Glucose-responsive molecular connector, and preparation method therefor and use thereof

Abstract

Provided in the present invention is a glucose-responsive molecular connector, and a preparation method therefor and the use thereof. A brand-new glucose-responsive molecular connector is formed by the esterification of a phenylboronic acid analogue and a 1,3-diol structure. The compound can be directly biolinked to insulin by amidation and has a very good glucose-responsive function, wherein glucose at a high concentration competes with the 1,3-diol structure for binding to phenylboronic acid, so that the solubility of insulin is increased and the release of insulin is promoted, and when the glucose is at a low concentration, only a small amount of insulin is in a free state, thereby reducing the occurrence of hypoglycemia. The compound achieves the glucose response of insulin by means of a brand-new mechanism, causing the glucose response effect more sensitive. Moreover, the compound has the advantages of simple structure, being easy to prepare and low cost, and has great clinical application value.

Description

A sugar-responsive molecular connector and its preparation method and application

[0001] This application claims priority to the prior Chinese application, application number: 202310793910.9, filing date June 30, 2023; the specification, claims, abstract and drawings of that application are all incorporated herein by reference as part of this application. Technical Field

[0002] The present invention belongs to the field of drug delivery, and specifically relates to an oral drug delivery carrier, and more particularly to a sugar-responsive molecular connector and a preparation method and application thereof. Background Art

[0003] Today, diabetes and its complications threaten the health of 422 million people worldwide, a number that continues to grow, projected to reach 592 million by 2035. Furthermore, the global annual cost of treating diabetes and its complications exceeds $500 billion. The sheer number of patients and the high cost of treatment indicate that diabetes has become a major global public health and safety issue.

[0004] Insulin replacement therapy is an integral part of the treatment of type 1 diabetes and is also essential for the treatment of advanced type 2 diabetes. In healthy individuals, endogenous insulin secretion from pancreatic β cells oscillates in sync with fluctuations in blood glucose levels, minimizing hyperglycemia and hypoglycemia. Although exogenous insulin replacement strategies are designed to mimic endogenous insulin secretion, the daily dose of insulin injection or infusion must be carefully adjusted based on the individual's physiology and lifestyle, including possible daily changes in stress, physical activity, and dietary intake. Because exogenous insulin is not regulated by endogenous feedback mechanisms, even with a carefully designed dosing regimen and even when patients strictly adhere to their doctor's prescribed diet and lifestyle, diabetic patients will inevitably experience symptoms of hyperglycemia or hypoglycemia.

[0005] Therefore, mimicking β-cell function by releasing insulin in a glucose-dependent manner is attractive for safe and effective insulin delivery. Self-regulating glucose-responsive insulin delivery systems have been extensively developed. Most of these systems rely on glucose in combination with three substances: glucose oxidase, glucose-binding protein, and phenylboronic acid.

[0006] Among them, phenylboronic acid shows good modification potential in the construction of rapid response and long-effect insulin delivery systems. Its ability to bind to sugars and potential charge conversion make phenylboronic acid an ideal material for insulin delivery.

[0007] However, in the prior art, phenylboronic acid or phenylboronic acid derivatives are usually compounded with polymers to prepare insulin delivery carriers. For example, patent CN115671050A uses polyacryl carbonate-polycaprolactone as raw materials, phenylboronic acid derivatives and dihydroxy to form phenylboronic acid ester complexes, and liver-targeted glucose-responsive nanoparticles are prepared by W / O / W emulsification and UV crosslinking technology. Other researchers have also prepared PBA group-modified poly N-(2-aminoethyl) acrylamide based on the above strategy, and used this material to load insulin. The researchers have verified the good sugar response performance and blood sugar control effect of this system in animals (mice and pigs). However, the above studies all prepared composite materials, which are cumbersome to prepare, inconvenient to use, and have poor actual results.

[0008] Therefore, it is necessary to find a more suitable oral insulin delivery carrier that has both good sugar response performance and blood sugar lowering effect, and can reduce the difficulty of preparation and use, improve the actual effect, and achieve long-term oral insulin delivery technology for blood sugar control.

[0009] Summary of the Invention

[0010] To address the above-mentioned problems, the present invention provides a sugar-responsive molecular connector, its preparation method, and application. A new sugar-responsive molecular connector compound is formed by esterifying a phenylboronic acid analog with a 1,3-diol structure. This compound can directly biolink to insulin through amidation and has excellent glucose-responsiveness. High concentrations of glucose can compete with the 1,3-diol structure to bind to phenylboronic acid, increasing insulin solubility and promoting insulin release. At low concentrations, only a very small amount of insulin is free, reducing the occurrence of hypoglycemia. This compound utilizes a new mechanism to achieve insulin sugar response, making the sugar response effect more sensitive, extending the treatment time, and more effectively controlling blood sugar. Furthermore, its simple structure, ease of preparation, and low cost make it highly valuable for clinical application.

[0011] In one aspect, the present invention provides a sugar-responsive molecular linker compound, which is formed by esterification of phenylboronic acid or a phenylboronic acid analog with an alcohol compound molecule.

[0012] The structural formula of the phenylboronic acid is as follows:

[0013] The sugar-responsive molecular linker compound provided by the present invention can also be understood as a structural unit with sugar-responsiveness. This structural unit is formed by esterification of phenylboronic acid or a phenylboronic acid analog with an alcohol compound molecule, can directly link with insulin and has sugar-responsiveness (Figure 1), and has the following general structural formula:

[0014] Among them, R1, R2, R3, and R4 are H, C1-C20 Alkyl, C1-C 20 Alkenyl, C1-C 20 Alkynyl, carboxyl, amino, substituted C1-C 20 Alkyl, substituted C1-C 20 Alkenyl, substituted C1-C 20 Alkynyl or substituted amino; the substituted group is selected from C1-C 20 Alkyloxy, C1-C 20 Alkenyloxy, C1-C 20 Alkynyloxy, hydroxy, amino or carboxyl, R5, R6, R7, R8, R9 are H, alkyl, alkenyl, alkynyl, carboxyl, amino, substituted alkyl, substituted alkenyl, substituted alkynyl or substituted amino; the substituted group is selected from alkyloxy, alkenyloxy, alkynyloxy, hydroxy, amino or carboxyl.

[0015] Furthermore, the phenylboronic acid analogues include phenylboronic acid modified with different groups; the modified groups include one or more of alkyl, halogen, nitro and amide; the alkyl is a straight chain or branched alkyl, and the carbon chain length of the alkyl is 2-100 carbons.

[0016] Furthermore, the phenylboronic acid or phenylboronic acid analogue is modified with a carboxyl group.

[0017] The present invention uses carboxyl-modified phenylboronic acid or phenylboronic acid analogs to construct a sugar-responsive molecular linker compound. Due to the carboxyl group in the structure, it can undergo an amidation reaction with the amino group on insulin, thereby achieving the link between insulin and the compound.

[0018] Furthermore, the carboxyl-modified phenylboronic acid or phenylboronic acid analogue includes one or more of the following:

[0019] Furthermore, the alcohol compound includes methanol, diol, triol, tetraol, polyhydroxy alcohol or aldehyde or acid and substituents of the above alcohols; the substituent is one or more of alkyl, halogen, nitro, amino, ether, alkenyl, thiol, aromatic and amide.

[0020] Furthermore, the alcohol compound is a compound with a diol structure, which can be a polyhydroxy alkane or olefin or fatty amine or fatty acid or mercaptoalkane or thioether or amide. Preferably, the analogue of the 1,2-diol structure is selected from one or more of the following groups:

[0021] or

[0022] or

[0023] The sugar-responsive molecular linker compound constructed in this invention exhibits excellent sugar-responsiveness. Through esterification of the phenylboronic acid ester and the diol structure, this ester bond can be cleaved by glucose molecules, thereby releasing insulin. Therefore, in the case of hyperglycemia, high concentrations of glucose and the diol structure competitively bind to the phenylboronic acid, cleaving the phenylboronic acid ester bond. This increases insulin solubility and promotes insulin release, resulting in excellent glucose-responsive release properties (Figure 1), allowing the body to quickly restore normal blood sugar levels. Under normal blood sugar conditions, only a very small amount of insulin remains free, reducing the risk of hypoglycemia.

[0024] Furthermore, the alcohol compound is a 1,3-diol compound, which may be a polyhydroxy alkane or olefin or fatty acid or amide, and is preferably selected from one or more of the following:

[0025] Studies have shown that the sugar-responsive molecular linker compound generated by reacting 1,3-diol with carboxyl-modified phenylboronic acid or phenylboronic acid analogs has better sugar-responsiveness, thereby being able to control blood sugar more effectively and for a longer period of time.

[0026] In some embodiments, the phenylboronic acid analog is preferably 4-carboxy-3-fluorophenylboronic acid:

[0027] The 1,3-diol compound is preferably:

[0028] The compound prepared using the above-mentioned phenylboronic acid analogue and the 1,3-diol compound has the following structure:

[0029] On the other hand, the present invention provides a method for preparing the sugar-responsive molecular linker compound as described above, which is prepared by mixing phenylboronic acid or a phenylboronic acid analog with an alcohol compound molecule for reaction, followed by purification.

[0030] Furthermore, the preparation method comprises the following steps: mixing carboxyl-modified phenylboronic acid or phenylboronic acid compounds with a 1,3-diol structure compound solution, heating and refluxing, and purifying to obtain the product.

[0031] In some embodiments, the molar ratio of the carboxyl-modified phenylboronic acid or phenylboronic acid compound to the 1,3-diol structure compound is preferably 1:(1-3).

[0032] In some embodiments, the solvent in the solution of the carboxyl-modified phenylboronic acid or phenylboronic acid compounds can be a conventional organic solvent in the art that can partially or completely dissolve the carboxyl-modified phenylboronic acid or phenylboronic acid compounds, preferably toluene or acetone.

[0033] In some embodiments, the solvent in the solution of the 1,3-diol structure compound can be a conventional organic solvent in the art that can dissolve the 1,3-diol structure compound, preferably toluene or acetone.

[0034] In some embodiments, the concentration of the carboxyl-modified phenylboronic acid or phenylboronic acid compound solution is preferably 1-200 mg / mL, for example 20 mg / mL.

[0035] In some embodiments, the concentration of the 1,3-diol structure compound solution is preferably 1 to 600 mg / mL, for example 20 mg / mL.

[0036] In some embodiments, the carboxyl-modified phenylboronic acid compound may be p-carboxylphenylboronic acid or o-carboxylphenylboronic acid substituted or unsubstituted with a substituent, wherein the substituent may be one or more of a halogen and a nitro group.

[0037] In some embodiments, the carboxyl-modified phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.

[0038] In some embodiments, the mixing method includes directly mixing the solution of the carboxyl-modified phenylboronic acid compound with the solution of the 1,3-diol structure compound.

[0039] In some embodiments, the heating reflux time is 1 to 8 hours, for example, 4 hours.

[0040] In some embodiments, the purification method comprises recrystallization and column chromatography.

[0041] In some embodiments, the recrystallization solvent is toluene or acetone; the column chromatography uses 200-400 mesh silica gel, and the developing solvent is 10-50% ethyl acetate / petroleum ether.

[0042] In another aspect, the present invention provides a sugar-responsive insulin, which is prepared by reacting the sugar-responsive molecular linker compound described above with insulin.

[0043] In another aspect, the present invention provides a method for preparing a sugar-responsive insulin, comprising the following steps:

[0044] (1) preparing sugar-responsive molecular linker compounds;

[0045] (2) Add insulin solution and react to obtain the product.

[0046] Furthermore, the method for preparing the sugar-responsive insulin comprises the following steps:

[0047] (a) first reacting the sugar-responsive molecular linker with N,N,N′,N′-tetramethyl-O-(N-succinimidyl)tetrafluoroborate and N,N-diisopropylethylamine in a solvent;

[0048] (b) then adding the mixture to an insulin solution for reaction, adjusting the pH to 8-10, and performing post-treatment to obtain the product;

[0049] In some embodiments, in step (a), the solvent can be conventional in the art, preferably anhydrous DMF.

[0050] In some embodiments, in step (a), the reaction is preferably carried out at room temperature.

[0051] In some embodiments, in step (a), the molar ratio of the sugar-responsive molecular linker to N,N,N′,N′-tetramethyl-O-(N-succinimidyl)tetrafluoroborate and N,N-diisopropylethylamine is preferably 1:(0.8-1.2):(1.1-1.5).

[0052] The addition of the N,N,N',N'-tetramethyl-O-(N-succinimidyl) tetrafluoroborate and N,N-diisopropylethylamine helps the sugar-responsive molecular linker and insulin to undergo an amidation reaction so as to be linked together.

[0053] In some embodiments, in step (b), the reaction is preferably carried out in an ice bath.

[0054] In some embodiments, in step (b), the concentration of the insulin solution is preferably 0.2-100 mg / mL.

[0055] In some embodiments, in step (b), the solvent of the insulin solution is preferably phosphate buffered saline (PBS) or dimethyl sulfoxide.

[0056] In some embodiments, in step (b), the pH of the insulin solution is preferably 8-10.

[0057] In some embodiments, in step (b), the post-treatment is preferably dialysis.

[0058] The dialysis can be performed in deionized water using a conventional dialysis bag in the art, and the molecular weight cut-off of the dialysis bag is preferably 1k to 3.5k.

[0059] In some embodiments, preferably, the dialysis time is 12 to 24 hours, for example, 12 hours.

[0060] In the present invention, the white flocculent precipitate formed after dialysis is the glucose-responsive insulin. The glucose-responsive insulin is soluble or easily soluble in weakly alkaline water.

[0061] The sugar-responsive insulin prepared by the present invention is an amorphous flocculent precipitate that can form an insulin reservoir upon subcutaneous injection. Sugar response is achieved through competitive, reversible binding of glucose with 1,3-diol and phenylboronic acid under physiological conditions (Figure 1). Under hyperglycemia, high concentrations of glucose and the 1,3-diol structure compete with phenylboronic acid, resulting in the cleavage of the phenylboronic acid ester bond, increasing insulin solubility and promoting insulin release. This gives insulin good glucose-responsive release properties, allowing the body to quickly restore normal blood sugar levels. Under normal blood sugar levels, only a very small amount of insulin is in a free state, reducing the occurrence of hypoglycemia.

[0062] In another aspect, the present invention provides use of the sugar-responsive molecular linker compound described above for preparing a reagent for linking and releasing insulin in a glucose-responsive manner.

[0063] In another aspect, the present invention provides a composition for delivering insulin, comprising the compound as described above and insulin.

[0064] In another aspect, the present invention provides use of the glucose-responsive insulin described above in the preparation of a drug for treating diabetes.

[0065] Furthermore, the diabetes may be type 1 diabetes or late-stage type 2 diabetes.

[0066] In some embodiments, the glucose-responsive insulin can be resuspended in PBS or physiological saline and administered by subcutaneous injection for treatment.

[0067] The present invention has the following beneficial effects:

[0068] 1. For the first time, a new sugar-responsive molecular connector compound was constructed. This compound can directly biolink to insulin through amidation to form a sugar-responsive insulin with excellent glucose responsiveness. It has a simple structure and is easy to prepare at low cost.

[0069] 2. Utilizing the principle that glucose can competitively bind to phenylboronic acid with a 1,3-diol structure, a novel mechanism has been proposed to achieve insulin glucose response. At high concentrations, glucose competes with the 1,3-diol structure, preferentially binding to phenylboronic acid. This leads to the cleavage of the phenylboronic acid ester bond, increasing insulin solubility and promoting insulin release. At low concentrations, only a very small amount of insulin remains free, reducing the risk of hypoglycemia. This mechanism makes glucose response more sensitive, prolongs treatment time, and effectively controls blood sugar.

[0070] 3. The sugar-responsive molecular linker compound is coupled to insulin to produce sugar-responsive insulin. Subcutaneous injection can form an insulin reservoir, achieving stable and rapid glucose-responsive insulin release. In diabetic mice, it can quickly regulate blood sugar, with a smooth and long-lasting effect. Compared with existing diabetes medications, it can significantly prolong the effective time, maintain blood sugar within the normal range, and almost no hypoglycemia occurs. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG1 is a schematic diagram of the formation of glucose-responsive insulin and the mechanism of glucose-responsive insulin release;

[0072] Figure 2 is a hydrogen nuclear magnetic resonance spectrum of 2-fluoro-4-(5-propyl-1,3,2-dioxaborolan-2-yl)benzoic acid synthesized in Example 1 (the deuterated reagent is DMSO-d6);

[0073] FIG3 is a MALDI-TOF mass spectrum of the sugar-responsive insulin FPRDBA-insulin synthesized in Example 2;

[0074] Figure 4 is a hydrogen nuclear magnetic resonance spectrum of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid synthesized in Example 3 (the deuterated reagent is DMSO-d6);

[0075] FIG5 is a MALDI-TOF mass spectrum of the sugar-responsive insulin FPTDBA-insulin synthesized in Example 4;

[0076] FIG6 is a hydrogen nuclear magnetic resonance spectrum of 4-(dihydroxyboryl)-N-dodecyl-2-fluorobenzamide synthesized in Example 5 (the deuterated reagent is DMSO-d6);

[0077] FIG7 is a hydrogen nuclear magnetic resonance spectrum of 2-{4-[(dodecylamino)carbonyl]-3-fluorophenyl}-5-methyl-1,3,2-dioxaborinane-5-carboxylic acid synthesized in Example 5 (the deuterated reagent is CDCl3);

[0078] FIG8 is a MALDI-TOF mass spectrum of the recombinant human insulin conjugate (DFPMDCA-insulin) synthesized in Example 6;

[0079] FIG9 is a proton nuclear magnetic resonance spectrum of C5M synthesized in Example 7;

[0080] FIG10 is a HPLC purification profile of C5M-INS synthesized in Example 8;

[0081] FIG11 is a proton nuclear magnetic resonance spectrum of C5P synthesized in Example 9;

[0082] FIG12 is a HPLC purification profile of C5P-INS synthesized in Example 10;

[0083] Figure 13 is a standard curve of insulin concentration-absorbance in Example 11;

[0084] FIG14 is a glucose-responsive insulin release curve of the glucose-responsive recombinant human insulin (FPTDBA-insulin) and the glucose-responsive recombinant human insulin (DFPMDCA-insulin) in Example 11;

[0085] FIG15 shows the blood glucose levels of type 1 diabetic mice treated with recombinant human insulin and the commercial long-acting insulin preparation glargine and phosphate buffered saline in Example 12;

[0086] FIG16 is a spectrum of pure C5F-INS obtained by HPLC purification in Example 13;

[0087] FIG17 is an ESI mass spectrum of Example 13;

[0088] FIG18 is a circular dichroism spectrum of Example 13;

[0089] FIG19 is a scanning electron microscope image of Example 13;

[0090] FIG20 is a MALDI-TOF mass spectrum of Example 13;

[0091] FIG21 is a solid-state NMR boron spectrum in Example 13;

[0092] FIG22 is a comparative diagram of the blood glucose control effects of the sugar-responsive insulins prepared using three groups of sugar-responsive molecular linker compounds investigated in Example 14;

[0093] FIG23 is a schematic diagram showing the blood glucose lowering results in diabetic mice using different concentrations of glucose-responsive insulin in Example 15;

[0094] FIG24 shows the blood glucose control results in mice after single and multiple injections of insulin glargine in Example 15;

[0095] FIG25 shows the survival rate of mice after injection of different insulins in Example 15;

[0096] FIG26 shows the results of an intraperitoneal glucose tolerance test on type I diabetic mice in Example 15, wherein the left figure shows the results of a glucose tolerance test after one day of glucose stimulation, and the right figure shows the results of a glucose tolerance test after two glucose stimulations, one day and five days later;

[0097] FIG27 is a graph showing the results of the verification of insulin release in response to sugar in type I diabetic mice in Example 15;

[0098] FIG28 shows the pharmacokinetic results of the sugar-responsive insulin analog (C5F-INS) in type I diabetic mice treated with the drug in Example 15, wherein the left figure shows the results of intravenous injection and the right figure shows the results of subcutaneous injection;

[0099] FIG29 shows the results of biosafety evaluation of type I diabetic mice treated with the sugar-responsive insulin analog (C5F-INS) in Example 15. DETAILED DESCRIPTION

[0100] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0101] The manufacturers or models of the reagents or instruments used in the following examples are shown in Table 1.

[0102] Table 1. Manufacturer or type of reagents or instruments

[0103] Example 1. Preparation of Sugar-Responsive Molecular Linker Compound FPRDBA

[0104] The reaction formula of the sugar-responsive molecular linker compound FPRDBA provided in this example is:

[0105] The preparation process is as follows:

[0106] Take 9.20g of 4-carboxy-3-fluorophenylboronic acid and 5.91g of 2-n-propyl-1,3-propanediol, place them in a 100mL round-bottom flask, add 50mL of acetone and heat under reflux for 4 hours under stirring. After cooling, filter and take the filtrate, remove ethyl acetate by vacuum rotary evaporation at 25°C and dry in a vacuum drying oven at 40°C for 24 hours to obtain the product 2-fluoro-4-(5-propyl-1,3,2-dioxaborohexane-2-yl)benzoic acid (FPRDBA). 1 The results were characterized by H-NMR (see Figure 2).

[0107] Example 2. Preparation of glucose-responsive insulin (FPRDBA-insulin)

[0108] This example uses the sugar-responsive molecular linker compound FPRDBA prepared in Example 1 to prepare sugar-responsive insulin FPRDBA-insulin. The preparation process is as follows:

[0109] (1) 6.9 mg of the sugar-responsive molecular linker compound FPRDBA and 7.8 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate prepared in Example 1 were placed in a 5 mL sample vial, 1.5 mL of anhydrous N,N-dimethylformamide was added, and the mixture was stirred until clear. 5.4 μL of N,N-diisopropylethylamine was added dropwise under an ice bath and the mixture was allowed to react at room temperature for 3 hours. The reaction progress was monitored by TLC spot plate using methanol-dichloromethane (v / v = 1 / 4) as the developing solvent.

[0110] (2) Take 50.0 mg of recombinant human insulin and place it in a 10 mL sample bottle. Add 5.00 mL of 1×PBS. While stirring, add 70 μL (10 μL×5) of 1 M hydrochloric acid solution and 70 μL (10 μL×5) of 1 M sodium hydroxide solution in sequence. Wait for 1 minute for the solution to become clear.

[0111] (3) Place the prepared recombinant human insulin solution in an ice bath and quickly add the fully reacted FPRDBA solution. During the reaction, adjust the pH to >8 with 1 M sodium hydroxide solution. Allow the reaction to proceed overnight in an ice bath.

[0112] (4) After the reaction, a regenerated cellulose dialysis bag (3500 mm) of appropriate length was added to the reaction solution. The solution was dialyzed against deionized water for 12 hours (3 × 4 L). After dialysis, the dialyzate was removed and freeze-dried to obtain the product. Characterization was performed by MALDI-TOF MS (Figure 3).

[0113] Example 3. Preparation of Sugar-Responsive Molecular Linker Compound FPTDBA

[0114] The reaction formula of the sugar-responsive molecular linker compound FPTDBA provided in this example is:

[0115] The preparation process is as follows:

[0116] Take 9.20g of 4-carboxy-3-fluorophenylboronic acid and 7.31g of 2-n-pentylpropane-1,3-diol, place them in a 100mL round-bottom flask, add 50mL of acetone and heat under reflux for 4 hours under stirring. After cooling, filter and take the filtrate, remove ethyl acetate by vacuum rotary evaporation at 25°C and dry in a vacuum drying oven at 40°C for 24 hours to obtain the product 2-fluoro-4-(5-pentyl-1,3,2-dioxaborohexane-2-yl)benzoic acid (FPTDBA). 1 The results were characterized by H-NMR (see Figure 4).

[0117] Example 4. Preparation of glucose-responsive insulin (FPTDBA-insulin)

[0118] This example uses the sugar-responsive molecular linker compound FPTDBA prepared in Example 3 to prepare the sugar-responsive insulin FPTDBA-insulin. The preparation process is as follows:

[0119] (1) 9.1 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid and 9.3 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate were placed in a 5 mL sample vial. 1.5 mL of anhydrous N,N-dimethylformamide was added and stirred until clear. 6.2 μL of N,N-diisopropylethylamine was added dropwise in an ice bath and the mixture was allowed to react at room temperature for 3 hours. The reaction progress was monitored by TLC using methanol-dichloromethane (v / v = 1 / 4) as the developing solvent.

[0120] (2) Take 60.0 mg of recombinant human insulin and place it in a 10 mL sample bottle. Add 6.00 mL of 1× PBS. Add 70 μL (10 μL × 5) of 1 M hydrochloric acid solution and 70 μL (10 μL × 5) of 1 M sodium hydroxide solution successively while stirring. Wait for 1 minute for the solution to become clear.

[0121] (3) Place the prepared recombinant human insulin solution in an ice bath and quickly add the fully reacted FPTDBA solution. During the reaction, adjust the pH to >8 with 1 M sodium hydroxide solution. Allow the reaction to proceed overnight in an ice bath.

[0122] (4) After the reaction, a regenerated cellulose dialysis bag (3500 mm) of appropriate length was added to the reaction solution. The solution was dialyzed against deionized water for 12 hours (3 × 4 L). After the dialysis was complete, the dialyzate was taken out and freeze-dried to obtain the product. Characterization was performed by MALDI-TOF MS (see Figure 5).

[0123] Example 5. Preparation of Sugar-Responsive Molecular Linker Compound DFPMDCA

[0124] The reaction formula of the sugar-responsive molecular linker compound DFPMDCA provided in this example is:

[0125] The preparation process is as follows:

[0126] (1) 10.00 g of 4-carboxy-3-fluorophenylboronic acid, 10.00 g of N-hydroxysuccinimide, and 21.20 g of 4-dimethylaminopyridine were placed in a 250 mL round-bottom flask. 100 mL of DMF was added and stirred until clear. The reaction system was placed in an ice bath. 16.65 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was slowly added under stirring and allowed to react at room temperature overnight. After the overnight reaction, DMF was removed by rotary evaporation under reduced pressure. 150 mL of ethyl acetate was added and the mixture was washed with 1 M hydrochloric acid solution (150 mL × 3). The organic phase was removed by rotary evaporation under reduced pressure at 25°C to remove ethyl acetate, washed with 1M hydrochloric acid solution (30 mL × 3), filtered, and the precipitate was dried in a vacuum drying oven at 40°C for 24 hours to obtain 1-({[4-(dihydroxyboryl)-2-fluorophenyl]carbonyl}oxy)tetrahydropyrrole-2,5-dione.

[0127] (2) 2.81 g of 1-({[4-(dihydroxyboryl)-2-fluorophenyl]carbonyl}oxy)tetrahydropyrrole-2,5-dione and 1.85 g of 1-aminododecylamine were placed in a 50 mL round-bottom flask, 15 mL of DMF was added, and the mixture was stirred until clear. The reaction was allowed to proceed at room temperature overnight.

[0128] After the reaction is completed, the reaction solution is added dropwise to 150 mL of deionized water. Centrifuge and take the precipitate. Dry in a vacuum oven at 40°C for 24 hours to obtain the product 4-(dihydroxyboryl)-N-dodecyl-2-fluorobenzamide (DFPBA). 1 The results were characterized by H-NMR (see Figure 6).

[0129] (3) Take 1.75 g of 4-(dihydroxyboryl)-N-dodecyl-2-fluorobenzamide and 1.34 g of 2,2-bis(hydroxymethyl)propionic acid, place them in a 50 mL round-bottom flask, add 20 mL of acetone and heat under reflux for 4 hours under stirring. After cooling, filter and take the filtrate, remove acetone by vacuum rotary evaporation at 30 ° C, and dry in a vacuum drying oven at 40 ° C for 24 hours to obtain the product 2-{4-[(dodecylamino)carbonyl]-3-fluorophenyl}-5-methyl-1,3,2-dioxaborinane-5-carboxylic acid (DFPMDCA), through 1 The results were characterized by H-NMR (see Figure 7).

[0130] Example 6. Preparation of glucose-responsive insulin (DFPMDCA-insulin)

[0131] This example uses the sugar-responsive molecular linker compound DFPMDCA prepared in Example 5 to prepare the sugar-responsive insulin DFPMDCA-insulin. The preparation process is as follows:

[0132] (1) 9.3 mg of 2-{4-[(dodecylamino)carbonyl]-3-fluorophenyl}-5-methyl-1,3,2-dioxaborinane-5-carboxylic acid and 5.6 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate were placed in a 5 mL sample vial, 1.5 mL of anhydrous DMF was added, and the mixture was stirred until clear. 4.0 μL of N,N-diisopropylethylamine was added dropwise in an ice bath and the mixture was allowed to react at room temperature for 6 hours. The reaction progress was monitored by TLC spot plate using methanol-dichloromethane (v / v = 1 / 4) as the developing solvent.

[0133] (2) Take 60.0 mg of recombinant human insulin and place it in a 10 mL sample vial. Add 1.00 mL of 1× PBS and 5.00 mL of dimethyl sulfoxide in an ice bath and stir until dissolved.

[0134] (3) Place the prepared recombinant human insulin solution in an ice bath and quickly add the fully reacted DFPMDCA solution. During the reaction, adjust the pH to >8 with 1 M sodium hydroxide solution. Allow the reaction to proceed overnight in an ice bath.

[0135] (4) After the reaction, a regenerated cellulose dialysis bag (3500 mm) of appropriate length was added to the reaction solution. The solution was dialyzed against deionized water for 12 hours (3 × 4 L). After the dialysis was complete, the dialyzate was taken out and freeze-dried to obtain the product. Characterization was performed by MALDI-TOF MS (see Figure 8).

[0136] Example 7. Preparation of Sugar-Responsive Molecular Linker Compound C5M

[0137] The reaction formula of the sugar-responsive molecular linker compound C5M provided in this example is:

[0138] The preparation process is as follows:

[0139] 9.20 g of 4-carboxy-3-methylphenylboronic acid and 7.31 g of 2-n-pentylpropane-1,3-diol were placed in a 100 mL round-bottom flask, 50 mL of acetone was added, and the mixture was heated under reflux for 4 hours under stirring. After cooling, the mixture was filtered and the filtrate was evaporated under reduced pressure at 25°C to remove ethyl acetate. The mixture was then dried in a vacuum drying oven at 40°C for 24 hours to obtain the product C5M. 1 The results were characterized by H-NMR (see Figure 9).

[0140] Example 8. Preparation of glucose-responsive insulin (C5M-INS)

[0141] This example uses the sugar-responsive molecular linker compound C5M prepared in Example 7 to prepare sugar-responsive insulin C5M-INS. The preparation process is as follows:

[0142] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample vial, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours.

[0143] Take 2 g of recombinant human insulin and place it in a 60 mL sample bottle. Add 20 mL of anhydrous dimethyl sulfoxide and stir until the solution is clear.

[0144] The completely reacted C5F solution was added dropwise to the recombinant human insulin solution and reacted for 1 hour.

[0145] After the reaction is completed, the solution is added dropwise to a precipitant of isopropyl acetate: methyl tert-butyl ether = 4:1 and stirred for one hour. The precipitate is filtered off and dried to obtain a crude product.

[0146] The purified product was then purified by HPLC to obtain pure C5M-INS (see FIG10 ).

[0147] Example 9. Preparation of Sugar-Responsive Molecular Linker Compound C5P

[0148] The reaction formula of the sugar-responsive molecular linker compound C5P provided in this example is:

[0149] The preparation process is as follows:

[0150] 9.20 g of 4-carboxy-3-methylphenylboronic acid and 7.31 g of 2-n-pentylpropane-1,3-diol were placed in a 100 mL round-bottom flask, 50 mL of acetone was added, and the mixture was heated under reflux for 4 hours under stirring. After cooling, the mixture was filtered and the filtrate was evaporated under reduced pressure at 25 ° C to remove ethyl acetate. The mixture was then dried in a vacuum drying oven at 40 ° C for 24 hours to obtain the product C5P. 1 The results were characterized by H-NMR (see Figure 11).

[0151] Example 10. Preparation of glucose-responsive insulin (C5P-INS)

[0152] This example uses the sugar-responsive molecular linker compound C5P prepared in Example 8 to prepare sugar-responsive insulin C5P-INS. The preparation process is as follows:

[0153] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample vial, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours.

[0154] Take 2 g of recombinant human insulin and place it in a 60 mL sample bottle. Add 20 mL of anhydrous dimethyl sulfoxide and stir until the solution is clear.

[0155] The completely reacted C5F solution was added dropwise to the recombinant human insulin solution and reacted for 1 hour.

[0156] After the reaction is completed, the solution is added dropwise to a precipitant of isopropyl acetate: methyl tert-butyl ether = 4:1 and stirred for one hour. The precipitate is filtered off and dried to obtain a crude product.

[0157] The purified product was then purified by HPLC to obtain pure C5P-INS (see FIG12 ).

[0158] Example 11: In vitro glucose response verification of glucose-responsive insulin

[0159] 1. In vitro glucose response verification of glucose-responsive insulin (FPTDBA-insulin)

[0160] A 1 mg / mL solution of sugar-responsive insulin (FPTDBA-insulin) (prepared in Example 4) was prepared using PBS (10 mM, pH 7.4) as a solvent. A 0.4 g / mL glucose solution was added to the solution to obtain different initial glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, and 1600 mg / dL). At 0 h, 0.25 h, 0.5 h, 1 h, 2 h, and 4 h, 30 μL of the complex was aspirated and centrifuged. Then, 4 μL of the supernatant was mixed with 200 μL of Coomassie Brilliant Blue. The absorbance at 595 nm was measured using a microplate reader, and the insulin concentration in the supernatant was calculated using a pre-established standard curve (see Figure 13 for the insulin concentration-absorbance standard curve).

[0161] In phosphate buffer (PBS) at pH 7.4, the insulin concentration in the supernatant was determined using the Coomassie Brilliant Blue method. The glucose-responsive insulin release of the recombinant insulin conjugate (FPTDBA-insulin) was evaluated at varying glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, and 1600 mg / dL). Addition of glucose to the PBS solution significantly increased the insulin release rate and equilibrium insulin concentration. At high glucose concentrations, free glucose competes for the binding sites of the 1,3-diol structure with the phenylboronic acid analog, enhancing insulin solubility and resulting in a faster insulin release rate (see Figure 14, left panel).

[0162] 2. In vitro glucose response verification of glucose-responsive insulin (DFPMDCA-insulin)

[0163] A 1 mg / mL solution of the recombinant insulin conjugate (DFPMDCA-insulin) (prepared in Example 5) was prepared in PBS (10 mM, pH 7.4). A 0.4 g / mL glucose solution was added to the solution to obtain different initial glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, and 1600 mg / dL). At 0 h, 0.25 h, 0.5 h, 1 h, 2 h, and 4 h, 30 μL of the complex was aspirated and centrifuged. Then, 4 μL of the supernatant was mixed with 200 μL of Coomassie Brilliant Blue. The insulin concentration in the supernatant was calculated using a microplate reader at 595 nm using a pre-established standard curve ( FIG13 ).

[0164] In phosphate buffer (pH 7.4), the insulin concentration in the supernatant was determined using the Coomassie Brilliant Blue method. The glucose-responsive insulin release properties of the recombinant insulin conjugate (DFPMDCA-insulin) were evaluated at varying glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, and 1600 mg / dL). Adding glucose to the PBS solution significantly increased the insulin release rate and equilibrium insulin concentration. At high glucose concentrations, free glucose competes for the binding sites of the 1,3-diol structure and the phenylboronic acid analog, enhancing insulin solubility and resulting in a faster insulin release rate (see Figure 14, right panel).

[0165] Example 12: Animal efficacy study

[0166] 1. Construction of type 1 diabetes mouse model

[0167] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected and fasted overnight. STZ solution was injected intraperitoneally in the dark at a dose of 120 mg / kg. Blood glucose was measured one and two weeks after injection. If blood glucose > 300 mg / dL, the model was successfully established. Otherwise, STZ solution was injected intraperitoneally again at a dose of 50 mg / kg two weeks later, and blood glucose was measured again one and two weeks later.

[0168] 2. In vivo hypoglycemic study in type 1 diabetic mice

[0169] A type 1 diabetic mouse model was established according to the above method, and type 1 diabetic mice with a blood sugar level of 400-500 mg / dL were selected for therapeutic effect evaluation. After subcutaneous injection of the sugar-responsive recombinant insulin FPTDBA-insulin (2.4 mg / kg) prepared in Example 4, the blood sugar level of the diabetic mice remained in the normal range below 200 mg / dL for more than 21 hours (the shortest was more than 7 hours), which was significantly longer than insulin and glargine insulin. Compared with insulin and commercially available long-acting insulin (glargine insulin), the sugar-responsive recombinant insulin showed a faster blood sugar lowering effect and a significantly more stable blood sugar control ability. No diabetic mice showed hypoglycemia (less than 50 mg / dL) (see Figure 15).

[0170] Example 13. Preparation of Sugar-Responsive Insulin (C5F-INS)

[0171] 1. Preparation of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid (sugar-responsive molecular linker compound FPTDBA)

[0172] Take 9.20g of 4-carboxy-3-fluorophenylboronic acid and 7.31g of 2-n-pentylpropane-1,3-diol, place them in a 100mL round-bottom flask, add 50mL of acetone and heat under reflux for 4 hours under stirring. After cooling, filter and take the filtrate, remove ethyl acetate by vacuum rotary evaporation at 25℃ and dry in a vacuum drying oven at 40℃ for 24 hours to obtain the product. 1 The results were characterized by H-NMR (see Figure 4).

[0173] 2. Preparation of insulin analog (C5F-INS) (C5F-INS is a purified product of FPTDBA-INS)

[0174] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborolan-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample vial, add 3.0 mL of anhydrous dimethyl sulfoxide and 78 μL of N,N-diisopropylethylamine, stir until clear, and react at room temperature for 3 hours.

[0175] Take 2 g of recombinant human insulin and place it in a 60 mL sample bottle. Add 20 mL of anhydrous dimethyl sulfoxide and stir until the solution is clear.

[0176] The fully reacted C5F solution was added dropwise to the recombinant human insulin solution and allowed to react for 1 hour. After the reaction, the solution was added dropwise to a precipitant containing isopropyl acetate and methyl tert-butyl ether (MTBE) in a ratio of 4:1 and stirred for 1 hour. The precipitate was filtered and dried to obtain the crude product. Purification by HPLC yielded pure C5F-INS (Figure 16).

[0177] The structure of the insulin was characterized by ESI mass spectrometry (Figure 17), circular dichroism (Figure 18), scanning electron microscopy (Figure 19), MALDI-TOF mass spectrometry (Figure 20), and solid-state boron nuclear magnetic resonance spectroscopy (Figure 21), demonstrating the successful preparation of glucose-responsive insulin.

[0178] Example 14. Comparison of the therapeutic effects of glucose-responsive insulin prepared from glucose-responsive molecular linker compounds with different structures

[0179] In the previous study, this example compared glucose-responsive insulins prepared with glucose-responsive molecular linker compounds of different structures. It was found that the glucose-responsive molecular linker compounds of different structures had different effects on the therapeutic effects of glucose-responsive insulin, and there were certain differences in the therapeutic effects. The three most typical ones were selected, namely, the glucose-responsive insulin C5M-INS prepared in Example 8, the glucose-responsive insulin C5P-INS prepared in Example 10, and the glucose-responsive insulin C5F-INS prepared in Example 13, and animal experiments were carried out. The experimental process is as follows:

[0180] A type 1 diabetes mouse model was established according to Example 12, and type 1 diabetic mice with blood glucose levels of 400-500 mg / dL were selected for evaluation of the therapeutic effect. After subcutaneous injection of 12 mg / kg of the glucose-responsive insulin C5M-INS prepared in Example 8, the glucose-responsive insulin C5P-INS prepared in Example 10, and the glucose-responsive insulin C5F-INS prepared in Example 13, the mice's blood glucose levels were measured to examine the blood glucose control effects of the three groups, as shown in Figure 22.

[0181] As can be seen from Figure 22, the three groups of sugar-responsive insulin all have good blood sugar control effects, but there are certain differences in the therapeutic effects of the three groups. Compared with the C5M-INS group and the C5P-INS group, although the difference is not obvious in the early stage, in the later stage, especially after 150 hours, the blood sugar control effect of the C5P-INS group is significantly better than that of the C5M-INS group. It can be seen that in the sugar-responsive molecular linker compound, the methyl modification on the phenylboronic acid is not conducive to the later therapeutic effect.

[0182] Compared with the C5F-INS group and the C5P-INS group, it can be seen that the C5F-INS group has the best blood sugar control effect and the longest duration. In almost the first 150 hours, blood sugar can be controlled below 100 mg / dL, and even a good therapeutic effect can be maintained for about 200 hours. It can be seen that compared with sugar-responsive molecular linker compounds with other structures, the sugar-responsive molecular linker compound with FPTDBA structure, the prepared sugar-responsive insulin C5F-INS (C5F-INS is a purified product of FPTDBA-INS) has better blood sugar control effect and more therapeutic advantages.

[0183] Example 15: Rodent efficacy study

[0184] 1. Construction of type 1 diabetes mouse model

[0185] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected and fasted overnight. STZ solution was injected intraperitoneally in the dark at a dose of 120 mg / kg. Blood glucose was measured one and two weeks after injection. If blood glucose > 300 mg / dL, the model was successfully established. Otherwise, STZ solution was injected intraperitoneally again at a dose of 50 mg / kg two weeks later, and blood glucose was measured again one and two weeks later.

[0186] 2. In vivo hypoglycemic study in type 1 diabetic mice

[0187] A type 1 diabetes mouse model was established according to the above method, and type 1 diabetic mice with blood glucose levels of 400-500 mg / dL were selected for treatment effect evaluation. After subcutaneous injection of the glucose-responsive insulin analog (C5F-INS), the blood glucose levels of the diabetic mice remained in the normal range below 200 mg / dL for more than 214 hours (Figure 23), which was significantly longer than that of glargine insulin (Figure 24). Compared with the commercially available long-acting insulin (glargine insulin), the glucose-responsive insulin analog (C5F-INS) showed a faster blood glucose lowering effect and more stable blood glucose control ability, and no diabetic mice experienced hypoglycemia (below 50 mg / dL).

[0188] At the same time, the dose-effect relationship showed that the sugar-responsive insulin analog (C5F-INS) did not cause hypoglycemia in the range of 4-12 mg / kg, and all mice survived. However, when glargine insulin was at 400 U / kg (approximately 13.8 mg / kg in mass units), 40% of the mice developed hypoglycemia. Moreover, when glargine insulin was used to control blood sugar for one week (i.e., injected once a day at 40 U / kg), all mice developed hypoglycemia, and 40% of the mice died due to severe hypoglycemia (Figure 25).

[0189] 3. Intraperitoneal glucose tolerance test in type 1 diabetic mice

[0190] A type 1 diabetic mouse model was established according to the above method, and type 1 diabetic mice with blood glucose of 400-500 mg / dL (n=5) were selected to evaluate the glucose control effect. On the first and fifth days after subcutaneous injection of a glucose-responsive insulin analog (C5F-INS) (12 mg / kg), the mice were intraperitoneally injected with 1.5 g / kg of glucose. The blood glucose of the mice was detected and recorded every 15 minutes for 2 hours. At the same time, a group of healthy C57BL / 6J mice were selected and intraperitoneally administered with 1.5 g / kg of glucose. The blood glucose of the mice was detected and recorded every 15 minutes for 2 hours as a control.

[0191] The results showed that when the mice were given a sugar stimulus on the first day after administration, their blood sugar levels were maintained within the normal blood sugar range (50-200 mg / dL), and the glucose control effect (assessed by the area under the curve of the blood sugar-time curve) was better than that of normal mice. When the mice were given a sugar stimulus on the fifth day after administration, their blood sugar levels showed a significant peak at 15 minutes, returned to normal (below 200 mg / dL) within an hour, and remained stable for 2 hours (Figure 26), showing a glucose control effect still better than that of normal mice.

[0192] 4. Verification of glucose-responsive insulin release in type 1 diabetic mice

[0193] A type 1 diabetic mouse model was established using the aforementioned method. In vivo glucose-responsive insulin release was evaluated in type 1 diabetic mice (n=5) with blood glucose levels of 400-500 mg / dL. On day 1 and day 5 after subcutaneous injection of a glucose-responsive insulin analog (C5F-INS) (12 mg / kg), mice were intraperitoneally injected with 3.0 g / kg of glucose. Blood glucose levels were measured and recorded every 15 minutes for 2 hours. Blood samples (20 μL of blood drawn from the orbital cavity and centrifuged for plasma) were collected at 0, 15, 30, 60, and 120 minutes.

[0194] The plasma insulin content was detected by recombinant human insulin ELISA kit.

[0195] The results showed that after sugar stimulation, the blood glucose of mice showed an obvious peak (higher than 300 mg / kg), and the insulin concentration in the plasma also changed synchronously with the change of blood glucose, with a peak at 30 minutes. On the first day after administration, the peak value of insulin increased by 4-5 times compared with the initial value; on the fifth day after administration, the peak value of insulin increased by 2-3 times. At the same time, when blood glucose gradually dropped to the initial value, the insulin concentration in the plasma also decreased back to the initial level, verifying the sugar-responsive insulin release (see Figure 27).

[0196] 5. Pharmacokinetics of glucose-responsive insulin analog (C5F-INS) in type 1 diabetic mice

[0197] A type 1 diabetic mouse model was established using the aforementioned method. Pharmacokinetic evaluation was performed in 5 type 1 diabetic mice (n=5) with blood glucose levels of 400-500 mg / dL. Following subcutaneous injection of a glucose-responsive insulin analog (C5F-INS) (12 mg / kg), blood samples (20 μL of blood drawn from the orbital cavity and centrifuged for plasma) were collected at predetermined time points. The mice were monitored continuously for 17 days (see Figure 28).

[0198] A group of healthy C57BL / 6J mice were simultaneously administered 1 nmol / kg of a glucose-responsive insulin analog (C5F-INS) intravenously. Blood samples (20 μL of blood was drawn from the eye sockets and centrifuged for plasma) were collected at predetermined time points and monitored continuously for 21 days (see Figure 28).

[0199] Plasma insulin concentration-time curve data were analyzed using a non-compartmental Winnonlin model. In diabetic mice, the half-life of the glucose-responsive insulin analog (C5F-INS) reached 79.0 ± 17.7 hours after subcutaneous injection. In healthy mice, the half-life of the glucose-responsive insulin analog (C5F-INS) reached 93.7 ± 9.5 hours after intravenous injection, a 1,000-fold increase over that of normal human insulin (half-life of approximately 5 minutes).

[0200] 6. Biosafety Evaluation of Type 1 Diabetic Mice Treated with a Sugar-Responsive Insulin Analog (C5F-INS)

[0201] A type 1 diabetic mouse model was established according to the above method, and type 1 diabetic mice (n=5) with a blood glucose level of 400-500 mg / dL were selected for biosafety evaluation. Blood samples were collected from mice before administration. After subcutaneous injection of a sugar-responsive insulin analog (C5F-INS) (12 mg / kg), blood samples were collected on the 7th day. Blood-related biochemical indices and routine blood tests were performed separately. At the same time, a group of healthy C57BL / 6J mice were selected, blood samples were collected, and blood-related biochemical indices and routine blood tests were also performed as controls. The results showed that after subcutaneous injection of a sugar-responsive insulin analog (C5F-INS), the various indicators of the mice were similar to those of healthy mice (see Figure 29).

[0202] The embodiments described in the present invention are all preferred embodiments and features. Any person skilled in the art can make some changes and variations based on the essence of the description of the present invention. These changes and variations are also considered to fall within the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. A sugar-responsive molecular linker compound, characterized in that: It is formed by esterification of phenylboronic acid or phenylboronic acid analogs with alcohol compound molecules.

2. The compound according to claim 1, characterized in that The compound has the following general structural formula: Where R1, R2, R3, and R4 are H, C1-C 20 Alkyl, C1-C 20 Alkenyl, C1-C 20 Alkynyl, carboxyl, amino, substituted C1-C 20 Alkyl, substituted C1-C 20 Alkenyl, substituted C1-C 20 Alkynyl or substituted amino; the substituted group is selected from C1-C 20 Alkyloxy, C1-C 20 Alkenyloxy, C1-C 20 Alkynyloxy, hydroxyl, amino or carboxyl, R5, R6, R7, R8, R9 are H, alkyl, alkenyl, alkynyl, carboxyl, amino, substituted alkyl, substituted alkenyl, substituted alkynyl or substituted amino respectively; the substituted group is selected from alkyloxy, alkenyloxy, alkynyloxy, hydroxyl, amino or carboxyl.

3. The compound according to claim 1, characterized in that The phenylboronic acid analogs include phenylboronic acid modified with different groups; the modified groups include one or more of alkyl, halogen, nitro and amide; the alkyl is a straight chain or branched chain alkyl, and the carbon chain length of the alkyl is 2-100 carbons.

4. The compound according to claim 1, characterized in that The phenylboronic acid or phenylboronic acid analog is modified with a carboxyl group.

5. The compound according to claim 4, characterized in that The carboxyl-modified phenylboronic acid or phenylboronic acid analogues include one or more of the following:

6. The compound according to claim 5, characterized in that The alcohol compounds include methanol, diols, triols, tetraols, polyhydroxy alcohols or aldehydes or acids and substituents of the above alcohols; the substituents are one or more of alkyl, halogen, nitro, amino, ether, alkenyl, thiol, aromatic and amide.

7. The compound according to claim 6, characterized in that The alcohol compound is a compound with a diol structure, including one or more of the following: or or 8. The compound according to claim 7, characterized in that The alcohol compound is a 1,3-diol compound, including one or more of the following:

9. The compound according to claim 8, characterized in that The phenylboronic acid or phenylboronic acid analog is preferably: The 1,3-diol compound is preferably:

10. The compound according to claim 9, characterized in that The compound has the following structure:

11. The method for preparing the sugar-responsive molecular linker compound according to any one of claims 1 to 10, characterized in that: The method is prepared by mixing phenylboronic acid or phenylboronic acid analogs with alcohol compound molecules for reaction and then purifying the mixture.

12. The preparation method according to claim 11, characterized in that: The purification includes recrystallization and column chromatography; the recrystallization solvent is toluene or acetone; the column chromatography uses 200-400 mesh silica gel, and the developing agent is 10-50% ethyl acetate / petroleum ether.

13. A sugar-responsive insulin, characterized in that The method is prepared by a mixed reaction of the sugar-responsive molecular linker compound as described in any one of claims 1 to 10 and insulin.

14. A method for preparing a sugar-responsive insulin, comprising the following steps: (1) preparing the sugar-responsive molecular linker compound according to any one of claims 1 to 10; (2) Add insulin solution and react to obtain the product.

15. Use of the sugar-responsive molecular linker compound according to any one of claims 1 to 10 for preparing a reagent for linking and releasing insulin in a glucose-responsive manner.

16. A composition for delivering insulin, characterized in that The composition comprises the compound according to any one of claims 1 to 10 and insulin.

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