CN111777629B

CN111777629B - Dithiobis-mannobiose-gold nanoprobe, preparation method and application

Info

Publication number: CN111777629B
Application number: CN202010787460.9A
Authority: CN
Inventors: 刘媛媛; 张景意; 李壹; 张舒萌; 徐馨琦; 何淑怡
Original assignee: Southeast university chengxian college
Current assignee: Southeast university chengxian college
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2022-03-29
Anticipated expiration: 2040-08-07
Also published as: CN111777629A

Abstract

The invention belongs to the field of glycochemistry and biology, and particularly relates to a dithiobis-mannobiose-gold nanoprobe, a preparation method and application thereof in virus receptor DC-SIGN/R recognition. A double-sulfur double-mannose-gold nano probe is characterized in that: modifying the surface of the gold nanoparticles with a dithiobis-mannobiose ligand by a ligand exchange method. According to the invention, based on click chemistry and ligand exchange, a mannobiose ligand is modified on the surface of a gold nanoparticle, a novel dithiobis-mannobiose-gold nanoprobe is constructed, and based on a fluorescence quenching strategy, the sugar binding mechanisms of tetramer lectins DC-SIGN and DC-SIGNR are researched, so that a foundation is laid for establishing a rapid and sensitive method and researching the binding affinity and thermodynamics between protein-sugar multivalent interactions.

Description

Dithiobis-mannobiose-gold nanoprobe, preparation method and application

Technical Field

The invention belongs to the field of glycochemistry and biology, and particularly relates to a dithiobis-mannobiose-gold nanoprobe, a preparation method and application thereof in virus receptor DC-SIGN/R recognition.

Technical Field

C-type lectins are a class containing calcium ions (Ca)²⁺) The carbohydrate recognition domain-dependent (CRDs) superfamily of proteins, which bind to the surface sugars of various viral glycoproteins, play a key role in many important biological processes, such as innate immunity and serum glycoprotein clearance (Biochemistry,2004,43, 3783-. In 2001, Feinberg topic group (Science,2001,294,2163-2166) reported in Science that two important type II membrane proteins consisting of C-type lectin receptors, DC-SIGN (dendritic Cell surface-specific intercellular adhesion factor-3-binding non-integrin factor) and DC-SIGNR (endothelial Cell receptor closely related to DC-SIGN), were bound to oligosaccharide in the CRD crystal structure (Cell,2000,100,587)-597). The two have high affinity to sugar-containing molecules, especially mannose, such as mannose existing on the surface of HIV and Ebola virus glycoprotein, and viral infection can be promoted by combining hydroxyl on a mannose pyran ring with CRD of protein.

DC-SIGN and DC-SIGNR are closely related to the occurrence of body infectious diseases as important recognition receptors of pathogenic microorganisms such as viruses and bacteria. Despite the extensive research performed by scientists over the past 20 years, the detailed protein structure remains unknown except for structural models based on solution X-ray scattering data (without X-ray crystallography validation).

The gold nanoparticles not only have size-dependent and adjustable fluorescence spectrum, high quantum efficiency and large Stokes shift, but also have the advantages of small particle size, no toxicity, good biocompatibility and the like which are not possessed by fluorescent dyes, quantum dots and the like, so that the gold nanoparticles become an ideal fluorescent probe.

The invention aims to prepare a dithiobis-mannobiose-gold nanoprobe, discusses the interaction with tetramer agglutinin DC-SIGN and DC-SIGNR, and lays a foundation for developing specific multivalent inhibition blocking DC-SIGN/R mediated virus infection.

Disclosure of Invention

The invention aims to provide a dithiobis-mannobiose-gold nanoprobe, a preparation method and application thereof in virus receptor DC-SIGN/R recognition.

A double-sulfur double-mannose-gold nano probe is characterized in that: modifying the surface of the gold nanoparticles with a dithiobis-mannobiose ligand by adopting a ligand exchange method, wherein the structure is shown as a formula I:

x is 1 to 1500.

The object of the invention can be achieved by the following measures:

the bis-mannose bis-gold nanoprobe (i.e., the substance of formula I) was prepared as follows: dissolving 1 mol of gold nanoparticles, 1-2000 mol of bis-sulfur bis-mannobiose ligand II and 1-2500 mol of tris (2-carboxyethyl) phosphine (TCEP) in ultrapure water, stirring at room temperature in a dark place for 12-24 h, concentrating, washing with water, and preparing a bis-sulfur bis-mannobiose-gold nanoprobe I, wherein the process comprises the following steps:

the disulfide bis-mannobiose ligand shown in the general formula II is prepared from mannobiose ligand III, disulfide ligand IV, tert-butyl trichloroacetimidate (TBTA), copper sulfate pentahydrate and sodium ascorbate through click chemistry. Wherein the molar ratio of the disulfide ligand IV to the mannobiose ligand III to the tert-butyl trichloroacetimidate (TBTA) to the copper sulfate pentahydrate to the sodium ascorbate is 1: 2-3: 0.1-0.3: 0.05-0.2: 0.1-1, and stirring the mixture at room temperature in a dark place by using a solvent methanol or ethanol, wherein the product is separated and purified by column chromatography. The specific reaction formula is as follows:

according to the synthesis references of mannobiose ligands shown in the general formula III J.Am.chem.Soc.2017,139,11833-11844, mannose and peracetyl mannose are used as raw materials and react with acetic anhydride, phosphorus tribromide, hydrazine acetate, trichloroacetonitrile and DBU respectively to prepare III-4 and III-5. Then reacting with boron trifluoride ethyl ether solution at low temperature to prepare III-3. Reference is made to the synthesis of III-5 for preparation of III-2. III-2 is sequentially reacted with H (OCH)₂CH₂)₃Cl and NaN₃And reacting to prepare III-1. Finally, III-1 is hydrolyzed under alkaline condition to prepare the mannobiose III, and the specific reaction formula is as follows:

the disulfide ligand IV is prepared by taking aspartic acid as a raw material, sequentially reacting with di-tert-butyl dicarbonate and alkynylamine IV-1 to prepare Boc-protected alkynylamine IV-3, then deprotecting, and condensing with lipoic acid through DCC, and the specific reaction formula is as follows:

the application of the bis-sulfur bis-mannobiose-gold nanoprobe in preparing a virus receptor DC-SIGN/R detection reagent.

Advantageous effects

According to the invention, based on click chemistry and ligand exchange, a mannobiose ligand is modified on the surface of a gold nanoparticle, a novel dithiobis-mannobiose-gold nanoprobe is constructed, and based on a fluorescence quenching strategy, the sugar binding mechanisms of tetramer lectins DC-SIGN and DC-SIGNR are researched, so that a foundation is laid for establishing a rapid and sensitive method and researching the binding affinity and thermodynamics between protein-sugar multivalent interactions.

Drawings

FIG. 1 shows a high resolution mass spectrum of a bis-sulfur bis-mannobiose ligand II (n-2).

FIG. 2 shows the UV-VIS absorption spectrum of the bis-mannose-gold nanoprobe I.

FIG. 3 is a dynamic light scattering characterization of the bis-mannose-gold nanoprobe I.

FIG. 4 is a graph showing the binding of the bis-mannose-gold nanoprobe I to DC-SIGN.

FIG. 5 is a graph showing the binding of the bis-mannobiose gold nanoprobe I to DC-SIGNR.

Detailed Description

The following specific examples are provided to further illustrate the invention.

The starting gold nanoparticles (5 nm in diameter, dispersed in PBS solution) were purchased from Sigma-Aldrich.

Synthetic examples

Example 1

Synthesis of Compound III-4

Dissolving mannose in acetic anhydride, adding a few drops of perchloric acid, stirring at room temperature for 1 hour, cooling to 10 ℃, dropwise adding phosphorus tribromide, and controlling the temperature to be 20-25 ℃. After the completion of the dropping, the mixture was stirred at room temperature for 1.5 hours. Cooling to 5 deg.C, adding sodium acetate solution, controlling temperature at 20-25 deg.C, and stirring for 20 min. Chloroform extraction, saturated sodium bicarbonate solution and water washing, drying with anhydrous sodium sulfate, filtration and concentration. Recrystallization from cold ether afforded compound III-4 as a white solid at m.p.132-133 ℃. High resolution mass spectrometry data: [ M + NH ]₄]⁺366.1395 (theoretical), 366.1400 (measured).

Example 2

Synthesis of Compound III-5

Under the protection of nitrogen, dissolving the total acetyl alpha-D-mannose in DMF, heating to 50 ℃, adding hydrazine acetate, stirring for 20 minutes, cooling, adding ethyl acetate, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering and concentrating. Dichloromethane was added, cooled to-10 ℃, trichloroacetonitrile and Diazabicyclo (DBU) were added dropwise, and the mixture was allowed to warm to room temperature and stirred for 2 hours. Concentrating, separating by column chromatography to obtain compound III-5 as colorless liquid. High resolution mass spectrometry data: [2M + Na ]]⁺1005.0198 (theoretical), 1005.0175 (measured).

Example 3

Synthesis of Compound III-3

The compounds III-4 and III-5 were co-distilled with toluene, followed by addition of anhydrous dichloromethane and 4A type molecular sieve, and stirring at room temperature for 2 hours. Cooling to-50 deg.C, adding boron trifluoride ether solution, slowly heating to room temperature, and reacting for 18 hr. Adding sodium bicarbonate, filtering, concentrating, separating by column chromatography, and making into desired dosage formCompound III-3, white solid, m.p.70-71 ℃. High resolution mass spectrometry data: [ M + Na ]]⁺701.1900 (theoretical), 701.1904 (measured).

Example 4

Synthesis of Compound III-2

Dissolving compound III-3 in DMF, heating to 50 deg.C, adding hydrazine acetate, stirring for 20 min, cooling, adding ethyl acetate, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, and concentrating. Adding dichloromethane, cooling to-10 ℃, dropwise adding trichloroacetonitrile and DBU, slowly raising the temperature to room temperature after dropwise adding, and stirring for 2 hours. Concentrating, separating by column chromatography to obtain compound III-2, yellow liquid. High resolution mass spectrometry data: [ M + Na ]]⁺802.0906 (theoretical), 802.0906 (measured).

Example 5

Synthesis of Compound III-1

The compound III-2 and toluene are steamed together, and then anhydrous dichloromethane is added,

Stirring the molecular sieve and 2-chloroethoxy-2-ethoxy diethanol at room temperature for 1.5 hours, cooling to-50 ℃, adding boron trifluoride diethyl etherate, and heating to 2 ℃ within 2 hours. Washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Adding anhydrous DMF and NaN₃And TBAI, stirred at 87 ℃ for 17 hours. Concentrating, separating by column chromatography to obtain compound III-1 as colorless liquid. High resolution mass spectrometry data: [ M + Na ]]⁺816.2586 (theoretical), 816.2589 (measured).

Example 6

Synthesis of mannobiose ligand III

Hydrolyzing the compound III-1 under alkaline condition to prepare the mannobiose ligand III as colorless liquid. Nuclear magnetic data (¹H NMR,400MHz, internal standard TMS, solvent CDCl₃) The following were used: delta ppm 5.03(d,1H, J)_1a,2a1.7Hz,H-1a),4.94(d,1H,J_1b,2b 1.7Hz,H-1b),3.98(dd,1H,J_2b,3b 3.4Hz,J_1b,2b 1.7Hz,H-2b),3.90(dd,1H,J_2a,3a3.4Hz,J_1a,2a 1.7Hz,H-2a),3.82-3.76(m,5H,H-3a,H-3b,H-6,H-6’,CH₂O),3.70-3.53(m,15H,C-4a,C-4b,C-5a,C-5b,C-6,C-6,CH₂O),3.42(t,2H,J 4.87Hz,CH₂-N); high resolution mass spectrometry data: [ M + Na ]]⁺522.2352 (theoretical), 522.1905 (measured).

Example 7

Synthesis of alkynylamine IV-1(n ═ 2)

Mixing diglycolamine 10mL, tetrahydrofuran 100mL and H₂O100mL, 24.0g of di-tert-butyl dicarbonate and 31.8g of sodium carbonate were added thereto, and after stirring at room temperature for 24 hours, the pH was adjusted to about 5 with 2mol/L hydrochloric acid, followed by extraction with ethyl acetate, drying over anhydrous sodium sulfate, filtration, rotary evaporation, and dissolution in 30mL of tetrahydrofuran. And (3) adding 3.93g NaH into the mixed solution in an ice bath, stirring the mixed solution for 10 minutes, adding 4.48mL of bromopropyne, stirring the mixed solution for 4 hours at room temperature, adjusting the pH to be about 6 to 7 by using 2M hydrochloric acid, extracting an aqueous layer by using ethyl acetate, drying the aqueous layer by using anhydrous sodium sulfate, performing rotary evaporation, and performing column chromatography separation to prepare the alkynylamine IV-1 as a light yellow liquid. Mass spectrometry data: [ M + H ]]⁺144.10 (theoretical), 144.12 (measured).

Example 8

Synthesis of Boc protected aspartic acid IV-2

2g of aspartic acid, 10mL of dioxane and 30mLH₂And mixing, adding 30mL of sodium hydroxide (1M) while stirring until the solution is clear, cooling in an ice bath, dropwise adding 10mL of dioxane solution of di-tert-butyl dicarbonate (3.6g), and stirring at room temperature overnight after dropwise adding. Rotary evaporation, addition of 20mL ethyl acetate, acidification with HCl (3M) to pH 2, drying over anhydrous sodium sulfate, filtration, rotary evaporation, column chromatography separation to afford Boc protected aspartic acid IV-2. Mass spectrometry data: [ M + H ]]⁺234.10 (theoretical), 234.11 (measured).

Example 9

Synthesis of Boc-protected alkynylamide IV-3(n ═ 2)

4.16g of compound IV-2, 11.03g of DCC and 30mL of dichloromethane are mixed, stirred and reacted for 1 hour at 0 ℃, 5.11g of alkynylamine IV-1 and a catalytic amount of DMAP are added, the temperature is slowly raised to room temperature, the mixture is stirred overnight, filtered, rotary-evaporated and separated by column chromatography, and Boc protected alkynylamide IV-3 is prepared as a yellow viscous liquid. Mass spectrometry data: [ M + H ]]⁺484.27 (theoretical), 484.26 (measured).

Example 10

Synthesis of alkynylamide IV-4(n ═ 2)

To a solution of compound IV-3(0.5g) in dichloromethane (10mL) at 0 ℃ was added 0.5mL of trifluoroacetic acid, stirred at 0 ℃ for 2.5 hours, rotary evaporated, and dissolved in 10mL of methanol. Cooling to 5 ℃, adding 0.03g of potassium carbonate, stirring for 30 minutes, extracting with ethyl acetate, drying with anhydrous sodium sulfate, performing rotary evaporation, and performing column chromatography separation to prepare the alkynylamide IV-4 as a light yellow viscous liquid. Mass spectrometry data: [ M + H ]]⁺384.21 (theoretical), 384.24 (measured).

Example 11

Synthesis of Dithiobis-mannobiose ligand II (n ═ 2)

Mixing disulfide ligand IV (0.38g, 1mmol) and mannobiose ligand III (1.0g, 2mmol) in methanol, adding TBTA (0.042g, 0.19mmol) and CuSO₄·5H₂O (0.028g, 0.11mmol) and sodium ascorbate (0.08g, 0.41mmol) are stirred at room temperature in the dark, and subjected to rotary evaporation and column chromatography separation to prepare the dithiobis-mannobiose ligand II. The results of high resolution mass spectrometry (FIG. 1) show: [ M + Na + K ]]²⁺815.7968 (theoretical), 815.8078 (measured).

Example 12

Synthesis of bis-mannobiose gold nanoprobe I (n ═ 2)

2mL of gold nanoparticles (91mM) in PBS was concentrated to 200. mu.L, and bis-sulfur-bis-mannobiose ligand II (0.3g, 0.36mol) and TCEP (0.124g, 0.432mol) were added thereto, followed by stirring overnight at room temperature in the dark. Concentrating, washing with water, preparing dithiobis-mannobiose-gold nanoprobe I, and characterizing by ultraviolet visible absorption spectrum (figure 2) and dynamic light scattering (figure 3), wherein lambda is_maxAt 530nm, the average particle size was 14.62 nm.

Example 13

Fluorescence quenching experiment

According to the experimental scheme shown in Table 1, the fluorescence spectrum of the bis-mannose-gold nanoprobe I + DC-SIGN/R and the fluorescence spectrum of the DC-SIGN/R (the excitation wavelength is 597nm) at different concentrations are respectively measured, and the fluorescence quenching efficiency QE% is calculated according to the formula (X-Y)/X (X, Y is the spectral peak area of the DC-SIGN/R, the sugar nanoprobe + DC-SIGN/R respectively). Using a Hill equation to fit and Origin software to draw a binding curve to obtain the binding affinity K_d. The results show that: the bonding force of the bis-mannose-gold nanoprobe I and the DC-SIGN is strong, the fluorescent quenching phenomenon is obvious after the two are mixed, and K_d5.74 (fig. 4); and binding to DC-SIGNRSlightly weak force, K_d55.30 (fig. 5). The buffer solutions used in the experiments were: NaCl (100mM), HEPES (20mM), CaCl₂(10mM), containing 1mg/mL BSA solution.

TABLE 1 fluorescence quenching Experimental protocol

Claims

1. A double-sulfur double-mannose-gold nano probe is characterized in that: modifying the surface of the gold nanoparticles with a dithiobis-mannobiose ligand by adopting a ligand exchange method, wherein the structure is shown as a formula I:

x is 1 to 1500, and n is 1 to 10.

2. The method for preparing the dithiobis-mannobiose-gold nanoprobe of claim 1, which is characterized by comprising the following steps:

dissolving 1 mole of gold nanoparticles, 1-2000 moles of bis-sulfur bis-mannobiose ligand II and 1-2500 moles of tris (2-carboxyethyl) phosphine in ultrapure water, stirring at room temperature in a dark place for 12-24 hours, concentrating, washing with water, and preparing a bis-sulfur bis-mannobiose-gold nanoprobe I, wherein the reaction formula is as follows:

wherein n is 1-10.

3. The method for preparing the bis-sulfur bis-mannobiose-gold nanoprobe according to claim 2, wherein the bis-sulfur bis-mannobiose ligand II is prepared by click chemistry from a mannobiose ligand III, a bis-sulfur ligand IV, tert-butyl trichloroacetimidate, copper sulfate pentahydrate and sodium ascorbate, and has the reaction formula:

wherein: n is 1-10;

the molar ratio of the disulfide ligand IV to the mannobiose ligand III to the tert-butyl trichloroacetimidate to the copper sulfate pentahydrate to the sodium ascorbate is 1: 2-3: 0.1-0.3: 0.05-0.2: 0.1-1, using methanol or ethanol as a solvent, stirring at room temperature in a dark place, and separating and purifying a product by column chromatography.

4. The use of the bis-sulfur bis-mannobiose-gold nanoprobe of claim 1 in the preparation of a virus receptor DC-SIGN/R detection reagent.