CN108456197A - Optoacoustic ratio nano-probe and the preparation method and application thereof for In vivo detection hydrogen sulfide - Google Patents
Optoacoustic ratio nano-probe and the preparation method and application thereof for In vivo detection hydrogen sulfide Download PDFInfo
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Abstract
The optoacoustic ratio nano-probe and the preparation method and application thereof that the invention discloses a kind of for In vivo detection hydrogen sulfide.The structural formula for being used for the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide is shown in formula I.The preparation method of the probe is mainly raw material by nucleophilic displacement of fluorine using Cy7 Cl, and knoevenagel condensation, amino azido reaction obtains probe.The problem of scattering of fluorescence signal that the present invention overcomes fluorescence probes in In vivo detection and tissue resorption, probe obtained has photoacoustic signal penetration depth big, realize the imaging of deep tissues, reaction speed is fast, specificity is good, the more accurate advantage of ratio test can be used for In vivo detection hydrogen sulfide, or for the sulfurated hydrogen detection in water body, food.
Description
Technical field
The invention belongs to tumor-marker analyte detection and imaging fields, more particularly to a kind of light for In vivo detection hydrogen sulfide
Acoustic ratio rate nano-probe and the preparation method and application thereof.
Background technology
Hydrogen sulfide is a kind of toxic gas with rotten egg smell, hydrogen sulfide still after nitric oxide and carbon monoxide it
The gasotransmitter of the 3rd kind of biosynthesis afterwards, hydrogen sulfide all plays an important role in the physiology course of many biologies, such as blood vessel
The regulation and control of the inhibition inflammation of the adjusting insulin signaling of the relaxation neurotransmission of smooth muscle and the perception effect of oxygen etc.；Its
Also there is the protective effect to heart and nerve in human body, and be effective neuromodulator, H2S physiological relevant concentrations
Range is differed from nanomole grade to a mM grade level.Under physiological concentration level, H2S participates in a series of physiological regulating control mistake
Journey, such as adjust antiotasis, myocardial contraction nerve conduction and insulin secretion etc..Cell cannot once maintain it normal
H2S concentration will cause the diseases such as artery and pulmonary hypertension, Alzheimer's disease, gastric mucosa damage and hepatic sclerosis.
[G.D.Yang,L.Y.Wu,B.Jiang,W.Yang,J.S.Qi,K.Cao,Q.H.Meng,A.K.Mustafa,W.T.Mu,
S.M.Zhang, S.H.Snyder, R.Wang, Science2008,322,587] in addition, H2S also can Scavenger of ROS and activity
Nitrogen species.Many researchs also show between three gasotransmitter hydrogen sulfide, nitric oxide and carbon monoxide also by various interactions
Acting regulatory the health and disease of human body.Hydrogen sulfide high expression in carcinoma of the colon and rectum tumour, can promote cancer cell multiplication, can
Using as a kind of tumor markers of carcinoma of the colon and rectum [(a) C.Szabo, C.Coletta, C.Chao, K.M ' odis,
B.Szczesny,A.Papapetropoulos and M.R.Hellmich,Proc.Natl.Acad.Sci.U.S.A.,2013,
110,12474.(b)B.Wu,H.Teng,G.Yang,L.Wu and R.Wang,Br.J.Pharmacol.,2012,167,
1492；(c)K.Zhao,S.Li,L.Wu,C.Lai and G.Yang,J.Biol.Chem.,2014,289,20824.].Therefore,
The detection of hydrogen sulfide seems quite important for being best understood from its biological function in living biological systems.
Currently, mainly the most commonly used is methylene blue laws and sulphion selectivity for detection and imaging for detection hydrogen sulfide
Electrode detection method, its shortcoming is that all there is homogenate that is destructive and needing sample, for the more physiological function details of hydrogen sulfide
Research, then need fast development of the new detection method with non-destructive testing technologies such as fluorescence imaging fluorescence probes, it is many
Novel detection method is come into being [a) Li, H.D.；Yao,Q.C.；Fan,J.L.；Jiang,N.；Wang,J.Y.；Xia,J.；
Peng,X.J.Chem.Commun.2015,51,16225-16228.b)C.Liu,J.Pan,S.Li,Y.Zhao,L.Y.Wu,
C.E.Berkman,A.R.Whorton and M.Xian,Angew.Chem.,IntEd.,2011,50,10327–10329c)
H.Peng,Y.Cheng,C.Dai,A.L.King,B.L.Predmore,D.J.Lefer and B.Wang,Angew.Chem.,
Int.Ed.,2011,50,9672–9675d)K.Sasakura,K.Hanaoka,N.Shibuya,Y.Mikami,Y.Kimura,
T.Komatsu,T.Ueno,T.Terai,H.Kimura and T.Nagano,J.Am.Chem.Soc.2011,133,18003–
18005]。
The major defect of such method is in In vivo detection, and biological tissue is to the physiological tissue of human body to ultrasonic signal
Scattering 3~4 orders of magnitude lower than general optical signalling, that is to say, that photoacoustic imaging can provide deeper imaging depth and more
High imaging resolution.Therefore, a species specific H is synthesized2S optoacoustics probe can detect H in deep tissues2S will be realized
To H in physiological tissue2S is more accurate, lossless, and high-resolution detection is of great significance.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of for In vivo detection vulcanization
The optoacoustic ratio nano-probe of hydrogen.
Another object of the present invention is to provide the systems of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide
Preparation Method.
Another object of the present invention is to provide answering for the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide
With.
The purpose of the invention is achieved by the following technical solution：A kind of optoacoustic ratio nanometer for In vivo detection hydrogen sulfide
Probe, structural formula are shown in formula I：
The preparation method of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide, includes the following steps：
(1) preparation of precursor：
1. m-aminophenol is dissolved in organic solvent, two t-butyl carbonates are then added, in 80 DEG C of items after stirring evenly
Back flow reaction is carried out under part, then the solution of acquisition is concentrated in vacuo, and obtains compound A；
2. under protective gas atmosphere, by step 1. in obtained compound A and sodium hydride be added to DMF (N, N- bis-
Methylformamide) in, it stirs evenly, adds Cy7-Cl, be stirred to react, solvent is removed under reduced pressure after reaction, obtain chemical combination
Object B；
3. under 0 DEG C, protective gas atmosphere, by step 2. in obtained compound B be added in anhydrous methylene chloride,
It stirs evenly, obtains mixed solution I；Trifluoroacetic acid is dissolved in dichloromethane, mixed solution I I is obtained；Then by mixed solution
II, which is added in mixed solution I, to be reacted, and compound C is obtained；
4. by step 3. in obtained compound C and sodium hydride be added in DMF, after mixing plus water, be stirred to react,
It waits for depressurizing removal solvent after reaction, obtains precursor compound D；The structural formula of precursor compound D is as shown in Formula II：
(2) preparation of probe
5. by step (1) 4. in obtained precursor compound D be added in hydrochloric acid solution, stir evenly, obtain under condition of ice bath
To mixed solution I II；
6. sodium nitrite is dissolved in the mixed solution I II obtained in being added drop-wise to step 5. after ice water, in 0~5 DEG C of condition
Under be stirred to react, obtain mixed solution I V；
7. sodium azide is dissolved in the mixed solution I V obtained in being added drop-wise to step 6. after ice water, under the conditions of 0~5 DEG C
It is stirred to react, obtains the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide.
Step 1. described in organic solvent be ethyl alcohol；Preferably absolute ethyl alcohol.
Step 1. described in the dosage of m-aminophenol be to match amino between 24~33.5mg by every milliliter of organic solvent
Phenol calculates.
Step 1. described in m-aminophenol and two t-butyl carbonates molar ratio be 1:1~2；Preferably 1:1.5.
Step 1. described in two t-butyl carbonate of addition realized preferably by following steps：First by two t-butyl carbonates
It is dissolved in organic solvent, is added dropwise again after stirring evenly.
Step 1. described in time of stirring be preferably 5 minutes.
Step 1. described in back flow reaction time be 5~8h；Preferably 5h.
Step 2. described in compound A, sodium hydride and Cy7-Cl molar ratio be 1：1~2：4；Preferably 1:1：4.
Step 2. described in the condition of stirring be preferably：It stirs 10~15 minutes at room temperature.
Step 2. described in Cy7-Cl be prepared preferably by following method：
A, under nitrogen protection, DMF is dissolved in dichloromethane, obtains DMF solution；
B, phosphorus oxychloride is dissolved in the dichloromethane under ice bath, the phosphorus oxychloride solution of acquisition is then added to step
In the DMF solution obtained in a, stirred under nitrogen atmosphere is uniform, obtains mixed solution V；
C, cyclohexanone is added in the mixed solution V obtained in step b, back flow reaction is carried out under the conditions of 80 DEG C, then will
The mixture obtained after reaction pours into ice water overnight, obtains compound E；
D, 2,3,3- tri-methyl indoles and iodoethane are dissolved in toluene, are stirred to react under the conditions of 100 DEG C, reaction terminates
Postcooling is filtered, washed to room temperature, obtains compound F；
E, the compound F and sodium acetate that are obtained in the compound E obtained in step c, step d are dissolved in acetic anhydride,
It is stirred to react under the conditions of 60 DEG C, is cooled to room temperature after reaction, be filtered, washed, be dried in vacuo, obtain Cy7-Cl.
DMF described in step a is preferably the DMF freezed.
The volume ratio of DMF and dichloromethane described in step a are preferably 1:1.
The volume ratio of phosphorus oxychloride and dichloromethane described in step b is preferably 1.05:1.
Phosphorus oxychloride and the volume ratio of the DMF described in step b are preferably 1.05:2.
The time of stirring described in step b is preferably 30min.
Cyclohexanone and the volume ratio of the phosphorus oxychloride described in step c are preferably 1:2.
The time of back flow reaction described in step c is preferably 3h.
The volume ratio of 2,3,3- trimethyls Yin and iodoethane described in step d are preferably 1:0.9.
2,3,3- trimethyl Yin dosages described in step d are to match 0.2ml 2,3,3- trimethyls Yin by every milliliter of toluene
It calculates.
The time being stirred to react described in step d is preferably 20h.
Washing described in step d is to be washed using ethyl acetate.
The molar ratio of compound E, compound F and sodium acetate described in step e are 0.6：1.3：0.6.
Compound E dosages described in step e are to match 0.2mmol compounds E by every ml acetic anhydride to calculate.
The time being stirred to react described in step e is preferably 3h.
Washing described in step e is to be washed successively with saturated sodium bicarbonate solution and water；Preferably by as follows
Step is realized：It is first washed with saturated sodium bicarbonate solution, until bubble-free occurs, is then washed with water and washs twice.
Step 2. described in the condition being stirred to react be preferably：25 DEG C of 2~3h of reaction.
2. the preparation method of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide is also wrapped later in step
Include the step of compound B that will be obtained is isolated and purified.
Described is isolated and purified to be isolated and purified using silicagel column.
Step 2. and 3. described in protective gas be preferably nitrogen.
Step 3. described in the dosage of compound B be to match 10mg compounds B by every milliliter of anhydrous methylene chloride to calculate.
Step 3. described in time of stirring be preferably 20~30min.
Step 3. described in mixed solution I I be added in mixed solution I preferably by following method realize：It will mix
Solution II is closed to be added in mixed solution I by syringe.
Step 3. described in the molar ratio of trifluoroacetic acid and compound B be 20:1.
Step 3. described in trifluoroacetic acid and dichloromethane volume ratio be 1:2~4；Preferably 1:3.
Step 3. described in time of reaction be preferably 6~8h.
3. the preparation method of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide is also wrapped later in step
Include the step of compound C that will be obtained is extracted, dried, removed solvent and isolate and purify：The chemical combination 3. obtained to step
Saturated sodium bicarbonate solution is added in object C, is extracted with dichloromethane, then is dried with anhydrous magnesium sulfate, pressurization removal
Solvent, obtained crude product purified by silica gel chromatographic column are isolated and purified, and compound C after purification is obtained.
The number of the extraction is preferably three times.
Step 4. described in compound C and sodium hydride molar ratio be 1:1~2；Preferably 1:1.2.
Step 4. described in the molar ratio of water and the compound C be 20~10：1；Preferably 20：1.
Step 4. described in the condition being stirred to react be preferably：50 DEG C of 5~7h of reaction.
3. the preparation method of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide is also wrapped later in step
Include the step of compound C that will be obtained is purified.
The purifying is to be purified using alumina column.
Step 4. described in reaction preferably using point board monitoring reaction, if there is bluish-green color dot and polarity greatly and raw material
As product.
Step 5. described in time of stirring be preferably 30~60min；Preferably 30min.
Step 5. described in the dosage of precursor compound D be based on matching 6mg precursor compounds D by every milliliter of hydrochloric acid solution
It calculates.
Step 5. described in hydrochloric acid solution be concentrated hydrochloric acid and ice water by volume 1:The solution that 4 proportionings obtain.
The concentrated hydrochloric acid is the concentrated hydrochloric acid of mass fraction 37%.
Step 6. and 7. described in dropwise addition be to be added dropwise using constant pressure funnel.
Step 6. described in sodium nitrite dosage by the molar ratio of sodium nitrite and the precursor compound D be 3~
5:1 proportioning calculates；Preferably press 3:1 proportioning calculates.
Step 6. described in the time being stirred to react be preferably 30min.
Step 7. described in sodium azide dosage by the molar ratio of sodium azide and the precursor compound D be 3~
5:1 proportioning calculates；Preferably press 3:1 proportioning calculates.
Step 7. described in the time being stirred to react be preferably 3~3.5h.
7. the preparation method of the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide is also wrapped later in step
Include the step of optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide that will be obtained is isolated and purified：First use dichloromethane
It is extracted, then is isolated and purified with silicagel column.
The application for the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide in detection field.
Described is detected as water body detection or food inspection.
The present invention has the following advantages and effects with respect to the prior art：
1, it the problem of scattering of fluorescence signal that the present invention overcomes fluorescence probes in In vivo detection and tissue resorption, provides
A kind of optoacoustic ratio image probe is used to detect the hydrogen sulfide in vivo tumor.The Response Mechanism of the probe is based on nitrine and sulphur
The redox reaction of the specificity of hydrogen radical ion, allows azido group to be reduced to amino, makes the conjugated system electronics of probe entirety
Cloud density increases, and red shift occurs so as to cause absorption spectrum before and after probe reaction, therefore become using the front and back absorption spectrum of its reaction
The property of change is designed to a kind of optoacoustic ratio image probe.
2, the probe that the present invention obtains has the following advantages that:Photoacoustic signal penetration depth is big, realize deep tissues at
Picture, reaction speed is fast, and specificity is good, and ratio test is more acurrate.The probe can be used for In vivo detection hydrogen sulfide, can be also used for
Vitro detection, such as the sulfurated hydrogen detection in water body, the sulfurated hydrogen detection etc. in food.
Description of the drawings
Fig. 1 is that the preparation in embodiment 1 of the present invention for the optoacoustic ratio probes of hydrogen sulfide in In vivo detection tumour is closed
At route map.
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of probe precursor compound D described in embodiment 1 of the present invention.
Fig. 3 is the inductivity coupled plasma mass spectrometry figure of probe precursor compound D described in embodiment 1 of the present invention.
Fig. 4 is the hydrogen nuclear magnetic resonance spectrogram of probe described in embodiment 1 of the present invention.
Fig. 5 is the inductivity coupled plasma mass spectrometry figure of probe described in embodiment 1 of the present invention.
Fig. 6 is that probe described in embodiment 2 of the present invention reacts front and back abosrption spectrogram with NaHS.
Fig. 7 is that probe described in embodiment 2 of the present invention reacts front and back fluorescence spectra with NaHS.
Fig. 8 be embodiment 3 of the present invention described in probe reacted with various concentration NaHS after photoacoustic imaging figure and
Photoacoustce signal intensity figure and optoacoustic rate value statistical chart；Wherein, figure a is the light after probe is reacted with various concentration NaHS
Acoustic imaging figure；Figure b is the photoacoustce signal intensity figure after probe is reacted with various concentration NaHS；It is that probe and difference are dense to scheme c
Spend the optoacoustic rate value statistical chart after NaHS reaction.
Fig. 9 be described in embodiment 4 of the present invention in probe intravenous injection into mice body after, mouse live body photoacoustic imaging figure with
Photoacoustce signal intensity figure；Wherein, figure a is photoacoustic imaging figure；Figure b is photoacoustce signal intensity figure.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1：A kind of preparation for the optoacoustic ratio probes of hydrogen sulfide in In vivo detection tumour.
1, a kind of specific synthesis for the optoacoustic ratio probes of hydrogen sulfide in In vivo detection tumour provided by the present invention
Step is as shown in Figure 1, specific as follows：
(1) the 1. preparation of I precursor：M-aminophenol (436mg, 4mmol) is dissolved in 13ml absolute ethyl alcohols, two carbonic acid uncles
Butyl ester (1.3g, 6mmol) is added dropwise after being dissolved in 5ml absolute ethyl alcohols, stirs 5 minutes, and 80 DEG C of reflux 5h, solution is concentrated in vacuo,
Obtain white solid A.
2. white solid A (81.9mg, 0.39mmol) and sodium hydride (9.36mg, 0.39mmol) being added, 2ml being housed
In the round-bottomed flask of DMF (n,N-Dimethylformamide), 10min is stirred at room temperature in mixture under nitrogen protection, by Cy7-Cl
(100mg, 0.156mmol) is added to after being dissolved in 1ml DMF in above-mentioned round-bottomed flask, and 2h is stirred at 25 DEG C, is removed under reduced pressure molten
Agent, silica gel post separation (volume ratio=100 of dichloromethane and methanol：3.5) dark green solid B is obtained.Wherein, the system of Cy7-Cl
Standby process is as follows：
A, the DMF 20ml of frost are dissolved under nitrogen protection in 20ml dichloromethane；
B, 10.5ml phosphorus oxychloride is dissolved in the 10ml dichloromethane under ice bath, then by the dichloromethane of phosphorus oxychloride
Solution is added dropwise in the DMF obtained in step a, stirred under nitrogen atmosphere 30min；
C, 5.26ml cyclohexanone is added in the mixed solution obtained in step b, mixture is stirred at 80 DEG C, and flow back 3h；
D, and then by the mixture obtained in step c it pours into ice water, overnight, yellow solid is obtained by filtration；
E, 2,3,3- tri-methyl indole 2ml and 1.8ml iodoethane are dissolved in 10ml toluene, 20h is stirred at 100 DEG C；
F, after reaction is cooled to room temperature, filtering, solid is washed to obtain pink solid with ethyl acetate；
G, the powder for taking the yellow solid obtained in 0.21g 1.2mmol steps d and being obtained in 0.81g 2.6mmol steps f
Color solid and 0.1632g 1.2mmol sodium acetates, are dissolved in 6ml acetic anhydrides；
H, the mixture obtained in step g is stirred into 3h at 60 DEG C；
I, it is cooled to room temperature, filters, washed with saturated sodium bicarbonate solution, until bubble-free occurs, then use solid
Twice, vacuum drying obtains dark green solid I to water washing；
J, dark green solid I is crossed into silicagel column (volume ratio=100 of dichloromethane and methanol：5), purifying obtains product
Cy7-Cl。
3. dark green solid B (50mg) is added in anhydrous methylene chloride (5ml), in 0 DEG C of stirred under nitrogen atmosphere
30min obtains the mixture of dark green solid B and dichloromethane；Then lead to after trifluoroacetic acid 1ml being dissolved in 3ml dichloromethane
It crosses syringe to be slowly added in mixture, allows reacting recovery to room temperature, stir 6h.The mixture after reaction is added after reaction
Enter into the saturated sodium bicarbonate solution of 50ml, three times with the extraction of 50ml dichloromethane, organic phase merges, dry with anhydrous magnesium sulfate
Dry, solvent is removed by pressurizeing, the isolated dark green solid C of crude product purified by silica gel chromatographic column.
4. compound C (35mg, 0.05mmol) and sodium hydride (1.44mg, 0.06mmol) are added to band round-bottomed flask
In, 2ml DMF are added as solvent, 100 μ l water are added (in offer Knoevenagel condensation reactions after stirring 5min
Hydrogen ion), 5h is stirred at 50 DEG C, is put board monitoring reaction process, solvent is depressurized away after complete reaction, with alumina column (two
Volume ratio=10 of chloromethanes and methanol：1) isolated I precursor compound D.
Characterize data：
δ=8.54 (d, J=4.0Hz, 1H) 1HNMR (Fig. 2) (500MHz, CDCl3), 7.45 (d, J=6.9Hz, 1H),
7.38 (t, J=7.5Hz, 2H), 7.26-7.17 (m, 2H), 7.08 (d, J=7.8Hz, 1H), 7.02 (s, 1H), 6.79 (s,
1H), 5.92 (d, J=14.2Hz, 1H), 4.09 (dd, J=13.3,6.1Hz, 2H), 2.82-2.70 (m, 2H), 2.68-2.59
(m, 2H), 1.94 (s, 2H), 1.77 (s, 6H), 1.46 (t, J=6.8Hz, 3H)
LC-MS schemes (3):calcd.for[M+]397.54,,found:397.20.
(2) preparation of probe I
Prepared hydrochloric acid solution (1ml 37% is added in I precursor compound D (30mg, 0.076mmol) obtained above
Concentrated hydrochloric acid：4ml ice water) in, 30min is stirred under ice bath, solution becomes atropurpureus.Take sodium nitrite (0.226mmol, 15.5mg)
It is dissolved in ice water, is added dropwise in above-mentioned atropurpureus solution with constant pressure funnel, keep the temperature 0~5 DEG C, stir 30min, mixed
Close solution.It takes sodium azide (14.69mg) to be dissolved in ice water (1ml), above-mentioned mixed solution is added dropwise with constant pressure funnel
In, 0~5 DEG C is kept the temperature, 30min is stirred.3h is stirred at room temperature, is extracted with dichloromethane.Finally cross silicagel column (dichloromethane and methanol
Volume ratio=25：1 elution) isolated bluish violet solid probe I.
Characterize data：
δ=8.54 (d, J=4.0Hz, 1H) 1HNMR (Fig. 4) (500MHz, CDCl3), 7.45 (d, J=6.9Hz, 1H),
7.38 (t, J=7.5Hz, 2H), 7.26-7.17 (m, 2H), 7.08 (d, J=7.8Hz, 1H), 7.02 (s, 1H), 6.79 (s,
1H), 5.92 (d, J=14.2Hz, 1H), 4.09 (dd, J=13.3,6.1Hz, 2H), 2.82-2.70 (m, 2H), 2.68-2.59
(m, 2H), 1.94 (s, 2H), 1.77 (s, 6H), 1.46 (t, J=6.8Hz, 3H)
LC-MS (Fig. 5):calcd.for[M+]423.54,,found:423.10.
Embodiment 2:Probe I reacts front and back absorption, fluorescence spectrum with NaHS.
I solution of probe and sodium hydrosulfide aqueous solution obtained in configuration embodiment 1, then by I solution of probe and sulphur hydrogen
Change sodium water solution hybrid reaction and measures its absorption after five minutes；Wherein, a concentration of 10 μM of reaction system middle probe I, sulphur hydrogenation
A concentration of 50 μM of sodium.Absorption spectrum as shown in fig. 6, fluorescence spectrum as shown in fig. 7, as can be seen from the figure probe with sulphur hydrogen
Changing before and after sodium reacts has fluorescence response.
Embodiment 3:Photoacoustic imaging figure and ratio image after probe I is reacted with various concentration NaHS.
Configuration concentration is 20 μM of I solution of probe totally 6 groups and a concentration of 0,20,40,60,80,100 μM of NaHS
Aqueous solution, then will I solution of probe and sodium hydrosulfide aqueous solution mix after reacted that (dosage of I solution of probe is 40 μ l, sulphur
The dosage of sodium hydride aqueous solution is respectively 1,2,3,4,5 μ l), every group of each autoreaction after five minutes, with optoacoustic computed tomography
Instrument measures the photoacoustic signal and optoacoustic X-Y scheme (Fig. 8) of 6 groups of solution.As can be seen from the figure photoacoustic signal with concentration increasing
The photoacoustce signal intensity of 532nm is added gradually to weaken, the photoacoustce signal intensity of 700nm gradually increases, two wave band photoacoustce signal intensities
Rate value gradually increases with the concentration of NaHS, shows that probe can be as the ratio optoacoustic probe of detection hydrogen sulfide.
Embodiment 4:The photoacoustic imaging figure in the living body of probe I and ratio image.
By (2mg/kg) tail vein injection of probe I to mouse, (3~4 weeks BABc mouse, average 2 weight 20g, buy in south
Medical university) in vivo after Isosorbide-5-Nitrae, 8,12, mouse tumor position is imaged using optoacoustic computed tomographic scanner afterwards for 24 hours,
It is imaged respectively in two wave bands of 532nm and 700nm.The results are shown in Figure 9, as can be seen from the figure as probe is in mouse
The optoacoustic ratio of Endogenous Hydrogen Sulfide is imaged after metabolism to tumor locus in vivo.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (10)
1. a kind of optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide, it is characterised in that structural formula is shown in formula I：
2. the preparation method described in claim 1 for the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, which is characterized in that
Include the following steps：
(1) preparation of precursor：
1. m-aminophenol is dissolved in organic solvent, two t-butyl carbonates are then added, after stirring evenly under the conditions of 80 DEG C
Back flow reaction is carried out, then the solution of acquisition is concentrated in vacuo, obtains compound A；
2. under protective gas atmosphere, by step 1. in obtained compound A and sodium hydride be added in DMF, stir evenly,
Cy7-Cl is added, is stirred to react, solvent is removed under reduced pressure after reaction, obtains compound B；
3. under 0 DEG C, protective gas atmosphere, by step 2. in obtained compound B be added in anhydrous methylene chloride, stir
Uniformly, mixed solution I is obtained；Trifluoroacetic acid is dissolved in dichloromethane, mixed solution I I is obtained；Then mixed solution I I is added
Enter and reacted into mixed solution I, obtains compound C；
4. by step 3. in obtained compound C and sodium hydride be added in DMF, after mixing plus water, be stirred to react, wait for anti-
Decompression removal solvent, obtains precursor compound D after answering；Wherein, the structural formula of precursor compound D is as shown in Formula II：
(2) preparation of probe
5. by step (1) 4. in obtained precursor compound D be added in hydrochloric acid solution, stir evenly, mixed under condition of ice bath
Close solution III；
6. sodium nitrite is dissolved in the mixed solution I II obtained in being added drop-wise to step 5. after ice water, stirred under the conditions of 0~5 DEG C
Reaction is mixed, mixed solution I V is obtained；
7. sodium azide is dissolved in the mixed solution I V obtained in being added drop-wise to step 6. after ice water, stirred under the conditions of 0~5 DEG C
Reaction, obtains the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide.
3. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 2
Be, step 2. described in Cy7-Cl be prepared via a method which to obtain：
A, under nitrogen protection, DMF is dissolved in dichloromethane, obtains DMF solution；
B, phosphorus oxychloride is dissolved in the dichloromethane under ice bath, then the phosphorus oxychloride solution of acquisition is added in step a
In obtained DMF solution, stirred under nitrogen atmosphere is uniform, obtains mixed solution V；
C, cyclohexanone is added in the mixed solution V obtained in step b, back flow reaction is carried out under the conditions of 80 DEG C, then will reaction
The mixture obtained afterwards pours into ice water overnight, obtains compound E；
D, 2,3,3- trimethyl Yin and iodoethane are dissolved in toluene, are stirred to react under the conditions of 100 DEG C, cool down after reaction
It to room temperature, is filtered, washed, obtains compound F；
E, the compound F and sodium acetate that are obtained in the compound E obtained in step c, step d are dissolved in acetic anhydride, in 60
It is stirred to react under the conditions of DEG C, is cooled to room temperature after reaction, be filtered, washed, be dried in vacuo, obtain Cy7-Cl.
4. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 3
It is：
The volume ratio of phosphorus oxychloride and the DMF described in step b is 1.05:2；
The volume ratio of cyclohexanone and the phosphorus oxychloride described in step c is 1:2；
The volume ratio of 2,3,3- trimethyls Yin and iodoethane described in step d are 1:0.9；
The molar ratio of compound E, compound F and sodium acetate described in step e are 0.6：1.3：0.6.
5. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 3
It is：
The time of back flow reaction described in step c is 3h；
The time being stirred to react described in step d is 20h；
Washing described in step d is to be washed using ethyl acetate；
The time being stirred to react described in step e is 3h；
Washing described in step e is to be washed successively with saturated sodium bicarbonate solution and water.
6. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 2
It is：
Step 1. described in m-aminophenol and two t-butyl carbonates molar ratio be 1:1~2；
Step 2. described in compound A, sodium hydride and Cy7-Cl molar ratio be 1：1~2：4；
Step 3. described in the molar ratio of trifluoroacetic acid and compound B be 20:1；
Step 4. described in compound C and sodium hydride molar ratio be 1:1~2；
Step 4. described in the molar ratio of water and the compound C be 20~10：1；
Step 6. described in the dosage of sodium nitrite by the molar ratio of sodium nitrite and the precursor compound D be 3~5:1 matches
Than calculating；
Step 7. described in the dosage of sodium azide by the molar ratio of sodium azide and the precursor compound D be 3~5:1 matches
Than calculating.
7. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 2
It is：
Step 1. described in organic solvent be absolute ethyl alcohol；
Step 5. described in hydrochloric acid solution be concentrated hydrochloric acid and ice water by volume 1:The solution that 4 proportionings obtain；The dense salt
Acid is the concentrated hydrochloric acid of mass fraction 37%.
8. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 2
It is：
Step 1. described in back flow reaction time be 5~8h；
Step 2. described in the condition being stirred to react be：25 DEG C of 2~3h of reaction；
Step 3. described in reaction time be 6~8h；
Step 4. described in the condition being stirred to react be：50 DEG C of 5~7h of reaction；
Step 6. described in the time being stirred to react be 30min；
Step 7. described in the time being stirred to react be 3~3.5h.
9. being used for the preparation method of the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide, feature according to claim 2
Be, step 7. after further include the optoacoustic ratio nano-probe for In vivo detection hydrogen sulfide that will be obtained detach it is pure
The step of change：It is first extracted with dichloromethane, then is isolated and purified with silicagel column.
10. the application described in claim 1 for the optoacoustic ratio nano-probe of In vivo detection hydrogen sulfide in detection field,
It is characterized in that：Described is detected as water body detection or food inspection.
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| CN116217515A (en) * | 2022-12-20 | 2023-06-06 | 华南师范大学 | Compound, preparation method thereof and application thereof in detection of cysteine |
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