CN114685311A - Azo aromatic compound, application thereof and reagent for enhancing Raman scattering signals - Google Patents

Azo aromatic compound, application thereof and reagent for enhancing Raman scattering signals Download PDF

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CN114685311A
CN114685311A CN202011590275.7A CN202011590275A CN114685311A CN 114685311 A CN114685311 A CN 114685311A CN 202011590275 A CN202011590275 A CN 202011590275A CN 114685311 A CN114685311 A CN 114685311A
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CN114685311B (en
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唐浴尘
高婷娟
张礼知
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Central China Normal University
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    • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
    • C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
    • C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
    • C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
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    • C07C255/65—Carboxylic acid nitriles containing cyano groups and nitrogen atoms further bound to other hetero atoms, other than oxygen atoms of nitro or nitroso groups, bound to the same carbon skeleton with the nitrogen atoms further bound to nitrogen atoms
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    • C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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Abstract

The invention relates to the field of analytical chemistry, and discloses an azo aromatic compound, application thereof and a reagent for enhancing Raman scattering signals, wherein the compound has a structure shown in a formula (I) or a formula (II). When the azo aromatic compound provided by the invention is used for enhancing Raman scattering signals, the sensitivity is improved by more than 2-4 orders of magnitude, and the azo aromatic compound has frequency adjustability of 10 different spectral bands. Formula (I):
Figure DDA0002868418630000011
formula (II):
Figure DDA0002868418630000012

Description

Azo aromatic compound, application thereof and reagent for enhancing Raman scattering signals
Technical Field
The invention relates to the field of analytical chemistry, in particular to an azo aromatic compound, application thereof and a reagent for enhancing Raman scattering signals.
Background
The development of modern spectroscopy and microscopy technology has promoted the more sensitive and specific observation of the dynamic process of molecular changes in living systems. In particular, the ability to directly observe a large number of different molecular species inside a cell is particularly important for understanding complex systems and processes, while imaging many species with high sensitivity and high selectivity at the cellular and subcellular level remains challenging. Raman microscopy has become a useful tool for performing multicolor live cell imaging, in contrast to other widely used imaging techniques, particularly fluorescence imaging.
Due to the inherent nature of molecular vibrations coupled with incident light, raman signals provide characteristic chemical bond information, are stable, have narrow spectral lines, are free of photobleaching phenomena, allow multicolor live cell imaging in a typical raman spectral range, and enable visual study of live cell dynamic processes through multicolor imaging. However, the lower sensitivity of raman imaging compared to fluorescence imaging limits its wide application. The cross section of Raman scattering is small, and the Raman scattering becomes a main obstacle for sensitive imaging of endogenous components or exogenous probes in living cells.
Currently, various strategies and techniques have been developed to enhance the raman signal of a compound under test, such as Surface Enhanced Raman Scattering (SERS), coherent anti-stokes raman scattering (CARS), Stimulated Raman Scattering (SRS), and Tip Enhanced Raman Scattering (TERS). Surface Enhanced Raman (SERS) requires the use of surface plasmons on nanoparticles, and its reproducibility is to be improved. SERS provides significant sensitivity and diversity for cell imaging, but SERS particles may result in lower permeability to living cells and adverse biological interference compared to small molecule probes. TERS requires precise control of the relative position of the metal nanotip and the surface to achieve signal enhancement. Non-linear optical methods including coherent anti-stokes raman (CARS) and Stimulated Raman (SRS) require complex and expensive instrumentation to build. Therefore, if the signal inherent to the raman dye itself is greatly enhanced at the molecular level, it will have a great influence on the research and application in the raman field. When examined using typical commercial raman microscopy systems, convenient cellular raman imaging has a great demand for small molecule signaling compounds with inherently strong raman scattering.
Since the study of 5-ethynyl-2' -deoxyuridine (EdU) -based cellular raman imaging has been reported, a number of alkyne-containing molecules have been used as bio-orthogonal raman probes for spontaneous or nonlinear raman microscopy imaging. However, when high-quality raman imaging is performed using spontaneous raman microscopy, the raman intensity of the C ≡ C stretching vibration still cannot meet the requirement of sensitivity. Currently, phenyl-terminated alkyne signal molecules are synthesized as polychromatic raman reporter molecules for SRS imaging, but as the length of the polyacetylene chain increases, the chemical stability of the molecule becomes problematic. At the same time, their solubility also decreases, limiting the efficiency of cell targeting and staining.
Resonance raman strategies provide a means to enhance raman sensitivity by coupling electrons with chromophores and the transition of vibrational energy levels. In this case, there is a degree of matching of the vibrational energy level transition with the electronic energy level transition, resulting in significant amplification of the raman scattering signal. Recently, a polydiacetylene-based raman dye has reached 4 orders of magnitude or more relative raman intensity at resonance relative to EdU. This strategy is based on increasing the conjugate length to red-shift the absorption wavelength. However, the research result cannot provide the adjustability of the raman frequency, and the signal molecule presents a strong fluorescence background when excited by electron resonance, which interferes with the detection of the raman signal at this time.
That is, when the excitation wavelength is close to the absorption wavelength of the compound, the electron transition can increase the probability of raman scattering, resulting in an enhanced raman signal, but the common problem caused thereby is that when the excitation wavelength is near the absorption peak of the compound, the raman signal cannot be accurately detected due to the accompanying strong fluorescence background.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks of the prior art and to provide a new class of molecules that enhance raman scattering.
In order to achieve the above object, a first aspect of the present invention provides an azo aromatic compound having a structure represented by formula (I) or formula (II):
formula (I):
Figure BDA0002868418610000021
formula (II):
Figure BDA0002868418610000022
formula (I1):
Figure BDA0002868418610000023
formula (I2):
Figure BDA0002868418610000024
formula (I3):
Figure BDA0002868418610000025
formula (I4):
Figure BDA0002868418610000026
formula (I5):
Figure BDA0002868418610000027
formula (I6):
Figure BDA0002868418610000028
wherein, in the formula (I) and the formula (II),
R11、R12、R14、R15each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a); r13Is selected from R1-CONH-、NH2-、(R1)(R2)N-、C1-6Alkoxy radical of (2), R1-COO-、R1-OCO-, nitro, cyano; or, R11And R15Each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a), R12、R13And R14Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-6At least one substituent of the alkyl group of (a);
R21、R22、R24and R25Each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy group of (a); r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and R1-COO-、R1-OCO-、NC-、(R1)(R2) N-, cyano; or R21And R25Each independently selected from H, C1-6Alkyl of R1-COO-、R1-OCO-、C1-6Alkoxy group of (a); r22、R23And R24Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-6At least one substituent of the alkyl group of (a);
l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R1and R2Each independently selected from H, C1-6Alkyl of (C)1-6alkoxy-C of1-3alkylene-hydroxy-C of1-3Alkylene-of (a), a group of formula (I6);
R3and R4Each independently selected from H, phenyl, from R1-OCO-substituted phenyl;
n, m, x, y and z are each independently integers from 1 to 10.
A second aspect of the invention provides the use of a compound as described in the first aspect hereinbefore in raman scattering signals.
In a third aspect, the present invention provides a reagent for enhancing raman scattering signal, the reagent comprising a functional compound for enhancing raman scattering signal, wherein the functional group of the functional compound is provided by the compound described in the first aspect.
The inventor of the invention finds that the compound of the invention can promote the ultraviolet visible absorption wavelength of the compound to generate red shift by introducing the conjugated azobenzene group, and can be matched with the Raman excitation wavelength to generate resonance Raman effect. In addition, the azobenzene structure introduced by the invention can generate a cis-trans isomerization phenomenon in an excited state, so that the energy of the excited state can be released out in a non-radiative transition mode, the symbiotic fluorescence is effectively quenched, and the enhanced Raman signal is obviously displayed.
Compared with 5-ethynyl 2' -deoxyuridine (EdU), the azo aromatic compound provided by the invention has the advantages that the sensitivity is improved by more than 2-4 orders of magnitude when the azo aromatic compound is used for enhancing a Raman scattering signal, and the azo aromatic compound has the frequency adjustability of 10 different spectral bands. The present invention also develops viable cell organelle targeting probes for 6-color imaging and 7-multiplex coding applications.
The azo aromatic compounds provided by the present invention enhance the coupling between the electron and vibrational transitions and/or improve the symmetry of the vibrational modes. These effects lead to the ultrasensitive raman signals of the azo aromatic compounds of the present invention, providing a platform for the design and synthesis of small molecule raman probes that enable the multicolor targeting and imaging of specific organelles/biomolecules in living cells by spontaneous raman microscopy.
The probe formed by the azo aromatic compound provided by the invention can visualize different types of organelles such as mitochondria, lysosomes and the like on different spectral bands of Raman shift in a single image.
The azo aromatic compound provided by the invention opens up a new prospect for simultaneously tracking the reaction between functional biomolecules, organelles and/or cell types in the process of intracellular and/or intercellular transformation in the future.
More specifically, the azo aromatic compounds provided by the present invention can be prepared byUltrasensitivity of spontaneous Raman microscopy for live cell imaging (RIE of about 10)4Level) and multi-color functions (selection from 10 frequency options). These molecules have emerged as frequency tunability and have developed viable cell organelle targeting probes for 6-color imaging.
Drawings
FIG. 1 is a Raman spectrum of 100. mu.M C3.1 in DMF solvent.
FIG. 2 is the spectral properties of compound E3.1. FIG. 2a shows the absorption spectrum and fluorescence spectrum of E3.1 at 5.0. mu.M. FIG. 2b shows the stretching vibration v (C) of benzene ring carbon-azo nitrogen under 532nm and 785nm laser light respectively for 100 μ M E3.1ph-N) mode raman spectra. FIG. 2c shows E3.1 at 1122cm-1At the Raman signal relative to the DMF solvent peak (at 867 cm)-1Point) and its concentration.
FIG. 3 shows the results of an immunoassay using E3.1 as the signal source. FIG. 3a is the Raman spectrum of the functionalized polystyrene microspheres bound with immunoglobulin under 532nm laser. The concentration of immunoglobulin was 10 each-10、10-11、10-12、10- 13mol/L. FIG. 3b shows immunoglobulins at 10-13To 10-6Relative raman intensity profile of the mol/L concentration range. Relative Raman intensity of 1122cm-1Raman signal and polystyrene 1001cm-1The ratio of the raman signals. Fig. 3c is a linear plot of relative raman signal versus concentration.
FIG. 4 shows the results of co-staining HeLa cells with azo-enhanced Raman and fluorescent probes. FIG. 4a shows the result of co-staining with mitochondrial probes. Fig. 4b is the co-staining result of lysosomal probe.
Figure 5 is a multicolor raman image of azo enhanced raman probe stained hela cells. Wherein, fig. 5a is the chemical structure of 6 azo enhanced raman probes. FIG. 5b is a six color Raman image of cells. Fig. 5c shows raman spectra of 6 raman probes in cells. Fig. 5d shows the cytotoxicity test results of 6 raman probes.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
Said C of the invention1-6Alkyl of (a) represents alkyl having a total number of carbon atoms of 1 to 6, including straight chain, branched chain and cyclic alkyl groups, including but not limited to methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl, cyclopentyl, n-hexyl, cyclohexyl.
Said C of the invention1-6The alkoxy group of (b) represents an alkoxy group having a total number of carbon atoms of 1 to 6, including linear alkoxy groups, branched alkoxy groups and cycloalkoxy groups, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, tert-butoxy, cyclobutoxy, n-pentoxy, isopentoxy, cyclopentoxy, n-hexoxy, cyclohexoxy. For "carboxyl substituted C1-6The alkoxy group of (1) has a similar explanation, except that "carboxyl-substituted C1-6Alkoxy of (A) means that at least one carboxyl group is substituted by C1-6At least one H on the alkoxy group of (a).
C of the invention1-6alkoxy-C of1-3Alkylene-of (A) represents R31-R32-a group of formula (I) wherein R31Is represented by C1-6Wherein R is32Is represented by C1-3An alkylene group of (a). "hydroxy-C1-3Alkylene- "of (a) has a similar interpretation as this.
The invention relates to compounds consisting of1-OCO-substituted phenyl represents at least one H atom in phenyl group represented by R1A substituent represented by-OCO-, and the specific substitution position is not particularly limited.
As previously mentioned, a first aspect of the present invention provides an azo aromatic compound having a structure represented by formula (I) or formula (II):
formula (I):
Figure BDA0002868418610000041
formula (II):
Figure BDA0002868418610000042
formula (I1):
Figure BDA0002868418610000043
formula (I2):
Figure BDA0002868418610000044
formula (I3):
Figure BDA0002868418610000045
formula (I4):
Figure BDA0002868418610000046
formula (I5):
Figure BDA0002868418610000051
formula (I6):
Figure BDA0002868418610000052
wherein, in the formula (I) and the formula (II),
R11、R12、R14、R15each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a); r13Is selected from R1-CONH-、NH2-、(R1)(R2)N-、C1-6Alkoxy radical of (2), R1-COO-、R1-OCO-, nitro, cyano; or, R11And R15Each independently selected from H, C1-6Alkyl group of (A) or (B),C1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a), R12、R13And R14Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-6At least one substituent of the alkyl group of (a);
R21、R22、R24and R25Each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy group of (a); r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and R1-COO-、R1-OCO-、NC-、(R1)(R2) N-, cyano; or R21And R25Each independently selected from H, C1-6Alkyl of R1-COO-、R1-OCO-、C1-6Alkoxy group of (a); r22、R23And R24Together with the parent nucleus benzene ring of the three to form an N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not a benzene ring, is optionally provided with C1-6At least one substituent of the alkyl group of (a); l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R1and R2Each independently selected from H, C1-6Alkyl of (C)1-6alkoxy-C of1-3alkylene-hydroxy-C of1-3Alkylene-of (a), a group of formula (I6);
R3and R4Each independently selected from H, phenyl, from R1-OCO-substituted phenyl;
n, m, x, y and z are each independently integers from 1 to 10.
Preferably, in the formulae (I) and (II),
R11、R12、R14、R15each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a); r13Is selected from R1-CONH-、NH2-、(R1)(R2)N-、C1-6Alkoxy radical of (2), R1-COO-、R1-OCO-, nitro, cyano; or, R11And R15Each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a), R12、R13And R14Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-4At least one substituent of the alkyl group of (a);
R21、R22、R24and R25Each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy group of (a); r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and R1-COO-、R1-OCO-、NC-、(R1)(R2) N-, cyano; or R21And R25Each independently selected from H, C1-4Alkyl of R1-COO-、C1-4Alkoxy of (2); r22、R23And R24Together with the parent nucleus benzene ring of the three to form an N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not a benzene ring, is optionally provided with C1-4At least one substituent of the alkyl group of (a); l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R1and R2Each independently selected from H, C1-4Alkyl of (C)1-4alkoxy-C of1-3alkylene-hydroxy-C of1-3Alkylene-of formula (I6);
R3and R4Each independently selected from H, phenyl, from R1-OCO-substituted phenyl;
n, m, x, y and z are each independently integers from 1 to 6.
More preferably, in the formula (I) and the formula (II),
R11、R12、R14、R15each independently selected from H, methyl, methoxy, -O (CH)2)5At least one of COOH；R13Is selected from (CH)3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2)N-、CH3CONH-、NH2-、CH3O-、CH3COO-, nitro, cyano; or, R11And R15Each independently selected from H, methyl, methoxy, -O (CH)2)5At least one of COOH, R12、R13And R14The three compounds and parent nucleus benzene rings of the three compounds form N-containing fused tricyclic, a methyl substituent optionally exists on the ring structure of the non-benzene ring of the N-containing fused tricyclic, and the ring structures of the non-benzene ring of the N-containing fused tricyclic are all six-membered rings;
R21、R22、R24and R25Each independently selected from H, methoxy, a group represented by formula (I6) -COO-; r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and CH3OCO-、NC-、(CH3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2) N-; or R21And R25Each independently selected from H, methoxy, a group represented by formula (I6) -COO-; r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and CH3OCO-、NC-、(CH3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2) N-, cyano; r22、R23And R24The three compounds and parent nucleus benzene rings of the three compounds form N-containing fused tricyclic, a methyl substituent optionally exists on the ring structure of the non-benzene ring of the N-containing fused tricyclic, and the ring structures of the non-benzene ring of the N-containing fused tricyclic are all six-membered rings; l is selected from a group shown as a formula (I3), a group shown as a formula (I4) and a group shown as a formula (I5);
R3and R4Each independently selected from H, phenyl, and CH3An OCO-substituted phenyl group;
n, m, x, y and z are each independently 1,2, 3, 4, 5 or 6.
According to a preferred embodiment, the azo aromatic compound has the structure shown in formula (I).
More preferably, the azo aromatic compound is selected from any one of the following compounds:
compound a 2.1:
Figure BDA0002868418610000061
compound a 2.2:
Figure BDA0002868418610000062
compound B2.1:
Figure BDA0002868418610000063
compound C2.1:
Figure BDA0002868418610000064
compound D2.1:
Figure BDA0002868418610000065
compound D2.2:
Figure BDA0002868418610000066
compound D2.3:
Figure BDA0002868418610000067
compound D2.4:
Figure BDA0002868418610000068
compound E1.1:
Figure BDA0002868418610000069
compound E1.2:
Figure BDA0002868418610000071
compound E1.3:
Figure BDA0002868418610000072
compound E2.1:
Figure BDA0002868418610000073
compound E2.2:
Figure BDA0002868418610000074
according to another preferred embodiment, the azo aromatic compound has the structure shown in formula (II).
More preferably, the azo aromatic compound is selected from any one of the following compounds:
compound a 3.1:
Figure BDA0002868418610000075
compound a 3.2:
Figure BDA0002868418610000076
compound B3.1:
Figure BDA0002868418610000077
compound B3.2:
Figure BDA0002868418610000078
compound C3.1:
Figure BDA0002868418610000079
compound C3.2:
Figure BDA00028684186100000710
compound E3.1:
Figure BDA0002868418610000081
compound F2.1:
Figure BDA0002868418610000082
the present invention is not particularly limited to a specific preparation method for preparing the aforementioned azo aromatic compound, and those skilled in the art can obtain a specific preparation method for the entire azo aromatic compound of the present invention based on the specific structural formula provided by the present invention in combination with a known synthesis method in the field of organic synthesis and a preparation method in some specific examples provided in the following examples of the present invention. The general preparation process for the preparation of the aforementioned azo aromatic compounds will not be described in detail herein and those skilled in the art should not be construed as limiting the invention.
As mentioned above, a second aspect of the present invention provides the use of the aforementioned azo aromatic compounds to enhance Raman scattering signals.
As described above, the third aspect of the present invention provides a reagent for enhancing raman scattering signal, the reagent comprising a functional compound for enhancing signal, wherein the functional group of the functional compound is provided by the compound of the first aspect.
Preferably, the concentration of said functional compound in said reagent is between 0.1 and 1000. mu. mol/l.
The present invention will be described in detail below by way of examples. In the following examples, the raw materials used are all common commercial products unless otherwise specified.
Preparation example 1: synthesis of group A Compounds (core groups are diynes)
Figure BDA0002868418610000083
To a 25mL single-neck flask were added 3mL of chloroform and 1mL of 1, 4-dioxane, followed by weighing 3mmol of phenylacetylene and 1mmol of methyl 4-acetylenecarboxylateDissolved therein, and 0.05mmol of copper powder and 0.2mmol of tetramethylethylenediamine were added. The mixture was stirred at 50 ℃ for 12 h. Then 20mL of dichloromethane is added, the mixture is washed by saturated ammonium chloride, distilled water and saturated brine in sequence, dried by anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, and a yellow solid product A1.1 is obtained after column chromatography (yield is 65%)1H NMR(400MHz,CDCl3)δ7.99(d,J＝8.3Hz,2H),7.55(d,J＝8.3Hz,2H),7.39-7.30(d,J＝8.3Hz,2H),6.65-6.54(d,J＝8.3Hz,2H),3.92(s,3H).13C NMR(101MHz,CDCl3)δ166.5,147.9,134.2,132.2,129.9,129.5,127.0,114.6,110.2,84.5,79.8,77.5,71.8,52.4.HRMS(ESI):calcd for C18H14NO2 +[M+H]+276.1019,found 276.1017.
0.15mmol of the product A1.1 is weighed and dissolved in 2mL of absolute ethyl alcohol, 1.5mmol of concentrated sulfuric acid and 0.6mmol of potassium nitrite (dissolved in 1mL of water) are added under the ice bath condition, after the ice bath is carried out for 30min, the pH value is adjusted to 5.0 by sodium acetate, and then the ethanol solution of N-ethyl N-hydroxyethylaniline (0.3mmol) is added for continuous reaction for 3 h. After the reaction was completed, 20mL of ethyl acetate was added, and the mixture was washed with distilled water and saturated brine, dried over anhydrous magnesium sulfate, and subjected to column chromatography to obtain a red solid product a2.2 (yield 47%).1H NMR(400MHz,Chloroform-d)δ8.01(d,J＝8.1Hz,2H),7.86(d,J＝8.8Hz,2H),7.81(d,J＝8.3Hz,2H),7.63(d,J＝8.2Hz,2H),7.59(d,J＝8.1Hz,2H),6.78(d,J＝8.8Hz,2H),4.32(t,J＝6.3Hz,2H),3.93(s,3H),3.67(t,J＝6.3Hz,2H),3.50(t,J＝6.9Hz,2H),1.25(d,J＝7.0Hz,3H).HRMS(ESI):calcd for C28H26N3O3 +[M+H]+452.1969,found 452.1972.
Figure BDA0002868418610000091
The same synthetic procedure as for A1.1 was repeated to give intermediate A1.2 as a pale yellow solid.1H NMR(400MHz,CDCl3)δ7.31(d,J＝8.4Hz,4H),6.58(d,J＝8.4Hz,4H),3.88(s,4H).13C NMR(101MHz,CDCl3)δ147.3,133.9,114.7,111.2,81.8,72.4.HRMS(ESI):calcd for C16H13N2 +[M+H]+233.1073,found 233.1083.
Weighing the product A1.2(0.15mmol, 1eq) and dissolving in 2mL of absolute ethanol, adding concentrated sulfuric acid (1.5mmol, 10eq) and potassium nitrite (0.6mmol, 4eq) (dissolving in 1mL of water) under the ice-bath condition, adjusting the pH to 5.0 with sodium acetate after ice-bath for 30min, adding an ethanol solution of N-ethyl N-hydroxyethylaniline (0.3mmol), and continuing the reaction for 3 h. After the reaction was completed, 20mL of ethyl acetate was added, and the mixture was washed with distilled water and saturated brine, dried over anhydrous magnesium sulfate, and subjected to column chromatography to obtain a red solid product a3.1 (yield 47%).1H NMR(400MHz,Chloroform-d)δ7.88(d,J＝8.5Hz,4H),7.82(t,J＝6.9Hz,4H),7.63(t,J＝9.1Hz,4H),6.83(d,J＝8.6Hz,4H),4.35(t,J＝6.0Hz,4H),3.71(t,J＝6.1Hz,4H),3.54(q,J＝7.1Hz,4H),1.24(d,J＝7.2Hz,6H).HRMS(ESI):calcd for C36H37N6O2 -[M+H]+585.2973,found 585.2980.
Preparation example 2: synthesis of group B Compounds (core groups are Triacetylenes)
Figure BDA0002868418610000092
A50 mL single neck flask was charged with 9mL of chloroform and 3mL of 1, 4-dioxane, followed by trimethylsilylacetylene (15mmol, 3eq) and methyl 4-acetylenecarboxylate (5mmol, 1eq) dissolved therein, and copper powder (0.25mmol, 0.05eq) and tetramethylethylenediamine (1mmol, 0.2eq) added. The mixture was stirred at 50 ℃ for 12 h. Then, 40mL of dichloromethane was added, and the mixture was washed with saturated ammonium chloride, distilled water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a pale yellow solid product 1 (yield 64%) after column chromatography.1H NMR(400MHz,Chloroform-d)δ8.03–7.91(m,2H),7.53(d,J＝8.4Hz,2H),3.91(s,3H),0.23(s,9H).13C NMR(101MHz,CDCl3)δ166.3,132.6,130.4,129.5,126.1,92.5,87.4,76.8,75.6,52.4,0.4.HRMS(ESI):calcd for C15H16O2SiNa+[M+Na]+279.0812,found 279.0825.
819mg of product 1 are dissolved in 25mL of acetonitrile, weighed against water (6.4mmol) and silver fluoride (3.2mmol), and after stirring the mixture for 20min in the absence of light, N-bromosuccinimide (3.2mmol) is added and stirring is continued for 3 h. After the reaction was completed, the solvent was distilled off under reduced pressure, diluted with 30mL of ethyl acetate, washed with distilled water and saturated brine successively, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a pale yellow solid product 2 (yield 85%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ7.98(d,J＝8.1Hz,2H),7.54(d,J＝8.2Hz,2H),3.91(s,3H).13C NMR(101MHz,CDCl3)δ166.3,132.8,130.5,129.5,125.7,76.9,73.0,65.1,52.4,46.4.HRMS(ESI):calcd for C12H8BrO2 +[M+H]+262.9702,found 262.9712.
Product 2(1mmol), 4-ethynylaniline (1.5mmol), bis (triphenylphosphine) palladium (II) chloride (0.1mmol) and cuprous iodide (0.1mmol) were weighed into a 25mL two-necked flask, and after 3 times of repeated nitrogen purging, 5mL tetrahydrofuran and 170. mu.L triethylamine were added, and the mixture was stirred at 50 ℃ for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, diluted with 20mL of ethyl acetate, washed with saturated ammonium chloride, distilled water and saturated brine in this order, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a yellow solid product B1.1 (yield 65%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ7.99(dd,J＝8.3,1.8Hz,2H),7.60–7.50(m,2H),7.39–7.30(m,2H),6.65–6.54(m,2H),5.38(s,2H),3.92(d,J＝1.7Hz,3H).13C NMR(101MHz,DMSO-d6)δ150.8,134.8,132.8,130.6,129.6,125.7,114.2,105.7,82.6,78.7,77.5,72.3,69.2,68.5,65.5,52.4.HRMS(ESI):calcd for C20H13NO2 +[M]+299.0941,found 299.0988.
The same diazotization step as for A1.1 is carried out on B1.1 to obtain B2.1 as an orange solid.1H NMR(400MHz,Chloroform-d)δ8.01(d,J＝8.1Hz,2H),7.86(d,J＝8.8Hz,2H),7.81(d,J＝8.3Hz,2H),7.63(d,J＝8.2Hz,2H),7.59(d,J＝8.1Hz,2H),6.78(d,J＝8.8Hz,2H),4.32(t,J＝6.3Hz,2H),3.93(s,3H),3.67(t,J＝6.3Hz,2H),3.50(t,J＝6.9Hz,2H),2.67(d,J＝6.3Hz,2H),2.63(d,J＝6.2Hz,2H),1.25(d,J＝7.0Hz,3H).HRMS(ESI):calcd for C30H26N3O3 +[M+H]+476.1969,found 476.1975.
Figure BDA0002868418610000101
5mL of tetrahydrofuran was added to a 25mL single-neck flask, and 4-ethynylaniline (6mmol) and di-tert-butyl dicarbonate (18mmol) were weighed and dissolved therein, and the mixture was stirred under reflux for 12 hours, and after completion of the reaction, the solvent was distilled off under reduced pressure. To this system were added 6mL of chloroform, 2mL of 1, 4-dioxane and 3mL of tetramethylethylenediamine, and trimethylsilylacetylene (14mmol) and copper powder (0.6mmol) were weighed and stirred at 50 ℃ for 12 hours. Then, 20mL of dichloromethane was added, and the mixture was washed with saturated ammonium chloride, distilled water and saturated brine, dried over anhydrous magnesium sulfate, and subjected to column chromatography to distill off the solvent under reduced pressure to obtain a pale yellow solid product 3 (yield 70%).1H NMR(400MHz,Chloroform-d)δ7.41(dd,J＝8.7,2.3Hz,2H),7.34(dd,J＝8.6,2.3Hz,2H),1.51(d,J＝2.3Hz,9H),0.23(d,J＝2.4Hz,9H).13C NMR(101MHz,CDCl3)δ152.3,139.5,133.6,118.0,115.2,90.2,88.0,81.0,76.9,73.4,28.3,0.4.HRMS(ESI):calcd forC18H24NO2Si+[M+H]+314.1571,found 314.1582.
1.34g of product 3 are dissolved in 30mL of acetonitrile, water (8.4mmol) and silver fluoride (4.2mmol) are weighed out, and after stirring the mixture for 20min in the dark, N-bromosuccinimide (4.2mmol) is added and stirring is continued for 3 h. After the reaction was completed, the solvent was distilled off under reduced pressure, diluted with 40mL of ethyl acetate, washed with distilled water and saturated brine successively, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a pale yellow solid product 4 (yield 81%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ7.41(dt,J＝9.9,6.2Hz,2H),7.33(t,J＝6.9Hz,2H),1.49(d,J＝6.6Hz,9H).13C NMR(101MHz,CDCl3)δ146.8,139.6,133.8,117.3,114.9,85.2,81.1,65.5,60.4,44.0,28.3.HRMS(ESI):calcd for C15H13BrNO2 -[M-H]-318.0135,found 318.0148.
Product 4(1mmol), 4-ethynylaniline (1.5mmol), bis (triphenylphosphine) palladium (II) chloride (0.1mmol) and cuprous iodide (0.1mmol) were weighed into a 25mL two-necked flask, and after repeatedly introducing nitrogen gas 3 times, 5mL of tetrahydrofuran and 170. mu.L of triethylamine were added, and the mixture was stirred at 50 ℃ for 12 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, diluted with 20mL of ethyl acetate, washed with saturated ammonium chloride, distilled water and saturated brine in this order, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a yellow solid product 5 (yield 60%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ7.48–7.40(m,2H),7.37–7.29(m,4H),6.58(d,J＝8.0Hz,2H),1.51(d,J＝1.6Hz,9H).13C NMR(101MHz,CDCl3)δ152.3,148.0,139.7,134.7,133.9,133.3,118.0,114.6,109.6,81.1,79.9,78.4,74.1,72.9,67.0,66.1,28.23.HRMS(ESI):calcd for C23H21N2O2 +[M+H]+357.1598,found 357.1580.
After the product 5 was dissolved in 3mL of dichloromethane, 1mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours, the solvent was removed by distillation under reduced pressure, and after a sufficient amount of triethylamine was added, the mixture was dissolved in 10mL of ethyl acetate, washed with distilled water and saturated brine successively, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure, and column chromatography was performed to give a yellow solid product B1.2 (yield 83%).1H NMR(400MHz,Chloroform-d)δ7.37–7.28(m,4H),6.62–6.53(m,4H),3.93(s,4H).13C NMR(101MHz,CDCl3)δ147.8,134.6,114.7,109.9,79.6,73.1,66.5.HRMS(ESI):calcd for C18H13N2 +[M+H]+257.1073,found 257.1070.
B1.2 is subjected to the same diazotization step as A2.1 to obtain B3.1.1H NMR(400MHz,Chloroform-d)δ7.88(d,J＝8.5Hz,4H),7.82(t,J＝6.9Hz,4H),7.63(t,J＝9.1Hz,4H),6.83(d,J＝8.6Hz,4H),4.35(t,J＝6.0Hz,4H),3.71(t,J＝6.1Hz,4H),3.54(q,J＝7.1Hz,4H),1.24(d,J＝7.2Hz,6H).HRMS(ESI):calcd for C38H37N6O2[M+H]+609.2973,found 609.2978.
Preparation example 3: synthesis of group C Compounds (core groups are tetraalkynes)
Figure BDA0002868418610000111
A50 mL single-neck flask was charged with 9mL of chloroform and 3mL of 1, 4-dioxane, and trimethylsilylacetylene (15mmol) and 4-ethynylaniline (5mmol) were weighed out and dissolved therein, and copper powder (0.25mmol) and tetramethylethylenediamine (1mmol) were added. The mixture was stirred at 50 ℃ for 12 h. Then, 40mL of dichloromethane was added, and the mixture was washed with saturated ammonium chloride, distilled water and saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to give product 6 as a tan solid (yield 60%).1H NMR(400MHz,Chloroform-d)δ7.28(d,J＝8.4Hz,2H),6.55(d,J＝8.4Hz,2H),3.90(s,2H),0.21(s,9H).13C NMR(101MHz,CDCl3)δ147.7,134.3,114.6,110.1,89.5,88.5,78.1,72.4,0.3.HRMS(ESI):calcd for C13H16NSi+[M+H]+214.10465,found 214.10468.
Product 6(1mmol) was weighed out and dissolved in 2mL dichloromethane and 2mL methanol, anhydrous potassium carbonate (10mmol) was added and stirred at room temperature for 3 h. After the reaction, 20mL of dichloromethane was added for dilution, and the mixture was washed with distilled water and saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to 1 mL. Separately, the product 2(1mmol), bis (triphenylphosphine) palladium (II) chloride (0.1mmol) and cuprous iodide (0.1mmol) were weighed into a 25mL two-necked flask, nitrogen gas was repeatedly introduced 3 times, and then a solution of the product 6, 5mL of tetrahydrofuran and triethylamine (1.2mmol) were added to stir the mixture at 50 ℃ for 12 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, diluted with 20mL of ethyl acetate, washed with saturated ammonium chloride, distilled water and saturated brine in this order, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a yellow solid product C1.1 (yield 52%) by column chromatography.1HNMR(400MHz,Chloroform-d)δ7.99(dd,J＝8.3,1.8Hz,2H),7.60–7.50(m,2H),7.39–7.30(m,2H),6.65–6.54(m,2H),5.38(s,2H),3.92(s,3H).13CNMR(101MHz,DMSO)δ165.7,151.6,135.3,133.4,131.1,129.7,124.9,114.1,104.3,82.2,77.1,72.6,69.1,66.8,66.2,63.5,60.5,52.6.HRMS(ESI):calcd forC22H13NO2 +[M]+323.0941,found 323.0962.
C2.1 was subjected to the same diazotization step as a2.1 to give C2.1. A red-colored solid, which is,1H NMR(400MHz,Chloroform-d)δ8.01(d,J＝8.1Hz,2H),7.86(d,J＝8.8Hz,2H),7.81(d,J＝8.3Hz,2H),7.63(d,J＝8.2Hz,2H),7.59(d,J＝8.1Hz,2H),6.78(d,J＝8.8Hz,2H),4.32(t,J＝6.3Hz,2H),3.93(s,3H),3.67(t,J＝6.3Hz,2H),3.50(t,J＝6.9Hz,2H),1.25(d,J＝7.0Hz,3H).HRMS(ESI):calcd for C32H26N3O3 +[M+H]+500.1969,found 500.1975.
Figure BDA0002868418610000121
product 6(1.2mmol) was weighed into 7mL of dichloromethane and 7mL of piperidine, and copper acetate monohydrate (2.4mmol) was added and stirred at room temperature for 3 h. After the completion of the reaction, the solvent was distilled off under reduced pressure, diluted with 10mL of ethyl acetate, washed with saturated ammonium chloride, distilled water and saturated brine in this order, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a yellow solid product C1.2 (yield 42%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ7.37–7.28(m,4H),6.62–6.53(m,4H),3.93(s,4H).13C NMR(101MHz,CDCl3)δ147.4,133.9,114.8,108.9,79.6,74.1,68.3,62.5.HRMS(ESI):calcd for C20H13N2 +[M+H]+281.1073,found 281.1087.
Diazotizing C1.2 with A2.1 to obtain C3.1. Deep red solid1H NMR(400MHz,DMSO-d6)δ7.79(d,J＝5.1Hz,12H),6.88(d,J＝8.5Hz,4H),4.24(d,J＝5.8Hz,4H),3.68(s,4H),3.51(s,4H),1.23(s,6H).HRMS(ESI):calcd for C40H37N6O2 +[M+H]+633.2973,found 633.2979.
Preparation example 4: synthesis of group D Compounds (Nitro group as core group)
Figure BDA0002868418610000122
Diazotizing 4-nitroaniline with the same diazotization step as A2.1 to obtain D2.3.
D2.3 (yield 73%) red solid.1H NMR(400MHz,CDCl3)δ8.30(d,J＝8.8Hz,2H),7.90(d,J＝8.8Hz,2H),7.40(s,1H),6.35(s,1H),4.65(s,2H),3.98(s,3H),3.90(s,3H).13C NMR(101MHz,CDCl3)δ157.3,156.3,147.1,144.5,142.0,133.9,124.7,122.6,97.8,97.3,56.7,55.9.HRMS(ESI):calcd for C14H15N4O4 +[M+H]+303.1088,found 303.1090.
Diazotizing 4-nitroaniline and 1,2, 4-trimethoxybenzene to obtain D2.1 by the same diazotization step as D2.3.
Figure BDA0002868418610000123
Orange solid1H NMR(400MHz,CDCl3)δ8.34(d,J＝8.9Hz,2H),7.96(d,J＝8.9Hz,2H),7.45(s,1H),6.64(s,1H),4.08(s,3H),4.02(s,3H),3.93(s,3H).13C NMR(101MHz,CDCl3)δ156.68,155.19,147.85,144.23,135.39,124.72,123.07,111.88,98.76,97.38,57.54,56.30,56.21.HRMS(ESI):calcd for C15H16N3O5 +[M+H]+318.1085,found 318.1086.
Diazotizing 4-nitroaniline and 6- (8-hydroxy julolidine) -caproic acid to obtain D2.4 by the same diazotization step as that of D2.3.
Figure BDA0002868418610000131
Purple solid1H NMR(400MHz,CDCl3)δ8.30(d,J＝8.5Hz,2H),7.83(d,J＝8.5Hz,2H),7.46(s,1H),4.21(t,J＝6.5Hz,2H),3.31(t,J＝5.6Hz,4H),2.90-2.70(m,4H),2.41(t,J＝7.4Hz,2H),2.00-1.90(m,4H),1.90-1.80(m,2H),1.79-1.68(m,2H),1.65-1.55(m,2H).13C NMR(101MHz,CDCl3)δ179.3,157.5,157.1,148.5,146.6,136.0,124.8,122.2,117.7,114.9,112.8,76.08,50.3,49.9,33.9,30.3,27.5,25.8,24.6,21.6,21.2,20.9.HRMS(ESI):calcd for C24H27N4O5 -[M-H]-451.1987,found 451.1952.
Preparation example 5: synthesis of group E Compounds (core group is azo)
Figure BDA0002868418610000132
Weighing 4-ethynylaniline (3mmol, 1eq) and dissolving in 10mL absolute ethyl alcohol, adding concentrated sulfuric acid (18mmol) and potassium nitrite (18mmol) (dissolved in 3mL water) under the ice bath condition, adding sulfamic acid (18mmol) after ice bath for 30min, adjusting the pH to 5.0 with sodium acetate after 5min, adding an ethanol solution of 2, 5-dimethoxyaniline (4.5mmol), and continuing to react for 3 h. After the reaction is finished, 60mL of ethyl acetate is added, the mixture is washed by distilled water and saturated saline solution in sequence, dried by anhydrous magnesium sulfate, the solvent is distilled off under reduced pressure, and a red solid product E1.1 is obtained after column chromatography.1H NMR(400MHz,Chloroform-d)δ8.16–8.06(m,2H),7.85(dd,J＝8.5,1.9Hz,2H),7.40(d,J＝1.7Hz,1H),6.35(d,J＝1.7Hz,1H),4.53(s,2H),3.96(d,J＝1.8Hz,3H),3.93(d,J＝1.7Hz,3H),3.88(d,J＝1.7Hz,3H).13C NMR(101MHz,CDCl3)δ166.9,156.4,155.4,143.4,141.9,133.7,130.5,122.1,97.9,56.8,55.8,52.2,21.1,14.2.HRMS(ESI):calcd for C16H18N3O4 +[M+H]+316.1292,found 316.1298.
The following products were obtained by the same procedure as above:
Figure BDA0002868418610000133
red solid.1H NMR(400MHz,CDCl3)δ7.86(d,J＝8.4Hz,2H),7.71(d,J＝8.4Hz,2H),7.39(s,1H),6.35(s,1H),4.60(s,2H),3.96(s,3H),3.89(s,3H).13C NMR(101MHz,CDCl3)δ156.0,155.9,144.1,141.9,133.8,133.1,122.8,119.2,111.4,97.8,97.5,56.7,55.9.HRMS(ESI):calcd for C15H15N4O2 +[M+H]+283.1190,found 283.1209.
Figure BDA0002868418610000134
A purple solid.1H NMR(400MHz,CDCl3)δ7.81(d,J＝8.5Hz,2H),7.71(d,J＝8.5Hz,2H),7.43(s,1H),4.19(t,J＝6.4Hz,2H),3.29(t,J＝5.8Hz,4H),2.81(t,J＝6.4Hz,2H),2.73(t,J＝6.3Hz,2H),2.40(t,J＝7.2Hz,2H),2.00-1.90(m,4H),1.85(p,J＝6.6Hz,2H),1.68-1.77(m,2H),1.65-1.54(m,2H).13C NMR(101MHz,CDCl3)δ179.4,156.8,156.1,148.2 135.7,133.1,122.4,119.3 117.5,114.8,112.8,110.7,75.9,50.2,49.9,34.0,27.5,25.8,24.6,21.6,21.3,21.1,21.0.HRMS(ESI):calcd for C25H28N4O3 -[M]-432.2167,found 432.2181.
Figure BDA0002868418610000141
Weighing the product E1.1(0.15mmol) and dissolving in 2mL of absolute ethanol, adding 10 wt% nitrosyl sulfuric acid (0.3mmol) under the ice bath condition, adjusting the pH to 5.0 with sodium acetate after 30min of ice bath, adding an ethanol solution of N-ethyl N-hydroxyethylaniline (0.3mmol), and continuing to react for 3 h. After the reaction, 20mL of ethyl acetate was added, and the mixture was washed with distilled water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a violet solid product E2.1 (yield 60%) by column chromatography. H NMR (400MHz, Chloroform-d) δ 7.89(d, J ═ 8.1Hz,2H),7.62(d, J ═ 8.0Hz,2H), 7.54-7.43 (m,3H),4.43(s,1H),4.23(t, J ═ 6.4Hz,2H),4.04(d, J ═ 4.1Hz,6H),3.28(t, J ═ 5.7Hz,4H),2.38(t, J ═ 7.3Hz,2H),1.96(s,4H),1.89(t, J ═ 7.3Hz,2H),1.72(t, J ═ 7.6Hz,2H),1.59(d, J ═ 8.4Hz,2H), ms (hresi: C for C), C, (C), (d), (C), (d), (C), (d), (4H), (d), (4H), (26H30N5O5 +[M+H]+492.2242,found 492.2248.
The following products were obtained by the same procedure as above:
Figure BDA0002868418610000142
1H NMR(400MHz,Chloroform-d)δ8.37(d,J＝8.5Hz,2H),8.04(d,J＝8.3Hz,2H),7.93(d,J＝8.7Hz,2H),7.48(d,J＝16.1Hz,2H),6.79(d,J＝8.8Hz,2H),4.33(s,2H),4.07(d,J＝18.5Hz,6H),3.69(d,J＝8.8Hz,4H),1.25(s,3H).HRMS(ESI):calcd forC24H27N6O5 +[M+H]+479.2037,found 479.2042.
Figure BDA0002868418610000143
weighing 4,4' -azodiphenylamine (1mmol) and dissolving in 3mL of absolute ethanol, adding concentrated sulfuric acid (12mmol) and potassium nitrite (12mmol) under the ice-bath condition (dissolving in 3mL of water), after ice-bath for 30min, adjusting the pH to 5.0 by using sodium acetate, adding an ethanol solution of N-ethyl N-hydroxyethylaniline (4mmol), and continuing to react for 3 h. After the reaction was completed, 30mL of ethyl acetate was added, and the mixture was washed with distilled water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a violet solid product E3.1 (yield 56%) by column chromatography.1H NMR(400MHz,Chloroform-d)δ8.06(d,J＝8.3Hz,4H),7.98(d,J＝7.8Hz,4H),7.93–7.85(m,4H),6.79(d,J＝9.0Hz,4H),4.33(t,J＝6.3Hz,4H),3.68(s,4H),3.52(d,J＝7.5Hz,4H),1.25–1.22(m,6H).13C NMR(101MHz,DMSO)δ172.7,154.5,152.5,151.3,143.4,126.0,124.4,123.4,112.0,62.0,48.7,45.4,12.5.HRMS(ESI):calcd for C32H37N8O2 +[M+H]+565.3034,found 565.3029.
Preparation example 6: synthesis of group F Compounds (core group polyene)
Figure BDA0002868418610000151
Compound 8(26.3mmol) was dissolved in 50mL of anhydrous ether and phosphorus tribromide (18mmol) was added dropwise in an ice bath. After the addition was complete, the reaction was stirred overnight. The reaction was poured into ice water and neutralized with sodium carbonate. The product was extracted with ether, the organic phase was washed with water and saturated brine, dried over sodium sulfate. After filtration, the mixture was distilled under reduced pressure to obtain compound 9 (yield: 84%).1H NMR(400MHz,CDCl3)δ6.35-6.25(m,2H),6.0-5.85(m,2H),4.12(d,J＝7.0Hz,4H).
Compound 9(9.0mmol) was dissolved in 30mL of toluene, and triethyl phosphite (20mmol) was added and stirred at room temperature for 12 h. The solvent and excess starting material were removed by distillation under the reduced pressure to give a pale yellow product 10 (yield 80%).1H NMR(400MHz,CDCl3)6.25-6.15(m,2H),5.76-5.66(m,2H),4.20-4.05(m,8H),2.68(dd,J＝9.6Hz,4.0Hz,4H),1.32(t,J＝7.0Hz,12H).
Figure BDA0002868418610000152
Weighing 4-aminobenzaldehyde (1mmol) and dissolving in 3mL of absolute ethanol, adding concentrated sulfuric acid (12mmol) and potassium nitrite (12mmol) under ice bath conditions (dissolving in 3mL of water), after ice bath for 30min, adjusting the pH value to 5.0 by using sodium acetate, adding an ethanol solution of N-ethyl N-hydroxyethylaniline (3mmol), and continuing to react for 3 h. After the reaction is finished, adding 30mL of ethyl acetate, washing with distilled water and saturated saline sequentially, drying with anhydrous magnesium sulfate, distilling off the solvent under reduced pressure, performing column chromatography to obtain a purple solid product 11, mixing the compound 10(0.22mmol) and the compound 11(0.1mmol), dissolving in 5mL of ethylene glycol dimethyl ether, and adding an ethylene glycol dimethyl ether solution of potassium tert-butoxide (0.3 mmol). Heating to react at reflux temperature or lower, adding water after 12 hr to separate out product, and column chromatographic separation to obtain red solid product F2.1 (yield 53%).1H NMR(400MHz,DMSO-d6)δ7.76(t,J＝8.0Hz,8H),7.47(d,J＝8.1Hz,4H),6.83(d,J＝8.9Hz,4H),5.32(t,J＝5.7Hz,2H),4.84(t,J＝5.3Hz,2H),4.58(d,J＝4.5Hz,4H),3.63–3.58(m,4H),3.54–3.45(m,8H),1.15(t,J＝6.9Hz,6H).13C NMR(101MHz,DMSO-d6)151.9,150.9,144.6,142.6,127.5,125.4,122.1,111.6,63.0,58.8,52.6,45.5,12.5.
Test example 1: characterization of Raman Spectroscopy Properties of Compounds
The compounds shown in table 1 were tested for raman shift and relative raman intensity using a confocal raman spectrometer. Each compound was tested in DMF solution at 867cm- 1The peak of (1) is an internal standard, and the concentration of the compound is adjusted to ensure that the peak intensity of the detected peak is 867cm from DMF-1The ratio of the peaks of (a) to (b) is between 0.1 and 10. The excitation light wavelength was 532nm, and the laser intensity of the remaining compounds tested was 5mW with an integration time of 10 times for 1 second each, except that the laser intensity of the rhodamine B and 6G tested was 0.025mW, and the integration time was 10 times for 1 second each. The results obtained are shown in Table 1.
Table 1 shows the Raman shifts and Raman intensities of the compounds
Figure BDA0002868418610000153
Figure BDA0002868418610000161
As can be seen from the results in Table 1, the Raman signal intensity of the compounds provided by the present invention was increased to a different extent as compared with that of EdU, and the highest signal intensity reached 10 of the EdU signal4Approximately twice as large, e.g., B3.2, C3.1, C3.2, E3.1.
Further, the raman spectrum of compound C3.1 in DMF is shown in fig. 1 (concentration 10)-4M, 532nm excitation). Fig. 2 shows the properties of the representative characteristic uv-visible absorption, fluorescence and raman spectra of compound E3.1. The maximum absorption wavelength of the compound E3.1 is 532nm, which is well matched with the common excitation wavelength of a commercial Raman spectrometer. The compound has extremely low fluorescence signal background under the excitation of a light source of 532 nm. 10 thereof-4Pulling of solutions of M excited under excitation light of 532nm and 785nmThe Manan signal proves 1120cm-1The raman signal at (a) is greatly enhanced in the case of electron resonance. The Raman signal of the compound has a good linear relation at a concentration of 1.0-500 mu M and taking a DMF solvent peak as an internal standard, and shows the capability of the compound for quantitative analysis. Wherein, FIG. 2a is the UV-VIS absorption spectrum of 5 μ M compound E3.1; FIG. 2b is a Raman spectrum of 100 μ M Compound E3.1 using 532nm and 785nm laser excitation and compared to solvent DMF; figure 2c is a linear plot of the relative raman intensity of compound E3.1 against the DMF solvent peak versus concentration.
Test example 2: the Raman molecules are used as signal markers for immunodetection
Immunoassay techniques are one of the most effective tools for the quantitative detection of biochemical markers such as proteins. The accuracy and operability of immunoassays have facilitated intensive research into the problems of disease diagnosis, food safety, and environmental protection. Usually, the immunoassay needs a very sensitive signal source, and the obtained resonance raman compound can be used as a signal emitting group and connected to an antibody, and the sandwich immunoassay method is adopted to test the effect of the resonance raman compound on the immunoassay.
The band compound E3.1 was first labeled on goat anti-rabbit IgG. 1mg (0.0013mmol) of synthesized compound E3.1, 1mg (0.0087mmol) of NHS and 1mg (0.0052mmol) of EDC are weighed out and dissolved in 200. mu.L of DMF, and 20. mu.L of MES buffer solution is added and activated for 30min at room temperature. Dissolving 1mg goat anti-rabbit IgG in 200 μ L PBS, shaking, placing 20 μ L in 0.5mL centrifuge tube, adding 20 μ L NaHCO3Solution, the solution after activation in a is diluted 10 times with DMF, and 4. mu.L of the solution is transferred and reacted for 5 hours at room temperature. After the reaction was complete, the resulting solution was transferred to a dialysis bag and 500. mu.L NaHCO was added3Dialyzing the solution for 3 times for more than 24h (50 mmol/L NaHCO solution as dialysate)3A solution). After dialysis, transferring the solution in the bag to a centrifuge tube to reach a constant volume of 1mL, thus obtaining the goat anti-rabbit IgG with the Raman label with the concentration of 100 mug/mL.
Polystyrene microspheres are used as carriers, and the diameter of the polystyrene microspheres is 13 mu m. The microspheres are dispersed in the solution, the concentration is 0.025g/mL, and the concentration of amino groups on the microspheres is 0.1 mmol/g.The specific operation steps are as follows: (i) the solution containing polystyrene microspheres was vigorously shaken to be uniformly dispersed in the solution, and 100. mu.L (amino group content: 2.5X 10) was pipetted using a pipette gun-4mmol, 1eq) this liquid was placed in a 1mL centrifuge tube, 0.5mL dichloromethane, 17. mu.L triethylamine and 1.2mg DMAP were added, and the reaction was shaken on a shaker at room temperature for 3 h. After the reaction, the mixture was centrifuged for 3min (5000r/min) to remove the supernatant. (ii) Weighing 11.2mg (0.1mmol) of NHS and 38.4mg (0.2mmol) of EDC, respectively dissolving in 1mL MES, transferring 100 μ L of NHS solution and 200 μ L of EDC in the polystyrene microsphere obtained in (i) by using a liquid transfer gun, shaking and activating on a shaking table at room temperature for 30min, centrifuging for 3min (5000r/min) after the activation is finished, removing the supernatant, and washing with MES for three times. Subsequently, 300. mu.L of PBS was added, and 30. mu.L of goat anti-rabbit IgG was pipetted and reacted at 4 ℃ with shaking for 3 hours. After the reaction, the mixture was centrifuged for 3min (5000r/min) to remove the supernatant. Washed 3 times with PBST. (iii) Weighing 50mg of skimmed milk powder, dissolving in 1mL of PBS, transferring 500. mu.L of the solution, mixing with the polystyrene microspheres in (ii), and sealing with shaking at 37 ℃ for 2h. After the sealing, the mixture was centrifuged for 3min (5000r/min) to remove the supernatant. Washed 3 times with PBST. (iv) Preparing a series of rabbit IgG with concentration gradient to make its concentration be 0 and 10 respectively-7、10-8、10-9、10-10、10-11、10-12、10-13And (5) fixing the volume of the polystyrene microspheres obtained in the step (iii) to 100 mu L, respectively putting 5 mu L of polystyrene microsphere solution into rabbit IgG solutions with different concentrations, and placing the solutions on a shaking table to carry out room temperature shaking reaction for 3 hours. After the reaction was completed, the reaction mixture was centrifuged for 3min (5000r/min), the supernatant was removed, and the reaction mixture was washed 3 times with PBST. (v) mu.L of synthetic goat anti-rabbit IgG with Raman signal was added at a concentration of 10. mu.g/mL. The mixture was placed on a shaker and reacted at room temperature for 3 hours with shaking. After the reaction is finished, centrifuging for 3min (5000r/min), removing supernatant, washing for 3 times by PBST, and finally washing for one time by clear water. (vi) And (v) placing the polystyrene microspheres obtained in the step (v) under a laser confocal Raman microscope to test the Raman spectrum of the polystyrene microspheres, wherein the excitation wavelength is 532nm, the laser intensity is 5mw, the integration time is 2s, and 10 microspheres are measured in each sample.
Using synthesized compound E3.1 as detection signal to make quantitative detection of rabbit IgG, after the completion of immunoassay, detectingThe obtained polystyrene microsphere has a spectrogram as shown in FIG. 3, wherein FIG. 3a shows that after immunodetection, the polystyrene microsphere has an excitation wavelength of 532nm and rabbit IgG concentrations of 10 from top to bottom-10、10-11、10-12And 10-13Raman spectrum of M, all spectra are measured at 1000cm with polystyrene microspheres-1The intensities below are normalized.
Because of the difference in the concentration of amino groups on the surface of each polystyrene microsphere, to ensure the accuracy of the results, the polystyrene microspheres are used at 1000cm-1The characteristic peak below is taken as a reference, and the peak is determined by using compound E3.1 at 1122cm-1Signal intensity at 1cm divided by polystyrene microspheres at 1001cm-1The signal intensity below is shown in fig. 3, and the results are plotted on the ordinate and on the abscissa, respectively, as relative raman signal intensity). Wherein, FIG. 3b shows that the concentration of rabbit IgG is 10-13～10-6Relative raman intensity of M. 3c rabbit IgG concentration of 10-13～10-10Linear fit curve between relative intensity and concentration at M.
As can be seen from FIG. 3, when the resonance Raman signal of the compound E3.1 of the present invention was used for immunoassay, the concentration range of 10 was achieved by using a linear curve-10～10-13Quantitative detection of rabbit IgG of M. Furthermore, when the concentration of rabbit IgG was 10-13M is about 1.5X 10 in terms of mass concentration-8mg/mL, the level of trace analysis (the content of the component to be detected is less than one millionth) is achieved. Therefore, the synthesized compound E3.1 has very sensitive signals and can be used as a signal source for immunodetection.
Test example 3: the Raman molecules are used as signal markers for cell imaging
Test example 3.1: synthesis of organelle-targeted Raman probes
Mitochondria (mitochondria) are organelles having a double-membrane structure, which are major sites for the respiration and energy production of cells, and are involved in life metabolic processes such as calcium cycle, protein synthesis, apoptosis, and the like. However, these processes generate large amounts of Reactive Oxygen Species (ROS), which can impair mitochondrial function. Therefore, synthesizing some probes for Mito plays an important role in monitoring mitochondrial function and studying mitochondrial related diseases. During mitochondrial respiration, proton pumps within the mitochondrial membrane transport protons to the mitochondrial membrane space, resulting in the formation of a negative potential across the membrane, and thus, some positively charged cationic compounds are able to specifically localize to the mitochondria. Such directing groups include Triphenylphosphine (TPP), positively charged pyridine and quinoline derivatives, rhodamine, and the like. Here, we use triphenylphosphine cation as mitochondrion localization group to synthesize cell imaging molecule, and the specific synthesis steps are as follows:
Figure BDA0002868418610000181
triphenylphosphine (3.82mmol) and 3-bromopropylamine hydrochloride (3.82mmol) were weighed out and dissolved in this, and 5mL of acetonitrile was added thereto, followed by heating and refluxing for 16 hours. After the reaction is finished, cooling to room temperature, adding a large amount of n-hexane to precipitate a product, filtering to obtain a crude product, dissolving the crude product in isopropanol, and recrystallizing by using petroleum ether to obtain the product 12 (yield 92%).1H NMR(400MHz,DMSO-d6)δ7.96-7.75(m,15H),3.99-3.85(m,2H),3.12-3.05(m,2H),1.99-1.84(m,2H).13C NMR(101MHz,DMSO-d6)δ135.6,134.1,130.9,118.5,39.3,20.6,18.9.HRMS(ESI):calcd for C21H23NP+[M-Br]+320.3872,found 320.3862.
A2.2(0.05mmol) was dissolved in 5mL of EDCM, succinic anhydride (0.10mmol), DMAP (0.01mmol) and triethylamine (0.1mmol) were added, and the mixture was refluxed for 3 hours. And after the reaction is finished, distilling under reduced pressure to remove the solvent, dissolving the product by using ethyl acetate, washing the product by using water and saturated saline solution, drying the product by using sodium sulfate, and performing suction filtration and then spin-drying on the solvent to obtain an orange solid intermediate product. This intermediate was dissolved in 2mL of DMF, HATU (0.2mmol) and DIEA (0.5mmol) were added, activated at room temperature for 10min, then 0.2mmol of product 12 was added and the reaction was continued for 12 h. After the reaction, 10mL of dichloromethane is added, the mixture is washed with distilled water and saturated brine for three times to remove DMF, and then the mixture is subjected to recrystallization by using a chloroform/diethyl ether mixed solventCrystallization gave the product Mito-A as a red solid (yield 40%).1H NMR(400MHz,CDCl3)δ8.01(d,J＝8.4Hz,2H),7.81-7.74(m,7H),7.68-7.58(m,17H),6.71(d,J＝9.2Hz,2H),4.14(t,J＝6.8Hz,2H),3.93(s,3H),3.62(t,J＝6.8Hz,2H),3.51-3.45(m,2H),3.28-3.21(m,2H),2.66-2.64(m,2H),2.59-2.56(m,2H),2.03-1.90(m,4H),1.14(t,J＝6.8Hz,3H).13C NMR(101MHz,CDCl3)δ172.9,172.7,166.3,153.2,150.4,143.6,135.2,135.2,133.4,133.3,133.2,1324,130.6,130.5,130.3,129.5,125.6,122.3,118.4,117.5,111.3,83.2,77.2,76.8,75.1,61.2,52.3,48.6,45.7,30.4,29.7,29.3,22.4,19.4,12.3.HRMS(ESI):calcd for C53H50N4O5P+[M-Br-]+853.3519,found 853.3512.
Repeating the above steps can yield mitochondrial raman dyes with a plurality of characteristic raman shifts as shown below.
Figure BDA0002868418610000182
A red solid.1H NMR(400MHz,CDCl3)δ8.84(s,2H),7.79(d,J＝5.1Hz,8H),7.75-7.55(m,34H),6.70(d,J＝8.8Hz,4H),4.14(t,J＝6.0Hz,4H),3.85-3.75(m,4H),3.60-3.50(m,4H),3.45-3.55(m,8H),2.40-2.68(m,8H),1.86(m,4H),1.21(t,J＝6.0Hz,6H).13C NMR(101MHz,CDCl3)δ173.0,172.7,143.7,135.0,135.0,133.8,133.4,133.3,131.6,130.5,130.4,125.6,125.0,122.3,119.7,118.6,117.7,111.3,110.1,61.1,48.7,45.7,42.2,30.5,29.4,23.4,22.6,12.3.HRMS(ESI):calcd for C88H86N8O6P2+[M-2Br-]2+706.3073,found 706.3052.
Figure BDA0002868418610000183
A purple solid.1H NMR(400MHz,CDCl3)δ8.28(d,J＝8.8Hz,2H),7.83(d,J＝8.8Hz,2H),7.80-7.63(m,15H),4.20(t,J＝6.5Hz,2H),3.65-3.61(m,2H),3.48-3.45(m,2H),3.30(t,J＝5.6Hz,4H),2.82(t,J＝6.4Hz,2H),2.73(t,J＝6.4Hz,2H),2.41(t,J＝7.4Hz,2H),2.00-1.90(m,4H),1.90-1.68(m,6H),1.65-1.55(m,2H).13C NMR(101MHz,CDCl3)δ174.6,157.5,146.4,136.1,135.1,135.1,133.5,133.4,130.6,130.5,124.8,122.2,118.6,117.7,117.5,114.8,113.0,76.3,50.2,50.0,36.2,30.4,29.8,29.3,27.5,27.2,25.9,25.7,25.5,21.6,21.2,20.9.HRMS(ESI):calcd for C45H49N5O4P+[M-Br-]+754.3522,found 754.3513.
Figure BDA0002868418610000191
A purple solid.1H NMR(400MHz,CDCl3)δ7.87-7.74(m,5H),7.69-7.39(m,15H),4.15(t,J＝6.4Hz,2H),3.42-3.38(m,2H),3.27(t,J＝5.8Hz,4H),2.78(t,J＝6.4Hz,2H),2.70(t,J＝6.3Hz,2H),2.34(t,J＝7.2Hz,2H),2.15-1.98(m,4H),2.00-1.90(m,4H),1.83(p,J＝6.6Hz,2H),1.77-1.68(m,2H),1.65-1.54(m,2H).13C NMR(101MHz,CDCl3)δ174.6,156.9,156.1,148.2,135.7,135.3,133.3,133.2,133.0,130.7,130.5,122.4,119.3,118.2,117.4,117.4,114.6,113.0,110.5,70.8,50.2,49.9,39.1,39.0,36.1,30.3,27.5,25.9,25.6,22.5,22.4,21.6,21.2,20.9,20.3,19.8.HRMS(ESI):calcd for C46H49N5O2P+[M-Br-]+734.3624,found 734.3618.
Figure BDA0002868418610000192
A purple solid.1H NMR(400MHz,CDCl3)δ8.17(d,J＝8.5Hz,2H),7.96(d,J＝8.5Hz,2H),7.84(d,J＝9.2Hz,2H),7.68-7.60(m,15H),7.48(s,1H),7.44(s,1H),6.71(d,J＝9.2Hz,2H),4.14-4.12(m,2H),4.11(s,3H),4.01(s,3H),3.95(s,3H),3.64-3.59(m,2H),3.53-3.39(m,4H),3.30-3.18(m,2H),2.67-2.60(m,2H),2.59-2.53(m,2H),1.90-1.80(m,2H),1.20(t,J＝6.8Hz,3H).13C NMR(101MHz,CDCl3)δ172.9,172.7,166.6,155.7,153.0,151.7,150.9,150.6,146.0,144.4,142.3,135.3,135.2,133.31,133.30,133.22,133.20,131.5,130.62,130.58,130.5,126.3,126.1,122.8,118.4,117.5,111.3,110.2,101.0,100.3,61.2,56.8,56.7,52.3,48.6,45.8,30.4,29.3,22.4,22.4,19.4,12.3.HRMS(ESI)calcd for C51H54N6O7P+[M-Br-]+893.3792,found 893.3789.
Figure BDA0002868418610000193
Dark purple solid.1H NMR(400MHz,CDCl3)δ8.99(s,2H),8.03(d,J＝8.7Hz,4H),7.94(d,J＝8.7Hz,4H),7.83(d,J＝9.0Hz,4H),7.79-7.52(m,30H),6.74(d,J＝9.0Hz,4H),4.18(t,J＝6.4Hz,4H),3.80-3.70(m,4H),3.62(t,J＝6.3Hz,4H),3.51-3.47(m,8H),2.75(t,J＝6.3Hz,4H),2.65(t,J＝6.3Hz,4H),2.02-1.97(m,4H),1.20(t,J＝6.4Hz,6H).13C NMR(101MHz,CDCl3)δ173.1,172.7,154.6,152.8,150.4,143.8,135.0,135.0,133.5,133.4,130.5,130.4,125.7,123.9,123.0,118.7,117.9,111.3,61.1,48.8,45.8,30.6,29.6,23.5,22.5,20.2,12.3.HRMS(ESI):calcd for C82H86N10O6P2 2+[M-2Br-]2+684.3104,found 684.3077.
In addition, we changed the recognition group of organelles and synthesized a small molecule Raman probe Lyso-D with lysosome targeting.
Figure BDA0002868418610000194
Dark red solid.1H NMR(400MHz,CDCl3)δ8.28(d,J＝8.5Hz,2H),7.83(d,J＝8.5Hz,2H),7.44(s,1H),6.32(s,1H),4.19(t,J＝6.5Hz,2H),3.34(dd,J＝11.4,5.3Hz,2H),3.30-3.25(m,4H),2.80(t,J＝6.0Hz,2H),2.72(t,J＝6.0Hz,2H),2.46(t,J＝6.0Hz,2H),2.26(s,6H),2.21(t,J＝7.2Hz,2H),1.99-1.90(m,4H),1.88-1.79(m,2H),1.76-1.67(m,2H),1.59-1.51(m,2H).13C NMR(101MHz,CDCl3)δ173.1,157.5,157.1,148.5,146.5,136.0,124.7,122.2,117.6,114.9,112.8,76.1,57.9,50.2,49.9,44.9,36.5,36.4,30.3,27.4,25.9,25.6,21.6,21.2,20.9；HRMS(ESI)calcd for C28H39N6O4 +[M+H]+523.3027,found 523.3031.
Test example 3.2: cell co-localization imaging:
cell imaging molecules synthesized by the cell imaging method and different Raman shifts have low toxicity to cells through cytotoxicity tests, so that the cell imaging molecules are applied to subsequent organelle imaging experiments. We took Mito-E3.1 and Lyso-D as examples to image the mitochondria and lysosomes, respectively, of HeLa cells, combining the currently mature commercial fluorescent dyes Mitotrack Red and Lysotrack Red to obtain the cellular image shown in FIG. 4 below: from left to right are respectively a Raman imaging effect picture (left), a fluorescence effect imaging effect picture (middle) and a superposition effect picture (right). Wherein FIG. 4a uses 1120cm Raman imaging-1Imaging the signal, and dyeing for 1h by 5 mu MAzo-3-Mito with the excitation wavelength of 532 nm; fluorescence imaging was performed using a 610nM wavelength signal, 200nM Mitotracker staining for 1 h. FIG. 4b Raman imaging using 1340cm-1Imaging the signal, exciting the wavelength to be 532nm, and dyeing the signal for 1h by 5 mu M lyso-D; fluorescence imaging 600nm wavelength signal, 100nM Lysotracker Red staining for 1 h.
Test example 3.3: multi-color imaging of cells
The Raman dyes synthesized by the method have mitochondrion targeting function, namely Mito-A, Mito-B, Mito-D and Mito-E1,Mito-E2,Mito E3The HeLa cells are dyed to obtain the following imaging graph, which shows that the azo enhanced multicolor Raman signal molecules have strong intensity, narrow spectral line and characteristics, and can be well used for cell imaging.
FIG. 5 is a schematic representation of a multi-color Raman image using super-strong Raman molecules, wherein FIG. 5a shows the molecular structures Mito-E2, Mito-E3, Mito-E1, Mito-D, Mito-B and Mito-A; fig. 5b is a raman image obtained after staining the hela cells mitochondria using the above raman dye, the incubation time is 2h, the laser intensity is 5mW, and the imaging time of each pixel is 30 ms. FIG. 5c is the cumulative Raman imaging spectrum of the cells, and FIG. 5d is the toxicity test of the Raman dye, with the staining concentration of 5 μ M incubation time of 2h.
The results of the invention show that the azo group connected by conjugation has obvious enhancement effect on endogenous Raman signals of various functional groups, and the signal molecules have excellent effects of strong signal, short imaging time, weak photobleaching and quenching effect and the like when being used for Raman imaging.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. An azo aromatic compound having a structure represented by formula (I) or formula (II):
formula (I):
Figure FDA0002868418600000011
formula (II):
Figure FDA0002868418600000012
formula (I1):
Figure FDA0002868418600000013
formula (I2):
Figure FDA0002868418600000014
formula (I3):
Figure FDA0002868418600000015
formula (I4):
Figure FDA0002868418600000016
formula (I5):
Figure FDA0002868418600000017
formula (I6):
Figure FDA0002868418600000018
wherein, in the formula (I) and the formula (II),
R11、R12、R14、R15each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a); r13Is selected from R1-CONH-、NH2-、(R1)(R2)N-、C1-6Alkoxy of R1-COO-、R1-OCO-, nitro, cyano; or, R11And R15Each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a), R12、R13And R14Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-6At least one substituent of the alkyl group of (a);
R21、R22、R24and R25Each independently selected from H, C1-6Alkyl of (C)1-6Alkoxy group of (a); r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and R1-COO-、R1-OCO-、NC-、(R1)(R2) N-, cyano; or R21And R25Each independently selected from H, C1-6Alkyl of R1-COO-、R1-OCO-、C1-6Alkoxy group of (a); r22、R23And R24Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, and the non-benzene ring of the N-containing fused tricyclic ring is optionally arranged on the ring structurePresence of a compound selected from C1-6At least one substituent of the alkyl group of (a);
l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R1and R2Each independently selected from H, C1-6Alkyl of (C)1-6alkoxy-C of1-3alkylene-hydroxy-C of1-3Alkylene-of (a), a group of formula (I6);
R3and R4Each independently selected from H, phenyl, from R1-OCO-substituted phenyl;
n, m, x, y and z are each independently integers from 1 to 10.
2. The azo aromatic compound according to claim 1, wherein, in the formulae (I) and (II),
R11、R12、R14、R15each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a); r13Is selected from R1-CONH-、NH2-、(R1)(R2)N-、C1-6Alkoxy radical of (2), R1-COO-、R1-OCO-, nitro, cyano; or, R11And R15Each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy, carboxyl substituted C1-6At least one of alkoxy groups of (a), R12、R13And R14Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-4At least one substituent of the alkyl group of (a);
R21、R22、R24and R25Each independently selected from H, C1-4Alkyl of (C)1-4Alkoxy group of (a); r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and R1-COO-、R1-OCO-、NC-、(R1)(R2) N-, cyano; or R21And R25Each independently selected from H, C1-4Alkyl of R1-COO-、C1-4Alkoxy group of (a); r22、R23And R24Together with the parent nucleus benzene ring of the three to form the N-containing fused tricyclic ring, wherein the ring structure of the N-containing fused tricyclic ring, which is not the benzene ring, is optionally provided with C1-4At least one substituent of the alkyl group of (a);
l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R1and R2Each independently selected from H, C1-4Alkyl of (C)1-4alkoxy-C of1-3Alkylene-, hydroxy-C of1-3Alkylene-of (a), a group of formula (I6);
R3and R4Each independently selected from H, phenyl, from R1-OCO-substituted phenyl;
n, m, x, y and z are each independently integers from 1 to 6.
3. The azo aromatic compound according to claim 1, wherein, in the formulae (I) and (II),
R11、R12、R14、R15each independently selected from H, methyl, methoxy, -O (CH)2)5At least one of COOH; r13Is selected from (CH)3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2)N-、CH3CONH-、NH2-、CH3O-、CH3COO-, nitro, cyano; or, R11And R15Each independently selected from H, methyl, methoxy, -O (CH)2)5At least one of COOH, R12、R13And R14The three compounds and parent nucleus benzene rings of the three compounds form N-containing fused tricyclic, a methyl substituent optionally exists on the ring structure of the non-benzene ring of the N-containing fused tricyclic, and the ring structures of the non-benzene ring of the N-containing fused tricyclic are all six-membered rings;
R21、R22、R24and R25Each of which isIndependently selected from H, methoxy, a group represented by formula (I6) -COO-; r23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and CH3OCO-、NC-、(CH3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2) N-; or R21And R25Each independently selected from H, methoxy, a group represented by formula (I6) -COO-; r is23Selected from H, a group shown as a formula (I1), a group shown as a formula (I2), nitro and CH3OCO-、NC-、(CH3CH2)(CH3OCH2CH2)N-、(CH3CH2)(HOCH2CH2) N-, cyano; r22、R23And R24The three compounds and parent nucleus benzene rings form N-containing fused tricyclic ring together, a methyl substituent optionally exists on the ring structure of the non-benzene ring of the N-containing fused tricyclic ring, and the ring structures of the non-benzene ring of the N-containing fused tricyclic ring are six-membered rings;
l is selected from the group represented by formula (I3), the group represented by formula (I4), the group represented by formula (I5);
R3and R4Each independently selected from H, phenyl, and CH3An OCO-substituted phenyl group;
n, m, x, y and z are each independently 1,2, 3, 4, 5 or 6.
4. The azo aromatic compound according to any one of claims 1 to 3, which has a structure represented by formula (I).
5. The azo aromatic compound according to claim 4, wherein the compound is selected from any one of the following compounds:
compound a 2.1:
Figure FDA0002868418600000041
compound a 2.2:
Figure FDA0002868418600000042
compound B2.1:
Figure FDA0002868418600000043
compound C2.1:
Figure FDA0002868418600000044
compound D2.1:
Figure FDA0002868418600000045
compound D2.2:
Figure FDA0002868418600000051
compound D2.3:
Figure FDA0002868418600000052
compound D2.4:
Figure FDA0002868418600000053
compound E1.1:
Figure FDA0002868418600000054
compound E1.2:
Figure FDA0002868418600000055
compound E1.3:
Figure FDA0002868418600000056
compound E2.1:
Figure FDA0002868418600000057
compound (I)E2.2：
Figure FDA0002868418600000058
6. The azo aromatic compound according to any one of claims 1 to 3, which has a structure represented by formula (II).
7. The azo aromatic compound according to claim 6, wherein the compound is selected from any one of the following compounds:
compound a 3.1:
Figure FDA0002868418600000061
compound a 3.2:
Figure FDA0002868418600000062
compound B3.1:
Figure FDA0002868418600000063
compound B3.2:
Figure FDA0002868418600000064
compound C3.1:
Figure FDA0002868418600000065
compound C3.2:
Figure FDA0002868418600000066
compound E3.1:
Figure FDA0002868418600000067
compound F2.1:
Figure FDA0002868418600000068
8. use of a compound according to any one of claims 1 to 7 for raman scattering signals.
9. An agent having a super-strong raman scattering signal, comprising a functional compound having a signal enhancing function, wherein the functional group of the functional compound is provided by the compound according to any one of claims 1 to 7.
10. The reagent according to claim 9, wherein the concentration of the functional compound is 0.1 to 1000. mu. mol/liter.
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