阅读说明：本技术 一种整合素αvβ3靶向的AIE荧光化合物及其制备与应用 (Integrin α v β 3 targeted AIE fluorescent compound and preparation and application thereof ) 是由 朱勍 蒋建泽 刘江 于 2019-09-24 设计创作，主要内容包括：本发明提供了一种式(VIII)所示的AIE荧光环肽c(RGDf-TPEK),及其制备方法与应用。在不破坏靶向结构和功能的前提下,实现了整合素αvβ3的AIE荧光探针的构建。相比现有的荧光分子和靶向肽连接的策略,本发明将AIE分子嵌合于整合素αvβ3靶向肽c(RGDfK)的结构中,可以在整合素αvβ3高表达的肿瘤细胞表面发生聚集,分子内旋转受限(RIR)产生AIE发光,具有发光强度高,背景低和灵敏度高的特性。<Image he="605" wi="700" file="DDA0002212862950000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention provides an AIE fluorescent cyclic peptide c (RGDf-TPEK) shown in a formula (VIII), a preparation method and application thereof, and the construction of an AIE fluorescent probe of integrin α v β 3 is realized on the premise of not damaging a target structure and functions compared with the existing strategy of connecting a fluorescent molecule and a target peptide, the AIE fluorescent cyclic peptide c (RGDfK) is characterized in that the AIE molecule is embedded in the structure of the integrin α v β 3 target peptide c (RGDfK), the aggregation can be generated on the surface of a tumor cell highly expressed by integrin α v β 3, the molecular rotation is limited (RIR) to generate AIE luminescence, and the AIE fluorescent cyclic peptide c (RGDf-TPEK) has the characteristics of high luminescence intensity, low background and high sensitivity.)

1. An integrin α v β 3 targeted AIE fluorescent compound has a structure shown in a formula (VIII):

2. a method of making the fluorescent compound of claim 1, the method comprising:

(1) the compound (II) and the diboron pinacol ester are subjected to PdCl in a DMF solvent at 70-80 ℃ under the protection of nitrogen₂(dppf) is used as a catalyst, the suzuki reaction is carried out in the presence of KOAc, and the compound (III) is obtained by separation and purification after the reaction is finished;

(2) compound (III) andtriphenylbromoethylene in THF and H₂In an O mixed solvent, Pd (pph3)4 is used as a catalyst under the protection of nitrogen at 70-80 ℃, and K is added₂CO₃Carrying out a suzuki reaction in the presence of the compound (IV), and separating and purifying after the reaction is finished to obtain a compound (IV);

(3) removing Boc protecting group from compound (IV) to obtain compound (I);

(4) protecting an amino group of a phenylalanine derivative shown as a formula (I) by Fmoc-OSU in a DMF solvent in the presence of DIEA to obtain a compound (V);

(5) solid-phase synthesis of a pentapeptide chain shown in the formula (VI) by adopting CTC resin for the compound (V), wherein the extension sequence from the C end to the N end of amino acid is Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, the compound (V), Fmoc-Asp (OtBu) -OH and Fmoc-Gly-OH;

(6) cleaving the resin from compound (VI) with 3% TFA in DCM, and then performing condensation cyclization in the presence of HOBt, EDC and DIEA to obtain compound (VII);

(7) removing all protecting groups of amino acid from the compound (VII) to obtain the fluorescent compound (VIII);

3. the method of claim 2, wherein in step (1), the compound (II), pinacol diboron, PdCl₂The ratio of the amounts of substances of (dppf) and KOAc was 1:1.5:0.06: 2.8.

4. As in claimThe process according to claim 2, wherein in the step (3), the compound (III), triphenylbromoethylene, Pd (pph)₃)₄、K₂CO₃The amount of substance(s) is 1:1.2:0.05: 3; THF H in solvent₂The volume ratio of O is 10: 1.

5. The method according to claim 2, characterized in that in step (4) compound (I): Fmoc-OSU: the ratio of the amount of DIEA material is 1:1.5: 3.

6. The method according to claim 2, wherein in step (6) compound (VI): HOBt: EDC: the ratio of the amount of DIEA material is 1:2:2: 3.

7. Use of the fluorescent compound of claim 1 in the preparation of a fluorescent probe for fluorescence confocal cell imaging.

8. The use of claim 7, wherein said fluorescent probe targets cancer cells with high integrin expression.

9. The use according to claim 7, wherein said cancer cells are A549 cells.

(I) technical field

The invention relates to an integrin α v β 3 targeted AIE fluorescent compound, and a preparation method and application thereof.

(II) background of the invention

The use of tumor cell labeling and imaging for cancer diagnosis and clinical treatment has been a continuing trend, which has prompted the development of cancer cell labeling probes. The effect of the labeling depends on the affinity of the targeting molecule for the cancer cell expression marker, and the biocompatibility and fluorescence properties of the fluorescent molecule.

RGD (arginine-glycine-aspartic acid) tripeptide sequence can mimic cell adhesion protein and bind with integrin, and its series of cyclo (RGDFK) is an effective selective inhibitor of α v β 3 integrin at present, on the basis, many tumor targeting drugs and labeled probes have been developed, including c (RGDFK) and traditional dyes such as coumarin, BODIPY, cyanine, etc., even binding with nanomaterials, but these probes all suffer from the high background of fluorescent dyes in biological solution, and it is difficult to distinguish non-specific probe fluorescence emission from fluorescence emission at target region, which adversely affects probe accuracy.

Disclosure of the invention

The invention relates to a targeting peptide c (RGD (f-TPE) K) with AIE fluorescence property, which is constructed by embedding an AIE fluorescent molecule in c (RGDfK), and aims to provide a novel integrin α v β 3 targeted AIE fluorescent probe, and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

an integrin α v β 3 targeted cyclic peptide fluorescent compound has a structure shown in a formula (VIII):

the c (RGD (f-TPE) K (VIII) has AIE luminescence characteristics, does not have fluorescence in a solvent with strong solubility, generates fluorescence by limiting intramolecular rotation (RIR) along with the increase of concentration in a solution with poor solubility, and has the maximum emission wavelength of 460 nm.

The invention also relates to a method for preparing the fluorescent compound, which comprises the following steps:

(1) the compound (II) and the diboron pinacol ester are subjected to PdCl in a DMF solvent at 70-80 ℃ under the protection of nitrogen₂(dppf) is used as a catalyst, the suzuki reaction is carried out in the presence of KOAc, and the compound (III) is obtained by separation and purification after the reaction is finished;

(2) compound (III) with triphenylbromoethylene in THF and H₂In an O mixed solvent, Pd (pph3)4 is used as a catalyst under the protection of nitrogen at 70-80 ℃, and K is added₂CO₃Carrying out a suzuki reaction in the presence of the compound (IV), and separating and purifying after the reaction is finished to obtain a compound (IV);

(3) removing the Boc protecting group of the compound (IV) by using HCl-1,4 dioxane to obtain a compound (I);

(4) AIE phenylalanine of formula (I) is protected at 50 ℃ with Fmoc-OSU in DMF solvent in the presence of DIEA to give compound (V). Other protected amino acids are: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Lys (Boc) -OH.

(5) And (5) carrying out solid phase synthesis on the compound (V) by adopting CTC resin to obtain a pentapeptide chain shown in the formula (VI). The special amino acid shown in the formula (V) is placed at the middle end of the linear peptide. The sequence of the amino acid C-terminal to N-terminal extension is: Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Compound (V), Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH;

(6) after the compound (VI) is separated from the resin by using a DCM solution of 3% TFA, condensation cyclization is carried out in the presence of HOBt, EDC and DIEA, the reaction time is 2 hours at normal temperature, and after the reaction is completed, the compound (VII) is separated and purified.

(7) Removing all amino acid protecting groups of the compound (VII) by using 95% TFA to obtain the fluorescent compound (VIII);

in the step (2), the compound (II), the diboron pinacol ester and PdCl₂The ratio of the amounts of substances of (dppf) and KOAc is preferably 1:1.5:0.06: 2.8. Specifically, the separation and purification method in the step (2) is column chromatography, and dichloromethane: and (3) taking a mixed solution with the volume ratio of the A being 30:1 as an eluent, collecting a target component, and drying to obtain the compound of the formula (III).

In the step (3), the compound (III), triphenylbromoethylene and Pd (pph)₃)₄、K₂CO₃The amount of the substance(s) is preferably 1:1.2:0.05: 3; THF H in solvent₂The volume ratio of O is 10: 1. Specifically, the separation and purification method in the step (3) is column chromatography, and dichloromethane: and (3) taking mixed liquor with the volume ratio of A to A of 15:1 as eluent, collecting target components, and drying to obtain the compound of the formula (IV).

In step (4), compound (I): Fmoc-OSU: the ratio of the amount of DIEA material is 1:1.5: 3.

In the step (5), the amino acid condensation conditions are as follows: resin: amino acids: HBTU: DIEA ═ 1:3:3: 3. The Fmoc protecting group was removed as a 20% solution of piperazine DMF.

Compound (VI) in step (6): HOBt: EDC: the ratio of the amount of DIEA material is 1:2:2: 3. Specifically, the separation method in step (6) uses thin layer chromatography, and the mobile phase is dichloromethane: methanol 24:1, product Rf 0.4-0.6.

The invention also relates to application of the fluorescent compound as a fluorescent probe in cell fluorescence confocal imaging, and particularly relates to the fluorescent probe targeting cancer cells highly expressed by integrin α v β 3.

Preferably, the cancer cells are a549 cells.

Compared with the prior strategy of connecting fluorescent molecules and targeting peptides, the invention leads the AIE molecules to be embedded in the structure of the targeting peptide c (RGDf-TPEK) of integrin α v β 3, can gather on the surface of tumor cells highly expressed by integrin α v β 3, and can generate AIE luminescence due to limited intramolecular rotation (RIR), and has the characteristics of high luminous intensity, low background and high sensitivity.

(IV) description of the drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the compound (III).

FIG. 2 is a nuclear magnetic hydrogen spectrum of Compound (IV).

FIG. 3 is a nuclear magnetic hydrogen spectrum of Compound (I).

FIG. 4 is a nuclear magnetic carbon spectrum of Compound (I).

FIG. 5 is a nuclear magnetic hydrogen spectrum of the compound (V).

FIG. 6 is a LC-MS diagram of Compound (VI).

FIG. 7 is a LC-MS diagram of Compound (VII).

FIG. 8 is a LC-MS diagram of Compound (VIII).

FIG. 9 is a graph showing the fluorescence spectra of compound (VIII) at a concentration of 40. mu.M in dimethylsulfoxide and PBS.

FIG. 10 is a graph of the fluorescence spectra of compound (VIII) at various concentrations in PBS (1% DMSO).

FIG. 11 is a photograph of fluorescence confocal images of compound (VIII) after 30min incubation in A549 cells.

FIG. 12 is a photograph of fluorescence confocal images of compound (VIII) after incubation in HEK-293 cells for 30 min.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: