CN109535097A - The molecular probe and its preparation method and application of one kind detection dihydrolipoic acid succinyltransferase - Google Patents
The molecular probe and its preparation method and application of one kind detection dihydrolipoic acid succinyltransferase Download PDFInfo
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Abstract
The present invention relates to the molecular probes and its preparation method and application of a kind of detection dihydrolipoic acid succinyltransferase.The probe can effectively detect the dihydrolipoic acid succinyltransferase in living cells as shown in following Formulas I at low concentrations.The probe not only has good selectivity dihydrolipoic acid succinyltransferase in cell, but also higher reactivity is shown to it, and can effectively be purified to the destination protein after viable cell labelling.
Description
Technical field
The present invention relates to chemical biology fields, and in particular to one kind detects dihydrolipoic acid amber in living cells
Molecular probe of acyltransferase and its preparation method and application.
Background technique
Dihydrolipoic acid succinyltransferase (DLST) is used as ketoglurate dehydrogenase complex (α-
Ketoglutarate dehydrogenase complex) core component, catalysis α-ketoglutaric acid be converted into amber
Key effect is played during acyl coenzyme A, is the important component of tricarboxylic acid cycle (TCA)[1-3].Research shows that DLST egg
It is white closely related with the generation of a variety of diseases, such as Alzheimer disease[4-6], leukaemia[7], cardiovascular disease[8]Deng, however due to
Lack effective tool small molecule, the functional study of DLST is greatly limited.Therefore, developing one kind can be used in
The molecular probe that DLST is detected in living cells is of great significance for the functional study of DLST.
It is one based on active Proteomic analysis (Activity-based protein profiling, ABPP) technology
The powerful chemical proteomics research tool of kind, it is using the chemical small molecule probe of active-site directed directly in complexity
The functional status of enzyme is monitored in biosystem[9-11].The core of ABPP is that design one can be in living cells or the protein of complexity
" bioactive molecule probe " (Activity-based spy of covalent reaction occurs in group with certain class enzyme active center amino acid residue
Needle, ABP), since the chemical modification of albumen occurs in its catalytic active center for ABP, we can be marked by probe
Horizontal height can read immediately the active function state of certain albuminoid enzyme in complicated protein group[12], this technology original
Managing has important reference for the design of DLST molecular probe.
Above-mentioned citation is as follows:
[1]Yang,L.；Shi,Q.；Ho,D.J.；Starkov,A.A.；Wille,E.J.；Xu,H.；Chen,H.L.；
Zhang,S.；Stack,C.M.；Calingasan,N.Y.；Gibson,G.E.；Beal,M.F.Mice deficient in
dihydrolipoyl succinyl transferase show increased vulnerability to
mitochondrial toxins.Neurobiol.Dis.,2009,36,320-330.
[2]Starkov,A.A.An update on the role of mitochondrialα-ketoglutarate
dehydrogenase in oxidative stress.Mol.Cell.Neurosci.,2013,55,13-16.
[3]Guo,H.；Madzak,C.；Du,G.；Zhou,J.Mutagenesis of conserved active site
residues of dihydrolipoamide succinyltransferase enhances the accumulation of
α-ketoglutarate in Yarrowia lipolytica.Appl.Microbiol.Biotechnol.,2016,100,
649-659.
[4]Gibson,G.E.；Blass,J.P.；Beal,M.F.；Bunik,V.Theα-ketoglutarate-
dehydrogenase complex.Mol.Neurobiol.,2005,31,43-63.
[5]Dumont,M.；Ho,D.J.；Calingasan,N.Y.；Xu,H.；Gibson,G.；Beal,
M.F.Mitochondrial dihydrolipoyl succinyltransferase deficiency accelerates
amyloid pathology and memory deficit in a transgenic mouse model of amyloid
deposition.Free Radic.Biol.Med.,2009,47,1019-1027.
[6]Gibson,G.E.；Starkov,A.；Blass,J.P.；Ratan,R.R.；Beal,M.F.Cause and
consequence:mitochondrial dysfunction initiates and propagates neuronal
dysfunction,neuronal death and behavioral abnormalities in age-associated
neurodegenerative diseases.Biochim.Biophys.Acta,2010,1802,122-134.
[7]Anderson,N.M.；Li,D.；Peng,H.L.；Laroche,F.J.F.；Mansour,M.R.；Gjini,
E.；Aioub,M.；Helman,D.J.；Roderick,J.E.；Cheng,T.；Harrold,I.；Samaha,Y.；Meng,L.；
Amsterdam,A.；Neuberg,D.S.；Denton,T.T.；Sanda,T.；Kelliher,M.A.；Singh,A.；Look,
A.T.；Feng,H.The TCA cycle transferase DLST is important for MYC-mediated
leukemogenesis.Leukemia,2016,30,1365-1374.
[8]Heggermont,W.A.；Papageorgiou,A.P.；Quaegebeur,A.；Deckx,S.；Carai,P.；
Verhesen,W.；Eelen,G.；Schoors,S.；van Leeuwen,R.；Alekseev,S.；Elzenaar,I.；
Vinckier,S.；Pokreisz,P.；Walravens,A.S.；Gijsbers,R.；Haute,C.V.D.；Nickel,A.；
Schroen,B.；van Bilsen,M.；Janssens,S.；Maack,C.；Pinto,Y.；Carmeliet,P.；Heymans,
S.Inhibition of MicroRNA-146a and Overexpression of Its Target Dihydrolipoyl
Succinyltransferase Protect Against Pressure Overload-Induced Cardiac
Hypertrophy and Dysfunction.Circulation,2017,136,747–761.
[9]Barglow,K.T.；Cravatt,B.F.Activity-based protein profiling for the
functional annotation of enzymes.Nat.Methods,2007,4,822–827.
[10]Cravatt,B.F.；Wright,A.T.；Kozarich,J.W.*Activity-Based Protein
Profiling:From Enzyme Chemistry to Proteomic Chemistry.Annu.Rev.Biochem.,
2008,77,383–414.
[11]Nomura,D.K.；Dix,M.M.；Cravatt,B.F.Activity-based protein profiling
for biochemical pathway discovery in cancer.Nat.Rev.Cancer,2010,10,630-638.
[12]Sanman,L.E.；Bogyo,M.Activity-Based Profiling of Proteases[J]
.Annu.Rev.Biochem.,2014,83,249-273.
Summary of the invention
One of the objects of the present invention is to provide a kind of chemical combination based on detection label dihydrolipoic acid succinyltransferase
Object；
It is a further object of the present invention to provide the midbody compounds of preparation above compound；
It is a further object of the present invention to provide the preparation methods of preparation above compound；
Further object of the present invention is to provide the composition containing above compound；
Further object of the present invention is to provide the purposes of above compound.
In order to achieve the above objectives, the application adopts the following technical solutions:
A kind of Compound Compound or its stereoisomer or its salt or its solvate, shown in following Formulas I:
Wherein R1Selected from any substituted alkylidene, imino group, any substituted or unsubstituted alkenylene, it is any replace or
Unsubstituted alkynylene, any substituted or unsubstituted cycloalkylidene, any substituted or unsubstituted arlydene or any substitution
Or unsubstituted inferior heteroaryl；Preferably, selected from any substituted or unsubstituted C1-6It is alkylidene, any substituted or unsubstituted
C1-6Arlydene；It is highly preferred that selected from methylene, ethylidene, propylidene, isopropylidene, butylidene, sub- sec-butyl, pentylidene,
Sub- neighbour's phenyl, it is sub- between phenyl, it is sub- between phenyl, sub- adjacent thienyl, it is sub- thienyl, sub- adjacent furyl, it is sub- furyl, sub- adjacent pyrrole
Piperidinyl, it is sub- between pyridyl group, it is sub- to pyridyl group, naphthylene, anthrylene,
R2Selected from hydrogen, alkyl, alkenyl, alkynyl, naphthenic base, aryl, heteroaryl；Preferably, it is selected from C1-6Alkyl or C1-6Virtue
Base；It is highly preferred that being selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, amyl or phenyl.
Most preferably, compound of formula I is selected from following compounds:
Probe 1:2- (mesyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 2:2- (ethylsulfonyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 3:2- (mesyl) -5- (3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 4:2- (ethylsulfonyl) -5- (3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 5:2- (mesyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 6:2- (ethylsulfonyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles.
A kind of compound of preparation of compounds of formula I, as shown in following formula 6:
Wherein R1And R2As described above.
A kind of compound of 6 compound of preparation formula, as shown in following formula 5:
Wherein R1And R2As described above.
A kind of compound of 5 compound of preparation formula, as shown in following formula 4:
Wherein R1And R2As described above.
A kind of compound of 4 compound of preparation formula, as shown in following formula 4:
Wherein R1As described above.
The preparation method of compound described in Formulas I, includes the following steps:
It preferably includes with the reaction step of 6 preparation of compounds of formula Compound I of formula；
It further preferably include with the reaction step of 5 preparation of compounds of formula of formula, 6 compound；
It still further preferably include with the reaction step of 4 preparation of compounds of formula of formula, 5 compound；
It still further preferably include with the reaction step of 3 preparation of compounds of formula of formula, 4 compound；
Most preferably, the preparation method includes: (1) with 3 preparation of compounds of formula of formula, 4 compound；(2) again with 4 chemical combination of formula
5 compound of object preparation formula；(3) again with 5 preparation of compounds of formula of formula, 6 compound；(4) again with 6 preparation of compounds of formula Compound I of formula
Reaction step.
Wherein it is preferred in step (1), 3 compound of formula, 4 chemical combination of preparation formula in the presence of KOH and dimethyl sulfate (second) esters
Object；Preferably, in step (2), 4 compound of formula, 5 compound of preparation formula in the presence of NBS and AIBN；Preferably, in step (3),
5 compound of formula, 6 compound of preparation formula in the presence of propargyl alcohol and NaH；Preferably, in step (4), 6 compound of formula is deposited in mCPBA
In lower preparation of compounds of formula I.
A kind of composition, containing above compound, and agriculturally available auxiliary agent or fungicide, insecticide or weeding
Agent；Or pharmaceutically available auxiliary agent.As known to those skilled in the art, the compound and fungicide, insecticide or weeding
When agent mixes, agriculturally available compound preparation is formed.
When the composition contains agriculturally available auxiliary agent, the dosage form of the composition is selected from missible oil (EC), pulvis
(DP), wettable powder (WP), granule (GR), aqua (AS), suspending agent (SC), ultra-low volume spray agent (ULV), solubility
Pulvis (SP), microcapsule formulations (MC), fumicants (FU), aqueous emulsion (EW), water-dispersible granules (WG).
When the composition contains pharmaceutically available auxiliary agent, the dosage form of the composition is selected from plain piece, film coating
Piece, sugar coated tablet, casing piece, dispersible tablet, capsule, granule, oral administration solution or oral administration mixed suspension.
The compound can be used for preventing and treating the purposes in terms of agricultural pest, it is preferable that the agricultural pest is to plant
Object is bacillary or fungal disease；It is highly preferred that the agricultural pest is plant leaf blight and plant canker；Most preferably
Ground, the agricultural pest are bacterial blight of rice, cucumber bacterial leaf-blight, tobacco bacterial wilt, konjaku bacterial leaf-blight, citrus ulcer
Disease, tobacco bacterial wilt, grape ulcer, canker of tomato, Prospect on Kiwifruit Bacterial Canker, apple canker, gray mold of cucumber, capsicum are withered
Wither disease, sclerotinia sclerotiorum, wheat scab, the late blight of potato, blueberry root-rot.
The compound, which can be used for preparing, treats or prevents Alzheimer disease or leukemia medicament；Preferably, it is making
In standby prevention Alzheimer disease or leukaemia in drug, the compound is prepared to the label detection dihydro sulphur in living cells
The reagent of sad succinyltransferase or purified reagent to the target protein after viable cell labelling.
It is further preferred that the preparation method of the compounds of this invention includes following specific steps:
(1) using containing methyl substituted benzoic acid and methanol, as raw material, back flow reaction 4-6h, has reacted under concentrated sulfuric acid catalyst
Methanol is removed in vacuum distillation afterwards, and different methyl formates 1 is obtained after column Chromatographic purification.
(2) using methyl formate as raw material, back flow reaction after 80% hydrazine hydrate is added.After system is cooling, there is white solid
It is precipitated, drying obtains different formylhydrazines 2 after washing.
(3) with the formylhydrazine of above-mentioned preparation, CS2And KOH is raw material, ethyl alcohol is that solvent refluxing reacts 36-48h, has been reacted
Ethyl alcohol is removed in vacuum distillation afterwards, obtains different mercaptan 3 after adjusting pH to 4 or so.
(4) dimethyl sulfate (second) esters are added using the mercaptan of above-mentioned preparation, KOH as raw material, thereto, stirring at normal temperature 2h, slightly
Product obtains corresponding thioether 4 through column Chromatographic purification.
(5) using the thioether of above-mentioned preparation, NBS as raw material, with CCl4For solvent, the back flow reaction under AIBN catalysis, reaction
CCl is removed in vacuum distillation after complete4, crude by column chromatography purifies to obtain different benzyl bromine substituted thioethers 5.
(6) using above-mentioned benzyl bromine substituted thioethers, propargyl alcohol as raw material, using DMF as solvent, 1.2 are added into system under ice bath
The NaH of times equivalent reacts 2h.It is extracted with saturated ammonium chloride and ethyl acetate, crude by column chromatography purifies to obtain different propine
Base substituted thioethers 6.
(7) using above-mentioned propinyl substituted thioethers as raw material, solvent is made with methylene chloride, is added under ice bath into system
MCPBA carries out oxidation reaction.Methylene chloride is removed in vacuum distillation, and crude by column chromatography purifies to obtain corresponding probe 1-6.
Wherein:
Column Chromatographic purification uses 200-300 mesh silica gel, and it is that petroleum ether is mixed with ethyl acetate that column, which chromatographs organic solvent used,
Solution.
A kind of molecular probe of dihydrolipoic acid succinyltransferase that detects in living cells of the present invention is to rice
Leaf spot bacteria and citrus processing have good bacteriostatic activity, IC of the probe 1-6 to rice leaf spot bacteria50Respectively
Are as follows: 3.58,7.95,5.51,13.94,3.99,6.80 μM, IC of the probe 1-4 to citrus processing50Respectively 17.53,
7.06、15.60、12.63μM。
The object of the invention is also to provide the purposes of the probe, and it is pungent that probe 1-6 is applied to dihydro sulphur in living cells
The detection of sour succinyltransferase.Experiment flow such as Fig. 1, specific implementation step are as follows:
(1) 10 μ L probe 1-6 are added to 990 μ L cell suspending liquids respectively, make its final concentration of 5 μM, 25 DEG C of incubation 2h.
(2) supernatant is abandoned after being centrifuged, is washed 3 times with PBS (pH 7.2), then cell is resuspended in 100 μ L PBS.
(3) sonicated cells under condition of ice bath respectively are centrifuged 30min, 12000rpm at 4 DEG C.
(4) supernatant protein is diluted to 1 μ g/ μ L, then takes 45 μ L.
(5) the 1 μ L biotin-N into supernatant respectively3, 1 μ L sodium ascorbate, 3 μ L BTTAA/CuSO4Mixed solution, 37 DEG C
Lower mixing 2h.
(6) 50 μ 2 × loading of L buffer are separately added into, boil 10min at 95 DEG C.
(7) 20 μ L are taken to carry out SDS-PAGE respectively in above-mentioned sample.
(8) by the protein delivery to pvdf membrane on gel, condition: transferring film 50min under 200mA electric current.
(9) it is washed film 3 times with the PBS solution (0.05%PBST) containing 0.05%tween-20, each 10min, then with 5%
Skimmed milk power closes 2h.
(10) it is washed film 3 times, each 10min with 0.05%PBST, the Streptavidin of HRP label is then added in ice bath
Lower incubation 2h.
(11) washed film 3 times, each 10min with 0.05%PBST, be added ECL luminescence reagent, then using chemiluminescence at
As system carries out imaging analysis.
Beneficial effects of the present invention:
Probe 1-6 of the present invention is that report for the first time can be used for the molecular probe of detection DLST albumen in living cells,
Not only the DLST in cell can effectively be detected at low concentrations, but also to intracellular DLST selection with higher
Property, and probe 1 and probe 2 can effectively purify the destination protein (DLST) after viable cell labelling.Probe 1-6 for
The functional study of DLST albumen is of great significance.
Detailed description of the invention
Fig. 1；Label process of the probe 1-6 to living cells
Fig. 2；In vivo marker of the probe 1-6 to Xoo cell
Fig. 3；Probe 1 marks the living body concentration dependent of Xoo
Fig. 4；Purifying of the probe 1 to DLST in Xoo cell
Fig. 5；In vivo marker of the probe 1-4 to Xac cell
Fig. 6；Probe 2 marks the living body concentration dependent of Xac
Fig. 7；Purifying of the probe 2 to DLST in Xac cell
Fig. 8；The concentration dependent label of 1 couple of probe recombinant expression DLST
Embodiment
Below by embodiment, the invention will be further described.It should be understood that the method for the embodiment of the present invention
It is only used for illustrating the present invention, rather than limiting the invention, to preparation side of the invention under concept thereof of the invention
The simple modifications of method belong to the scope of protection of present invention.All raw materials and solvent used in embodiment are commercially available phase
Answer the product of purity.
Embodiment 1: the preparation of probe 1-6
(1) first of 20 times of equivalents is added in 250mL round-bottomed flask in the benzoic acid for taking 0.20mol difference to replace thereto
Pure and mild 98% concentrated sulfuric acid of 4mL is then refluxed for reaction 4-6h.Vacuum distillation uses silica gel mixed sample after removing methanol, is mentioned by column chromatography
It is pure to obtain compound 1, yield 96-99%.
(2) it takes 0.15mol compound 1 in 100mL round-bottomed flask, 80% hydrazine hydrate of 2.5 times of equivalents is added thereto,
Back flow reaction 2h.After system is cooling, there is white solid precipitation, drying obtains compound 2, yield 77-91% after washing.
(3) it takes 0.15mol compound 2 in 250mL round-bottomed flask, KOH the and 100mL second of 2 times of equivalents is added thereto
2 times of equivalent CS are added dropwise after stirring and dissolving for alcohol2, back flow reaction 24-36h after stirring at normal temperature 8h.It is evaporated under reduced pressure after removing ethyl alcohol in ice
Bath is lower to adjust pH to 4-5 with 5% dilute hydrochloric acid, obtains crude product 3 after suction filtration.
(4) take 0.10mol crude product 3 in 100mL round-bottomed flask, the 50mL that the KOH containing 0.20mol is added thereto is water-soluble
Liquid is slowly added dropwise 2 times of equiv. sulfuric acid diformazan (second) esters, 2h is stirred at room temperature.Yellow solid is obtained after system is filtered, and takes a small amount of second
Silica gel mixed sample after acetoacetic ester dissolution, obtains compound 4, two step yield 36-53% after column Chromatographic purification.
(5) take 0.03mol compound 4 in 100mL round-bottomed flask, thereto the dry CCl of 50mL4, then it is added 1.2 times
The NBS and 0.1 times of equivalent AIBN, back flow reaction 48h of equivalent.Reaction system is filtered to be evaporated under reduced pressure after removing insoluble matter and is removed
CCl4, take a small amount of CH2Cl2Silica gel mixed sample after dissolved solid obtains compound 5, yield 43-77% through column Chromatographic purification.
(6) take 5mmol compound 5 and 1.1 times equivalent propargyl alcohol in 100mL round-bottomed flask, thereto be added 20mL without
Then water DMF, stirring and dissolving under ice bath are added 1.2 times of equivalent NaH and react 2h.It is molten that saturation 20mL ammonium chloride is added into system
Liquid and the extraction of 60mL ethyl acetate, water phase merges organic phase after being extracted 2 times with 50mL ethyl acetate, after solvent is removed in vacuum distillation
With silica gel mixed sample, compound 6, yield 43-66% are obtained after column Chromatographic purification.
(7) 5mmol compound 6 is taken to be dissolved in the dry CH of 15mL2Cl2, then stirring and dissolving under ice bath is added 3 into system
The mCPBA of times equivalent reacts 2h.Reaction system suction filtration is removed into solid insoluble, part CH is removed in vacuum distillation2Cl2, remaining
System silica gel mixed sample obtains target product probe 1-6, yield 40-70% after column Chromatographic purification.
The structural characterization of probe 1-6 is as follows:
Probe 1:2- (mesyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H
NMR(500MHz,CDCl3) δ 8.26 (dd, J=6.6,2.2Hz, 1H), 8.10 (ddd, J=8.5,4.8,2.3Hz, 1H), 7.25
(t, J=8.9Hz, 1H), 4.74 (s, 2H), 4.30 (d, J=2.4Hz, 2H), 3.53 (s, 3H), 2.52 (t, J=2.4Hz,
1H).13C NMR(125MHz,CDCl3)δ165.89,164.48,162.43,162.23,129.89,129.57,127.10,
118.53,116.95,78.98,75.54,64.53,58.24,43.08.HRMS(ESI)calculated for
C13H11FN2O4S[M+H]+m/z 311.0496,found 311.0493.
Probe 2:2- (ethylsulfonyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H
NMR(500MHz,CDCl3) δ 8.26 (dd, J=6.6,2.3Hz, 1H), 8.10 (ddd, J=8.5,4.8,2.3Hz, 1H), 7.25
(t, J=8.9Hz, 1H), 4.75 (s, 2H), 4.31 (d, J=2.4Hz, 2H), 3.62 (q, J=7.4Hz, 2H), 2.52 (t, J=
2.4Hz, 1H), 1.55 (t, J=7.4Hz, 3H)13C NMR(125MHz,CDCl3)δ165.90,164.46,162.42,
161.42,129.90,129.56,127.08,118.59,116.92,78.98,75.50,64.50,58.23,50.16,
6.92.HRMS(ESI)calculated for C14H13FN2O4S[M+H]+m/z 325.0653,found 325.0648.
Probe 3:2- (mesyl) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 8.12 (d, J=8.4Hz, 2H), 7.55 (d, J=8.5Hz, 2H), 4.70 (s, 2H), 4.26 (d, J=
2.4Hz, 2H), 3.53 (s, 3H), 2.51 (t, J=2.4Hz, 1H)13C NMR(125MHz,CDCl3)δ166.57,162.17,
143.50,128.49,127.96,121.44,79.21,75.28,70.78,57.88,43.08.HRMS(ESI)calculated
for C13H12N2O4S[M+H]+m/z 293.0591,found 293.0587.
Probe 4:2- (ethylsulfonyl) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 8.11 (d, J=8.3Hz, 2H), 7.55 (d, J=8.1Hz, 2H), 4.70 (s, 2H), 4.25 (d, J=
2.3Hz, 2H), 3.61 (q, J=7.4Hz, 2H), 2.50 (t, J=2.4Hz, 1H), 1.54 (t, J=7.4Hz, 3H)13C NMR
(125MHz,CDCl3)δ166.59,161.37,143.47,128.47,127.94,121.53,79.22,75.25,70.79,
57.87,50.17,6.91.HRMS(ESI)calculated for C14H14N2O4S[M+H]+m/z 307.0747,found
307.0744.
Probe 5:2- (mesyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3) δ 7.93 (d, J=8.5Hz, 1H), 7.54 (d, J=8.5Hz, 1H), 4.97 (s, 2H), 4.30
(d, J=2.4Hz, 2H), 3.54 (s, 3H), 2.52 (t, J=2.4Hz, 1H)13C NMR(125MHz,CDCl3)δ164.55,
162.69,141.97,136.88,135.66,132.18,129.01,121.33,79.12,75.33,66.34,58.53,
43.07.HRMS(ESI)calculated for C13H10N2O4SCl2[M+H]+m/z 360.9811,found360.9807.
Probe 6:2- (ethylsulfonyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3) δ 7.93 (d, J=8.5Hz, 1H), 7.54 (d, J=8.5Hz, 1H), 4.97 (s, 2H), 4.30
(d, J=2.4Hz, 2H), 3.62 (q, J=7.5Hz, 2H), 2.52 (t, J=2.4Hz, 1H), 1.56 (t, J=7.4Hz, 3H)
.13C NMR(125MHz,CDCl3)δ164.60,161.94,141.92,136.84,135.64,132.18,129.00,
121.42,79.12,75.30,66.33,58.51,50.25,6.94.HRMS(ESI)calculated for
C14H12N2O4SCl2[M+H]+m/z 374.9968,found 374.9966.
6 structural characterization of formula is as follows:
6-1:2- (methyl mercapto) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 8.09 (dd, J=6.8,2.2Hz, 1H), 7.96 (ddd, J=8.5,4.9,2.3Hz, 1H), 7.17 (t,
J=9.1Hz, 1H), 4.72 (s, 2H), 4.27 (d, J=2.4Hz, 2H), 2.78 (s, 3H), 2.51 (t, J=2.4Hz, 1H)13C
NMR(125MHz,CDCl3)δ165.27,165.04,163.56,161.54,128.74,128.42,126.10,120.14,
116.53,79.13,75.36,64.72,58.05,14.76.HRMS(ESI)calculated for C13H11FN2O2S[M+H]+
m/z 279.0598,found 279.0600.
6-2:2- (ethylmercapto group) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 8.09 (dd, J=6.7,2.2Hz, 1H), 7.96 (ddd, J=8.6,4.9,2.3Hz, 1H), 7.17 (t,
J=9.0Hz, 1H), 4.71 (s, 2H), 4.27 (d, J=2.4Hz, 2H), 3.31 (q, J=7.4Hz, 2H), 2.50 (t, J=
2.4Hz, 1H), 1.51 (t, J=7.4Hz, 3H)13C NMR(125MHz,CDCl3)δ164.91,164.58,163.57,
161.55,128.77,128.35,126.09,120.19,116.52,79.14,75.33,64.75,58.03,27.15,
14.82.HRMS(ESI)calculated for C14H13FN2O2S[M+H]+m/z 293.0755,found293.0757.
6-3:2- (methyl mercapto) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
CDCl3) δ 7.98 (d, J=8.3Hz, 2H), 7.48 (d, J=8.0Hz, 2H), 4.66 (s, 2H), 4.22 (d, J=2.4Hz,
2H), 2.77 (s, 3H), 2.49 (t, J=2.4Hz, 1H)13C NMR(125MHz,CDCl3)δ165.74,165.15,141.37,
128.40,126.85,123.16,79.36,75.15,70.97,57.66,14.75.HRMS(ESI)calculated for
C13H12N2O2S[M+H]+m/z 261.0692,found 261.0692.
6-4:2- (ethylmercapto group) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
CDCl3) δ 7.99 (d, J=6.7Hz, 2H), 7.48 (d, J=7.4Hz, 2H), 4.67 (s, 2H), 4.22 (t, J=2.1Hz,
2H), 3.32 (q, J=7.4Hz, 2H), 2.49 (dd, J=4.4,2.2Hz, 1H), 1.51 (t, J=7.4Hz, 3H)13C NMR
(125MHz,CDCl3)δ165.60,164.46,141.34,128.40,126.85,123.21,79.36,75.13,70.98,
57.65,27.14,14.84.HRMS(ESI)calculated for C14H14N2O2S[M+H]+m/z 275.0849,found
275.0849.
6-5:2- (methyl mercapto) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H
NMR(400MHz,CDCl3) δ 7.82 (d, J=8.5Hz, 1H), 7.46 (d, J=8.5Hz, 1H), 4.95 (s, 2H), 4.28 (d, J
=2.4Hz, 2H), 2.78 (s, 3H), 2.50 (t, J=2.4Hz, 1H)13C NMR(100MHz,CDCl3)δ166.12,
163.65,140.21,135.99,134.97,131.51,128.59,122.91,79.20,75.08,66.39,58.34,
14.64.HRMS(ESI)calculated for C13H10N2O2SCl2[M+H]+m/z 328.9913,found 328.9915.
6-6:2- (ethylmercapto group) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；1H
NMR(400MHz,CDCl3) δ 7.82 (d, J=8.5Hz, 1H), 7.46 (d, J=8.5Hz, 1H), 4.95 (s, 2H), 4.28 (d, J
=2.4Hz, 2H), 3.32 (q, J=7.4Hz, 2H), 2.50 (t, J=2.4Hz, 1H), 1.52 (t, J=7.4Hz, 3H)13C
NMR(100MHz,CDCl3)δ165.47,163.51,140.17,135.95,134.96,131.51,128.59,122.94,
79.21,75.08,66.40,58.34,27.09,14.78.HRMS(ESI)calculated for C14H12N2O2SCl2[M+H]+
m/z 343.0069,found 343.0072.
5 structural characterization of formula is as follows:
5-1:2- (methyl mercapto) -5- (3- (bromomethyl) -4- fluorine) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
DMSO-D6) δ 8.21 (d, J=7.0Hz, 1H), 8.03-7.96 (m, 1H), 7.48 (t, J=9.2Hz, 1H), 4.80 (s, 2H),
2.77(s,3H).13C NMR(125MHz,DMSO-D6)δ165.42,164.50,163.71,161.69,130.28,129.76,
127.53,120.50,117.83,26.40,14.88.HRMS(ESI)calculated for C10H8FN2OSBr[M+H]+m/
z302.9598,found 302.9600.
5-2:2- (ethylmercapto group) -5- (3- (bromomethyl) -4- fluorine) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
CDCl3) δ 8.07 (dd, J=7.0,2.2Hz, 1H), 7.97 (ddd, J=8.6,4.8,2.3Hz, 1H), 7.20 (t, J=
9.0Hz, 1H), 4.54 (s, 2H), 3.33 (q, J=7.4Hz, 2H), 1.52 (t, J=7.4Hz, 3H)13C NMR(125MHz,
CDCl3)δ164.77,164.47,163.51,161.47,129.93,129.06,126.58,120.59,116.91,27.18,
24.51,14.79.HRMS(ESI)calculated for C11H10FN2OSBr[M+H]+m/z 316.9754,
found316.9759.
5-3:2- (methyl mercapto) -5- (4- (bromomethyl)) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3)δ
7.98 (d, J=8.4Hz, 2H), 7.51 (d, J=8.3Hz, 2H), 4.51 (s, 2H), 2.78 (s, 3H)13C NMR(125MHz,
CDCl3)δ165.42,165.39,141.40,129.82,127.15,123.63,32.39,14.75.HRMS(ESI)
calculated for C10H9N2OSBr[M+H]+m/z 284.9692,found 284.9695.
5-4:2- (ethylmercapto group) -5- (4- (bromomethyl)) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3)δ
7.98 (d, J=8.4Hz, 2H), 7.51 (d, J=8.3Hz, 2H), 4.51 (s, 2H), 3.32 (q, J=7.4Hz, 2H), 1.52
(t, J=7.4Hz, 3H)13C NMR(125MHz,CDCl3)δ165.28,164.71,141.38,129.81,127.15,
123.68,32.39,27.15,14.83.HRMS(ESI)calculated for C11H11N2OSBr[M+H]+m/z
298.9848,found 298.9851.
5-5:2- (methyl mercapto) -5- (bis- chloro- 3- (bromomethyl) of 2,4-) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 7.82 (d, J=8.5Hz, 1H), 7.48 (d, J=8.5Hz, 1H), 4.83 (s, 2H), 2.79 (s, 3H)
.13C NMR(125MHz,CDCl3)δ166.34,163.48,139.03,135.85,134.79,131.26,128.83,
123.15,27.35,14.76.HRMS(ESI)calculated for C10H7N2OSCl2Br[M+H]+m/z 352.8912,
found 352.8914.
5-6:2- (ethylmercapto group) -5- (bis- chloro- 3- (bromomethyl) of 2,4-) phenyl -1,3,4- oxadiazoles；1H NMR
(500MHz,CDCl3) δ 7.84 (d, J=8.6Hz, 1H), 7.48 (d, J=8.5Hz, 1H), 4.84 (s, 2H), 3.34 (q, J=
7.4Hz, 2H), 1.54 (t, J=6.8Hz, 3H)13C NMR(125MHz,CDCl3)δ165.69,163.33,138.99,
135.83,134.76,131.26,128.83,123.18,27.37,27.19,14.86.HRMS(ESI)calculated for
C11H9N2OSCl2Br[M+H]+m/z 366.9069,found 366.9071.
4 structural characterization of formula is as follows:
4-1:2- (methyl mercapto) -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,DMSO-D6)
δ 7.91 (d, J=7.1Hz, 1H), 7.85-7.78 (m, 1H), 7.35 (t, J=9.1Hz, 1H), 2.76 (s, 3H), 2.31 (s,
3H).13C NMR(125MHz,DMSO-D6)δ165.12,164.91,164.15,162.16,130.37,126.82,126.59,
119.92,116.83,14.84,14.51.HRMS(ESI)calculated for C10H9FN2OS[M+H]+m/z225.0492,
found 225.0493.
4-2:2- (ethylmercapto group) -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；1H NMR(400MHz,CDCl3)δ
7.87 (d, J=7.2Hz, 1H), 7.84-7.77 (m, 1H), 7.11 (t, J=8.9Hz, 1H), 3.31 (q, J=7.4Hz, 2H),
2.34 (s, 3H), 1.51 (t, J=7.4Hz, 3H)13C NMR(100MHz,CDCl3)δ165.09,164.56,164.25,
162.06,130.12,126.27,126.09,119.69,116.04,27.07,14.74,14.53.HRMS(ESI)
calculated for C11H11FN2OS[M+H]+m/z 239.0649,found 239.0647.
4-3:2- (methyl mercapto) -5- (4- methyl) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3)δ7.88
(d, J=8.2Hz, 2H), 7.28 (d, J=8.5Hz, 2H), 2.77 (s, 3H), 2.41 (s, 3H)13C NMR(125MHz,
CDCl3)δ166.07,164.74,142.24,129.82,126.69,120.97,21.74,14.75.HRMS(ESI)
calculated for C10H10N2OS[M+H]+m/z 207.0587,found 207.0588.
4-4:2- (ethylmercapto group) -5- (4- methyl) phenyl -1,3,4- oxadiazoles；1H NMR(400MHz,CDCl3)δ7.88
(d, J=8.2Hz, 2H), 7.28 (d, J=8.0Hz, 2H), 3.30 (q, J=7.4Hz, 2H), 2.41 (s, 3H), 1.50 (t, J=
7.4Hz,3H).13C NMR(100MHz,CDCl3)δ165.86,163.95,142.13,129.73,126.62,120.95,
27.05,21.64,14.78.HRMS(ESI)calculated for C11H12N2OS[M+H]+m/z 221.0743,
found221.0744.
4-5:2- (methyl mercapto) -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
CDCl3) (s, the 3H) of δ 7.66 (d, J=8.5Hz, 1H), 7.41 (d, J=8.5Hz, 1H), 2.78 (s, 3H), 2.5613C NMR
(125MHz,CDCl3)δ165.97,164.10,138.75,136.70,134.40,128.80,127.98,122.31,18.15,
14.73.HRMS(ESI)calculated for C10H8N2OSCl2[M+H]+m/z 274.9807,found 274.9807.
4-6:2- (ethylmercapto group) -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,
CDCl3) δ 7.67 (d, J=8.5Hz, 1H), 7.41 (d, J=8.5Hz, 1H), 3.32 (q, J=7.4Hz, 2H), 2.56 (s,
3H), 1.52 (t, J=7.4Hz, 3H)13C NMR(125MHz,CDCl3)δ165.31,163.95,138.72,136.68,
134.37,128.80,127.98,122.35,27.15,18.14,14.87.HRMS(ESI)calculated for
C11H10N2OSCl2[M+H]+m/z 288.9964,found 288.9965.
3 structural characterization of formula is as follows:
3-1:2- sulfydryl -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,DMSO-D6)1H
NMR (500MHz, DMSO-D6) δ 14.74 (s, 1H), 7.84 (d, J=6.8Hz, 1H), 7.79-7.68 (m, 1H), 7.35 (t, J
=9.1Hz, 1H), 2.31 (s, 3H) δ 177.39,163.82,161.83,159.88,129.64,126.19,118.82,
116.22,14.01.
3-2:2- sulfydryl -5- (4- methyl) phenyl -1,3,4- oxadiazoles；1H NMR(400MHz,CDCl3)δ11.14(s,
1H), 7.82 (d, J=8.3Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 2.43 (s, 3H)13C NMR(100MHz,CDCl3)δ
178.24,161.98,143.53,130.11,126.66,119.74,21.91.
3-3:2- sulfydryl -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles；1H NMR(500MHz,CDCl3)δ
7.41 (s, 1H), 7.32 (d, J=8.3Hz, 1H), 7.26 (d, J=8.3Hz, 1H), 2.49 (s, 3H)13C NMR(125MHz,
CDCl3)δ167.83,137.70,135.96,132.99,132.47,127.99,127.22,17.93.
Embodiment 2: probe 1-6 503nhibiting concentration (IC50) test
Probe 1-6 is made into the toxic fluid nutrient medium of 5 respective concentrations, takes 5mL in test tube, is contained with microplate reader measurement
Medicine aseptic liquid nutrient medium OD600Value is added 40 μ L and contains rice leaf spot bacteria or citrus processing NB fluid nutrient medium
(3g containing beef peptone in every liter of culture medium, peptone 5g, yeast powder 1g, glucose 10g, pH 7.0-7.2), then 28 DEG C,
Shaking table culture 36-48h under the conditions of 180rpm measures the OD of each concentration bacterium solution with microplate reader600Value.
Correct OD value=value of OD containing bacterium culture medium-aseptic culture medium OD value
Culture is compareed after preventive effect %=(control medium bacterium solution OD value-toxic culture medium OD value of correction after correction)/correction
Value × 100 base bacterium solution OD
Inhibiting rate data conversion is converted into logarithm (x) at probit value (y), drug concentration (μ g/mL), in Excel number
According to regression analysis is carried out in processing software, virulence regression equation (y=ax+b) and related coefficient (R) are obtained, calculates medicament to disease
Opportunistic pathogen EC50Value, the results are shown in Table 1 and table 2.According to the above method, the bacteriostatic activity of probe 1-6 is shown in Tables 1 and 2
IC of the 1 probe 1-6 of table to rice leaf spot bacteria50Value
Higher bacteriostatic activity is shown to rice leaf spot bacteria from probe 1-6 known to 1 active testing result of table,
IC50Respectively 3.58 ± 0.03,7.95 ± 0.09,5.51 ± 0.03,13.94 ± 0.03,3.99 ± 0.08,6.80 ± 0.16 μ
M is suitable with comparison medicament methylsulfonyl bacterium azoles (3.51 ± 0.04 μM).
IC of the 2 probe 1-4 of table to citrus processing50Value
Higher bacteriostatic activity is shown to citrus processing from probe 1-4 known to 2 active testing result of table,
IC50Respectively 17.53 ± 0.10,7.06 ± 0.25,15.60 ± 0.14,12.63 ± 0.33 μM, with comparison medicament methylsulfonyl bacterium
Azoles (8.59 ± 0.16 μM) is suitable.
IC of 3 probe of table to tobacco ralstonia solanacearum50Value
From probe 2 (224.59 ± 14.55 μM) known to 3 active testing result of table to the activity and methylsulphur of tobacco ralstonia solanacearum
(65.06 ± 6.98 μM) of acyl bacterium azoles are compared about 4 times of decline, and probe 3 (84.67 ± 6.77 μM) is to tobacco ralstonia solanacearum activity and first
Sulphonyl bacterium azoles (65.06 ± 6.98 μM) is suitable.
Embodiment 3: probe 1-6 is in living cells to the labelling experiment of DLST
1, in vivo marker of the probe 1-6 to rice leaf spot bacteria (Xoo)
2mL OD is taken respectively600=0.6 Xoo bacterium solution, abandons supernatant after centrifugation, then with 0.99mL PBS (pH=7.2) weight
Outstanding cell.10 μ L probe 1-6 are separately added into, making its final concentration is 5 μM, and 10 μ L DMSO, 25 DEG C of incubation 2h are added in control group.
Supernatant is abandoned after centrifugation, PBS is added 100 μ L PBS and cell, sonicated cells under ice bath are resuspended after washing 3 times.By egg after centrifugation
It is white to be diluted to 1 μ g/ μ L, 45 μ L supernatants are taken respectively, are then respectively adding biotin-N3, sodium ascorbate and BTTAA/CuSO4It is mixed
It closes solution and carries out click reaction.50 μ L 2 × loading buffer are added into sample respectively again, are boiled at 95 DEG C
10min.20 μ L samples are taken to carry out SDS-PAGE respectively, then the transferring film 50min under 200mA constant current, PBST is taken off after washing film with 5%
Rouge milk powder closes 2h.The Streptavidin (HRP-Streptavidin) that HRP label is added after film is cleaned with PBST is incubated for 2h,
After washing film with PBST again, chemical illuminating reagent is added, imaging analysis (such as Fig. 2) is carried out by chemiluminescence imaging system.From
In Fig. 2 it can be seen that probe 1-6 under 5 μM of concentration can DLST albumen in significant notation cell, and in control group experiment not
DLST can be marked.
2, concentration dependent labelling experiment of the probe 1 to Xoo cell
2mL OD is taken respectively600=0.6 Xoo bacterium solution, abandons supernatant after centrifugation, then with 0.99mL PBS (pH=7.2) weight
Outstanding cell.It is separately added into the probe 1 of 10 μ L various concentrations, making its final concentration is respectively 0.5,1,2.5,5,10 μM, and control group adds
Enter 10 μ L DMSO, is incubated for 2h at 25 DEG C respectively.Supernatant is abandoned after centrifugation, PBS is added 100 μ L PBS and is resuspended carefully after washing 3 times
Born of the same parents, sonicated cells under ice bath.Albumen is diluted to 1 μ g/ μ L after centrifugation, 45 μ L supernatants is taken respectively, is then respectively adding
biotin-N3, sodium ascorbate and BTTAA/CuSO4Mixed solution carries out click reaction, and 50 μ L 2 are added into sample respectively
× loading buffer, boils 10min at 95 DEG C.20 μ L samples are taken to carry out SDS-PAGE respectively, then in 200mA constant current
Lower transferring film 50min, PBST are washed after film and are closed 2h with 5% skimmed milk power.The strepto- parent of HRP label is added after film is cleaned with PBST
Be incubated for 2h with plain (HRP-Streptavidin), after washing film with PBST again, chemical illuminating reagent be added, by chemiluminescence at
As system carries out imaging analysis (such as Fig. 3).As can be seen from Figure 3 probe 1 has concentration to DLST under 0.5-10 μM of concentration
The label effect of dependence, and to DLST selectivity with higher.
3, purification experiment of the probe 1 to DLST in Xoo cell
20mL OD is taken respectively600=0.6 Xoo bacterium solution, abandons supernatant after centrifugation, then use 0.99mL PBS (pH=7.2)
Cell is resuspended.10 μ L probes 1 are added in experimental group, make its final concentration of 100 μM, 10 μ L DMSO are added in control group, respectively at 25 DEG C
Lower incubation 2h.Supernatant is abandoned after centrifugation, PBS is added 1000 μ L PBS and cell, sonicated cells under ice bath are resuspended after washing 3 times.
The albumen of experimental group and blank group is diluted to same concentrations after centrifugation, 950 μ L supernatants is taken respectively, is then respectively adding
biotin-N3, sodium ascorbate and BTTAA/CuSO4Mixed solution makes 1000 μ L of its final volume, then mixes 2h at 37 DEG C.
The pre- cold acetone of 5 times of volumes is added into system respectively, is centrifuged after -80 DEG C of freezing 2h, abandons supernatant, precipitating is washed 3 with methanol
Secondary, albumen precipitation 1mL 0.2%SDS/PBS will be dissolved after solvent by volatilizing, and be then respectively adding 1mL Streptavidin MagneSphere
(streptavidin bead, Promega), mixes 2h at room temperature.Sample is placed on magnetic frame respectively, supernatant is abandoned, by magnetic bead
It is washed 3 times with 6M urea, 1%SDS/PBS, PBS respectively, is then respectively adding 30 μ L PBS and 30 2 × loading of μ L
Buffer boils 10min at 95 DEG C.It takes 40 μ L samples to carry out SDS-PAGE respectively, then carries out coomassie brilliant blue staining (such as
Fig. 4), as can be seen from Figure 4 probe 1 effectively can carry out enriching and purifying to the DLST albumen in Xoo cell, and control group then can not
DLST is effectively purified.
4, in vivo marker of the probe 1-4 to citrus processing (Xac)
2mL OD is taken respectively600=0.6 Xac bacterium solution, abandons supernatant after centrifugation, then with 0.99mL PBS (pH=7.2) weight
Outstanding cell.10 μ L probe 1-4 are separately added into, making its final concentration is 50 μM, and 10 μ L DMSO, 25 DEG C of incubation 2h are added in control group.
Supernatant is abandoned after centrifugation, PBS is added 100 μ L PBS and cell, sonicated cells under ice bath are resuspended after washing 3 times.By egg after centrifugation
It is white to be diluted to 1 μ g/ μ L, 45 μ L supernatants are taken respectively, are then respectively adding biotin-N3, sodium ascorbate and BTTAA/CuSO4It is mixed
It closes solution and carries out click reaction, 50 μ L 2 × loading buffer are added into sample respectively, boil 10min at 95 DEG C.
20 μ L samples are taken to carry out SDS-PAGE respectively, the then transferring film 50min under 200mA constant current, PBST are washed after film with 5% skimmed milk power
Close 2h.The Streptavidin (HRP-Streptavidin) that HRP label is added after film is cleaned with PBST is incubated for 2h, uses again
After PBST washes film, chemical illuminating reagent is added, imaging analysis (such as Fig. 5) is carried out by chemiluminescence imaging system.It can from Fig. 5
With find out probe 1-4 under 50 μM of concentration can DLST albumen in significant notation Xac cell, and fail to mark in control group experiment
Remember DLST.
5, concentration dependent labelling experiment of the probe 2 to Xac cell
2mL OD is taken respectively600=0.6 Xac bacterium solution, abandons supernatant after centrifugation, then with 0.99mL PBS (pH=7.2) weight
Outstanding cell.It is separately added into the probe 2 of 10 μ L various concentrations, making its final concentration is respectively 12.5,25,50,75,100 μM, control group
10 μ L DMSO are added, are incubated for 2h at 25 DEG C respectively.Supernatant is abandoned after centrifugation, PBS is added 100 μ L PBS and is resuspended carefully after washing 3 times
Born of the same parents, sonicated cells under ice bath.Albumen is diluted to 1 μ g/ μ L after centrifugation, 45 μ L supernatants is taken respectively, is then respectively adding
biotin-N3, sodium ascorbate and BTTAA/CuSO4Mixed solution carries out click reaction, and 50 μ L 2 are added into sample respectively
× loading buffer, boils 10min at 95 DEG C.20 μ L samples are taken to carry out SDS-PAGE respectively, then in 200mA constant current
Lower transferring film 50min, PBST are washed after film and are closed 2h with 5% skimmed milk power.The strepto- parent of HRP label is added after film is cleaned with PBST
Be incubated for 2h with plain (HRP-Streptavidin), after washing film with PBST again, chemical illuminating reagent be added, by chemiluminescence at
As system carries out imaging analysis (such as Fig. 6).As can be seen from Figure 6 probe 2 has DLST dense under 12.5-100 μM of concentration
The label effect of dependence is spent, and to DLST selectivity with higher.
6, purification experiment of the probe 2 to DLST in Xac cell
20mL OD is taken respectively600=0.6 Xac bacterium solution, abandons supernatant after centrifugation, then use 0.99mL PBS (pH=7.2)
Cell is resuspended.10 μ L probes 2 are added in experimental group, make its final concentration of 200 μM, 10 μ L DMSO are added in control group, respectively at 25 DEG C
Lower incubation 2h.Supernatant is abandoned after centrifugation, PBS is added 1000 μ L PBS and cell, sonicated cells under ice bath are resuspended after washing 3 times.
The albumen of experimental group and blank group is diluted to same concentrations after centrifugation, 950 μ L supernatants is taken respectively, is then respectively adding
biotin-N3, sodium ascorbate and BTTAA/CuSO4Mixed solution makes 1000 μ L of its final volume, then mixes 2h at 37 DEG C.
The pre- cold acetone of 5 times of volumes is added into system respectively, is centrifuged after -80 DEG C of freezing 2h, abandons supernatant, precipitating is washed 3 with methanol
Secondary, albumen precipitation 1mL 0.2%SDS/PBS will be dissolved after solvent by volatilizing, and be then respectively adding 1mL Streptavidin MagneSphere
(Streptavidin bead), mixes 2h at room temperature.Sample is placed on magnetic frame respectively, supernatant is abandoned, magnetic bead is used into 6M respectively
Urea, 1%SDS/PBS, PBS are washed 3 times, 30 μ L PBS and 30 μ L 2 × loading buffer are then respectively adding, 95
10min is boiled at DEG C.It takes 40 μ L samples to carry out SDS-PAGE respectively, then coomassie brilliant blue staining (such as Fig. 7) is carried out, from Fig. 7
In it can be seen that probe 2 effectively can carry out enriching and purifying to the DLST albumen in Xac cell, control group can not then carry out DLST
Effectively purifying.
Embodiment 4: the concentration dependent labelling experiment of 1 couple of probe recombinant expression DLST
It takes 100ng to recombinate rice leaf spot bacteria DLST (44 μ L PBS of in) respectively, it is dense to be then respectively adding 1 μ L difference
The probe 1 of degree, making its final concentration is respectively 0.5,1,2.5,5,10 μM, and 1 μ L DMSO is added in control group, is incubated at 25 DEG C respectively
Educate 2h.Then biotin-N is added into system respectively3, sodium ascorbate and BTTAA/CuSO4Mixed solution mixes at 37 DEG C
Close 2h.50 μ L 2 × loading buffer are added into system respectively again, boil 10min at 95 DEG C.40 μ L samples are taken respectively
Product carry out SDS-PAGE, then the transferring film 50min under 200mA constant current, and PBST is washed after film and closed 2h with 5% skimmed milk power.By film
The Streptavidin (HRP-Streptavidin) that HRP label is added after being cleaned with PBST is incubated for 2h, after washing film with PBST again,
Chemical illuminating reagent is added, imaging analysis (such as Fig. 8) is carried out by chemiluminescence imaging system.As can be seen from Figure 8 probe 1
Under 0.5-10 μM of concentration, there is the mode of action of concentration dependent to recombinant expression DLST.
The above description is only a preferred embodiment of the present invention, and embodiments of the present invention are not by the limit of above-described embodiment
System.For those skilled in the art, still can continue to make improvement without departing from the principle of the present invention, these change
Into should also be included within protection scope of the present invention.
Claims (13)
1. a kind of compound or its stereoisomer or its salt or its solvate, it is characterised in that shown in following Formulas I:
Wherein R1It does not take selected from any substituted alkylidene, imino group, any substituted or unsubstituted alkenylene, any substitution or
The alkynylene in generation, any substituted or unsubstituted cycloalkylidene, any substituted or unsubstituted arlydene any replace or not
Substituted inferior heteroaryl；Preferably, selected from any substituted or unsubstituted C1-6Alkylidene, any substituted or unsubstituted C1-6It is sub-
Aryl；It is highly preferred that selected from methylene, ethylidene, propylidene, isopropylidene, butylidene, sub- sec-butyl, pentylidene, sub- adjacent benzene
Base, it is sub- between phenyl, it is sub- between phenyl, sub- adjacent thienyl, it is sub- thienyl, sub- adjacent furyl, it is sub- furyl, sub- adjacent pyridyl group,
Pyridyl group, Asia are to pyridyl group, naphthylene, anthrylene, sub- halogenophenyl between Asia；Most preferably, it is selected from
R2Selected from hydrogen, alkyl, alkenyl, alkynyl, naphthenic base, aryl, heteroaryl；Preferably, it is selected from C1-6Alkyl or C1-6Aryl；More
Preferably, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, amyl or phenyl are selected from.
2. compound according to claim 1 or its stereoisomer or its salt or its solvate, it is characterised in that choosing
From following compounds:
Probe 1:2- (mesyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 2:2- (ethylsulfonyl) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 3:2- (mesyl) -5- (3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 4:2- (ethylsulfonyl) -5- (3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 5:2- (mesyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
Probe 6:2- (ethylsulfonyl) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles.
3. a kind of compound for preparing claim 1 compounds of formula I, it is characterised in that as shown in following formula 6:
Wherein R1And R2As described in claim 1；Preferably, 6 compound of formula are as follows:
6-1:2- (methyl mercapto) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
6-2:2- (ethylmercapto group) -5- (the fluoro- 3- of 4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
6-3:2- (methyl mercapto) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
6-4:2- (ethylmercapto group) -5- (4- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
6-5:2- (methyl mercapto) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles；
6-6:2- (ethylmercapto group) -5- (2,4- bis- chloro- 3- (propinyl oxygroup) methyl) phenyl -1,3,4- oxadiazoles.
4. a kind of compound of 6 compound of preparation formula, it is characterised in that as shown in following formula 5:
Wherein R1And R2As described in claim 1, it is preferable that 5 compound of formula are as follows:
5-1:2- (methyl mercapto) -5- (3- (bromomethyl) -4- fluorine) phenyl -1,3,4- oxadiazoles；
5-2:2- (ethylmercapto group) -5- (3- (bromomethyl) -4- fluorine) phenyl -1,3,4- oxadiazoles；
5-3:2- (methyl mercapto) -5- (4- (bromomethyl)) phenyl -1,3,4- oxadiazoles；
5-4:2- (ethylmercapto group) -5- (4- (bromomethyl)) phenyl -1,3,4- oxadiazoles；
5-5:2- (methyl mercapto) -5- (bis- chloro- 3- (bromomethyl) of 2,4-) phenyl -1,3,4- oxadiazoles；
5-6:2- (ethylmercapto group) -5- (bis- chloro- 3- (bromomethyl) of 2,4-) phenyl -1,3,4- oxadiazoles.
5. a kind of compound of 5 compound of preparation formula, it is characterised in that as shown in following formula 4:
Wherein R1And R2As described in claim 1；Preferably, 4 compound of formula are as follows:
4-1:2- (methyl mercapto) -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；
4-2:2- (ethylmercapto group) -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；
4-3:2- (methyl mercapto) -5- (4- methyl) phenyl -1,3,4- oxadiazoles；
4-4:2- (ethylmercapto group) -5- (4- methyl) phenyl -1,3,4- oxadiazoles；
4-5:2- (methyl mercapto) -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles；
4-6:2- (ethylmercapto group) -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles.
6. a kind of compound of 4 compound of preparation formula, it is characterised in that as shown in following formula 3:
Wherein R1As described in claim 1；Preferably, 3 compound of formula are as follows:
3-1:2- sulfydryl -5- (3- methyl -4- fluorine) phenyl -1,3,4- oxadiazoles；
3-2:2- sulfydryl -5- (4- methyl) phenyl -1,3,4- oxadiazoles；
3-3:2- sulfydryl -5- (the chloro- 3- methyl of 2,4- bis-) phenyl -1,3,4- oxadiazoles.
7. the preparation method of compound described in claim 1, it is characterised in that include the following steps:
It preferably includes with the reaction step of 6 preparation of compounds of formula Compound I of formula；
It further preferably include with the reaction step of 5 preparation of compounds of formula of formula, 6 compound；
It still further preferably include with the reaction step of 4 preparation of compounds of formula of formula, 5 compound；
It still further preferably include with the reaction step of 3 preparation of compounds of formula of formula, 4 compound；
Most preferably, the preparation method includes: (1) with 3 preparation of compounds of formula of formula, 4 compound；(2) again with 4 compound system of formula
Standby 5 compound of formula；(3) again with 5 preparation of compounds of formula of formula, 6 compound；(4) again with the reaction of 6 preparation of compounds of formula Compound I of formula
Step.
8. preparation method according to claim 7, it is characterised in that: preferably, in step (1), 3 compound of formula is in KOH
With 4 compound of preparation formula in the presence of dimethyl sulfate (second) esters；
Preferably, in step (2), 4 compound of formula, 5 compound of preparation formula in the presence of NBS and AIBN；Preferably, step (3)
In, 5 compound of formula, 6 compound of preparation formula in the presence of propargyl alcohol and NaH；Preferably, in step (4), 6 compound of formula exists
Preparation of compounds of formula I in the presence of mCPBA.
9. a kind of composition, it is characterised in that containing compound of any of claims 1 or 2, and agriculturally available auxiliary agent
Or fungicide, insecticide or herbicide；Or pharmaceutically available auxiliary agent；When the composition contains agriculturally available auxiliary agent
When, the dosage form of the composition be selected from missible oil (EC), pulvis (DP), wettable powder (WP), granule (GR), aqua (AS),
Suspending agent (SC), ultra-low volume spray agent (ULV), soluble powder (SP), microcapsule formulations (MC), fumicants (FU), aqueous emulsion
(EW), water-dispersible granules (WG)；When the composition contains pharmaceutically available auxiliary agent, the dosage form of the composition is selected from
Plain piece, thin membrane coated tablet, sugar coated tablet, casing piece, dispersible tablet, capsule, granule, oral administration solution or oral administration mixed suspension.
10. purposes of the compound of any of claims 1 or 2 in terms of preventing and treating agricultural pest, it is preferable that the agricultural disease pest
Evil is vegetative bacteria or fungal disease；It is highly preferred that the agricultural pest is plant leaf blight and plant canker；Most
Preferably, the agricultural pest is bacterial blight of rice, tobacco bacterial wilt, cucumber bacterial leaf-blight, konjaku bacterial leaf-blight, citrus
Canker, grape ulcer, canker of tomato, Prospect on Kiwifruit Bacterial Canker, apple canker, gray mold of cucumber, capsicum wilt, oil
Dish sclerotiniose, wheat scab, the late blight of potato, blueberry root-rot.
11. compound of any of claims 1 or 2 is in terms of preparation treats or prevents Alzheimer disease or leukemia medicament
Purposes；Preferably, in preparation prevention Alzheimer disease, leukaemia or cardiovascular disease medicine, the compound is produced
At the reagent of label detection dihydrolipoic acid succinyltransferase in living cells or to the target protein after viable cell labelling
Purified reagent.
12. a kind of method for preventing and treating agricultural pest, it is characterised in that: make compound of formula I of any of claims 1 or 2, or power
Benefit requires 9 composition to act on nuisance or its living environment；Preferably, the agricultural pest is vegetative bacteria or true
Fungal disease；It is highly preferred that the agricultural pest is bacterial blight of rice, cucumber bacterial leaf-blight, konjaku bacterial leaf-blight, citrus
Canker, tobacco bacterial wilt, grape ulcer, canker of tomato, Prospect on Kiwifruit Bacterial Canker, apple canker.
13. the method for protecting the plants from agricultural pest infringement comprising wherein make plant and claims 1 or 2 institute
The compound for the Formulas I stated or the method and step contacted with the composition of claim 9.
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