effect to fluorophor fluoro fluorescein, when double disulfide bond connect When object is broken, polypeptide is separated with fluorophor, and fluorescence can restore.From the result of cell imaging as it can be seen that when being incubated for 4h, Fluorescence in the imaging of the probe of 2uM or 5uM concentration is all weaker, illustrates that fraction fracture does not occur or only occurs for probe.When When being incubated for for 24 hours, the fluorescence in the imaging of the probe of 2uM or 5uM concentration is significantly increased, and illustrates that probe is largely resolved It splits, so that fluorescence restores.The result of fluorescence imaging is consistent with the result of in-vitro simulated release, such double disulfide bond connection Object has non-linear reduction responsiveness.
Fig. 4 provides the fluorescence spectrum of 10mM GSH reduction fluorescence probe F2.

Claims (5)

1. restoring chemical linkers of the response type containing double disulfide bond, it is characterised in that its chemical general formula are as follows:
It is by the compound SH-M containing double sulfydryls1- SH and SH-M2Double disulfide bond connection that-SH is formed by respective double sulfydryls The dimer constituted；Wherein SH is sulfydryl, and S is sulphur atom, M1And M2It is the residues Structures for removing thiol portion；
SH-M1- SH is selected from double sulfhydryl compounds with following chemical general formula:
SH-M2- SH is selected from double sulfhydryl compounds with following chemical general formula:
2,3- dimercaprol dimercaptopropanol, 1,2- dimercapto propylamine, has Cys-Xaa-Cys or Cys-Xaa- at 2,3- dimercapto propionic acid One of the polypeptide of Xaa-Cys sequence being made of 3-30 amino acid residue；
Ar indicates phenyl in the above chemical general formula；R1Indicate singly-bound；Y indicates carboxyl, amino, hydroxyl, azido, sulfonic group；R2Table Show hydrogen, and a is 3；R3、R4It is each independently selected from the alkylidene of singly-bound, 1~3 carbon atom；R5Indicate hydrogen；
Work as SH-M1When-SH is carboxyl diphenyl disulfide phenol, SH-M2- SH cannot be carboxyl diphenyl disulfide phenol；Residue Xaa is selected in the polypeptide From amino acids Glycine (Gly, G), alanine (Ala, a), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, P), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), glutamine (Gln, Q), methionine (Met, M), serine (Ser, S), It is threonine (Thr, T), proline (Pro, P), histidine (His, H), a kind of in arginine (Arg, R).
2. reduction chemical linkers of the response type containing double disulfide bond as described in claim 1, it is characterised in that form dimer Double sulfhydryl compound SH-M1- SH and SH-M2- SH chemical general formula are as follows:
3. reduction chemical linkers of the response type containing double disulfide bond as described in claim 1, it is characterised in that the reduction response The preparation method of chemical linkers of the type containing double disulfide bond, it is living by two sulphur, two pyridine using the compound containing double sulfydryls as raw material Change or the preparation of the method for dimethylsulfoxide oxidation, the solvent that dimethylsulfoxide oxidation uses are dimethyl sulfoxide and 7.4 phosphoric acid buffer of pH The volume ratio of liquid mixed solution, the dimethyl sulfoxide and 7.4 phosphate buffer of pH is 20 ︰ 80, aoxidizes sulfhydryl compound mole Concentration control is 1mM；Two sulphur, two pyridine is (1~10) ︰ 1, two sulphur with the Sulfhydryl Groups inventory molar ratio that can each react The solvent that two pyridines use is one of methanol, ethyl alcohol, acetonitrile, water, dimethyl sulfoxide, n,N-Dimethylformamide.
4. a kind of method based on synergistic effect regulation disulfide bond redox responsiveness between key, the method includes providing as weighed Benefit require 1 described in one kind reduction chemical linkers and preparation method thereof of the response type containing double disulfide bond, the method is by containing double The compound SH-M of sulfydryl1- SH and SH-M2Double disulfide bond that-SH is formed by respective double sulfydryls.
5. as described in claim 1 reduction chemical linkers of the response type containing double disulfide bond synthesis have controlled drug delivering, The purposes of the molecule of release and bio-sensing function.
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