CN104592200A - Chiral paramagnetic probe using 1,4,7,10-tetraazacyclododecane as skeleton - Google Patents

Abstract

A chiral paramagnetic probe with a skeleton of 1,4,7,10-tetraazacyclododecane, its chemical name is 1,4,7,10-tetraazacyclododecane-1- (2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex, the chemical formula is C ₃₀ H ₄₀ N ₅ O ₈ STm, the chemical structure formula is: . The advantages of the present invention are: 1) specific chirality, single conformation, only one set of peaks in the spectrum, high sensitivity; 2) high chemical selectivity; 3) electrical neutrality, little influence on macromolecules; 4) good water solubility , many coordination sites, strong stability, and high degree of toothing; 5) extremely rigid, and has a good immobilization effect on macromolecules; 6) the probe is connected to the protein through a stable thioether bond, which can be used for intracellular measurement. It can play an important role in promoting the paramagnetic research of proteins.

Description

A chiral paramagnetic probe based on 1,4,7,10-tetraazacyclododecane

technical field

The invention belongs to high-performance probes in chemical biology research, in particular to a chiral paramagnetic probe with 1,4,7,10-tetraazacyclododecane (cyclen) as the skeleton.

Background technique

The spatial structure of proteins is the basis of their biological functions. Due to its unique properties, nuclear magnetic resonance under the paramagnetic effect has played an increasingly powerful role in the study of the structure and function of biological macromolecules (such as proteins, DNA, RNA, etc.) in recent years; the paramagnetic effect can provide other techniques Long-range structural information of macromolecules that is difficult to obtain by means; functionalized paramagnetic tags are one of the most important technical means for modifying proteins in paramagnetic technology.

The quality of the paramagnetic label plays a decisive role in the study of the structure and dynamics of biological macromolecules using paramagnetic NMR. Paramagnetic tags are generally some small molecular probes with dual functional groups, one end is used for chelation of paramagnetic metal ions, and the other end is used for linking with proteins. After linking the paramagnetic label to the biomacromolecule, the paramagnetic effect can be used to reveal the action mechanism and conformational changes of the biomacromolecule, and the high-resolution structure of the interaction between the small molecule and the macromolecule (see: J. Biomol. NMR , 2010 , 46 : 101-112), which will play an important guiding role in the screening and optimization of effective drug lead molecules.

A good label usually needs to meet the following conditions: 1) The rigidity of the label connection. When the label is combined with a paramagnetic metal ion, if the relative position of the paramagnetic label and the protein changes, the PCS (pseudocontact chemical shift) and RDC (residual dipole coupling) will be significantly reduced; 2) The optical purity is single, and the metal The combination of ions and tags with multiple potential chiral centers will produce enantiomers, resulting in multiple sets of NMR signals, which will make the spectrum complex and difficult to interpret; 3) The stability of the tag and protein linkage.

Su et al. ( Chembiochem , 2006 , 7: 1599–1604.), first activated the sulfhydryl group of the protein to form a disulfide bond with Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and the excess DTNB is removed by dialysis; then an equal amount of label is added, and the sulfhydryl group in the label reacts with the activated disulfide bond to form a new disulfide bond, finally connecting the polypeptide label with the protein (see Figure 1 for the reaction process). The leaving 5-mercapto-2-nitrobenzoic acid (TNB) is obviously yellow, which provides great convenience for the detection of the reaction process. This labeling method has a high yield (>70%), because the LBT polypeptide label is very acidic, and its pI value will change significantly after linking to the protein, so the linking product and unlinked protein can be separated by an ion exchange column. However, disulfide bonds are unstable, which hinders the widespread application of such tags.

Prudencio et al. ( Chem. Eur. J. , 2004 , 10(3): 3252～3260.) reported the compound L-1 with two active sulfhydryl groups (see Figure 2), which consists of DTPA bisanhydride and S-(2 -Aminoethyl) methane thiosulfonic acid reaction, the synthesis method is simple. This label can react with two cysteine residues at a distance of 8-10 Å on the protein at the same time, forming two disulfide bonds, which increases the rigidity of the label, limits the movement of the label relative to the protein, and avoids the averaging of PCS , there is still an obvious PCS at a distance of 4nm from the paramagnetic metal ion. However, due to the presence of multiple chiral centers, at least 5 paramagnetic signal peaks will be generated when paramagnetic metal ions are added dropwise after linking with the protein, resulting in a very complicated ¹ H- ¹⁵ N HSQC spectrum.

Li et al. ( Chem. Commun. , 2012 , 48: 2704–2706.) reported for the first time that the compound L-2 (see Figure 2) was labeled onto the protein by the Michael addition reaction of the terminal ethylenic bond and the protein side chain sulfhydryl group. The addition products contain thioether linkages, which are stable under reducing and alkaline conditions. However, compound L-2 has only three coordination sites, and after the lanthanide metal ion coordinates with it, it can also combine with other coordination atoms.

Vlasie et al. ( Chem. Eur. J. , 2007 , 13(6): 1715～1723.) reported compound L-3 (see Figure 2), which is linked by two disulfide bonds and two sulfhydryl groups on the protein, This type of attachment increases the rigidity of the tag and reduces the motion of the paramagnetic tag relative to the protein. However, the author found that after L-2 was connected to the protein, paramagnetic metal ions were added dropwise, and some residues in HSQC had two paramagnetic signals, indicating that after the label and the paramagnetic metal ion were coordinated, there was more than one conformation.

Keizers et al. ( J. Am. Chem. Soc. , 2007 , 129: 9292-9293; J. Am. Chem. Soc. , 2008 , 130: 14802-14812.) reported compounds L-4 and L-5 (see Figure 2), they introduced two pyridyl nitrogen oxides into the cyclen, and the introduction of pyridyl nitrogen oxides changed the coordination environment of L-4 and L-5 with lanthanide metal ions. There is only one set of PCS in the ¹ H- ¹⁵ N HSQC spectrum after they are linked to the protein, which indicates that L-4 and L-5 have only one conformation after they are coordinated with lanthanide metal ions. L-4 is connected to the protein through two disulfide bonds, which limits its movement relative to the protein, so the measured PCS and RDC values are much larger than those of L-4. The disadvantage of label L-5 is its own net charge, because it shows +3 valence after coordination with lanthanide metal ions, these three additional positive charges will cause some problems when studying protein-protein or protein-ligand recognition Unknown effects.

Liu et al. ( J. Am. Chem. Soc. , 2012 , 134: 17306–17313.) designed and synthesized the label L-6 (see Figure 2), which contains two p-nitrophenol groups, and the para-nitro group enhances With the acidity of phenol, the protons will leave after coordination with lanthanide metal ions, and the overall complex has a valence of +1. Compared with L-5, it has two less positive charges. After the compound L-6 is connected with the protein, a set of PCS is also generated. It is worth noting that when the label L-6 was linked to the yeast cytochrome C mutant, and the lanthanide metal ions were added dropwise, two sets of fronts appeared in most of the amino acid residues in the spectrum, and the peak intensity and chemical shift value varied with pH. Different changes. However, there is only one set of PCS for label L-5 attached to the same site of the same protein. This is because the oxygen atom charge density of p-nitrophenol in L-6 is smaller than that of pyridine nitrogen oxide in L-5. Circumstances lead to the participation of water molecules in the coordination of lanthanide metal ions, but this is not the fundamental reason why L-6 has two sets of PCS, because L-6 does not have two sets of fronts when it is connected to other proteins. The real reason is that in the yeast cytochrome C mutant, there is a histidine residue near the tag attachment site, and the nitrogen atom in the histidine imidazole ring can form hydrogen bonds with the water molecules participating in the coordination, destroying the network. The symmetry of the compound produces diastereomers. The difference in pH will affect the change of hydrogen bonds, resulting in the mutual conversion of the two diastereomers. Therefore, two sets of different PCS data can be obtained with the same ligand by adjusting the pH.

Graham et al. ( Bioconjugate chem. , 2011 , 22: 2118–2125.) introduced chiral amides into DOTA derivatives, so that compound L-7 (see Figure 2) has only one conformation after coordination with lanthanide metal ions , due to the relatively large space volume, which restricts its movement relative to the protein, a relatively large PCS is observed.

Loh et al. ( Bioconjugate chem. , 2013 , 24: 260–268. ) successfully labeled DOTA derivative L-8 (see Figure 2 ) onto proteins by means of click chemistry. The specific method is to introduce the unnatural amino acid p -azido-L-phenylalanine (AzF) into the protein, Cu(I) acts as a catalyst, and the tag L-8 and AzF undergo a cycloaddition reaction to covalently link to the protein on ubiquitin. Compared with the commonly used disulfide bond connection, the triazole ring has better rigidity and chemical stability, but this reaction needs to be carried out under anaerobic conditions, which increases the complexity of the experiment, and the catalyst Cu(I) will lead to Precipitation of protein samples.

Contents of the invention

The purpose of the present invention is to provide a kind of chiral paramagnetic probe with 1,4,7,10-tetraazacyclododecane as skeleton for above-mentioned technical analysis and existing problem, and this paramagnetic probe can be used with Selective connection at specific sites of the protein, specific chirality, single conformation, only one set of peaks in the spectrum, high sensitivity; high chemoselectivity; electrical neutrality, little impact on macromolecules; good water solubility, coordination points Multiple, strong stability, high degree of toothing; extremely rigid, good immobilization effect on macromolecules; probes and proteins are connected by stable thioether bonds, which can be used for intracellular measurement.

Technical scheme of the present invention:

A chiral paramagnetic probe with a skeleton of 1,4,7,10-tetraazacyclododecane, its chemical name is 1,4,7,10-tetraazacyclododecane-1- (2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex, the chemical formula is C ₃₀ H ₄₀ N ₅ O ₈ STm, the chemical structure formula is:

.

A preparation method of the chiral paramagnetic probe with 1,4,7,10-tetraazacyclododecane as the skeleton, the steps are as follows:

1) Synthesis of 2,6-lutidine nitrogen oxide

Mix 2,6-lutidine, glacial acetic acid, and 30wt% hydrogen peroxide in a volume ratio of 15:80:25, keep stirring at 80 ^o C and reflux for 4-12 hours until the conversion of raw materials is complete, and depressurize the resulting mixture Concentrate to one-third and pour into ice-water mixture. The dosage ratio of 2,6-lutidine to ice-water mixture in ice-water mixture is 15.0mL: 100g, and then use potassium carbonate solid to adjust the system to pH> 8, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 2,6-lutidine nitrogen oxide as a light yellow oil;

2) Synthesis of 2,6-dimethyl-4-nitropyridine nitrogen oxide

Cool the above-mentioned 2,6-lutidine nitrogen oxide ice bath to below 10 ^o C, slowly add 98wt% concentrated sulfuric acid, 95wt% fuming nitric acid, 2,6-bis The dosage ratio of picoline nitrogen oxide, concentrated sulfuric acid and fuming nitric acid is 15.2g: 22mL: 22mL. After adding, remove the ice bath, heat to 110 ^o C, stir and reflux for 3-6h until the reaction is complete; put the reaction solution in Dilute on ice cubes, the ratio of 2,6-lutidine nitrogen oxide to ice cubes is 15.2g: 200g, adjust the system to pH 8-10 with solid sodium carbonate, and a large number of yellow solids are precipitated; add water to the mixture In the mixture, the ratio of 2,6-lutidine nitrogen oxide to the mixed solution was 15.2g:500mL, heated to 40 ^o C, stirred to dissolve the formed inorganic salt, filtered out the yellow precipitate, dried to constant weight with an infrared lamp, and obtained Yellow solid 2,6-dimethyl-4-nitropyridine nitrogen oxide;

3) Synthesis of 2-methyl-4-nitro-6-hydroxymethylpyridine

Mix the above-mentioned 2,6-dimethyl-4-nitropyridine nitrogen oxide with dichloromethane, lower the ice bath to below 10 ^o C, slowly add trifluoroacetic anhydride, 2,6-dimethyl - The ratio of 4-nitropyridine nitrogen oxide, dichloromethane and trifluoroacetic anhydride is 9.2g: 100mL: 20mL, then remove the ice bath, heat and stir under reflux for 9-18h until the reaction is complete, spin dry and pour it out Put into ice water, the mass ratio of 2,6-dimethyl-4-nitropyridine nitrogen oxide to ice water is 9.2:80, adjust the system to pH 8 with solid potassium carbonate, stir at room temperature for 5 hours until the hydrolysis is complete, Potassium hydroxide was added to adjust the pH to 12, followed by extraction with dichloromethane, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, and spinning to obtain a light yellow solid 2-methyl-4-nitro-6- Hydroxymethylpyridine;

4) Synthesis of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine

Mix the above 2-methyl-4-nitro-6-hydroxymethylpyridine with sodium benzenesulfinate and acetonitrile, 2-methyl-4-nitro-6-hydroxymethylpyridine and sodium benzenesulfinate , The dosage ratio of acetonitrile is 3.0g: 7.0g: 60mL, heated to 85 ^o C under the protection of argon, stirred and refluxed for 2-4d until the conversion of raw materials is complete, after filtering, wash the filter residue with acetonitrile, combine the filtrate and washing liquid, add 5.0g of silica gel , after spinning to dryness and column separation, a white solid 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine was obtained;

5) Synthesis of 2-methyl-4-phenylsulfonyl-6-bromomethylpyridine

Mix the above 2-methyl-4-phenylsulfonyl-6-hydroxymethylpyridine with chloroform, lower the ice bath to below 10 ^o C, slowly add the mixture of phosphorus tribromide and chloroform dropwise under stirring, 2-methyl Base-4-phenylsulfone-6-hydroxymethylpyridine, phosphorus tribromide, and chloroform were used in amounts of 2.3g, 1.4mL, and 35+15mL, respectively, and then the ice bath was removed, and the reaction was carried out at 50 ^o C for 2-5h until The raw material conversion is complete, the reaction solution is diluted into ice water, the mass ratio of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine to ice water is 2.3:80, and the system is adjusted to a pH of 8-9, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, spin-dried, and column separated to obtain 2-methyl-4-phenylsulfone-6-bromomethylpyridine as a white solid;

6) Synthesis of 1, 1 ^, -ethylenebis-2-imidazoline

Mix commercially available triethylenetetramine, N,N-dimethylformamide dimethyl acetal, and toluene with a purity of 70%; The volume ratio is 30:60:100, heat to 85 ^o C, stir and reflux for 2-4h, spin off the toluene until a white solid precipitates, cool to room temperature, a large amount of solid precipitates, filter and wash with tetrahydrofuran, dry to constant weight with an infrared lamp , to obtain white solid 1, 1 ^, -ethylenebis-2-imidazoline;

7) Synthesis of intermediates by ring formation reaction

Mix the above 1,1 ^, -ethylenebis-2-imidazoline, 1,2-dibromoethane, potassium carbonate and acetonitrile, 1,1 ^, -ethylenebis-2-imidazoline, 1,2 - The ratio of dibromoethane, potassium carbonate and acetonitrile is 8.0g: 5.8mL: 5.0g: 400mL, heated and stirred to reflux for 3h under the protection of argon, and filtered after the system is cooled to 50 ^o C, and the filtrate is spin-dried to obtain Yellow solid intermediate;

8) Synthesis of 1,4,7,10-tetraazacyclododecane

Mix the above intermediate with potassium hydroxide and water, the ratio of intermediate, potassium hydroxide and water is 10.0g: 16.8g: 80mL, heat up to 110 ^o C, stir and reflux for 2h until the hydrolysis is complete, spin off the water until there is a white The solid was precipitated, cooled to room temperature and crystallized, and after filtration, the filter residue was dried to constant weight with an infrared lamp to obtain a white solid 1,4,7,10-tetraazacyclododecane;

9) Synthesis of 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine

Mix the above 1,4,7,10-tetraazacyclododecane and chloroform, and slowly add the 2-methyl-4-phenylsulfone- 6-bromomethylpyridine in chloroform, 1,4,7,10-tetraazacyclododecane, 2-methyl-4-phenylsulfone-6-bromomethylpyridine, chloroform and anhydrous potassium carbonate The dosage ratio is 2.9g: 3.7g: 150mL: 5.0g, react for 36-60h until the conversion of 2-methyl-4-phenylsulfone-6-bromomethylpyridine is complete, then add anhydrous potassium carbonate, continue to react for 5h , filtered, spin-dried, and column separated to obtain light yellow solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene) pyridine;

10) Synthesis of ethyl p-toluenesulfonyl lactate

Mix L-ethyl lactate, dichloromethane and p-toluenesulfonyl chloride, cool down in an ice bath, stir, slowly add triethylamine dropwise, the amount of L-ethyl lactate, dichloromethane, triethylamine, p-toluenesulfonyl chloride The ratio is 12.5g: 80mL: 18.6mL: 20.2g. After removing the ice bath, continue to react at room temperature for 3h, filter out triethylamine hydrochloride, and add 10% of the aforementioned triethylamine dropwise triethylamine to the filtrate , react at room temperature for 8-10 hours to complete the conversion of raw materials, then add distilled water, the ratio of distilled water to L-ethyl lactate is 150mL: 12.5g, sequentially extract with dichloromethane, dry with anhydrous sodium sulfate, filter, spin dry, After column separation, a colorless liquid L-ethyl p-toluenesulfonyl lactate was obtained;

11) 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Synthesis of ethyl propionate

The 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine prepared in step 9), anhydrous potassium carbonate, The above L-toluenesulfonyl lactylate ethyl ester mixed with acetonitrile, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene) The ratio of pyridine, anhydrous potassium carbonate, L-ethyl p-toluenesulfonyl lactylate, and acetonitrile is 1.02g: 1.64g: 3.25g: 40mL, react at 55-60 ^o C for 50-60h under the protection of argon, and filter The filtrate was spin-dried and separated by a column to obtain light yellow semi-solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine - ethyl 2,3,4-tri(S)-propionate;

12) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid

The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Mixture of ethyl propionate, sodium hydroxide, water and ethanol, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene ) The dosage ratio of pyridine-2,3,4-tri(S)-propionate, sodium hydroxide, water and ethanol is 1.50g:0.50g:10mL:10mL, stir at room temperature for 8-10h to complete the hydrolysis, Then use H ⁺ ion exchange resin to adjust the system to pH<4, filter and wash the filter residue with methanol, combine the filtrates and concentrate to dryness to obtain white solid 1,4,7,10-tetraazacyclododecane-1 -(2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid;

13) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid anion-Tm ³⁺ complex

The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O mixed with water, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6- Methylene) pyridine-2,3,4-tri(S)-propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O and water in the ratio of 0.1263g: 0.1839g: 8.0mL, heated to 100 ^o C Stir and reflux for 45 hours to make the reaction complete. After cooling to room temperature, add water, adjust the system to pH 8 with a potassium hydroxide solution with a concentration of 1mol/L, stir for 5 minutes, centrifuge to take the supernatant, add chromatographically pure methanol, and microwave Shake to make it fully dissolved, filter the inorganic salt and spin dry to obtain the target product 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6 -methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex.

The advantages of the present invention are: 1) specific chirality, single conformation, only one set of peaks in the spectrum, high sensitivity; 2) high chemical selectivity; 3) electrical neutrality, little influence on macromolecules; 4) good water solubility , many coordination points, strong stability, and high degree of toothing; 5) extremely rigid, good for immobilizing macromolecules; 6) the probe and protein are connected by a stable thioether bond, which can be used for intracellular measurement. These advantages make It can play an important role in promoting the paramagnetic research of proteins.

Description of drawings

Figure 1 shows the process of protein activation and connection.

Figure 2 shows the reported paramagnetic probes.

Fig. 3 is a synthetic route of the chiral paramagnetic probe of the present invention.

Figure 4 shows the connection method between protein and paramagnetic probe.

Fig. 5 is the ¹⁵ N-HSQC spectrum of 0.1 mM ¹⁵ N-labeled ubiquitin G47C.

Fig. 6 is the ¹⁵ N-HSQC spectrum of 0.1 mM ¹⁵ N-labeled ubiquitin G47C-Tm-T ₁ .

Detailed ways

Example:

A chiral paramagnetic probe with a skeleton of 1,4,7,10-tetraazacyclododecane, its chemical name is 1,4,7,10-tetraazacyclododecane-1- (2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex, the chemical formula is C ₃₀ H ₄₀ N ₅ O ₈ STm, the chemical structure formula is:

,

Its preparation method is shown in Figure 3, and the steps are as follows:

1) Synthesis of 2,6-lutidine nitrogen oxide, that is, compound 2 in the route

Mix 2,6-lutidine, glacial acetic acid, and 30wt% hydrogen peroxide in a volume ratio of 15:80:25, keep stirring at 80 ^o C and reflux for 4-12 hours until the conversion of raw materials is complete, and depressurize the resulting mixture Concentrate to one-third and pour into ice-water mixture. The dosage ratio of 2,6-lutidine to ice-water mixture in ice-water mixture is 15.0mL: 100g, and then use potassium carbonate solid to adjust the system to pH> 8, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 2,6-lutidine nitrogen oxide as a light yellow oil with a yield of 95.6%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.13 (2H, d, J = 7.6Hz), 7.05 (1H, t, J = 7.6Hz), 2.52 (6H, s); ¹³ C-NMR ( 100 MHz, CDCl ₃ ) δ ppm: 148.96, 124.46, 123.92, 18.23.

2) Synthesis of 2,6-dimethyl-4-nitropyridine nitrogen oxide, that is, compound 3 in the route

Cool the above-mentioned 2,6-lutidine nitrogen oxide ice bath to below 10 ^o C, slowly add 98wt% concentrated sulfuric acid, 95wt% fuming nitric acid, 2,6-bis The dosage ratio of picoline nitrogen oxide, concentrated sulfuric acid and fuming nitric acid is 15.2g: 22mL: 22mL. After adding, remove the ice bath, heat to 110 ^o C, stir and reflux for 3-6h until the reaction is complete; put the reaction solution in Dilute on ice cubes, the ratio of 2,6-lutidine nitrogen oxide to ice cubes is 15.2g: 200g, adjust the system to pH 8-10 with solid sodium carbonate, and a large number of yellow solids are precipitated; add water to the mixture In the mixture, the ratio of 2,6-lutidine nitrogen oxide to the mixed solution was 15.2g:500mL, heated to 40 ^o C, stirred to dissolve the formed inorganic salt, filtered out the yellow precipitate, dried to constant weight with an infrared lamp, and obtained Yellow solid 2,6-dimethyl-4-nitropyridine nitrogen oxide, yield 65.2%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 8.04 (2H, s), 2.59 (6H, s); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 150.32, 117.92, 18.56.

3) Synthesis of 2-methyl-4-nitro-6-hydroxymethylpyridine, that is, compound 4 in the route

Mix the above-mentioned 2,6-dimethyl-4-nitropyridine nitrogen oxide with dichloromethane, lower the ice bath to below 10 ^o C, slowly add trifluoroacetic anhydride, 2,6-dimethyl - The ratio of 4-nitropyridine nitrogen oxide, dichloromethane and trifluoroacetic anhydride is 9.2g: 100mL: 20mL, then remove the ice bath, heat and stir under reflux for 9-18h until the reaction is complete, spin dry and pour it out Put into ice water, the mass ratio of 2,6-dimethyl-4-nitropyridine nitrogen oxide to ice water is 9.2:80, adjust the system to pH 8 with solid potassium carbonate, stir at room temperature for 5 hours until the hydrolysis is complete, Potassium hydroxide was added to adjust the pH to 12, followed by extraction with dichloromethane, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, and spinning to obtain a light yellow solid 2-methyl-4-nitro-6- Hydroxymethylpyridine, yield 80.3%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.87 (1H, s), 7.81 (1H, s), 4.90 (2H, s), 3.43 (1H, s), 2.74 (3H, s).

4) Synthesis of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine, that is, compound 5 in the route

Mix the above 2-methyl-4-nitro-6-hydroxymethylpyridine with sodium benzenesulfinate and acetonitrile, 2-methyl-4-nitro-6-hydroxymethylpyridine and sodium benzenesulfinate , The dosage ratio of acetonitrile is 3.0g: 7.0g: 60mL, heated to 85 ^o C under the protection of argon, stirred and refluxed for 2-4d until the conversion of raw materials is complete, after filtering, wash the filter residue with acetonitrile, combine the filtrate and washing liquid, add 5.0g of silica gel , spin-dried and column separation, a white solid 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine was obtained with a yield of 82.0%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.98 (2H, d), 7.69 (1H, s), 7.65 (1H, t), 7.59 (2H, t), 7.55 (1H, s), 4.80 (2H, s), 2.65 (3H, s).

5) Synthesis of 2-methyl-4-phenylsulfonyl-6-bromomethylpyridine, that is, compound 6 in the route

Mix the above 2-methyl-4-phenylsulfonyl-6-hydroxymethylpyridine with chloroform, lower the ice bath to below 10 ^o C, slowly add the mixture of phosphorus tribromide and chloroform dropwise under stirring, 2-methyl Base-4-phenylsulfone-6-hydroxymethylpyridine, phosphorus tribromide, and chloroform were used in amounts of 2.3g, 1.4mL, and 35+15mL, respectively, and then the ice bath was removed, and the reaction was carried out at 50 ^o C for 2-5h until The raw material conversion is complete, the reaction solution is diluted into ice water, the mass ratio of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine to ice water is 2.3:80, and the system is adjusted to a pH of 8-9, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, spin-dried, and column separated to obtain a white solid 2-methyl-4-phenylsulfone-6-bromomethylpyridine with a yield of 96.3 %. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.98 (2H, d), 7.73 (1H, s), 7.68 (1H, t), 7.60 (2H, t), 7.54 (1H, s), 4.55 (2H, s), 2.65 (3H, s).

6) Synthesis of 1, 1 ^, -ethylene bis-2-imidazoline, that is, compound 8 in the route

Mix commercially available triethylenetetramine, N,N-dimethylformamide dimethyl acetal, and toluene with a purity of 70%; The volume ratio is 30:60:100, heat to 85 ^o C, stir and reflux for 2-4h, spin off the toluene until a white solid precipitates, cool to room temperature, a large amount of solid precipitates, filter and wash with tetrahydrofuran, dry to constant weight with an infrared lamp , a white solid 1, 1 ^, -ethylenebis-2-imidazoline was obtained with a yield of 73.9%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 6.81 (2H, s), 3.86 (4H, td), 3.27 (8H, t); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 157.32 , 55.06, 48.41, 46.43.

7) Synthesis of an intermediate by ring formation reaction, that is, intermediate 9 in the route

Mix the above 1,1 ^, -ethylenebis-2-imidazoline, 1,2-dibromoethane, potassium carbonate and acetonitrile, 1,1 ^, -ethylenebis-2-imidazoline, 1,2 - The ratio of dibromoethane, potassium carbonate and acetonitrile is 8.0g: 5.8mL: 5.0g: 400mL, heated and stirred to reflux for 3h under the protection of argon, and filtered after the system is cooled to 50 ^o C, and the filtrate is spin-dried to obtain Yellow solid intermediate, yield 77.7%. ¹ H-NMR (400 MHz, D ₂ O) δ ppm: 4.63 (1H, s), 3.71-3.94 (4H, m), 3.30-3.53 (6H, m), 2.51-3.22 (6H, m); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 162.05, 73.02, 54.28, 52.42, 45.63, 44.40.

8) Synthesis of 1,4,7,10-tetraazacyclododecane, that is, compound 10 in the route

Mix the above intermediate with potassium hydroxide and water, the ratio of intermediate, potassium hydroxide and water is 10.0g: 16.8g: 80mL, heat up to 110 ^o C, stir and reflux for 2h until the hydrolysis is complete, spin off the water until there is a white The solid precipitated, cooled to room temperature and crystallized, and after filtration, the filter residue was dried to constant weight with an infrared lamp to obtain a white solid 1,4,7,10-tetraazacyclododecane with a yield of 73.7%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 2.68 (16H, s); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 46.5.

9) Synthesis of 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine, namely compound 13 in the route

Mix the above 1,4,7,10-tetraazacyclododecane and chloroform, and slowly add the 2-methyl-4-phenylsulfone- 6-bromomethylpyridine in chloroform, 1,4,7,10-tetraazacyclododecane, 2-methyl-4-phenylsulfone-6-bromomethylpyridine, chloroform and anhydrous potassium carbonate The dosage ratio is 2.9g: 3.7g: 150mL: 5.0g, react for 36-60h until the conversion of 2-methyl-4-phenylsulfone-6-bromomethylpyridine is complete, then add anhydrous potassium carbonate, continue to react for 5h , filtered, spin-dried, and column separated to obtain light yellow solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene) Pyridine, yield 29.4%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 8.00-7.98 (2H, d), 7.82 (1H, s), 7.65-7.60 (1H, t), 7.58-7.52 (2H, t), 7.43 ( 1H, s), 3.82 (2H, s), 2.86 (4H, brs), 2.69 (8H, s), 2.62 (4H, brs); 2.58 (3H, s); 1.25 (3H, brs).

10) Synthesis of ethyl p-toluenesulfonyl lactylate, that is, compound 12 in the route

Mix L-ethyl lactate, dichloromethane and p-toluenesulfonyl chloride, cool down in an ice bath, stir, slowly add triethylamine dropwise, the amount of L-ethyl lactate, dichloromethane, triethylamine, p-toluenesulfonyl chloride The ratio is 12.5g: 80mL: 18.6mL: 20.2g. After removing the ice bath, continue to react at room temperature for 3h, filter out triethylamine hydrochloride, and add 10% of the aforementioned triethylamine dropwise triethylamine to the filtrate , react at room temperature for 8-10 hours to complete the conversion of raw materials, then add distilled water, the ratio of distilled water to L-ethyl lactate is 150mL: 12.5g, sequentially extract with dichloromethane, dry with anhydrous sodium sulfate, filter, spin dry, After column separation, a colorless liquid ethyl p-toluenesulfonyl lactylate was obtained with a yield of 85.1%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.82 (2H, d), 7.35 (2H, d), 4.39. (1H, q), 4.11 (2H, q), 2.45 (3H, s), 1.51 (3H, d), 1.21 (3H, t); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 169.0, 145.0, 133.3, 129.7, 129.0, 74.1, 61.8, 21.6, 18.4, 13.9.

11) 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of ethyl propionate, i.e. compound 14 in the route

The 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine prepared in step 9), anhydrous potassium carbonate, The above L-toluenesulfonyl lactylate ethyl ester mixed with acetonitrile, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene) The ratio of pyridine, anhydrous potassium carbonate, L-ethyl p-toluenesulfonyl lactylate, and acetonitrile is 1.02g: 1.64g: 3.25g: 40mL, react at 55-60 ^o C for 50-60h under the protection of argon, and filter The filtrate was spin-dried and separated by a column to obtain light yellow semi-solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine -2,3,4-tri(S)-ethyl propionate, yield 85.5%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 8.02 (3H, brs), 7.60 (4H, brs), 4.16 (6H, brs), 3.88-3.37 (5H, m), 2.96 (6H, brs) , 2.64 (13H, brs); 1.27 (13H, brs).

12) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid, i.e. compound 15 in the route

The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Mixture of ethyl propionate, sodium hydroxide, water and ethanol, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene ) The dosage ratio of pyridine-2,3,4-tri(S)-propionate, sodium hydroxide, water and ethanol is 1.50g:0.50g:10mL:10mL, stir at room temperature for 8-10h to complete the hydrolysis, Then use H ⁺ ion exchange resin to adjust the system to pH<4, filter and wash the filter residue with methanol, combine the filtrates and concentrate to dryness to obtain white solid 1,4,7,10-tetraazacyclododecane-1 -(2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid, yield 88.6%. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ppm: 7.97-7.92 (2H, d), 7.89 (1H, s), 7.78 (1H, s), 7.72-7.66 (1H, t), 7.62-7.55 ( 2H, t), 4.21-3.47 (5H, m), 3.46-2.76 (12H, m), 2.75-2.44 (7H, m), 1.47 (3H, s); 1.27 (3H, s); 1.19-1.12 ( 3H, d); ¹³ C-NMR (100 MHz, CDCl ₃ ) δ ppm: 161.1, 155.7, 150.9, 137.4, 135.2, 130.0, 128.1, 120.9, 119.7, 61.5, 60.8, 55.7, 55.3, 48.8, 46.8, 45.8 , 45.1, 43.0, 22.8, 21.4, 10.1, 9.2, 7.8. MS-ESI (-): 632.3 (M-1); 1265.6 (2M-1).

13) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid anion-Tm ³⁺ complex, i.e. compound 16 in the route

The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O mixed with water, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6- Methylene) pyridine-2,3,4-tri(S)-propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O and water in the ratio of 0.1263g: 0.1839g: 8.0mL, heated to 100 ^o C Stir and reflux for 45 hours to make the reaction complete. After cooling to room temperature, add water, adjust the system to pH 8 with a potassium hydroxide solution with a concentration of 1mol/L, stir for 5 minutes, centrifuge to take the supernatant, add chromatographically pure methanol, and microwave Shake to make it fully dissolved, filter the inorganic salt and spin dry to obtain the target product 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6 -Methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex 0.1290g, yield 92.5%. MS-ESI (-): 798.2 (M-1).

Linkage of tags to proteins:

1) Prepare a 0.1mM ubiquitin G47C solution with a pH of 6.4, prepare it with 2-(N-morpholine)ethanesulfonic acid buffer, contain 10% D ₂ O, and collect ¹ H- ¹⁵ N HSQC spectra;

2) Prepare 1.0mM ubiquitin G47C solution, prepare it with 2-(N-morpholine)ethanesulfonic acid buffer, add 100mM tris(2-carboxyethyl)phosphine solution (3 equiv) and mix well, then add 100.0mM The T ₁ solution (8 equiv) was mixed thoroughly, adjusted to pH 8.0, left to stand at room temperature for 24 hours, and unreacted tris (2-carboxyethyl) phosphine and probe T ₁ were removed using a desalting column to obtain a purified protein- Probe Ligation Complex. Dilute the ligation product ubiquitin G47C-Tm-T ₁ to 0.1mM with 2-(N-morpholine)ethanesulfonic acid buffer (containing 10% D ₂ O), adjust the pH to 6.4, and collect ¹ H- ¹⁵ N HSQC spectrum picture.

All of the above NMR experiments were sampled at 25°C with a Bruker-600MHz superconducting NMR spectrometer. The experimental data were all processed by Topspin, Sparky and Corel DRAW software.

Fig. 5 is a ¹ H- ¹⁵ N HSQC diagram of protein ubiquitin G47C before linking with a probe.

Fig. 6 is the ¹ H- ¹⁵ N HSQC chart of ubiquitin G47C-Tm-T ₁ complex.

Comparing Figure 5 and Figure 6, it can be found that: T ₁ can produce a strong paramagnetic relaxation enhancement effect, which broadens the spectral peak line width and weakens the peak intensity of the residues located near it in the spatial structure, and is positively correlated with the distance . The residue with weakened spectral peak intensity is located near G47C, which indicates that this probe can specifically react with sulfhydryl groups, and can be well applied to studies such as paramagnetic NMR and electron magnetic resonance.

Claims (2)

1. A chiral paramagnetic probe with 1,4,7,10-tetraazacyclododecane as a skeleton, characterized in that: the chemical name is 1,4,7,10-tetraazacyclododecane Dioxane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex with chemical formula C ₃₀ H ₄₀ N ₅ O ₈ STm, the chemical structure formula is: . 2. a kind of preparation method taking 1,4,7,10-tetraazacyclododecane as the chiral paramagnetic probe of skeleton as claimed in claim 1, it is characterized in that the steps are as follows: 1) Synthesis of 2,6-lutidine nitrogen oxide Mix 2,6-lutidine, glacial acetic acid, and 30wt% hydrogen peroxide in a volume ratio of 15:80:25, keep stirring at 80 ^o C and reflux for 4-12 hours until the conversion of raw materials is complete, and depressurize the resulting mixture Concentrate to one-third and pour into ice-water mixture. The dosage ratio of 2,6-lutidine to ice-water mixture in ice-water mixture is 15.0mL: 100g, and then use potassium carbonate solid to adjust the system to pH> 8, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 2,6-lutidine nitrogen oxide as a light yellow oil; 2) Synthesis of 2,6-dimethyl-4-nitropyridine nitrogen oxide Cool the above-mentioned 2,6-lutidine nitrogen oxide ice bath to below 10 ^o C, slowly add 98wt% concentrated sulfuric acid, 95wt% fuming nitric acid, 2,6-bis The dosage ratio of picoline nitrogen oxide, concentrated sulfuric acid and fuming nitric acid is 15.2g: 22mL: 22mL. After adding, remove the ice bath, heat to 110 ^o C, stir and reflux for 3-6h until the reaction is complete; put the reaction solution in Dilute on ice cubes, the ratio of 2,6-lutidine nitrogen oxide to ice cubes is 15.2g: 200g, adjust the system to pH 8-10 with solid sodium carbonate, and a large number of yellow solids are precipitated; add water to the mixture In the mixture, the ratio of 2,6-lutidine nitrogen oxide to the mixed solution was 15.2g:500mL, heated to 40 ^o C, stirred to dissolve the formed inorganic salt, filtered out the yellow precipitate, dried to constant weight with an infrared lamp, and obtained Yellow solid 2,6-dimethyl-4-nitropyridine nitrogen oxide; 3) Synthesis of 2-methyl-4-nitro-6-hydroxymethylpyridine Mix the above-mentioned 2,6-dimethyl-4-nitropyridine nitrogen oxide with dichloromethane, lower the ice bath to below 10 ^o C, slowly add trifluoroacetic anhydride, 2,6-dimethyl - The ratio of 4-nitropyridine nitrogen oxide, dichloromethane and trifluoroacetic anhydride is 9.2g: 100mL: 20mL, then remove the ice bath, heat and stir under reflux for 9-18h until the reaction is complete, spin dry and pour it out Put into ice water, the mass ratio of 2,6-dimethyl-4-nitropyridine nitrogen oxide to ice water is 9.2:80, adjust the system to pH 8 with solid potassium carbonate, stir at room temperature for 5 hours until the hydrolysis is complete, Potassium hydroxide was added to adjust the pH to 12, followed by extraction with dichloromethane, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, and spinning to obtain a light yellow solid 2-methyl-4-nitro-6- Hydroxymethylpyridine; 4) Synthesis of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine Mix the above 2-methyl-4-nitro-6-hydroxymethylpyridine with sodium benzenesulfinate and acetonitrile, 2-methyl-4-nitro-6-hydroxymethylpyridine and sodium benzenesulfinate , The dosage ratio of acetonitrile is 3.0g: 7.0g: 60mL, heated to 85 ^o C under the protection of argon, stirred and refluxed for 2-4d until the conversion of raw materials is complete, after filtering, wash the filter residue with acetonitrile, combine the filtrate and washing liquid, add 5.0g of silica gel , after spinning to dryness and column separation, a white solid 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine was obtained; 5) Synthesis of 2-methyl-4-phenylsulfonyl-6-bromomethylpyridine Mix the above 2-methyl-4-phenylsulfonyl-6-hydroxymethylpyridine with chloroform, lower the ice bath to below 10 ^o C, slowly add the mixture of phosphorus tribromide and chloroform dropwise under stirring, 2-methyl Base-4-phenylsulfone-6-hydroxymethylpyridine, phosphorus tribromide, and chloroform were used in amounts of 2.3g, 1.4mL, and 35+15mL, respectively, and then the ice bath was removed, and the reaction was carried out at 50 ^o C for 2-5h until The raw material conversion is complete, the reaction solution is diluted into ice water, the mass ratio of 2-methyl-4-phenylsulfone-6-hydroxymethylpyridine to ice water is 2.3:80, and the system is adjusted to a pH of 8-9, sequentially extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, spin-dried, and column separated to obtain 2-methyl-4-phenylsulfone-6-bromomethylpyridine as a white solid; 6) Synthesis of 1, 1 ^, -ethylenebis-2-imidazoline Mix commercially available triethylenetetramine, N,N-dimethylformamide dimethyl acetal, and toluene with a purity of 70%; The volume ratio is 30:60:100, heat to 85 ^o C, stir and reflux for 2-4h, spin off the toluene until a white solid precipitates, cool to room temperature, a large amount of solid precipitates, filter and wash with tetrahydrofuran, dry to constant weight with an infrared lamp , to obtain white solid 1, 1 ^, -ethylenebis-2-imidazoline; 7) Synthesis of intermediates by ring formation reaction Mix the above 1,1 ^, -ethylenebis-2-imidazoline, 1,2-dibromoethane, potassium carbonate and acetonitrile, 1,1 ^, -ethylenebis-2-imidazoline, 1,2 - The ratio of dibromoethane, potassium carbonate and acetonitrile is 8.0g: 5.8mL: 5.0g: 400mL, heated and stirred to reflux for 3h under the protection of argon, and filtered after the system is cooled to 50 ^o C, and the filtrate is spin-dried to obtain Yellow solid intermediate; 8) Synthesis of 1,4,7,10-tetraazacyclododecane Mix the above intermediate with potassium hydroxide and water, the ratio of intermediate, potassium hydroxide and water is 10.0g: 16.8g: 80mL, heat up to 110 ^o C, stir and reflux for 2h until the hydrolysis is complete, spin off the water until there is a white The solid was precipitated, cooled to room temperature and crystallized, and after filtration, the filter residue was dried to constant weight with an infrared lamp to obtain a white solid 1,4,7,10-tetraazacyclododecane; 9) Synthesis of 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine Mix the above 1,4,7,10-tetraazacyclododecane and chloroform, and slowly add the 2-methyl-4-phenylsulfone- 6-bromomethylpyridine in chloroform, 1,4,7,10-tetraazacyclododecane, 2-methyl-4-phenylsulfone-6-bromomethylpyridine, chloroform and anhydrous potassium carbonate The dosage ratio is 2.9g: 3.7g: 150mL: 5.0g, react for 36-60h until the conversion of 2-methyl-4-phenylsulfone-6-bromomethylpyridine is complete, then add anhydrous potassium carbonate, continue to react for 5h , filtered, spin-dried, and column separated to obtain light yellow solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene) pyridine; 10) Synthesis of ethyl p-toluenesulfonyl lactate Mix L-ethyl lactate, dichloromethane and p-toluenesulfonyl chloride, cool down in an ice bath, stir, slowly add triethylamine dropwise, the amount of L-ethyl lactate, dichloromethane, triethylamine, p-toluenesulfonyl chloride The ratio is 12.5g: 80mL: 18.6mL: 20.2g. After removing the ice bath, continue the reaction at room temperature for 3h, filter out triethylamine hydrochloride, and add the aforementioned triethylamine in the amount of 10% triethylamine dropwise to the filtrate , react at room temperature for 8-10 hours to complete the conversion of raw materials, then add distilled water, the ratio of distilled water to L-ethyl lactate is 150mL: 12.5g, sequentially extract with dichloromethane, dry with anhydrous sodium sulfate, filter, spin dry, After column separation, a colorless liquid L-ethyl p-toluenesulfonyl lactate was obtained; 11) 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Synthesis of ethyl propionate The 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine prepared in step 9), anhydrous potassium carbonate, The above L-toluenesulfonyl lactylate ethyl ester mixed with acetonitrile, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene) The ratio of pyridine, anhydrous potassium carbonate, L-ethyl p-toluenesulfonyl lactylate, and acetonitrile is 1.02g: 1.64g: 3.25g: 40mL, react at 55-60 ^o C for 50-60h under the protection of argon, and filter The filtrate was spin-dried and separated by a column to obtain light yellow semi-solid 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine - ethyl 2,3,4-tri(S)-propionate; 12) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Mixture of ethyl propionate, sodium hydroxide, water and ethanol, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6-methylene ) The dosage ratio of pyridine-2,3,4-tri(S)-propionate, sodium hydroxide, water and ethanol is 1.50g:0.50g:10mL:10mL, stir at room temperature for 8-10h to complete the hydrolysis, Then use H ⁺ ion exchange resin to adjust the system to pH<4, filter and wash the filter residue with methanol, combine the filtrates and concentrate to dryness to obtain white solid 1,4,7,10-tetraazacyclododecane-1 -(2-Methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S)-propionic acid; 13) 1,4,7,10-Tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) -Synthesis of propionic acid anion-Tm ³⁺ complex The above 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfonyl-6-methylene)pyridine-2,3,4-tri(S) - Propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O mixed with water, 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6- Methylene) pyridine-2,3,4-tri(S)-propionic acid, Tm(NO ₃ ) ₃ ^. 6H ₂ O and water in the ratio of 0.1263g: 0.1839g: 8.0mL, heated to 100 ^o C Stir and reflux for 45 hours to make the reaction complete. After cooling to room temperature, add water, adjust the system to pH 8 with a potassium hydroxide solution with a concentration of 1mol/L, stir for 5 minutes, centrifuge to take the supernatant, add chromatographically pure methanol, and microwave Shake to make it fully dissolved, filter the inorganic salt and spin dry to obtain the target product 1,4,7,10-tetraazacyclododecane-1-(2-methyl-4-phenylsulfone-6 -methylene)pyridine-2,3,4-tri(S)-propionic acid anion-Tm ³⁺ complex.