CN109568607B - Gadolinium-based metal fullerene water-soluble nitrene derivative and preparation method and application thereof - Google Patents

Gadolinium-based metal fullerene water-soluble nitrene derivative and preparation method and application thereof Download PDF

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CN109568607B
CN109568607B CN201811649095.4A CN201811649095A CN109568607B CN 109568607 B CN109568607 B CN 109568607B CN 201811649095 A CN201811649095 A CN 201811649095A CN 109568607 B CN109568607 B CN 109568607B
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gadolinium
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何睿
毋青男
刘艳杰
王合中
臧振锋
王朝霞
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Abstract

The invention discloses a gadolinium-based metal fullerene water-soluble nitrene derivative and a preparation method and application thereof. The gadolinium-based metal fullerene water-soluble nitrene derivative is prepared from paramagnetic gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)mNH2Or N3(CH2)m‑1COOH is prepared through nitrene reaction, and the structural general formula is as follows: gd (Gd)aNb@C2n[N(CH2)mNH2]xOr GdaNb@C2n[N(CH2)m‑ 1COOM]xIn the structural general formula, a is 1-3; b is 0-3; n is 30-45; m is 2-6; x is 8-30; m is H, NH4Na or K. The invention provides a method for preparing gadolinium-based metal fullerene water-soluble nitrene derivatives through nitrene reaction. The gadolinium-based metal fullerene water-soluble nitrene derivative has good water solubility and good paramagnetic property, can be used as a magnetic resonance imaging contrast agent, has a very obvious contrast enhancement effect when being used for MRI image imaging, and has an important application prospect in medical diagnosis and treatment, particularly in MRI image-guided diagnosis and treatment.

Description

Gadolinium-based metal fullerene water-soluble nitrene derivative and preparation method and application thereof
Technical Field
The invention belongs to the field of carbon nano materials, and relates to a gadolinium-based metal fullerene water-soluble nitrene derivative as well as a preparation method and application thereof.
Background
Having a high longitudinal relaxation rate (R)1) Paramagnetic metal fullerene T with low biotoxicity1MRI contrast agents of type have been developed for approximately 20 years. Among them, gadolinium-based metallofullerene MRI contrast agents are most commonly reported in the literature as molecular imaging probes. These MRI probes are all single-step or multi-step modified Gd @ C with hydroxylation, amination, Bingel-Hirsch or Prato and the like60、Gd@C82、ScxGd3-xN@C80、Gd3N@C80、Gd3N@C84And (3) derivatives. Of these derivatives, Gd @ C82(OH)40The labeled cells are used as molecular probes for MRI cell imaging for the first time; gd @ C82O6(OH)16(NHCH2CH2COOH)8The receptor protein is coupled for the first time and is used as a molecular probe for identifying the fluorescent protein; gd binding to small peptide IL-13 recognizing brain glioma3N@C80(OH)26(CH2CH2COOH)16And Gd3N@C80O12(OH)10(NH2)7(NO2)2Are reported as tumor diagnosis and treatment reagents with integrated diagnosis and treatment functions. In addition, Gd @ C82O14(OH)14(NH2)6The label-free fluorescent dye can form an MRI-fluorescence bimodal molecular imaging probe, and the MRI-fluorescence bimodal molecular imaging probe can also be used as an antioxidant for eliminating ROS free radicals and has the potential of tumor inhibition. Currently, the bottleneck exists in the application research of the paramagnetic gadolinium-based metal fullerene derivative as a nano diagnosis and treatment reagent, and the key point is that a synthetic method for economically, reasonably and massively preparing a novel paramagnetic gadolinium-based metal fullerene water-soluble derivative is lacked, so that sufficient products are difficult to provide for the subsequent biological effect research.
The invention aims to provide a paramagnetic gadolinium-based metal fullerene water-soluble derivative and a preparation method thereof by adopting a solvent-free strategy reaction technology, directly dispersing solid paramagnetic gadolinium-based metal fullerene by using an excess azide compound in a liquid state and carrying out nitrene reaction. The method provides a novel research idea for developing a novel gadolinium-based metal fullerene water-soluble nitrene derivative used as an MRI contrast agent.
Disclosure of Invention
The invention aims to solve the first technical problem of providing a gadolinium-based metal fullerene water-soluble nitrene derivative with a carbon cage externally connected with a side chain end bonded with a hydrophilic functional group, wherein the derivative has good water solubility and paramagnetic performance, and a reactive group exists at the side chain end.
The second technical problem to be solved by the invention is to provide a method for preparing the gadolinium-based metal fullerene water-soluble nitrene derivative with paramagnetism.
The third technical problem to be solved by the invention is to provide the application of the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative as a magnetic resonance imaging contrast agent.
The following is a detailed description of the technical solution adopted by the present invention to solve the above technical problems.
In one aspect, the invention provides a paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative, which is prepared from paramagnetic gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)mNH2Or N3(CH2)m-1COOH is prepared through nitrene reaction, and the structural general formula is as follows:
GdaNb@C2n[N(CH2)mNH2]xor GdaNb@C2n[N(CH2)m-1COOM]x
In the structural general formula, a is 1-3; b is 0-3; n is 30-45; m is 2-6; x is 8-30; m is H, NH4Na or K.
The paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative molecule takes a paramagnetic gadolinium-based metal fullerene carbon cage as a core, at least 8 hydrophilic side chains are arranged outside the carbon cage as connecting arms, each connecting arm is bonded with the carbon cage through a nitrogen bridge, each connecting arm is a short straight saturated carbon chain formed by a plurality of carbon atoms, and the tail end of each connecting arm is further bonded with a hydrophilic functional group-NH2or-COOM, thereby ensuring water solubility throughout the derivative molecule. The number of the connecting arms can be determined according to different azide and gadolinium-based metal fullerene varieties, and when the number of the connecting arms is less than 8, the connecting arms are extremely poor in water solubility and even completely insoluble in water; when the number of the connecting arms is more than 30, the carbon cage structure of the product is unstable, and even the inner package metal is leaked due to cage opening; therefore, the number of the connecting arms is preferably between 8 and 30, and Gd @ C is preferable82Particularly preferably 14 to 24 derivatives.
The paramagnetic gadolinium-based metal fullerene is a spherical or spheroidal structure with each molecule consisting of 60-90 carbon atoms and contains 1-3 Gd atoms or Gd atoms insideaNbThe paramagnetic gadolinium-based metal fullerene is preferably Gd @ C82、Gd@C60、Gd2@C80、Gd3N@C80Or Gd3N@C84More preferably Gd @ C82、Gd3N@C80Or Gd3N@C84And with Gd @ C82Is optimal.
The molecular structure of the azide is a saturated aliphatic straight carbon chain with a carbon chain containing 2-6 carbon atoms and one end bonded with a diazide group (-N)3) And the other end is bonded with hydrophilic amino (-NH)2) Or carboxyl (-COOH) with the structural general formula of N3(CH2)mNH2Or N3(CH2)m-1COOH is preferably 2-azidoethylamine, 2-azidoacetic acid, 3-azidopropylamine, 3-azidopropionic acid, 4-azidobutyric acid or 6-azidohexanoic acid.
The cycloaddition reaction of fullerene and azide group-containing compound is a well-known technique, but the paramagnetic gadolinium-based metal fullerene is rarely reported to produce water-soluble derivatives by the nitrene reaction.
In another aspect, the present invention provides a method for preparing a gadolinium-based metal fullerene water-soluble nitrene derivative having paramagnetism through nitrene reaction.
The structural general formula of the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative is GdaNb@C2n[N(CH2)mNH2]xOr GdaNb@C2n[N(CH2)m-1COOH]xThe preparation method comprises the following steps:
(1) gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)mNH2Or N3(CH2)m-1Respectively feeding COOH into a reactor, and stirring under an inert gas atmosphere, wherein the ratio of the amount of the substances of the gadolinium-based metal fullerene to the amount of the substances of the azide is 1:100 to 1000;
(2) heating the solid-liquid reaction system obtained in the step (1) from room temperature to 40-80 ℃ at a heating rate of 2-5 ℃/min, preserving heat for 0.5-2 hours, then heating to 110-150 ℃ at a heating rate of 0.5-2 ℃/min, preserving heat for 10-48 hours, stopping heating, and tracking and monitoring the whole reaction process by TLC;
(3) adding an organic solvent A into the reaction product, washing and filtering to remove most of unreacted azide, extracting the residue with water, filtering the extracting solution, and drying to obtain a solidified crude product;
(4) the crude product is dissolved in water again, dialyzed by a dialysis bag with MW & lt 500 & gt and 10000, and freeze-dried to obtain a pure paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative solid product.
Further, in the step (1), the charge ratio of the azide to the gadolinium-based metal fullerene must be within a specified range, that is, the ratio of the amount of the substances is 100 to 1000: 1. below the specified feed ratio of 100: 1 lower limit, the reaction system is easily cured rapidly and is difficult to continue and the number of derivatives added is small, resulting in difficulty in obtaining a water-soluble product; over 1000: 1, the subsequent treatment is complicated, and a purified product is not easy to obtain; the water-soluble paramagnetic gadolinium-based metal fullerene nitrene derivative can be obtained at relatively low cost and high yield within the range of the specified charge ratio.
Further, in the step (2), the temperature control operation is very critical, the reaction is difficult to occur and form a water-soluble product when the temperature is lower than 110 ℃, the reaction is too violent when the temperature is higher than 150 ℃, and a great runaway risk exists, wherein the temperature control range is preferably 110-150 ℃, and more preferably 110-120 ℃.
Further, in the step (3), washing may be performed by using a conventional reagent, such as diethyl ether, dichloromethane, chloroform, ethyl acetate, etc., to remove most of the unreacted azide.
Furthermore, in the step (4), the refining method adopts freeze drying after dialysis, so that the purification is realized, the treatment mode has few steps and the operation is simple.
The invention also provides another method for preparing a paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative through nitrene reaction, wherein the structural general formula of the paramagnetic gadolinium-based metal fullerene nitrene derivative is GdaNb@C2n[N(CH2)m- 1COOM]xM is Na or K or NH4The preparation method comprises the following steps:
(a) gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)m-1Respectively feeding COOH into a reactor, and stirring under an inert gas atmosphere, wherein the ratio of the amount of the substances of the gadolinium-based metal fullerene to the amount of the substances of the azide is 1:100 to 1000;
(b) heating the solid-liquid reaction system obtained in the step (1) from room temperature to 40-80 ℃ at a heating rate of 2-5 ℃/min, preserving heat for 0.5-2 hours, then heating to 110-150 ℃ at a heating rate of 0.5-2 ℃/min, preserving heat for 10-48 hours, stopping heating, and tracking and monitoring the whole reaction process by TLC;
(c) adding an organic solvent B into the reaction product for washing and filtering, removing most of unreacted azide, redissolving the residue in an alkaline solution, wherein the alkaline solution is NaOH or KOH aqueous solution or ammonia water, dialyzing by using a dialysis bag with MW & lt 500 & gt and 10000, and drying to obtain a pure paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative solid product.
Further, in the step (a), the charge ratio of the azide to the gadolinium-based metal fullerene must be within a specified range, that is, the ratio of the amount of the substances is 100 to 1000: 1. below the specified feed ratio of 100: 1 lower limit, the reaction system is easily cured rapidly and is difficult to continue and the number of derivatives added is small, resulting in difficulty in obtaining a water-soluble product; over 1000: 1, the subsequent treatment is complicated, and a purified product is not easy to obtain; the water-soluble paramagnetic gadolinium-based metal fullerene nitrene derivative can be obtained at relatively low cost and high yield within the range of the specified charge ratio.
Further, in the step (b), the temperature control operation is very critical, when the temperature is lower than 110 ℃, the reaction is difficult to occur and a water-soluble product is formed, when the temperature is higher than 150 ℃, the reaction is too violent, and a great runaway risk exists, wherein the temperature control range is preferably 110-150 ℃, and more preferably 110-120 ℃.
Further, in the step (c), washing may be performed by using a conventional reagent, such as diethyl ether, dichloromethane, chloroform, ethyl acetate, etc., to remove most of the unreacted azide. In the present invention, the organic solvents in steps (3) and (c) are represented by organic solvent a and organic solvent B, which are only used for distinguishing different steps, and do not indicate that different solvents must be used for organic solvent a and organic solvent B, and in fact, they are independent of each other, and can be selected by those skilled in the art according to the needs.
Further, in the step (c), the alkaline solution is preferably NaOH or KOH aqueous solution or ammonia water with the concentration of more than 0.1mol/L and less than or equal to 1mol/L, and more preferably NaOH or KOH aqueous solution with the concentration of 0.5 mol/L.
Further, the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative prepared in the step (c) can regulate and control the number of side chains and the width of the distribution range through varieties of azide and paramagnetic gadolinium-based metal fullerene.
In a third aspect, the invention provides the application of the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative as a magnetic resonance imaging contrast agent.
In the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative molecule obtained by the invention, the surface of a carbon cage is bridged with a plurality of terminal bonds-NH through nitrogen atoms2or-COOM hydrophilic side chain, so that the whole cage-type molecule has water solubility and simultaneously shows good paramagnetic property, and can be used as a magnetic resonance imaging contrast agent. In addition, by means ofSide chain terminal having reactive-NH2or-COOM functional group which can be used as a reaction intermediate for linking various markers such as paramagnetic, optical and enzyme markers and biomacromolecules such as saccharides, nucleic acid, polypeptide and antibody through amidation coupling reaction, thereby constructing the functionalized nano diagnosis and treatment reagent for being applied to the technical field of nano diagnosis and treatment.
Compared with the prior art, the invention has the beneficial effects that:
(A) the compound provided by the invention is a paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative with long and short side chain carbon chains, multiple and different chain end alkene hydrophilic groups, and the product library of the metal fullerene water-soluble derivative can be greatly expanded.
(B) The novel method for preparing the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative on a large scale has the characteristics that: the synthesis process is simple and efficient, and the purification and separation method is simple and is easy to realize large-scale industrial preparation.
(C) The reaction system of the solid-liquid phase reaction method provided by the invention does not need to add an organic solvent to disperse the paramagnetic gadolinium-based metal fullerene, and the azide of the liquid serving as a reactant is similar to the derivative in polarity, so that the azide is favorably dispersed in the liquid phase reaction system, and finally the derivative with higher addition number is generated until a water-soluble derivative product is generated.
(D) The paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative obtained by the invention has reactive group-NH at the tail end of the side chain externally connected to the carbon cage2or-COOM, which can be coupled with various labels such as magnetism, light, enzyme, etc., and biofunctional molecules such as saccharides, nucleic acids, polypeptides, antibodies, etc., through amidation condensation reaction.
(E) The obtained paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative has good water solubility and good paramagnetic property, can be used as a magnetic resonance imaging contrast agent, has a very obvious contrast enhancement effect when being used for MRI image imaging, and has an important application prospect in medical diagnosis and treatment, particularly in MRI image-guided diagnosis and treatment.
Drawings
FIG. 1 Gd @ C in example 182(NCH2CH2COOH)22MALDI-TOF MS spectrum of the aqueous solution.
FIG. 2 Gd @ C in example 182(NCH2CH2COOH)22FT-IR spectrum of the powder.
FIG. 3 Gd @ C in example 182(NCH2CH2COOH)22Thermogravimetric analysis of the powder.
FIG. 4 Gd @ C in example 382(NCH2CH2NH2)16FT-IR spectrum of the powder.
FIG. 5 Gd @ C82(NCH2COOH)24(A) And Gd @ C82(NCH2CH2COOH)22(B) The proton longitudinal relaxation rate of (a).
Detailed Description
The following examples are provided to aid in a further understanding of the invention, and the scope of the invention is not limited to the examples set forth.
The experimental methods described in the following examples are all conventional methods; the reagents and materials are commercially available. Wherein Gd @ C82Is from Xiamen Funan New Material science and technology limited company, and the purity is higher than 99.0 percent.
Example 1: preparation of Gd @ C82Water-soluble derivative Gd @ C with azidopropionic acid82(NCH2COOH)22
Mixing 10mg of Gd @ C82With liquid N3CH2CH2Sequentially feeding COOH into a three-mouth reaction bottle according to the stoichiometric ratio of 1:500, and stirring under the protection of nitrogen; firstly, slowly heating to 60 ℃ at the heating rate of 2 ℃/min, preserving heat for 1 hour, then heating to 120 ℃ at the heating rate of 0.5 ℃/min, preserving heat for 48 hours, and stopping heating; adding ethyl acetate, washing, filtering, repeatedly extracting the residue with deionized water to colorless, filtering the extractive solution, and drying to obtain solidified crude product; redissolving in deionized water, dialyzing with MW 3500 dialysis bag, filtering with filter membrane, and freeze drying to obtain pure paramagnetic Gd @ C82Solid products of water-soluble derivatives of polyalanine.
As shown in the figure1, the ion peak with m/z of 1142 in MALDI-TOF MS is attributed to Gd @ C82The carbon cage parent unit is generally considered that the external side chain of the metal fullerene water-soluble derivative carbon cage is stripped from the metal fullerene carbon cage due to the laser desorption effect in the MALDI-TOF MS experiment process, the molecular ion peak of the derivative is not generated, and only the ion peak of the metal fullerene unit exists. Due to Gd @ C82Insoluble in the aqueous phase, the water-soluble derivative is judged to be Gd @ C82A derivative of (1).
As shown in FIG. 2, the infrared spectrum was 3420cm-1The strong absorption peak is attributed to O-H stretching vibration and 1720cm-1The absorption peak belongs to the range of C ═ O and 1100cm-1Belongs to C-OH telescopic vibration and is 2930cm-1The weak absorption peak is attributed to C-H stretching vibration at 1640cm-1The strong absorption peak of the compound belongs to Gd @ C82And C, stretching and vibrating the residual C in the carbon cage. IR spectrum proves Gd @ C82The molecule of the poly-alanine water-soluble derivative has-COOH.
As shown in FIG. 3, the results of TG-DTG-DSC simultaneous analysis showed Gd @ C82The mass percentage of the poly-alanine water-soluble derivative molecules in the temperature range of 110-515 ℃ is reduced from 93.89% to 35.33%, and the mass percentage reduction in the temperature range (57.80%) is completely attributed to Gd @ C82Poly-alanine water-soluble derivative molecule external connection group-NCH2CH2The total loss of COOH, while the mass remaining at 515 ℃ is attributed to Gd @ C82Carbon cage, wherein the formula weight of the carbon cage is 1142, and all the carbon cage is externally connected with side chain (-NCH)2CH2COOH) is 87x, and x is calculated to be 21.47, from which Gd @ C is obtained82The average molecular formula of the poly-alanine water-soluble derivative is Gd @ C82(NCH2CH2COOH)22。
Example 2: preparation of Gd @ C82Water-soluble derivative Gd @ C with azidoacetic acid82(NCH2COOH)24
Mixing 10mg of Gd @ C82With liquid N3CH2Sequentially feeding COOH into a three-port reaction bottle, and stirring under the protection of nitrogen; firstly, the temperature is slowly raised to 80 ℃ at the temperature raising rate of 2 ℃/min, the temperature is kept for 2 hours, and then the temperature is raised to 0.5 DEG CHeating up to 120 ℃ at a temperature rise rate per minute, preserving heat for 48 hours, and stopping heating; the reaction mixture was washed with diethyl ether under the same conditions as in inventive example 1. Obtaining pure paramagnetic Gd @ C82Solid products of water-soluble derivatives of polyglycine. The thermogravimetric analysis indicates that the average molecular formula is Gd @ C82(NCH2COOH)24。
Example 3: preparation of Gd @ C82Water-soluble derivative Gd @ C with azidoethylamine82(NCH2CH2NH2)20
Mixing 10mg of Gd @ C82With liquid N3CH2CH2NH2Sequentially feeding materials into a three-port reaction bottle, protecting with nitrogen, and stirring; firstly, slowly heating to 60 ℃ at the heating rate of 2 ℃/min, preserving heat for 2 hours, then heating to 125 ℃ at the heating rate of 0.5 ℃/min, preserving heat for 48 hours, and stopping heating; adding ethyl acetate for washing; the other conditions were the same as in inventive example 1. Obtaining pure paramagnetic Gd @ C82Water-soluble derivative Gd @ C of polyethylene diamine82(NCH2CH2NH2)20The solid product of (4).
As shown in FIG. 4, the infrared spectrum is 3385cm-1The strong absorption peak is attributed to N-H stretching vibration and 1388cm-1The absorption peak belongs to NH bending vibration at 1037cm-1Is classified into C-NH2The stretching vibration is 2935 and 2850cm-1The weak absorption peak is attributed to C-H symmetric and antisymmetric telescopic vibration at 1654cm-1The strong absorption peak of the compound belongs to Gd @ C82And C, stretching and vibrating the residual C in the carbon cage. IR spectrum proves Gd @ C82Existence of-NH in the molecule of water-soluble derivative of polyethylene diamine2。
Example 4: preparation of Gd @ C82Metal fullerene based sodium polyglyconate water soluble derivative Gd @ C with azidoacetic acid82(NCH2COONa)14
Mixing 10mg of Gd @ C82With liquid N3CH2Sequentially feeding COOH into a three-mouth reaction bottle according to the stoichiometric ratio of 1:100, protecting with nitrogen, and mechanically stirring; firstly, the temperature is slowly raised to 40 ℃ at the temperature rise rate of 2 ℃/min, the temperature is preserved for 0.5 hour, and then the temperature is changed into the temperature of 0.5 ℃Heating up to 110 ℃ at the min heating rate, preserving the heat for 10 hours, and stopping heating; washing with diethyl ether, filtering, dissolving the residue in 0.5mol/LNaOH solution, dialyzing with MW 3500 dialysis bag, filtering with filter membrane, freeze drying, and refining to obtain Gd @ C containing sodium glycinate82The solid product of the water-soluble nitrene derivative is characterized by the thermogravimetric analysis that the average molecular formula is Gd @ C82(NCH2COONa)10。
Example 5: preparation of Gd @ C82Metal fullerene based poly (caproic acid) sodium water-soluble derivative Gd @ C with azido caproic acid82(NCH2CH2CH2CH2CH2COONa)10
Mixing 10mg of Gd @ C82With liquid N3CH2CH2CH2CH2CH2Sequentially feeding COOH into a three-mouth reaction bottle according to the stoichiometric ratio of 1:250, protecting with nitrogen, and mechanically stirring; firstly, slowly heating to 80 ℃ at the heating rate of 2 ℃/min, preserving heat for 2 hours, then heating to 140 ℃ at the heating rate of 0.5 ℃/min, preserving heat for 24 hours, and stopping heating; adding ethyl acetate, washing, filtering, dissolving the residue in 0.5mol/LNaOH solution, filtering with filter membrane, dialyzing with dialysis bag MW 3500, freeze drying, refining to obtain metal fullerene group polyhexamine sodium water-soluble derivative, and performing thermogravimetric analysis to show that the average molecular formula is Gd @ C82(NCH2CH2CH2CH2CH2COONa)10。
Comparative example
With reference to example 1, the difference is that: gd @ C82With liquid N3CH2CH2After COOH is mixed, directly heating to reflux temperature, and after about 30-60 min, quickly curing the system, forcibly terminating the reaction, and stopping heating; washing with ethyl acetate, filtering, extracting the residue with deionized water for 3-5 times, filtering the extractive solution, concentrating, dialyzing with MW 3500 dialysis bag, filtering with filter membrane, and freeze drying to obtain Gd @ C82Water-soluble polyalanine derivative Gd @ C82(NCH2CH2COOH) x.
Example 6：Gd@C82Specific embodiment of relaxation performance test of nitrene derivative used as MRI contrast agent
Relaxation performance experiments were performed on a 200 mega animal MRI machine. Respectively configuring Gd @ C82(NCH2COOH)24And Gd @ C82(NCH2CH2COOH)22A series of samples of different concentrations. And (2) adopting inversion recovery spin echo imaging sequence scanning, wherein the sequence parameters are as follows: repetition time 10s, echo time 13.5ms, imaging area 3.5 × 3.5cm2The thickness of the chip is 1.0mm, the data matrix is 64 multiplied by 64, and a series of proper TI values are selected from 0.05-15 s. Obtaining the longitudinal relaxation time T of all samples to be tested by fitting a three-parameter single exponential function1. T at different concentrations through its series1Value fitting of its proton longitudinal relaxation rate, R1The value is obtained. FIG. 5 is Gd @ C82(NCH2COOH)24And Gd @ C82(NCH2CH2COOH)22 Longitudinal relaxation rate 1/T1The slope of the change trend is R relative to the concentration change trend graph1The results are shown in Table 1. In general, the shorter the carbon chain in the linker arm, the higher the relaxation rate, and the greater the number of additions, the higher the relaxation rate.
TABLE 1
Sample name R1(mM-1S-1)
Gd@C82(NCH2COOH)24 26.29
Gd@C82(NCH2CH2COOH)22 17.34

Claims (8)

1. A method for preparing a gadolinium-based metal fullerene water-soluble nitrene derivative through nitrene reaction is characterized in that: the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative is prepared from paramagnetic gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)mNH2Or N3(CH2)m-1COOH is prepared through nitrene reaction, and the structural general formula is as follows:
GdaNb@C2n[N(CH2)mNH2]xor GdaNb@C2n [N(CH2)m-1COOM]x
In the general structural formula, a = 1-3; b =0 to 3; n = 30-45; m = 2-6; x = 8-30; m is H, NH4Na or K;
when the structural general formula of the paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative is GdaNb@C2n[N(CH2)mNH2]xOr GdaNb@C2n [N(CH2)m-1COOH]xThe preparation method comprises the following steps:
(1) gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)mNH2Or N3(CH2)m-1Respectively feeding COOH into a reactor, and stirring under an inert gas atmosphere, wherein the ratio of the amount of the substances of the gadolinium-based metal fullerene to the amount of the substances of the azide is 1:100 to 1000;
(2) heating the solid-liquid reaction system obtained in the step (1) from room temperature to 40-80 ℃ at a heating rate of 2-5 ℃/min, preserving heat for 0.5-2 hours, then heating to 110-150 ℃ at a heating rate of 0.5-2 ℃/min, preserving heat for 10-48 hours, stopping heating, and tracking and monitoring the whole reaction process by TLC;
(3) adding an organic solvent A into the reaction product, washing and filtering to remove most of unreacted azide, extracting the residue with water, filtering the extracting solution, and drying to obtain a solidified crude product;
(4) re-dissolving the crude product in water, dialyzing by using a dialysis bag with MW =500-10000, and freeze-drying to obtain a pure paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative solid product;
when the structural general formula of the paramagnetic gadolinium-based metal fullerene nitrene derivative is GdaNb@C2n [N(CH2)m-1COOM]xWherein M is Na or K or NH4The preparation method comprises the following steps:
(a) gadolinium-based metal fullerene GdaNb@C2nWith azide N3(CH2)m-1Respectively feeding COOH into a reactor, and stirring under an inert gas atmosphere, wherein the ratio of the amount of the substances of the gadolinium-based metal fullerene to the amount of the substances of the azide is 1:100 to 1000;
(b) heating the solid-liquid reaction system obtained in the step (1) from room temperature to 40-80 ℃ at a heating rate of 2-5 ℃/min, preserving heat for 0.5-2 hours, then heating to 110-150 ℃ at a heating rate of 0.5-2 ℃/min, preserving heat for 10-48 hours, stopping heating, and tracking and monitoring the whole reaction process by TLC;
(c) adding an organic solvent B into the reaction product, washing and filtering to remove most of unreacted azide, redissolving the residue in an alkaline solution, wherein the alkaline solution is NaOH or KOH aqueous solution or ammonia water, dialyzing by using a dialysis bag with MW =500-10000, and drying to obtain a pure paramagnetic gadolinium-based metal fullerene water-soluble nitrene derivative solid product.
2. The method of claim 1, wherein: and (C) in the step (2) or (b), after heat preservation is carried out for 0.5-2 hours at the temperature of 40-80 ℃, slowly heating to the temperature of 110-120 ℃ for heat preservation reaction.
3. The method of claim 1 or 2, wherein: the organic solvent A and the organic solvent B are respectively and independently selected from one or a mixture of any of the following: diethyl ether, dichloromethane, trichloromethane and ethyl acetate.
4. The method of claim 1 or 2, wherein: in the step (c), the alkaline solution is NaOH or KOH aqueous solution or ammonia water with the concentration of more than 0.1mol/L and less than or equal to 1 mol/L.
5. The method of claim 1 or 2, wherein: the paramagnetic gadolinium-based metal fullerene is Gd @ C82 、Gd@C60、Gd2@C80、Gd3N@C80Or Gd3N@C84。
6. The method of claim 5, wherein: the paramagnetic gadolinium-based metal fullerene is Gd @ C82，x=14~24。
7. The method of claim 1 or 6, wherein: the azide is 2-azidoethylamine, 2-azidoacetic acid, 3-azidopropylamine, 3-azidopropionic acid, 4-azidobutyric acid or 6-azidohexanoic acid.
8. The method of claim 5, wherein: the azide is 2-azidoethylamine, 2-azidoacetic acid, 3-azidopropylamine, 3-azidopropionic acid, 4-azidobutyric acid or 6-azidohexanoic acid.
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