CN100473653C - Near infrared meso-position nitrogen and sulfur substituted hepta-methyl-cyanine fluorochrome for bioanalysis - Google Patents
Near infrared meso-position nitrogen and sulfur substituted hepta-methyl-cyanine fluorochrome for bioanalysis Download PDFInfo
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- CN100473653C CN100473653C CNB2005100466252A CN200510046625A CN100473653C CN 100473653 C CN100473653 C CN 100473653C CN B2005100466252 A CNB2005100466252 A CN B2005100466252A CN 200510046625 A CN200510046625 A CN 200510046625A CN 100473653 C CN100473653 C CN 100473653C
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Abstract
The invention relates to a near-infrared new methenyl fluorescence dye, which is made by displacement at mid-position of methine colors by nucleophilic reagent containing nitrogen and sulfur. Mid-position nitrogen derivative replaces methine colors to generate ICT effect, Stokes displacement (70-170nm) and fine fluorescence. It can be used as high fidelity fluorescence labeling probe of biomolecules such as protein, sugar and DNA. When mid-position sulfer derivative replaces methine colours, PET phenomenon occurs in molecule.
Description
Technical field
The invention belongs to the used fluorescence dye of bioluminescence analysis technical field.
Background technology
Along with the develop rapidly of bio-science, fluorescence analysis is more and more in the application of aspects such as gene sequencing, genetic expression and clinical diagnosis.Medically, be the enzymatic immunoassay of core and the standard approach that fluorescence polarization immunoassay has become medical diagnosis with the fluorescence technique.On cytobiology, fluorescence spectrum is used to the position and the situation of movement of composition in the track cells.In addition, cell distinguishes and classifies also be that dependence is the flow cytometry of core with the fluorescence technique.The exploitation of fluorescence dye is the tool a crucial factor of development fluorescence analysis.Fluorescence dye is applied in the bioanalysis, and early stage significant achievement is that the DNA automated sequencing technology is utilized the sign of 4 kinds of different fluorescence dyes, can obtain the sequence of C, G, A, T four base analogs automatically fast in the eighties mid-term.This achievement also becomes the important milestone of modern molecular biosciences analytical technology development.Afterwards along with laser technology, Computer Processing technology and fluorescence spectrometry innovation continuous advancement in technology, fluorescence dye detects at gene chip, immunoassay, the identification of tumour cell early diagnosis and metal ions in cells etc. has all obtained using widely aspect many, and the annual progress that lot of documents and these aspects of patent report are all arranged.
For satisfying bioanalysis application with rapid changepl. never-ending changes and improvements demand to fluorescence dye, research and develop out the novel fluorescence dyestuff that more has the good fluorescence spectrum property, remain the key and the core of fluorescence analysis development.Use at present many fluorescence dyes such as rhodamine class, fluoresceins, BODIPY class and cyanines class, wherein the commercialization of some kind.But the most of spectrum of these commercial dyestuffs all is in the UV, visible light district, and biological sample is from very strong absorption is arranged in this zone, can show the autofluorescence of certain intensity during fluoroscopic examination, cause strong fluorescence background, thereby greatly reduce the sensitivity that detects.Near infrared fluorescent dye has been subjected to attention widely in recent years owing to can avoid this problem.Even have the expert to think, be exactly near infrared fluorescent dye and be the molecular probe of basic design from what the culture dish conceptual phase developed into most critical effect the Research of Animal Model for Study phase process in fluorescence technique with them.Only in the near-infrared region, fluorescence analysis could be used in the histocyte body fully.
Seven methine cyanine dyes are this respect research and the focus used as topmost near-infrared fluorescent group.The seven methine cyanine dyes that exploitation has the good light spectral property play crucial effects to development bioluminescence sign technology.
At the light stability of this class dyestuff and the shortcoming of poorly water-soluble, forefathers have obtained a lot of achievements on the Molecular Structure Design of dyestuff.The first, the fragrant parent nucleus at two ends has a variety ofly, comprises thiazole, thiophene, 2-quinoline, 4-quinoline and 3H-indoline etc., but it is best with the indoline to be that parent nucleus is proved to be performance.The second, the methine chainpropagation causes light stability decline to be confirmed.GaborPatonay etc. introduce the structural unit of a tetrahydrobenzene on the methine chain, the synthetic dyestuff is considered to respect to straight chain seven methine dyess better light stability is arranged, and fluorescence also is enhanced simultaneously.Three, AS Wangger etc. introduces sulfonic group in molecule, has improved the water-soluble of dyestuff.Also has the effect that improves stability simultaneously.This method has become the water-soluble the most frequently used method that improves this class dyestuff.
However, still there are some problem and shortage in long wavelength's seven methine indole cyanine dyes in the application of bioluminescence analysis at present.
The first, the light stability of seven methine cyanine dyes needs further to improve.
The second, present polymethin cyanine dyestuff all is a symmetrical structure generally, lacks single fluorescent mark reactive activity site.When fluorescent mark, fluorescent cyanogen dye is preferably in and contains a single carboxylic group in the molecule, and being used for specifically, derivatization reaction obtains the ideal fluorescent probe molecule.And be the polymethin cyanine dyestuff that is contained single carboxyl like this, general method is to synthesize unsymmetrical cyanine dye, introduces a carboxyl at an end of molecule.But this method makes the synthetic of dyestuff become complicated and difficult with separating to purify.
Three, also there is weak point in the spectrum property of cyanine dyes: fluorescence lifetime is short; Fluorescence quantum yield is low; Autohemagglutination etc. often appears in the aqueous solution.Particularly the Stokes shift of cyanine dyes is very little, and about 20nm, this causes cyanine dyes to have the fluorescence self-quenching of certain degree on the one hand in solution, and this is that little Stokes shift forms the result that spectrum is handed over lid.The normal appearance too causes measuring error near emitting detection wave length because of the excitation light source wavelength in fluoroscopic examination on the other hand.Recent two pieces of documents are all spoken of common polymethin cyanine dyestuff and are had the little weakness of Stokes shift.Document is as follows: (1) Tolosa, L.; Nowaczyk, K.; Lakowicz, J.An Introduction to Laser Spectroscopy, 2nd ed.; Kluwer:New York, 2002. (2) Zhang Z., Achilefu S.Synthesis and Evaluation of Polyhydroxylated Near-InfraredCarbocyanine Molecular Probes.Org.Lett.2004,6 (12): 2067-2070.
Summary of the invention
The seven methine cyanine dyes (general structure I) that the objective of the invention is selected long wavelength, design synthesize the near infrared seven methine cyanine dyes of the novel structure that is applicable to bioluminescence and indicates, excellent property as goal in research.
In the general formula I:
X＝II；
R
1, R
2=(CH
2)
nR
8, (CH
2)
mOR
9, (CHR
10CH
2O)
pR
9Or CH
2C
6H
4R
8
R
3, R
4, R
8=H, SO
3R
11Or CO
2R
12
R
5=(CH
2)
nR
8, (CH
2)
mOR
9Or (CHR
10CH
2O)
pR
9
R
6=(CH
2)
nR
8, (CH
2)
mOR
9, (CHR
10CH
2O)
pR
9, a position or contraposition C
6H
4R
17
R
9=H or C
1-18Alkyl;
R
10=H or CH
3
R
11=H or M; M=Na, K, N (R
13R
14R
15R
16);
R
12=H, M, R
7Or C
1-18Alkyl;
R
13, R
14, R
15, R
16=H, C
1-18Alkyl; (CH
2)
mOR
9Or (CHR
10CH
2O)
pR
9
R
17=H, NH
2, NHCOR
9Or (CH
2)
nR
8
n、m、p＝0-18。
The first, the present invention has at first designed one seven methine indoles cyanines parent dyestuff, structure has taken into full account the previous work achievement, in dye structure with indoline as fragrant parent nucleus, it is water-soluble to introduce the sulfonic group increase on indoline, introduces hexa-atomic bridged ring structure at the methine middle-of-chain and improves stability.
The second, the present invention's meta of being selected in seven methine cyanine dyes replaces, and synthesizes seven novel methine cyanine dyes, and with the fluorescence spectrum performance of the reactive monoazo dyestuffs center as the present invention's research.
In the structure of seven recently common methine indole cyanine dyes, because the methine chain is introduced chloride hexa-atomic bridged ring structure and is improved light stability, the site that is used for derivatization reaction that on the structure of dye molecule, has produced a uniqueness so simultaneously, be in the chlorine atom of methine chain " meta " exactly, can be replaced by nucleophilic reagent.
It is less relatively that seven methine cyanine dyes this " meta " replace forefathers' research, and concentrate on Sauerstoffatom or sulphur atom replacement meta chlorine atom.This class derivative dye has solved a difficult problem of introducing the single-activity site in molecule, can be applied in the bioluminescence analysis as the novel fluorescence probe more conveniently.The present invention studies spectrum property and the application thereof that meta nitrogen, sulphur replace seven methine cyanine dyes emphatically.
The general structure that the near infrared meta nitrogen that the present invention studied, sulphur replace the Heptamethine cyanines fluorescence dye is I ':
General formula I ' in:
X '=II ', III ', IV ' or V ';
R '
1, R '
2=(CH
2)
nR '
7, (CH
2)
mOR '
8, (CHR '
9CH
2O)
pR '
10, CH
2C
6H
4R '
11Or cyclohexyl;
R '
3, R '
4, R '
7=H, SO
3R '
12, CO
2R '
13Or NCS;
R '
5=(CH
2)
nR '
7, (CH
2)
mOR '
8, (CHR
9CH
2O)
pR '
10, CH
2C
6H
4R '
11, cyclohexyl, a position or contraposition C
6H
4R '
14
R '
8, R '
9, R '
10=H, C
1-18Alkyl or cyclohexyl;
R '
11The SO of=H, a position, contraposition
3R '
12Or CO
2R '
13
R '
12=H or M; M=Na, K or N (R '
15R '
16R '
17R '
18);
R '
13=H, M, R '
6, C
1-18Alkyl or cyclohexyl;
R '
14=H, NH
2, NHCOR '
10, (CH
2)
nR '
7, (CH
2)
mOR '
8, (CHR '
9CH
2O)
pR '
10, CH
2C
6H
4R '
11Or cyclohexyl;
R '
15, R '
16, R '
17, R '
18=H, C
1-18Alkyl, cyclohexyl, (CH
2)
qOR '
9Or (CHR '
9CH
2O)
rR '
10
n、m、p、q、r＝0-18。
1, dyestuff is synthetic
Starting raw material para-orientation phenylhydrazine and 3-methyl-2-butanone are refluxed in Glacial acetic acid, and indolization generates the indoles intermediate, down quaterisation takes place with alkylating reagent in nitrogen protection again and generates the intermediate indoles quaternary ammonium salt that alkyl replaces.Dehydration reaction must chloride hexa-atomic endocyclic seven methine 3H-indoles cyanines parent dyestuffs in propyl carbinol/toluene with condensing agent 2-chloro-1-formyl radical-3-methylol tetrahydrobenzene then:
Chloride bridged ring parent dyestuff and different amino or the sulfydryl nucleophilic reagents of containing are under the anhydrous and oxygen-free condition, stirring reaction in DMF, product are poured into and are then separated out a large amount of solids in the ether, after filtration after the drying, adopt the reverse phase filler post with water/methyl alcohol gradient elution, obtain the target dyestuff.
2, the feature of dyestuff
It is different fully with common polymethin cyanine dyestuff that meta azepine derivatives of the present invention replaces the spectrum property of seven methine cyanine dyes.Be mainly reflected in: the first, the Stokes shift maximum of reactive monoazo dyestuffs can reach 167nm, can reach 70nm at least, and than big a lot of about the 25nm of common cyanine dyes, and the fluorescence quantum yield maximum can surpass 0.4.The second, the spectrum of reactive monoazo dyestuffs obviously broadens with respect to common polymethin cyanine dyestuff, especially absorption spectrum.Three, absorption spectrum and emmission spectrum lose the mirror image symmetric relation.The width of absorption spectrum is obviously greater than emmission spectrum.Having these three features in spectrum is that dye molecule exists intramolecular charge to shift the external manifestation of (ICT) excited state.Dyestuff has big Stokes shift and has very significant meaning for fluorescence analysis.The numerous fluorescence dyes that are used for fluorescence analysis now, most Stokes shifts are no more than 30nm.As rhodamine B is 23nm in methanol solution, and BODIPY class dyestuff is 20nm approximately.The problem that little Stokes shift causes, the one, make the dyestuff self-quenching, be also referred to as the self-absorption of dyestuff.This is because the absorption spectrum of the dyestuff of little Stokes shift and emmission spectrum have very big friendship lid, and promptly dyestuff has part emission luminous energy to be absorbed by himself, causes fluorescence intensity to descend like this.Another problem is to cause the measuring error of fluorescence analysis.This is because little Stokes shift makes excitation wavelength and fluoroscopic examination wavelength too approaching, and excitation wavelength easily causes the scattered light interference to the fluoroscopic examination slit.Thereby this fluorescence dye with big Stokes shift has important application.This class cyanine fluorochrome of the present invention can be used as biomolecules fluorescence labeling probes such as high-sensitive protein, sugar, DNA.
It is similar to common polymethin cyanine dyestuff that meta sulfur derivatives of the present invention replaces the spectrum of seven methine cyanine dyes, but find that there is intramolecularly PET phenomenon in this class derivative dye.This phenomenon is seldom found near infrared fluorophore.The PET effect of dyestuff is directly related with the power supply capacity of meta sulphur substituted radical.Wherein, the strongest dye sheet of substituting group power supply capacity reveals the most effective intramolecularly PET, the fluorescence quantum yield minimum.Outer protonation that this dyestuff is carried out and the fluorometric titration of metal ion experiment find that in adding cationic finite concentration scope, the fluorescence of dyestuff strengthens.This result has effectively verified the existence of intramolecularly PET.To the fluorometric titration experimental result of light metal trivalent aluminium ion, find that the fluorescence intensification factor is greater than 10 times.This is the most effective near infrared fluorescent probe of finding so far.This lays the foundation for the near-infrared fluorescent metal ion probe of design based on this class dyestuff.
Table 1 is that two meta azepine derivatives replace dyestuff IIa, and IIb and chloride parent dyestuff Ib are at the aqueous solution (1 * 10
-6M) spectroscopic data in.Can see that wherein the Stokes shift ratio of alkylamino replacement derivative dye IIb reaches 140nm.What is more important is more than the chloride parent dyestuff of their the fluorescence intensity ratio Ib twice eager to excel in whatever one does.
Fig. 1 provides the spectrogram of dyestuff IIb in the aqueous solution, and Fig. 2 is the spectrogram of chloride parent dyestuff Ib in the aqueous solution.By more as can be seen, first, the spectrum of reactive monoazo dyestuffs IIb obviously broadens with respect to the parent dyestuff, absorption spectrum especially.The second, absorption spectrum and emmission spectrum lose the mirror image symmetric relation.The width of absorption spectrum is obviously greater than emmission spectrum.Three, the Stokes shift of reactive monoazo dyestuffs IIb reaches 140nm, and is more a lot of greatly than common cyanine dyes.
Table 2 is that the derivative dye IIIa-d that replaces of meta sulphur and parent dyestuff Ib are 1 * 10
-6Spectroscopic data in the aqueous solution of M.Meta sulphur replaces as can be seen influences the result to the dyestuff spectrographic.
(1) compare with the parent dyestuff, the absorption of derivative dye and emission peak shape remain unchanged, and Stokes shift does not have considerable change yet, and maximum absorption and emission wavelength have red shift slightly.Wherein the IIIa-c red shift surpasses 10nm, and IIId red shift 3-5nm.Reason may be that phenyl ring has prolonged conjugated system among the IIIa-c, and IIId has only the p-π resonance effect of sulphur.
(2) fluorescence intensity is in proper order: IIId〉Ib〉IIIa〉IIIc〉IIIb.It is very big to the fluorescence quantum yield influence to illustrate that sulphur replaces the substituent structure in back.The introducing of inhaling the carboxymethyl of electricity strengthens dyestuff IIId fluorescence.The introducing of phenyl ring to a certain degree weakens dyestuff IIIa fluorescence, and amino-benzene then makes dyestuff IIIb fluorescent quenching, then makes dyestuff IIIc fluorescence return to the level that approaches dyestuff IIIa but introduce acetyl amino phenyl.
We can infer easily that intramolecularly PET has taken place for it from the substituent structure of dyestuff IIIb and minimum fluorescence quantum yield.As other PET probe, the nonbonding electron of a high energy after the optical excitation on the N atom can be transferred on the HOMO of fluorophore, thus cancellation the fluorescence of molecule.
Dyestuff IIIb has been proved the strong PET process that taken place, and this fluorescence quantum yield from it (Φ f=0.0065) well below IIIa (Φ f=0.038) and IIIc (Φ f=0.036) as can be seen.
In like manner, dyestuff IIa is except having big Stokes shift (87nm), can infer that its intramolecular charge that excited state has taken place shifts outside (ICT),, can infer that also PET process to a certain degree takes place its intramolecularly according to its weak fluorescence quantum efficiency (Φ f=0.025).
The fluorescent quenching that strong PET will make dyestuff takes place in the PET principle according to discussing previously, when donor amino by protonated or with after metallic cation combines, fluorescence recovers.This fluorescence recovers the enhancing process can realized easily for short wavelength's fluorophore, but for near-infrared fluorescent group, this process is limited by objective factor, is difficult to realize.
Fortunately, behind protonation or the transition metal ion, dyestuff IIIb has observed comparatively significantly fluorescence enhancing outside.And parent dyestuff Ib and dyestuff IIIa, IIIc can only observe fluorescent quenching.See Fig. 3-7.This has confirmed to exist really in this quasi-molecule the PET phenomenon on the one hand.Lay a good foundation for this class near-infrared region PET fluorescent probe of research and design on the other hand.
In the fluorometric titration experiment, add proton or add transition-metal cation to dyestuff IIIb above after certain concentration, fluorescence also cancellation will occur.Illustrate that dyestuff IIIb is adding under the cationic condition, also exists two kinds of opposite effects.The one, positively charged ion combines with amino, has suppressed the PET process, the enhancement effect of fluorescence that causes.The 2nd, this two cationoids inductive fluorescent quenching effect.Strong acid makes cyanine dyes fade, and the energy that bumps between transition-metal cation and dye molecule shifts, cancellation fluorescence.In cationic low strength range, enhancement effect of fluorescence has occupied leading role.But simultaneous quenching effect makes design PET proton probe and transition metal ion probe become infeasible, because be difficult to obtain big fluorescence intensification factor and good linear relationship.
In addition, also attempt lanthanide metal ion, in dyestuff IIIb, added Ce (NO
3)
3After, find that also fluorescence strengthens the process of back cancellation earlier.But find in the test, relative Zn ion, it is more difficult that this class lanthanide metal ion fluorescence strengthens, and quenching phenomenon just begins to take place under very low concentration.This cancellation selectivity stems from generation
Dye molecule and the energy between metal ion that the type collision energy shifts are flux matched.This also is the theoretical basis of the fluorescent quenching probe of design transition metal ion.
But design metal ion PET probe, just need avoid this quenching effect.Fig. 8 has provided dyestuff IIIb and has added Al ionic fluorometric titration spectrum.Can see that dyestuff IIIb is after adding the Al ion, the fluorescence enhancing reaches 12 times.The energy that similar heavy metal ion can not take place between this presentation of results light metal ion and the cyanine dyes shifts, thereby has avoided the fluorescent quenching effect.So this class dyestuff is not suitable for being designed to the near infrared PET fluorescent probe of heavy metal ion, but can be used for light metal ion or neutral micromolecular PET fluoroscopic examination fully.
Description of drawings
Fig. 1 is that dyestuff IIb is at the aqueous solution (1 * 10
-6M) absorption in and emmission spectrum, X-coordinate are wavelength (nm), and the ordinate zou left side is an absorption value, and the right is an emissive porwer.
Fig. 2 is that dyestuff Ib is at the aqueous solution (1 * 10
-6M) absorption in and emmission spectrum, X-coordinate are wavelength (nm), and the ordinate zou left side is an absorption value, and the right is an emissive porwer.
Fig. 3 is dyestuff IIIb fluorescence emission spectrum (λ ex=795nm) in the aqueous solution that contains different concns HCl, and X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence; HCl concentration: a:0mM, b:0.096mM, c:0.0192mM, d:0.480mM, e:0.720mM, f:0.960mM, g:1.200mM;
Fig. 4 is dyestuff Ib fluorescence emission spectrum (λ ex=767nm) in the aqueous solution that contains different concns HCl, and X-coordinate is wavelength (nm), and ordinate zou is a relative intensity of fluorescence; HCl concentration: a:0mM, b:0.048mM, c:0.096mM, d:0.240mM, e:0.960mM, f:2.400mM, g:4.800mM, h:9.600mM;
Fig. 5 is dyestuff IIIa at different concns ZnSO
4Buffered soln in fluorescence emission spectrum (λ ex=795nm), buffer system is urotropine-hydrochloric acid, pH=7.04, X-coordinate are wavelength (nm), ordinate zou is a relative intensity of fluorescence; ZnSO
4Concentration: a:0mM, b:0.04mM, c:0.08mM, d:0.20mM, e:0.40mM, f:0.80mM, g:2.00mM;
Fig. 6 is dyestuff IIIb at different concns ZnSO
4Buffered soln in fluorescence emission spectrum (λ ex=795nm), buffer system is urotropine-hydrochloric acid, pH=7.04, X-coordinate are wavelength (nm), ordinate zou is a relative intensity of fluorescence; ZnSO
4Concentration: a:0mM, b:0.08mM, c:0.16mM, d:0.32mM, e:0.60mM, f:0.80mM, g:1.20mM, h:1.40mM;
Fig. 7 is dyestuff IIIc at different concns ZnSO
4Buffered soln in fluorescence emission spectrum (λ ex=795nm), buffer system is urotropine-hydrochloric acid, pH=7.04, X-coordinate are wavelength (nm), ordinate zou is a relative intensity of fluorescence; ZnSO
4Concentration: a:0mM, b:0.10mM, c:0.20mM, d:0.40mM, e:0.80mM, f:2.00mM, g:4.00mM;
Fig. 8 is dyestuff IIIb at different concns Al
2(SO
4)
3Buffered soln in fluorescence emission spectrum (λ ex=795nm), buffer system is urotropine-hydrochloric acid, pH=7.04, X-coordinate are wavelength (nm), ordinate zou is a relative intensity of fluorescence; Al2 (SO4) 3 concentration: 0mM---22.0mM.
Embodiment
The synthetic route of chloride bridged ring parent dyestuff Ib
(1) intermediate 2,3,3-trimethylammonium-3H-indoline-5-potassium sulfonate synthetic
In the 1000ml there-necked flask, add the 150ml Glacial acetic acid successively, 84ml (0.8mol) 3-methyl-2-butanone and 50g (0.26mol) phenyl hydrazine-p-sulfonic acid.Mixture heating up refluxed 3 hours, and reactant is poured in the beaker, had pink solid to separate out after leaving standstill cooling.Filter, vacuum-drying gets 2,3,3-trimethylammonium-3H-indoline-5-sulfonate 48 grams, and productive rate 75%, Rf=0.58, developping agent are propyl carbinol: acetate: water (2:1:5).
In the flask, add 50ml methyl alcohol successively at the bottom of the 250ml garden, 50ml Virahol, 48g (0.2mol) 2,3,3-trimethylammonium-3H-indoline-5-sulfonate and 12.3 gram (0.22mol) KOH.Mixture heating up refluxes, and pink is decorporated rapidly, and cooling has a large amount of yellow solids to separate out.Filter, vacuum-drying gets intermediate 2,3,3-trimethylammonium-3H-indoline-5-potassium sulfonate 54 grams, productive rate 97%.
(2) intermediate N benzyl-2,3,3-trimethylammonium-3H-indoline-5-sulfonic acid synthetic
In the 100ml round-bottomed flask; add 15.6g (0.06mol) 2,3 successively, 3-trimethylammonium-3H-indoline-5-potassium sulfonate; 9.8g (0.063mol) bromotoluene; toluene 65ml, heating reflux reaction is 5 hours under nitrogen protection, obtains pink solid; ultrasonicly make that solid enters in the liquid on the wall; filter, use toluene wash, vacuum-drying.
(3) condensing agent 2-chloro-1-formyl radical-3-methylol tetrahydrobenzene is synthetic
Mixed solution with the 35ml exsiccant methylene dichloride of 37ml phosphorus oxychloride under ice bath (0-5 ℃) is added dropwise to 80ml by exsiccant methylene dichloride and N, and (1:1 in mixed solution V/V), and then dropwise adds the 10g pimelinketone to dinethylformamide.Remove ice bath, reaction solution is heated to backflow, behind the reaction 3h,, pour in the 200g trash ice then in batches with the ice-water bath cooling.Placement is spent the night, and obtains red solid, filters, and washing is extremely yellow on a small quantity in batches with freezing acetone for solid.Product places refrigerator to preserve under nitrogen protection, and is standby.
(4) the synthetic and sign of chloride parent dyestuff Ib:
In the 250ml round-bottomed flask, add 8mmol quaternary ammonium salt 5 and 4mmol (692mg) condensing agent 6 (2-chloro-1-formyl radical-3-methylol tetrahydrobenzene) successively; add propyl carbinol again: toluene (7:3) 300ml; add water trap; add toluene solvant in the water trap; under the argon shield; reflux 5 hours, solution becomes scarlet by light red gradually, occurs a large amount of green color component at last.Rotary evaporation is removed propyl carbinol and benzene, adds methyl alcohol, ultrasonic dissolution, adding has a large amount of solids to separate out with respect to the ether in the quantity of methyl alcohol 1.5-2 times high degree of agitation immediately, filters with the sand core funnel, the ether washing, water recrystallization twice after the crude product drying, sealing is kept in Dark Place.Dyestuff Ib:
1H NMR (400MHz, DMSO-d
6): δ 1.58 (m, 2H, CH
2), 1.72 (s, 12H, CH
3), 2.09 (t, 4H, CH
2), 5.52 (s, 4H, CH
2), 6.36-6.40 (d, 2H J=13.6Hz, CH), 7.26-7.40 (m, 12H, CH), 7.62-7.64 (d, 2H, CH), 7.85 (s, 2H, CH), 8.22-8.25 (d, 2H, J=13.6Hz, CH) .Q-TOFMS:M
-1Calculated value: 793.2173, test value: 793.2186.
Embodiment 2
Synthetic and the sign of target dyestuff IIa:
Parent dyestuff Ib is in advance 50-60 ℃ of following vacuum-drying 8 hours, and the underpressure distillation in advance of nucleophilic reagent aniline is refining.Used glassware descended dry 3 hours at 120 ℃ in advance, after three mouthfuls of round-bottomed flasks of 50mL cool off under nitrogen atmosphere, added nucleophilic reagent and magnetic stir bar of 100mg (0.12mmol) raw material dyestuff Ib and 1.2mmol.Add the about 15mL of dry DMF with syringe under the nitrogen atmosphere.Reaction mixture is incubated 100 ℃ and stirred 2 hours under nitrogen protection.Reaction solution is poured in the 300mL ether of quick stirring, and a large amount of green solid precipitations generate.Filter out filtrate with sand core funnel, obtain solid and dissolve from funnel with methyl alcohol, evaporate to dryness methyl alcohol obtains the dyestuff crude product and purifies with the reverse phase filler post.
Dyestuff IIa,
1H NMR (400MHz, DMSO-d6): δ 1.35 (s, 12H, CH
3), 1.72-1.74 (m, 2H, CH
2), 2.39-2.42 (t, 4H, CH
2), 5.32 (s, 4H, CH
2), 5.99-6.02 (d, 2H J=13.6Hz, CH), 6.79-6.83 (t, 1H, CH), and 6.97-6.99 (d, 2H, CH), 7.14-7.16 (d, 2H, CH), and 7.20-7.22 (d, 4H, CH), 7.24-7.28 (t, 4H, CH), and 7.30-7.34 (t, 4H, CH), 7.55-7.57 (d, 2H, CH), 7.63 (s, 2H, CH), 7.90-7.93 (d, 2H, J=13.6Hz, CH) .MS, m/z:851.3 (M-)
Embodiment 3
Synthetic and the sign of target dyestuff IIb:
Synthetic method is with example 2, and wherein nucleophilic reagent uses γ-An Jidingsuan, and temperature of reaction is 40 ℃.
Dyestuff IIb,
1H NMR (400MHz, DMSO-d6): δ 0.86 (m, 2H, CH
2), 1.25 (m, 4H, CH
2), 1.63 (s, 12H, CH
3), 1.93 (m, 2H, CH
2), 2.29 (t, 4H, CH
2), 5.20 (s, 4H, CH
2), 5.72 (d, 2HJ=13.2Hz, CH), 7.00-7.02 (d, 2H, CH), 7.25 (m, 6H, CH), 7.34 (m, 4H, CH), 7.53 (m, 4H, CH), 7.64 (s, 2H, CH) .MS, m/z:861.3 (M-).
Embodiment 4
Synthetic and the sign of target dyestuff IIIa:
Synthetic method is with example 2, and wherein nucleophilic reagent uses thiophenol, and temperature of reaction is a room temperature.
Dyestuff IIIa,
1H NMR (400MHz, DMSO-d
6): δ 1.48 (s, 12H, CH
3), 1.80 (m, 2H, CH
2), 2.56 (t, 4H, CH
2), 5.48 (s, 4H, CH
2), 6.36-6.39 (d, 2H J=13.2Hz, CH), 7.13-7.17 (t, 1H, CH), 7.23-7.26 (t, 6H, CH), 7.29-7.33 (t, 6H, CH), 7.34-7.38 (t, 4H, CH), 7.59-7.61 (d, 2H, CH), 7.74 (s, 2H, CH), 8.59-8.62 (d, 2H, J=13.2Hz, CH) .Q-TOFMS:M
-1Calculated value: 867.2596, test value: 867.2598.
Embodiment 5
Synthetic and the sign of target dyestuff IIIb:
Synthetic method is with example 2, and wherein nucleophilic reagent uses the p-aminophenyl thiophenol, and temperature of reaction is a room temperature.
Dyestuff IIIb,
1H NMR (400MHz, DMSO-d6): δ 1.58 (s, 12H, CH
3), 1.72 (m, 2H, CH
2), 2.55 (t, 4H, CH
2), 5.46 (s, 4H, CH
2), 6.31-6.35 (d, 2H J=13.6Hz, CH), 6.50-6.52 (d, 3H, CH), and 6.95-6.98 (d, 2H, CH), 7.05-7.07 (d, 2H, CH), and 7.23-7.25 (d, 3H, CH), 7.28-7.31 (t, 3H, CH), and 7.33-7.37 (m, 3H, CH), 7.60-7.62 (d, 2H, CH), 7.77 (s, 2H, CH), 8.69-8.73 (d, 2H, J=13.6Hz, CH) .Q-TOFMS:M-1 calculated value: 882.2705, test value: 882.2742.
Embodiment 6
Synthetic and the sign of target dyestuff IIIc:
Synthetic method is with example 2, and wherein nucleophilic reagent uses the acetparaminosalol thiophenol, and temperature of reaction is a room temperature.
Dyestuff IIIc,
1H NMR (400MHz, DMSO-d6): δ 1.51 (s, 12H, CH
3), 1.79 (m, 2H, CH
2), 1.98 (s, 3H, CH
3), 2.55 (t, 4H, CH
2), 5.47 (s, 4H, CH
2), 6.35-6.38 (d, 2H J=13.6Hz, CH), 7.17-7.19 (d, 2H, CH), 7.23-7.25 (d, 4H, CH), 7.29-7.31 (t, 4H, CH), 7.34-7.38 (t, 4H, CH), and 7.51-7.54 (d, 2H, CH), 7.59-7.61 (d, 2H, CH), 7.75 (s, 2H, CH), 8.60-8.63 (d, 2H, J=13.6Hz, CH), 9.95 (s, 1H, NH) .13C NMR (400MHz, DMSO-d6): δ 20.5,23.9,25.7,27.3,47.0,48.9,102.7,110.7,119.9,120.3,126.3,126.6,126.9,127.8,129.0,130.0,134.1,135.0,137.6,140.4,142.4,145.4,145.6,151.1,168.3, the 172.7.Q-TOFMS:M-1 calculated value: 924.2811, test value: 924.2845.
Embodiment 7
Synthetic and the sign of target dyestuff IIId:
Synthetic method is with example 2, and wherein nucleophilic reagent uses Thiovanic acid, and temperature of reaction is a room temperature.
Dyestuff IIId,
1H NMR (400MHz, DMSO-d
6): δ 1.63 (m, 2H, CH
2) 1.71 (s, 12H, CH
3), 2.40 (t, 4H, CH
2), 5.48 (s, 4H, CH
2), 6.31-6.34 (d, J=13.6Hz, 2H, CH), 7.26-7.27 (m, 8H, CH), 7.33-7.35 (d, 4H, CH), 7.61 (d, 2H, CH), 7.80 (s, 2H, CH), 8.79-8.82 (d, 2H, J=13.2Hz, CH).
13C NMR (400MHz, DMSO-d
6): δ 20.5,25.6, and 27.6,47.0,49.0,102.3,110.5,120.0,126.3,126.6,127.8,129.0,133.5,135.0,140.3,142.7,144.8,145.6,157.3, the 172.4.Q-TOFMS:M-1 calculated value: 849.2338, test value: 849.2297.
Table 1 dyestuff IIa, IIb and parent dyestuff Ib are at the aqueous solution (1 * 10-6M) spectroscopic data in
aMolar extinction coefficient unit is cm-1M
bThe measurement solvent of fluorescence quantum yield is methyl alcohol, and the reference dyestuff is the methanol solution (φ of rhodamine Bf＝0.69).
The derivative dye that table 2 sulphur replaces and parent dyestuff are at the aqueous solution (1 * 10-6M) spectroscopic data in.
aMolar extinction coefficient unit is cm-1M
bThe measurement solvent of fluorescence quantum yield is dimethyl sulfoxide (DMSO), and the reference dyestuff is the DMSO solution (φ of IR-125f＝0.13).
Claims (1)
1. the class near infrared meta that is used for the bioluminescence analysis replaces seven methine cyanine dyes, it is characterized in that this dyestuff has following array structure
General formula I:
In the general formula:
X＝II；
R
1, R
2=(CH
2)
nR
8, (CH
2)
mOR
9, (CHR
10CH
2O)
pR
9Or CH
2C
6H
4R
8
R
3, R
4, R
8=H, SO
3R
11Or CO
2R
12
R
5=(CH
2)
nR
8, (CH
2)
mOR
9Or (CHR
10CH
2O)
pR
9
R
6=(CH
2)
nR
8, (CH
2)
mOR
9, (CHR
10CH
2O)
pR
9, a position or contraposition C
6H
4R
17
R
9=H or C
1-18Alkyl;
R
10=H or CH
3
R
11=H or M; M=Na, K, N (R
13R
14R
15R
16);
R
12=H, M, R
7Or C
1-18Alkyl;
R
13, R
14, R
15, R
16=H, C
1-18Alkyl; (CH
2)
mOR
9Or (CHR
10CH
2O)
pR
9
R
17=H, NH
2, NHCOR
9Or (CH
2)
nR
8
n、m、p＝0-18。
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| ITSV20060002A1 (en) | 2006-01-19 | 2007-07-20 | Ferrania Technologies Spa | FLUORESCENT DYE OF CIANIN TYPE |
| CN100486966C (en) * | 2006-04-07 | 2009-05-13 | 山东师范大学 | Near infrared fluorescent probe for detecting hydroxyl free radicals, synthesis method and use thereof |
| WO2008127139A1 (en) * | 2007-04-11 | 2008-10-23 | Institut Molekulyarnoi Biologii Im. V.A. Engeldardta Rossiiskoi Akademii Nauk | Indicyanine dyes and the derivatives thereof for analysing biological micromolecules |
| US20110313129A1 (en) * | 2009-02-11 | 2011-12-22 | Life Technologies Corporation | Large stokes shift dyes |
| CN102268191B (en) * | 2010-06-06 | 2013-08-14 | 史春梦 | Heptamethine indocyanine dye, synthetic method thereof and applications thereof |
| CN102492417A (en) * | 2011-12-07 | 2012-06-13 | 天津工业大学 | Mercury ion fluorescent probe and preparation method thereof |
| CN103146220B (en) * | 2012-10-25 | 2014-08-06 | 西安电子科技大学 | Symmetric pentamethyl cyanine dye and application thereof to molecular imaging |
| CN103529009A (en) * | 2013-11-01 | 2014-01-22 | 厦门大学 | Method for detecting hydrogen sulfide in serum through near-infrared fluorescent probe Cy-Cl |
| CN103911016B (en) * | 2014-03-28 | 2016-04-06 | 西安电子科技大学 | A kind of method prepared conjugated bridge chain meta and replace cyanine dye |
| CN105111773B (en) * | 2015-08-19 | 2017-06-27 | 大连理工大学 | One class amino cyanine fluorochrome and its preparation method and application |
| CN106496094B (en) * | 2016-11-01 | 2019-05-31 | 东南大学 | A kind of Heptamethine cyanines near-infrared fluorescent molecular probe and its preparation method and application |
| CN106496093B (en) * | 2016-11-01 | 2019-05-28 | 东南大学 | A kind of near-infrared fluorescent molecular probe and its synthetic method and application |
| CN107057398B (en) * | 2017-04-21 | 2019-08-16 | 中国药科大学 | A kind of application of seven methines cyanine dye and its accurate diagnosing and treating of tumour |
| CN108033907A (en) * | 2017-11-14 | 2018-05-15 | 中国医学科学院生物医学工程研究所 | A kind of Heptamethine cyanines active fluoro probe and preparation method and application |
| CN108728080B (en) * | 2018-05-25 | 2021-02-05 | 中国科学院烟台海岸带研究所 | Organic compound for detecting histamine and application thereof |
| CN108659142B (en) * | 2018-05-29 | 2020-09-29 | 苏州百源基因技术有限公司 | Fluorescence-labeled polysaccharide and preparation method and application thereof |
| CN108624081B (en) * | 2018-05-29 | 2020-03-10 | 苏州百源基因技术有限公司 | Fluorescent dye and preparation method and application thereof |
| CN111196896B (en) * | 2019-12-25 | 2021-09-28 | 中国药科大学 | Water-soluble heptamethine cyanine near-infrared dye with tumor targeting property and application thereof |
| CN113683602B (en) * | 2021-09-08 | 2022-06-10 | 中国人民解放军陆军军医大学 | Heptamethine cyanine micromolecule for multi-modal treatment of hypoxic tumors, and preparation method and application thereof |
| CN116023326A (en) * | 2023-01-09 | 2023-04-28 | 大连理工大学 | Nitrogen aryl pyridine cyanine dye derivative fluorescent probe, and preparation method and application thereof |
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