CN102827252A

CN102827252A - One kind of magnetic resonance imaging developers and two-photon imaging developers and preparation method thereof

Info

Publication number: CN102827252A
Application number: CN2012103425042A
Authority: CN
Inventors: 梁高林; 曹春艳
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2012-09-14
Filing date: 2012-09-14
Publication date: 2012-12-19
Anticipated expiration: 2032-09-14
Also published as: CN102827252B

Abstract

The invention discloses a preparation method of one kind of magnetic resonance imaging developers and two-photon imaging developers. The preparation method is characterized by comprising the steps of: firstly, synthesizing a lysine-cysteine-arginine-arginine-valine-arginine peptide fragment and a valine-arginine-cysteine-arginine-lysine-arginine peptide fragment, connecting the fragments with 2-amino-6-cyanobenzothiazole and then connecting a macrocyclic compound, and reacting with metal ions in a buffer solution to obtain five compounds, wherein the final product A1 of a first compound, the final product A2 of a second compound and the final product A5 of a fifth compound are magnetic resonance imaging developers, and the final product A3 of a third compound and the final product A4 of a fourth compound are two-photon imaging developers. Because of being micromolecules, the magnetic resonance imaging developers and two-photon imaging developers have the advantages of good hydrophilia, easiness of preparation and high absorption; and the final product A1 of the first compound and the final product of the third compound have peptide fragments with specific enzyme specificity recognition, and have the advantage of high targeting.

Description

One type of nuclear magnetic resonance developer and two-photon imaging developer and preparation method thereof

Technical field

The invention belongs to the developer technical field, be specifically related to the developer of nuclear magnetic resonance and developer of two-photon imaging and preparation method thereof.

Background technology

In recent years, nano material as image probe by wide coverage and application." American Chemical Society " son periodical (J.Am.Chem.Soc., 2007, P.3848) Vol.129 has reported a kind of quantum dot preparation that is used for nuclear magnetic resonance and optical imagery simultaneously.Though this preparation has imaging effect preferably; But because this preparation of preparation at first will synthesize the kernel of CdSe nanoparticle; Not only want the shell of synthesizing nano-particle then, also will modify water miscible organic cpds, comparison difficulty so preparation is got up; And low owing to having the cellular uptake rate at external synthetic nanoparticle, there are not shortcomings such as specific targeted molecular, also limited the development of this type preparation.GDCh's " applied chemistry " (Angew.Chem.Int.Ed., 2008, Vol.47 (15); P.2804) introduced a kind of " switching mode " near infrared gold nano preparation and preparation method thereof; Though this preparation has good biocompatibility,, also to modify the peptide section of target specific recognition because it is that optical molecular is modified at the gold nano surface; Synthetic more loaded down with trivial details, stability neither be fine.In a word, existing these nanometer developer ubiquities that the preparation difficulty is high, and external synthetic nano particle cell uptake ratio is low, are pumped out easily and shortcoming such as target difficulty, form the bottleneck into restriction biology imaging research.

Summary of the invention

The objective of the invention is to propose one type of nuclear magnetic resonance developer and two-photon imaging developer and preparation method thereof; To obtain the nanometer developer of the real-time controlled self-assembly of ability; Overcome traditional nanometer developer and prepare the difficulty height, shortcomings such as the cellular uptake rate is low, target difficulty.

The preparation method of nuclear magnetic resonance developer of the present invention and two-photon imaging developer is characterized in that:

Earlier synthetic respectively two sections peptide sections, press: with 1 mmole 2-chlorine trityl chloride resin at 4-6 milliliter N, in the dinethylformamide after swelling 4-8 minute; Add 2 mmole N-fluorenylmethyloxycarbonyls-N'-tertbutyloxycarbonyl-L-Methionin, add 2 mmole N again, the N-diisopropylethylamine; React after 2-3 hour,, cut the protection base of Methionin with 100 microliter methanol reaction 10-20 minute; Add second amino acid cysteine reaction of activatory 1.6 mmoles 2-3 hour; Cut the protection base of halfcystine, add the 3rd amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour, cut arginic protection base; Add the 4th amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add activatory 1.6 mmole five amino acid Xie Ansuans reaction 2-3 hour, cut the protection base of Xie Ansuan; Add the 6th amino acids Arginine reaction of activatory 1.6 mmoles 3-4 hour again; Cut the protection base of last amino acids Arginine, added 2-3 mmole aceticanhydride reaction 20-30 minute, use at last that to contain volumetric concentration be that the methylene dichloride of 1% trifluoroacetic acid downcuts above-mentioned synthetic peptide section from this resin; Separate out the frozen centrifugation upper strata ether that inclines with ether layer, treat solvent evaporates do after resulting white solid powder promptly be Methionin-halfcystine-l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section; Press again: 1 mmole 2-chlorine trityl chloride resin at 4-6 milliliter N, after swelling 4-8 minute, is added first amino acid Xie Ansuan of 2 mmoles in the dinethylformamide; Add 2 mmole N again, the N-diisopropylethylamine reacts after 2-3 hour; With 100 microliter methanol reaction 10-20 minute, cut the protection base of Xie Ansuan, add second amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add the 3rd amino acid cysteine reaction of activatory 1.6 mmoles 2-3 hour, cut the protection base of halfcystine; Add the 4th amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add activatory 1.6 mmole five amino acid Methionins reaction 2-3 hour, cut the protection base of Methionin; Add the 6th amino acids Arginine reaction of activatory 1.6 mmoles 3-4 hour again; Cut the protection base of last amino acids Arginine, added 2-3 mmole aceticanhydride reaction 20-30 minute, using volumetric concentration at last is that the dichloromethane solution of 1% trifluoroacetic acid downcuts above-mentioned synthetic peptide section from this resin; Adopt ether layer to separate out the frozen centrifugation upper strata ether that inclines, treat solvent evaporates do after resulting white solid powder promptly be Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section;

Above-mentioned synthetic Methionin-halfcystine-l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section is got 0.1 mmole to be dissolved in the 2-4 milliliter tetrahydrofuran solvent; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, after activation half a hour; Add 0.12 mmole 2-amino-6-cyanic acid benzothiazole; Maintain the temperature at 0-10 ℃ one hour, stirring at room reaction 8-9 hour separates through performance liquid chromatography and to purify; Being collected in ultraviolet 320 nanometers has the component of strong absorption, is first kind of head product X ₁Again above-mentioned synthetic Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section being got 0.1 mmole is dissolved in the 2-4 milliliter tetrahydrofuran solvent; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, after activation half a hour; Add 0.12 mmole 2-amino-6-cyanic acid benzothiazole; Maintain the temperature at 0-10 ℃ one hour, stirring at room reaction 8-9 hour separates through performance liquid chromatography and to purify; Being collected in ultraviolet 320 nanometers has the component of strong absorption, is second kind of head product Y ₁

Elder generation is with first kind of head product X of above-mentioned preparation ₁Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of purified product X ₂Again with second kind of head product Y ₁Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of purified product Y ₂

With 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added first kind of purified product X of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product X first kind time ₃With 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added second kind of purified product Y of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Y second kind time ₃

First kind purified product X with above-mentioned preparation ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of repurity product X ₄Again with second kind purified product Y ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of repurity product Y ₄

First kind of repurity product X with above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of first kind of compound ₁

Second kind of repurity product Y with above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of second kind of compound ₂

First kind of repurity product X with above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Europium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of the third compound ₃

Second kind of repurity product Y with above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Europium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of the 4th kind of compound ₄

The final product A of the 5th kind of compound ₅Synthesis step following:

Press N-tertbutyloxycarbonyl-N'-fluorenylmethyloxycarbonyl-L-Methionin 0.1 mmole is dissolved in 2 milliliters of tetrahydrofuran solvents; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, add 0.12 mmole 2-amino-6-cyanic acid benzothiazole after half a hour; Maintain the temperature at 0-10 ℃ one hour; Stirring at room reaction 8-9 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption promptly to get the 5th kind of head product Z ₁Then with the 5th kind of head product Z ₁Be dissolved in 10 ml volumes concentration and be in the methylene dichloride of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of purified product Z ₂

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the N-diisopropylethylamine is with 0.1 mmole N-(tertbutyloxycarbonyl l) S-sulphur ethyl-L-halfcystine

After the activation 5 minutes, add the 5th kind of purified product Z of the above-mentioned preparation of 0.1 mmole ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Z the 5th kind time ₃

The 5th kind purified product Z with above-mentioned preparation ₃Be dissolved in 10 ml volumes concentration and be 50% N, the N of N-diisopropylethylamine is in the dinethylformamide solution; Behind the stirring reaction five minutes; Add in the 4-5 milliliter trifluoroacetic acid and unnecessary N, N-diisopropylethylamine, ice bath immediately; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of repurity product Z ₄

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.5 mmole N, the N-diisopropylethylamine is with 0.1

mmole tri-tert

1,4; 7,10-tetraazacyclododecanand-1,4; 7, the activation of 10-tetraacethyl added the 5th kind of repurity product Z of the above-mentioned preparation of 0.1 mmole after 5 minutes ₄In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of four purified product Z ₅

With the 5th kind of four purified product Z ₅Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of five purified product Z ₆

Get the 5th kind of five purified product Z of above-mentioned preparation ₆0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of six purified product Z ₇

Get the 5th kind of six purified product Z of above-mentioned preparation ₇0.1 it is in 7.4 the phosphoric acid buffer that mmole is dissolved in pH; Add 0.4 mmole trichloroethyl phosphate; Stirring at room 1 hour is separated purification through performance liquid chromatography, and being collected in ultraviolet 323 nanometers has the component of strong absorption to be the final product A of the 5th kind of compound ₅

Wherein the used amino acid of solid phase all is as alpha-amino group protection base with 9-fluorenylmethyloxycarbonyl

; The sulfydryl of halfcystine is protected by uncle's butylthio; L-arginine side chain amino is by 2; 2; 4; 6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl

protection; Wherein the reagent of activated amino acid is I-hydroxybenzotriazole and the benzotriazole-N with the amount concentration of amino acid commaterial, N, N', N'-tetramethyl-urea hexafluorophosphate; When whenever connecting an amino acid and cutting the protection base, all to adopt Caesar to test (kaiser test) reagent detection imino-and whether exist: show blue as if positive, promptly show and sheared; If negative displaing yellow shows that then amino acid connects.

Thus, synthesize five kinds of particular compound among the preparation method of above-mentioned nuclear magnetic resonance developer of the present invention and two-photon imaging developer, can adopt structural formula to be expressed as respectively:

The final product A of above-mentioned first kind of compound ₁Final product A with the third compound ₃Structure in contain l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section, be the compound that the self-assembly condensation reaction can shear be taken place by the furin specific recognition; The final product A of second kind of compound ₂Final product A with the 4th kind of compound ₄Structure in do not contain this amino acid peptide section, so can not be discerned by enzyme spcificity, so can be respectively as the final product A of first kind of compound ₁Final product A with the third compound ₃Control compound; Through performance liquid chromatography experimental verification, the final product A of said the 5th kind of compound ₅Be final product A by first kind of compound ₁The product that the self-assembly condensation reaction generates takes place;

Five kinds of compounds of above-mentioned synthetic all are to be received on 2-amino-6-cyanic acid benzothiazole by amino acid or amino acid fragment, behind the deprotection base, connect big ring tri-tert 1; 4,7,10-tetraazacyclododecanand-1; 4,7, the 10-tetraacethyl; In buffered soln, add metals ion at last, their building-up process and reaction mechanism are similar, therefore can be classified as the compound of same reaction type; But on purposes, the final product A of said first kind of compound ₁, second kind of compound final product A ₂Final product A with the 5th kind of compound ₅Containing the paramagnetic metal ion gadolinium, is the developer of nuclear magnetic resonance, the final product A of said the third compound ₃Final product A with the 4th kind of compound ₄The metals ion europium that contains red fluorescence is the developer that is used for the two-photon imaging.

Its remarkable advantage of the developer that the present invention prepares is: the final product A of first kind of compound ₁Final product A with the third compound ₃It is the small molecules developer; They can be in the target spot of cell intelligence be self-assembled into nano material, accumulate and be not pumped out at imaging region, and have good accumulation imaging effect; Thereby overcome general small molecules nuclear magnetic resonance agent and the sensitivity of two-photon preparation is not high; Plasma half-life is short, needs the shortcoming of high density, has improved imaging sensitivity greatly; And different with traditional developer be the final product A of first kind of compound of developer of the present invention ₁Final product A with the third compound ₃Containing can be by the amino acid fragment of furin identification; This kind of enzyme is only expressed at specific tumors cell inner height; Therefore initiatively targeted imaging is regional; Be different from general nano particle and lean on the enhancing infiltration of tumor tissues and keep effect to reach the purpose of passive target imaging region, can fix a point to arrive cancerous area then and be self-assembled into nano particle, therefore imaging developer of the present invention has height target property.

In a word, the developer of the present invention design have the high and high target of cellular uptake rate to advantage, can generate nanoparticle in vivo, improved the partial preparation concentration of biological tissue, solved the problem of traditional developer muting sensitivity.

Description of drawings

Fig. 1 is first kind of repurity product X of synthetic among the embodiment 1 ₄Nucleus magnetic hydrogen spectrum figure;

Fig. 2 is second kind of repurity product of synthetic Y among the embodiment 1 ₄Nucleus magnetic hydrogen spectrum figure;

Fig. 3 is the final product A of first kind of compound among the embodiment 2 ₁High-efficient liquid phase chromatogram after the external condensation;

Fig. 4 is the final product A of first kind of compound among the embodiment 2 ₁Collect the ground substance assistant laser parsing ionization massspectrum figure that four main performance liquid chromatography peaks obtain after the external condensation;

Fig. 5 is the final product A of first kind of compound among the embodiment 2 ₁The transmission electron microscope that external condensation forms nanoparticle characterizes;

Fig. 6 is the final product A of first kind of compound among the embodiment 2 ₁Uv-absorbing figure before and after vitro enzyme is cut at 500-700nm;

Fig. 7 is the final product A of first kind of compound among the embodiment 3 ₁The transmission electron microscope of cultivating altogether after 8 hours with breast cancer cell characterizes;

Fig. 8 is the final product A of second kind of compound among the embodiment 3 ₂The transmission electron microscope of cultivating altogether after 8 hours with breast cancer cell characterizes;

Fig. 9 is the final product A of the third compound among the embodiment 3 ₃The two-photon imaging microscope of cultivating altogether after 8 hours with breast cancer cell characterizes;

Figure 10 is the final product A of the 4th kind of compound among the embodiment 3 ₄Characterize with the two-photon imaging microscope of co-culture of cells after 8 hours;

Figure 11 is for injecting the final product A of first kind of compound respectively among the embodiment 3 ₁Final product A with second kind of compound ₂Nude mice characterize in the nuclear magnetic resonance of different time points.

Embodiment

The concrete compound method of five kinds of developers is provided among the following embodiment 1, and embodiment 2 is the final product A of first kind of compound of experiment in vitro checking ₁Can be self-assembled into the example of nanoparticle in external generation condensation reaction then, embodiment 3 is the imaging effect of cell and this type of developer of animalcule checking.

Embodiment 1:

The final product A of first kind of compound in the present embodiment ₁Synthetic route following:

Earlier Methionin-halfcystine-l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section is synthesized; Concrete steps are following: with 0.33 mmole 2-chlorine trityl chloride resin at 2 milliliters of N; Swelling added first amino acid N-fluorenylmethyloxycarbonyl of 0.66 mmole-N'-tertbutyloxycarbonyl-L-Methionin after eight minutes in the dinethylformamide in reactor drum

Add 0.66 mmole N, the N-diisopropylethylamine reacted after two hours, reacted 20 minutes with 30 microliter methanol; Cut first amino acid protection base, Caesar tests and shows blue, adds second amino acid cysteine reaction of activatory 0.55 mmole two hours, and Caesar tests displaing yellow; Cut the protection base, Caesar tests and shows blue, adds the 3rd amino acids Arginine reaction of activatory 0.55 mmole three hours, and Caesar tests displaing yellow; Cut the protection base, Caesar tests and shows blue, adds the 4th amino acids Arginine reaction of activatory 0.55 mmole three hours; Caesar tests displaing yellow, cuts the protection base, and Caesar tests and shows blue; Add activatory 0.55 mmole five amino acid Xie Ansuan reaction two hours, Caesar tests displaing yellow, cuts the protection base; Caesar tests and shows blue, adds the 6th amino acids Arginine reaction of activatory 0.55 mmole four hours, and Caesar tests displaing yellow; Cut the protection base, Caesar tests and shows blue, adds 0.66 mmole aceticanhydride and reacts 20 minutes; Use at last that to contain volumetric concentration be that the methylene dichloride of 1% trifluoroacetic acid downcuts the peptide section from resin, adopt ether layer to go out the frozen centrifugation upper strata ether that inclines, treat that obtaining the white solid powder after solvent evaporates is done promptly is the synthetic peptide section of wanting; Above-mentioned synthetic peptide section is taken 0.1 mmole to be dissolved in 2 milliliters of tetrahydrofuran solvents; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, add 0.12 mmole 2-amino-6-cyanic acid benzothiazole after half a hour; Maintain the temperature at 0 ℃ to 10 ℃ one hour; Stirring at room reaction 8 hours separates through performance liquid chromatography and to purify, and being collected in ultraviolet 320 nanometers has the component of strong absorption kind of the head product X that promptly wins ₁

Then with first kind of head product X ₁Be dissolved in 10 ml volumes concentration and be in the methylene dichloride of 95% trifluoroacetic acid the stirring at room reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of purified product X ₂

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the N-diisopropylethylamine is with 0.1

mmole tri-tert

1,4; 7,10-tetraazacyclododecanand-1,4; 7, the activation of 10-tetraacethyl added first kind of purified product X of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product X first kind time ₃

First kind purified product X with above-mentioned preparation ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of repurity product X ₄

Get first kind of repurity product X of above-mentioned preparation ₄0.1 mmole is dissolved in 5 ml waters; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride 6.5, and stirring at room was reacted 5 hours; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of first kind of compound ₁

The final product A of second kind of compound ₂Synthetic route following:

Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section is synthesized, and concrete steps are following: at 2 milliliters of N, swelling added first amino acid Xie Ansuan of 0.66 mmole after five minutes in the dinethylformamide in reactor drum with 0.33 mmole 2-chlorine trityl chloride resin; Add 0.66 mmole N, the N-diisopropylethylamine reacts after two hours; Reacted 20 minutes with 30 microliter methanol, cut first amino acid protection base, Caesar tests and shows blue; Add second amino acids Arginine reaction of activatory 0.55 mmole three hours, Caesar tests displaing yellow, cuts the protection base; Caesar tests and shows blue, adds the 3rd amino acid cysteine reaction of activatory 0.55 mmole three hours, and Caesar tests displaing yellow; Cut the protection base, Caesar tests and shows blue, adds the 4th amino acids Arginine reaction of activatory 0.55 mmole three hours; Caesar tests displaing yellow, cuts the protection base, and Caesar tests and shows blue; Add activatory 0.55 mmole five amino acid Methionin reaction three hours, Caesar tests displaing yellow, cuts the protection base; Caesar tests and shows blue, adds the 6th amino acids Arginine reaction of activatory 0.55 mmole four hours, and Caesar tests displaing yellow; Cut the protection base, Caesar tests and shows blue, adds 0.66 mmole aceticanhydride and reacts 20 minutes; Use at last that to contain volumetric concentration be that the methylene dichloride of 1% trifluoroacetic acid downcuts the peptide section from resin, adopt ether layer to go out the frozen centrifugation upper strata ether that inclines, treat that obtaining the white solid powder after solvent evaporates is done promptly is Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section;

Above-mentioned synthetic peptide section is taken 0.1 mmole to be dissolved in 2 milliliters of tetrahydrofuran solvents; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, add 0.12 mmole 2-amino-6-cyanic acid benzothiazole after half a hour; Maintain the temperature at 0 ℃ to 10 ℃ one hour; Stirring at room reaction 8 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption promptly to get second kind of head product Y ₁

To go up the second kind of head product Y that obtains in the step then ₁Be dissolved in 10 ml volumes concentration and be in the methylene dichloride of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of purified product Y ₂

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.5 mmole N, the N-diisopropylethylamine is with 0.1 mmole tri-tert 1,4; 7,10-tetraazacyclododecanand-1,4; 7, the activation of 10-tetraacethyl added second kind of purified product Y of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂, stirring at room reaction 3 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Y second kind time ₃

Second kind purified product Y with above-mentioned preparation ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of repurity product Y ₄

Get second kind of repurity product Y of above-mentioned preparation ₄0.1 mmole is dissolved in 5 ml waters; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride 6.5, and stirring at room was reacted 5 hours; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has strong absorbent components to be the final product A of second kind of compound ₂

Get first kind of repurity product X of above-mentioned preparation ₄0.1 mmole is dissolved in 5 ml waters; The pH value of regulating this solution with yellow soda ash adds 1 mmole Europium trichloride 6.5, and stirring at room was reacted 5 hours; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of the third compound ₃

Get second kind of repurity product Y of above-mentioned preparation ₄0.1 mmole is dissolved in 5 ml waters; The pH value of regulating this solution with yellow soda ash adds 1 mmole Europium trichloride 6.5, and stirring at room was reacted 5 hours; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of the 4th kind of compound ₄

The final product A of the 5th kind of compound ₅Synthetic route following:

Get N-tertbutyloxycarbonyl-N'-fluorenylmethyloxycarbonyl-L-Methionin

0.1 mmole is dissolved in 2 milliliters of tetrahydrofuran solvents; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, add 0.12 mmole 2-amino-6-cyanic acid benzothiazole after half a hour; Maintain the temperature at 0 ℃ to 10 ℃ one hour; Stirring at room reaction 8 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption promptly to get the 5th kind of head product Z ₁

Then with the 5th kind of head product Z ₁Be dissolved in 10 ml volumes concentration and be in the methylene dichloride of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of purified product Z ₂

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate and 0.5 mmole N, the N-diisopropylethylamine is with 0.1 mmole N-(tertbutyloxycarbonyl l)-S-sulphur ethyl-L-halfcystine

After the activation 5 minutes, add the 5th kind of purified product Z of the above-mentioned preparation of 0.1 mmole ₂In, stirring at room reaction 3 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Z the 5th kind time ₃

The 5th kind purified product Z with above-mentioned preparation ₃Be dissolved in 10 ml volumes concentration and be 50% N; The N of N-diisopropylethylamine; Stirring reaction adds 5 milliliters of trifluoroacetic acids in the dinethylformamide solution after five minutes; Ice bath separates purification through performance liquid chromatography immediately, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of repurity product Z ₄

mmole tri-tert

1,4; 7,10-tetraazacyclododecanand-1,4; 7, the activation of 10-tetraacethyl added the 5th kind of repurity product Z of the above-mentioned preparation of 0.1 mmole after 5 minutes ₄In, stirring at room reaction 3 hours separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of four purified product Z ₅

The 5th kind of four purified product Z with above-mentioned preparation ₅Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of five purified product Z ₆

Get the 5th kind of five purified product Z of above-mentioned preparation ₆0.1 mmole is dissolved in 5 ml waters; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride 6.5, and stirring at room was reacted 5 hours; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of six purified product Z ₇

Among the above-mentioned preparation method of the present invention; Wherein the used amino acid of solid phase all is as alpha-amino group protection base with 9-fluorenylmethyloxycarbonyl

; The sulfydryl of halfcystine is protected by uncle's butylthio; L-arginine side chain amino is by 2; 2; 4,6,7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl

Because metals ion gadolinium and europium have paramagnetism, can't resolve with magnetic nuclear resonance method after the reaction, therefore can only obtain first kind of repurity product X ₄With second kind of repurity product Y ₄Nuclear magnetic spectrogram, obtain nucleus magnetic hydrogen spectrum figure as depicted in figs. 1 and 2 after adopting German Brooker company (bruker) Brooker nuclear-magnetism software to resolve:

First kind of repurity product X ₄Molecular formula is C ₆₂H ₁₀₃N ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1490.8, the gained mass spectrum is m/z1490.6.Visible by accompanying drawing 1, first kind of repurity product X ₄Nucleus magnetic hydrogen spectrum (d-methyl alcohol, 300MHz): CD ₃OD, 300MHz): 9.10 (s, 1H), 8.58 (d, 1H), 8.24 (d-d, 1H, J=9.0Hz), 7.31 (s, 1H), 4.83-4.56 (m; 3H), 4.51 (m, 2H), 4.38-4.09 (m, 6H), 4.00 (m, 12H, J=21Hz), 3.83-3.44 (m, 18H), 3.43-3.24 (m; 6H), 2.63 (m, 2H), 2.32-2.14 (m, 6H), 2.14-2.00 (m, 8H), 1.93 (s, 1H), 1.87 (s; 1H), 1.77 (s, 6H), 1.70 (m, 3H), 1.58 (t, 16H, J=14.1Hz), 1.37 (m, 6H).

Second kind of repurity product Y ₄Molecular formula is C ₆₂H ₁₀₃N ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1490.8, the gained mass spectrum is m/z 1490.7.Visible by accompanying drawing 2, the nucleus magnetic hydrogen spectrum of second kind of repurity product Y4 (d-methyl alcohol, 300MHz): 9.10 (s, 1H), 8.58 (d, 1H), 8.24 (d-d, 1H, J=9.0Hz), 7.31 (s; 1H), 5.81 (m, 2H), 4.91-4.80 (m, 1H), 4.83-4.56 (m, 3H), 4.51 (m, 2H), 4.38-4.09 (m; 4H), 4.00 (m, 12H, J=21Hz), 3.82-3.46 (m, 18H), 3.46-3.35 (m, 2H), 3.24 (s, 3H); 2.63 (m, 3H), 2.32-2.14 (m, 6H), 2.14-2.00 (m, 8H), 1.93 (s, 1H), 1.87 (s, 1H); 1.77 (s, 5H), 1.70 (m, 4H), 1.58 (t, 15H, J=14.1Hz), 1.37 (m, 6H).

Can confirm first kind of repurity product X by above-mentioned nucleus magnetic resonance and mass spectrum ₄Structure, the final product A of first kind of compound just ₁Main body frame, with the molecular formula of Gadolinium trichloride reaction back gained compound be C ₆₂H ₁₀₀GdN ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1645.6, the gained mass spectrum is m/z 1645.4, can confirm as the final product A of first kind of compound ₁

Can confirm second kind of repurity product Y by above-mentioned nucleus magnetic resonance and mass spectrum ₄Structure, the final product A of second kind of compound just ₂Main body frame, with the molecular formula of Gadolinium trichloride reaction back gained compound be C ₆₂H ₁₀₀GdN ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1645.6, the gained mass spectrum is m/z 1645.6, can confirm as the final product A of second kind of compound ₂

Can confirm first kind of repurity product X by above-mentioned nucleus magnetic resonance and mass spectrum ₄Structure, also be the final product A of the third compound simultaneously ₃Main body frame, with the molecular formula of Europium trichloride reaction back gained compound be C ₆₂H ₁₀₀EuN ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1640.6, the gained mass spectrum is m/z 1640.5, can confirm as the final product A of the third compound ₃

Can confirm second kind of repurity product Y by above-mentioned nucleus magnetic resonance and mass spectrum ₄Structure, also be the final product A of the 4th kind of compound simultaneously ₄Main body frame, with the molecular formula of Europium trichloride reaction back gained compound be C ₆₂H ₁₀₀EuN ₂₃O ₁₄S ₃, [(M+H) ⁺]: 1640.6, the gained mass spectrum is m/z 1640.3, can confirm as the final product A of the 4th kind of compound ₄

The final product A of the 5th kind of compound ₅Molecular formula be C ₆₆H ₈₄Gd ₂N ₁₈O ₁₈S ₄, [(M+H) ⁺]: 1861.3, the gained mass spectrum is m/z 1860.9, can confirm as the final product A of the 5th kind of compound ₅

Thus, five kinds of particular compound have been synthesized among the preparation method of above-mentioned nuclear magnetic resonance developer of the present invention and two-photon imaging developer: the final product A of first kind of compound ₁, second kind of compound final product A ₂, the third compound final product A ₃, the 4th kind of compound final product A ₄Final product A with the 5th kind of compound ₅, can adopt structural formula to be expressed as respectively:

Because final product A at first kind of compound ₁Final product A with the third compound ₃Structure in contain l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section, be can be, and furin be expressed at some cancer cell camber by the aminoacid sequence of furin identification, the aminoacid sequence that is identified can be by proteolytic cleavage.Because condensation reaction can take place in the reactive group that exposes; And the compound that hydrophilic variation generates can self-assembly generate nanoparticle; Therefore the precursor small molecules of imaging developer of the present invention has the peptide section of certain enzyme specific recognition, thereby initiatively targeted imaging is regional.Utilize nuclear magnetic resonance of the present invention and two-photon imaging developer and preparation method thereof can controlled self-assembled nano structures, to be used for the active imaging research of certain enzyme.The final product A of second kind of compound ₂Final product A as first kind of compound ₁Control compound, do not contain in the structure by the amino acid fragment of furin identification, therefore the final product A of second kind of compound ₂Can not shear even get into cell yet, just can not generate nanoparticle yet, so the final product A of second kind of compound ₂Final product A than first kind of compound ₁The nuclear magnetic resonance weak effect some; In like manner, the final product A of the 4th kind of compound ₄Be the final product A of the third compound ₃Control compound.Fig. 3 is the final product A of first kind of compound ₁The performance liquid chromatography experiment of condensation reaction takes place; Solid line is the condensation high-efficient liquid phase chromatogram of compound before; The high-efficient liquid phase chromatogram of compound after the condensation is for taking place in dotted line, and dotted line shown and mainly contain four reaction product peaks, collects four peaks and carries out high resolution mass spectrum and test; As shown in Figure 4, the molecular weight at four peaks all with the final product A of the 5th kind of compound ₅Molecular weight identical.Possibly be because the cause that different isomerss exists.Therefore by performance liquid chromatography and mass spectrum experimental verification, can draw the final product A of the 5th kind of compound ₅Be the final product A of first kind of compound ₁The product that condensation reaction generates taking place, but because the efficient that furin is sheared is lower, studies for ease and synthetic separately.

Five kinds of compounds of above-mentioned synthetic all are to be received on 2-amino-6-cyanic acid benzothiazole by amino acid or amino acid fragment, behind the deprotection base, connect big ring tri-tert 1; 4,7,10-tetraazacyclododecanand-1; 4,7, the 10-tetraacethyl; In buffered soln, add metals ion at last, building-up process and reaction mechanism are similar, therefore can be classified as a compounds.

On purposes, the final product A of said first kind of compound ₁, second kind of compound final product A ₂Final product A with the 5th kind of compound ₅Contain the paramagnetic metal ion gadolinium, the compound of gadolinium is typical mr probe, so they are developers of nuclear magnetic resonance; The final product A of said the third compound ₃Final product A with the 4th kind of compound ₄The metals ion europium that contains red fluorescence is the developer that is used for the two-photon imaging.

The final product A of 2: the first kinds of compounds of embodiment ₁The external confirmatory experiment of condensation reaction takes place

That adopt in the present embodiment experiment in vitro is the final product A of first kind of compound ₁Concentration is 100 μ mol/L; In concentration is that the TV of the not woods enzyme of 100mmol/L 4-HEPES damping fluid, 1mmol/L trichloroethyl phosphate, 1mmol/L calcium chloride and 5 μ L is in the solution of 100 μ L; Hatch for 30 ℃ and be condensed into dimer in 17 hours, and be self-assembled into nanoparticle.

Fig. 5 has provided the final product A of first kind of compound in the present embodiment ₁The transmission electron microscope that external condensation forms nanoparticle characterizes; Fig. 6 is the final product A of first kind of compound among the embodiment 2 ₁Vitro enzyme is cut the uv-absorbing figure of front and back at 500-700nm.

Wherein Fig. 5 is transmission electron microscope (TEM) sign of the nanoparticle that preparation forms to the inventive method; The nanometer ball that the product that adopts the inventive method to prepare is illustrated in figure 5 as black interconnects; The mean sizes of nanoparticle is 57nm, and size is homogeneous relatively.Fig. 6 is the variation of enzyme-added front and back uv-absorbing in the 500-700nm scope.By finding out among Fig. 6, the curve b before the below enzyme is cut shows that it does not absorb at 500-700nm, and the curve a after the top enzyme is cut shows that it has absorption at 500-700nm, and this has shown the generation of condensation reaction and the formation of nanoparticle.

Embodiment 3: the generation of checking condensation reaction on cell levels

At first cultivate human malignant breast carcinomas cell strain MDA-MB-468, concrete operations are following: malignant galactophore JEG-3 MDA-MB-468 uses the DMEM high glucose medium of the bovine serum albumin that contains volumetric concentration 10% to cultivate, and cell is 5%CO containing volumetric concentration ₂37 ° of C thermostat containers of air ambient in cultivate, change a subculture every day.

Through the section after transmission electron microscope photo observation of cell and the compound cultivation; Concrete operations are following: with malignant galactophore JEG-3 MDA-MB-468 cell kind in the culture plate of two 6 centimetres of sizes; About 6,000,000 cells, the described in the above volumetric concentration that contains is 5%CO ₂37 ° of C constant incubators of air ambient in cultivated 12 hours after, in two dish cells, add the final product A of first kind of compound respectively ₁Final product A with second kind of compound ₂Cultivated eight hours.Sop up substratum, with phosphate buffered saline buffer flushing three times, each five minutes; Adding trypsinase then digests cell from the culture plate bottom; Again with phosphate buffered saline buffer flushing three times, each five minutes, add 2.5% LUTARALDEHYDE fixing half a hour in phosphate buffered saline buffer; Sample presentation dyes and cuts into slices to test center.Fig. 7 is the final product A of first kind of compound ₁With the transmission electron microscope photo of co-culture of cells, from photo, can find out near the golgi body in the tenuigenin has large stretch of little black nano ball, and size is than homogeneous.Through measuring, the mean diameter that obtains this nanoparticle is 20 nanometers.Fig. 8 is the final product A of second kind of compound ₂With the transmission electron microscope photo of co-culture of cells, because the final product A of second kind of compound ₂Contain and therefore condensation reaction can not be taken place, at the final product A of second kind of compound by the amino acid fragment of furin identification ₂With the gathering that does not have nanoparticle in the section of co-culture of cells.From Fig. 8, do not find the black nano particle of similar Fig. 7.

Under the two-photon imaging microscope, observe the final product A of the third compound ₃Final product A with the 4th kind of compound ₄The cellular uptake situation, concrete operations are following: with malignant galactophore JEG-3 MDA-MB-468 cell kind in 6 orifice plates of deckglass are housed, about 1,000,000 cells in every hole, the described in the above volumetric concentration that contains is 5%CO ₂37 ° of C constant incubators of air ambient in cultivated 12 hours after, in different holes, add the final product A of the third compound respectively ₃Final product A with the 4th kind of compound ₄Cultivated eight hours.After sopping up substratum, with phosphate buffered saline buffer flushing three times, each five minutes; Add 4% Paraformaldehyde 96 fixedly after half a hour, with phosphate buffered saline buffer flushing three times, each five minutes.Take out deckglass, on slide glass, with the nail varnish mounting after, in the observation of two-photon microscopically, Fig. 9 is the final product A of the third compound to the glycerine that adds 1 microlitre 50% at phosphate buffered saline buffer ₃Two-photon imaging microscope photo with co-culture of cells in tenuigenin, has bright spot limpid in sight, represents fluorescence intensity stronger; And the obvious gathering of fluorescence is arranged, and representative has generated nanoparticle.Figure 10 is the final product A of the 4th kind of compound ₄Two-photon imaging microscope photo with co-culture of cells.In tenuigenin, have only fuzzy speck, can indistinctly feel through naked eyes, represent fluorescence intensity a little less than, tangible fluorescence also useless is assembled, representative does not have the generation of nanoparticle; Can analyze from the significant difference of Fig. 9 and Figure 10 fluorescence intensity and to obtain: the final product A of the third compound ₃Behind cellular uptake, condensation reaction takes place generated nanoparticle, so the final product A of the third compound ₃Final product A than the 4th kind of compound ₄Cellular uptake effective.

After carrying out cell experiment, verify the effect of this nuclear magnetic resonance probe in animal body.At first malignant galactophore cancer cells MDA-MB-468 (high expression level furin) is set up tumor model on the nude mice of heavily about 20g.Treat that tumor growth is to 300-700mm ³Size, the tail vein is given the final product A of first kind of compound of 0.03mmol/kg respectively in the nude mouse of two same treating processess ₁Final product A with second kind of compound ₂Before and after injection, respectively nude mice is carried out MRI scan, after compound arrives tumor locus, absorbed and in cell, be self-assembled into " intelligence " nanostructure by tumour cell.Final product A as first kind of compound ₁Control experiment, the injection second kind of compound final product A ₂Nude mice because contain and can not can not in tumour cell, be assembled into nanoparticle by the amino acid fragment of furin identification, but the final product A of second kind of compound ₂The magnetic resonance imaging contrast that still can be used as a kind of routine uses, and therefore injects the final product A of second kind of compound ₂The contrast gradient of the formed nuclear magnetic resonance of nude mice than the final product A of first kind of compound ₁A little less than.Figure 11 is for injecting the nuclear magnetic resonance figure of different contrast medium, the final product A of first kind of compound of wherein top line display injection in different time points ₁Nude mice magnetic resonance radiography figure, below the final product A of second kind of compound of delegation representative injection ₂Nude mice nuclear magnetic resonance radiography figure, primary injection volume is 0.06mmol/kg, after 50 minutes two nude mices is injected once more the compound of same dosage.Shown in figure 11, the zone that white arrow is pointed to is the tumor region of nude mice, before injection, and the figure shown in 0 minute just, tumor tissues and viscera tissue on every side almost can not be distinguished, and brightness is also very low, injects the final product A of first kind of compound ₁After 50 minutes, the tumor region of nude mice and the brightness of internal organs have tangible increase, and the quantification result calculated shows 50% brightness increase, and the contrast of tumour and internal organs is also apparent in view; The final product A of second kind of compound of injection ₂Tumor region and the brightness of internal organs of nude mice increase is also arranged, but because the final product of second kind of compound does not have target property, the amplitude that therefore increases has only 30%.The increase of tumor model high-contrast like this shows the final product A of first kind of compound ₁Be a kind of imaging effect preparation preferably, the final product A of second kind of compound ₂Also have imaging effect, but owing to lack target property, than the final product A of first kind of compound ₁Imaging effect poor slightly.

Its remarkable advantage of the developer that the present invention prepares is: the final product A of first kind of compound ₁Final product A with the third compound ₃It is the small molecules developer; They can be in the target spot of cell intelligence be self-assembled into nano material, accumulate and be not pumped out at imaging region, and have good accumulation imaging effect; Thereby overcome general small molecules nuclear magnetic resonance agent and the sensitivity of two-photon preparation is not high; Plasma half-life is short, needs the shortcoming of high density, has improved imaging sensitivity greatly; And different with traditional developer be the final product A of first kind of compound of developer of the present invention ₁Final product A with the third compound ₃Containing can be by the amino acid fragment of furin identification; This kind of enzyme is only expressed at specific tumors cell inner height; Initiatively targeted imaging is regional; Therefore imaging developer of the present invention has height target property, and general nano particle can only lean on the enhancing infiltration of tumor tissues and keep effect to reach the passive target imaging region.

Claims

1. the final product A of first kind of compound in one type of nuclear magnetic resonance developer and the two-photon imaging developer ₁The preparation method, it is characterized in that:

Press: 1 mmole 2-chlorine trityl chloride resin at 4-6 milliliter N, after swelling 4-8 minute, is added 2 mmole N-fluorenylmethyloxycarbonyls-N'-tertbutyloxycarbonyl-L-Methionin in the dinethylformamide; Add 2 mmole N again, the N-diisopropylethylamine reacts after 2-3 hour; With 100 microliter methanol reaction 10-20 minute, cut the protection base of Methionin, add second amino acid cysteine reaction of activatory 1.6 mmoles 2-3 hour; Cut the protection base of halfcystine, add the 3rd amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour, cut arginic protection base; Add the 4th amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add activatory 1.6 mmole five amino acid Xie Ansuans reaction 2-3 hour, cut the protection base of Xie Ansuan; Add the 6th amino acids Arginine reaction of activatory 1.6 mmoles 3-4 hour again; Cut the protection base of last amino acids Arginine, added 2-3 mmole aceticanhydride reaction 20-30 minute, use at last that to contain volumetric concentration be that the methylene dichloride of 1% trifluoroacetic acid downcuts above-mentioned synthetic peptide section from this resin; Separate out the frozen centrifugation upper strata ether that inclines with ether layer, treat solvent evaporates do after resulting white solid powder promptly be Methionin-halfcystine-l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section;

Above-mentioned synthetic Methionin-halfcystine-l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section is got 0.1 mmole to be dissolved in the 2-4 milliliter tetrahydrofuran solvent; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, after activation half a hour; Add 0.12 mmole 2-amino-6-cyanic acid benzothiazole; Maintain the temperature at 0-10 ℃ one hour, stirring at room reaction 8-9 hour separates through performance liquid chromatography and to purify; Being collected in ultraviolet 320 nanometers has the component of strong absorption, is first kind of head product X ₁

Elder generation is with first kind of head product X of above-mentioned preparation ₁Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of purified product X ₂

Again with 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added first kind of purified product X of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product X first kind time ₃

Then with first kind of above-mentioned preparation purified product X ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of repurity product X ₄

At last with first kind of repurity product X of above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of first kind of compound ₁

2. the final product A of first kind of compound of nuclear magnetic resonance developer of the said method of claim 1 preparation ₁, it is characterized in that structural formula is expressed as:

Containing l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section in its structure, is and to shear the compound that the self-assembly condensation reaction takes place by the furin specific recognition; Containing the paramagnetic metal ion gadolinium, is the developer of nuclear magnetic resonance.

3. the final product A of second kind of compound in one type of nuclear magnetic resonance developer and the two-photon imaging developer ₂The preparation method, it is characterized in that:

Press: 1 mmole 2-chlorine trityl chloride resin at 4-6 milliliter N, after swelling 4-8 minute, is added first amino acid Xie Ansuan of 2 mmoles in the dinethylformamide; Add 2 mmole N again, the N-diisopropylethylamine reacts after 2-3 hour; With 100 microliter methanol reaction 10-20 minute, cut the protection base of Xie Ansuan, add second amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add the 3rd amino acid cysteine reaction of activatory 1.6 mmoles 2-3 hour, cut the protection base of halfcystine; Add the 4th amino acids Arginine reaction of activatory 1.6 mmoles 2-3 hour; Cut arginic protection base, add activatory 1.6 mmole five amino acid Methionins reaction 2-3 hour, cut the protection base of Methionin; Add the 6th amino acids Arginine reaction of activatory 1.6 mmoles 3-4 hour again; Cut the protection base of last amino acids Arginine, added 2-3 mmole aceticanhydride reaction 20-30 minute, using volumetric concentration at last is that the dichloromethane solution of 1% trifluoroacetic acid downcuts above-mentioned synthetic peptide section from this resin; Adopt ether layer to separate out the frozen centrifugation upper strata ether that inclines, treat solvent evaporates do after resulting white solid powder promptly be Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section;

Again above-mentioned synthetic Xie Ansuan-l-arginine-halfcystine-l-arginine-Methionin-l-arginine peptide section being got 0.1 mmole is dissolved in the 2-4 milliliter tetrahydrofuran solvent; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, after activation half a hour; Add 0.12 mmole 2-amino-6-cyanic acid benzothiazole; Maintain the temperature at 0-10 ℃ one hour, stirring at room reaction 8-9 hour separates through performance liquid chromatography and to purify; Being collected in ultraviolet 320 nanometers has the component of strong absorption, is second kind of head product Y ₁

Then with second kind of head product Y ₁Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of purified product Y ₂

Then with 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added second kind of purified product Y of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Y second kind time ₃

Again with second kind purified product Y ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of repurity product Y ₄

At last with second kind of repurity product Y of above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of second kind of compound ₂

4. the final product A of second kind of compound of nuclear magnetic resonance developer of the said method of claim 3 preparation ₂, it is characterized in that structural formula is expressed as:

Wherein containing the paramagnetic metal ion gadolinium, is the developer of nuclear magnetic resonance.

5. the final product A of the third compound in one type of nuclear magnetic resonance developer and the two-photon imaging developer ₃The preparation method, it is characterized in that:

Then with 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added first kind of purified product X of the above-mentioned preparation of 0.1 mmole after 5 minutes ₂In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product X first kind time ₃

Again with first kind of above-mentioned preparation purified product X ₃Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be first kind of repurity product X ₄

At last with first kind of repurity product X of above-mentioned preparation ₄0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Europium trichloride at 6-7, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the final product A of the third compound ₃

6. the final product A of the third compound of two-photon imaging developer of the said method of claim 5 preparation ₃, it is characterized in that structural formula is expressed as:

Containing l-arginine-l-arginine-Xie Ansuan-l-arginine peptide section in its structure, is to be sheared the compound that the self-assembly condensation reaction takes place by the furin specific recognition; Containing the metals ion europium, is the developer of two-photon imaging.

7. the final product A of the 4th kind of compound in one type of nuclear magnetic resonance developer and the two-photon imaging developer ₄The preparation method, it is characterized in that:

Again with second kind of head product Y ₁Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be second kind of purified product Y ₂

With 0.1 mmole tri-tert 1,4,7; 10-tetraazacyclododecanand-1,4,7; The 10-tetraacethyl is with 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the activation of N-diisopropylethylamine added among second kind of purified product Y2 of the above-mentioned preparation of 0.1 mmole after 5 minutes; Stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be purified product Y second kind time ₃

8. the final product A of the 4th kind of compound of developer of the two-photon imaging of the said method of claim 7 preparation ₄, it is characterized in that structural formula is expressed as:

Wherein containing the metals ion europium, is the developer of two-photon imaging.

9. the final product A of the 5th kind of compound in one type of nuclear magnetic resonance developer and the two-photon imaging developer ₅The preparation method, it is characterized in that:

Press, with N-tertbutyloxycarbonyl-N'-fluorenylmethyloxycarbonyl-L-Methionin

0.1 mmole is dissolved in 2 milliliters of tetrahydrofuran solvents; Add 0.15 mmole N-methylmorphine, add 0.12 mmole isobutyl chlorocarbonate when being cooled to zero degree, add 0.12 mmole 2-amino-6-cyanic acid benzothiazole after half a hour; Maintain the temperature at 0-10 ℃ one hour; Stirring at room reaction 8-9 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption promptly to get the 5th kind of head product Z ₁Then with the 5th kind of head product Z ₁Be dissolved in 10 ml volumes concentration and be in the methylene dichloride of 95% trifluoroacetic acid stirring reaction 3 hours, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of purified product Z ₂

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N', N'-tetramethyl-urea hexafluorophosphate and 0.3-0.5 mmole N, the N-diisopropylethylamine is with 0.1 mmole N-(tertbutyloxycarbonyl l)-S-sulphur ethyl-L-halfcystine

With 0.1 mmole I-hydroxybenzotriazole and 0.1 mmole benzotriazole-N, N, N'; N'-tetramethyl-urea hexafluorophosphate and 0.5 mmole N, the N-diisopropylethylamine is with 0.1 mmole tri-tert 1,4; 7,10-tetraazacyclododecanand-1,4; 7, the activation of 10-tetraacethyl added the 5th kind of repurity product Z of the above-mentioned preparation of 0.1 mmole after 5 minutes ₄In, stirring at room reaction 3-5 hour separates purification through performance liquid chromatography, and being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of four purified product Z ₅With the 5th kind of four purified product Z ₅Be dissolved in 10 ml volumes concentration and be in the dichloromethane solution of 95% trifluoroacetic acid stirring reaction 3-5 hour, and separated through performance liquid chromatography and purify, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of five purified product Z ₆

Get the 5th kind of five purified product Z of above-mentioned preparation ₆0.1 mmole is dissolved in the 3-5 ml water; The pH value of regulating this solution with yellow soda ash adds 1 mmole Gadolinium trichloride 6.5, and stirring at room was reacted 3-5 hour; Separate purification through performance liquid chromatography, being collected in ultraviolet 320 nanometers has the component of strong absorption to be the 5th kind of six purified product Z ₇

10. the final product A of the 5th kind of compound of nuclear magnetic resonance developer of the said method of claim 9 preparation ₅, it is characterized in that structural formula is expressed as: