Sugar-responsive molecular connector as well as preparation method and application thereof

Abstract

According to the sugar-responsive molecular connector and the preparation method and application thereof provided by the invention, a brand new sugar-responsive molecular connector compound is formed by esterification and combination of a phenylboronic acid analogue and a 1, 3-diol structure, and the compound can be directly biologically linked with insulin through amidation and has a very good glucose-responsive function; high-concentration glucose can be competitively combined with a 1, 3-diol structure and phenylboronic acid, so that the solubility of insulin is increased, and the release of insulin is promoted; when the concentration is low, only a small part of insulin is in a free state, so that the occurrence of hypoglycemia is reduced. The compound utilizes a brand new mechanism to realize sugar response of insulin, so that the sugar response effect is more sensitive, and the compound is simple in structure, easy to prepare, low in cost and extremely high in clinical application value.

Description

[0001] This application claims priority to the prior Chinese application, application number: 202310793910.9, and 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 in particular 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, and this number is still increasing, and is expected to rise to 592 million by 2035. In addition, the annual global treatment costs related to diabetes and its complications have exceeded US$500 billion. The large number of patients and the high cost of treatment indicate that it has become a major global public health security issue.

[0004] Insulin replacement therapy is an integral part of the treatment of type 1 diabetes and is also a necessary means of treating advanced type 2 diabetes. In healthy individuals, endogenous insulin secretion from pancreatic β cells oscillates in sync with fluctuations in blood glucose levels, thereby 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 changes in stress, physical activity, and dietary intake that may occur every day. Since exogenous insulin is not regulated by endogenous feedback mechanisms, even with a carefully designed dosing regimen, and even when patients strictly follow the diet and lifestyle prescribed by their doctors, diabetic patients will inevitably experience symptoms of hyperglycemia or hypoglycemia.

[0005] Therefore, it is attractive to mimic the function of β cells by releasing insulin in a glucose-dependent manner for safe and effective insulin delivery. Self-regulating glucose-responsive insulin delivery systems have been widely developed. Most of these systems rely on glucose and 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-acting insulin delivery systems. Its ability to bind to sugars and potential charge transformation 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, such as patent CN115671050A, which 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 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 the system in animals (mice and pigs). However, the above studies all prepared composite materials, the preparation process is cumbersome, the use is not convenient, and the actual effect is not good.

[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 oral insulin delivery technology for long-term blood sugar control. Summary of the invention

[0009] To solve the above problems, the present invention provides a sugar-responsive molecular connector and its preparation method and application. A new sugar-responsive molecular connector compound is formed by esterifying phenylboronic acid analogs with 1,3-diol structures. The compound can be directly biolinked with insulin through amidation and has very good glucose response function; high concentrations of glucose can compete with 1,3-diol structures to bind to phenylboronic acid, increase insulin solubility, and promote insulin release; at low concentrations, only a small part of insulin is in a free state, reducing the occurrence of hypoglycemia. The compound uses a new mechanism to achieve insulin sugar response, making the sugar response effect more sensitive, prolonging the treatment time, and more effectively controlling blood sugar. Moreover, it has a simple structure, is easy to prepare, and has low cost, and is of great clinical application value.

[0010] 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.

[0011] The structural formula of the phenylboronic acid is as follows:

[0012]

[0013] The sugar-responsive molecular linker compound provided by the present invention can also be understood as a structural unit with sugar-responsive ability, which is formed by esterification of phenylboronic acid or phenylboronic acid analogs with alcohol compound molecules, and can be directly linked to insulin and has the ability to respond to sugar ( Figure 1 ), having the following general structural formula:

[0014]

[0015] 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.

[0016] Furthermore, 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.

[0017] Furthermore, the phenylboronic acid or phenylboronic acid analog is modified with a carboxyl group.

[0018] The present invention uses carboxyl-modified phenylboronic acid or phenylboronic acid analogs to construct a sugar-responsive molecular linker compound. Due to the presence of a carboxyl group in the structure, an amidation reaction can be carried out with the amino group on insulin, thereby achieving the link between insulin and the compound.

[0019] Furthermore, the carboxyl-modified phenylboronic acid or phenylboronic acid analogue includes one or more of the following:

[0020]

[0021] 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.

[0022] Furthermore, the alcohol compound is a compound with a diol structure, which may be a polyhydroxy alkane or olefin or a fatty amine or a fatty acid or a mercaptoalkane or a thioether or an amide. Preferably, the analogue of the 1,2-diol structure is selected from one or more of the following groups:

[0023]

[0024] or

[0025]

[0026] or

[0027]

[0028] The sugar-responsive molecular linker compound constructed by the present invention has very good sugar-responsiveness. Through esterification of phenylboronic acid ester and diol structure, the ester bond can be cut by glucose molecule, thereby releasing insulin. Therefore, under the condition of hyperglycemia, high concentration of glucose and diol structure competitively bind to phenylboronic acid, resulting in the cutting of phenylboronic acid ester bond, increasing insulin solubility, promoting insulin release, and making insulin have good glucose-responsive release performance ( Figure 1 ), can make the body quickly restore normal blood sugar; under normal blood sugar conditions, only a small amount of insulin is in a free state, reducing the occurrence of hypoglycemia.

[0029] Further, the alcohol compound is a 1,3-diol compound, which may be a polyhydroxy alkane or olefin or fatty acid or amide, preferably, one or more selected from the following:

[0030]

[0031] Studies have shown that the sugar-responsive molecular linker compound generated by the reaction of 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.

[0032] In some embodiments, the phenylboronic acid analog is preferably 4-carboxy-3-fluorophenylboronic acid:

[0033]

[0034] The 1,3-diol compound is preferably:

[0035]

[0036] The compound prepared by using the above-mentioned phenylboronic acid analogue and the 1,3-diol compound has the following structure:

[0037]

[0038] 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 phenylboronic acid analogs with alcohol compound molecules for reaction and then purifying them.

[0039] 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.

[0040] In some embodiments, the molar ratio of the carboxyl-modified phenylboronic acid or phenylboronic acid-based compound to the 1,3-diol structure compound is preferably 1:(1-3).

[0041] In some embodiments, the solvent in the solution of the carboxyl-modified phenylboronic acid or phenylboronic acid compounds may 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.

[0042] In some embodiments, the solvent in the solution of the 1,3-diol structure compound may be a conventional organic solvent in the art that can dissolve the 1,3-diol structure compound, preferably toluene or acetone.

[0043] In some embodiments, the concentration of the carboxyl-modified phenylboronic acid or phenylboronic acid compound solution is preferably 1-200 mg / mL, such as 20 mg / mL.

[0044] In some embodiments, the concentration of the 1,3-diol structure compound solution is preferably 1 to 600 mg / mL, such as 20 mg / mL.

[0045] 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.

[0046] In some embodiments, the carboxyl-modified phenylboronic acid compound is 4-carboxy-3-fluorophenylboronic acid.

[0047] 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.

[0048] In some embodiments, the heating reflux time is 1 to 8 hours, such as 4 hours.

[0049] In some embodiments, the purification method comprises recrystallization and column chromatography.

[0050] 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.

[0051] In another aspect, the present invention provides a sugar-responsive insulin, which is prepared by a mixed reaction of the sugar-responsive molecular linker compound described above and insulin.

[0052] In another aspect, the present invention provides a method for preparing a sugar-responsive insulin, comprising the following steps:

[0053] (1) preparing sugar-responsive molecular linker compounds;

[0054] (2) Add insulin solution and react to obtain the product.

[0055] Furthermore, the method for preparing the sugar-responsive insulin comprises the following steps:

[0056] (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;

[0057] (b) then adding it to the insulin solution for reaction, adjusting the pH to 8-10, and post-treating to obtain the product;

[0058] In some embodiments, in step (a), the solvent may be conventional in the art, preferably anhydrous DMF.

[0059] In some embodiments, in step (a), the reaction is preferably carried out at room temperature.

[0060] 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).

[0061] 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.

[0062] In some embodiments, in step (b), the reaction is preferably carried out in an ice bath.

[0063] In some embodiments, in step (b), the concentration of the insulin solution is preferably 0.2-100 mg / mL.

[0064] In some embodiments, in step (b), the solvent of the insulin solution is preferably phosphate buffered saline (PBS) or dimethyl sulfoxide.

[0065] In some embodiments, in step (b), the pH of the insulin solution is preferably 8-10.

[0066] In some embodiments, in step (b), the post-treatment is preferably dialysis.

[0067] The dialysis can be performed in deionized water using a conventional dialysis bag in the art, and the molecular weight cutoff of the dialysis bag is preferably 1k to 3.5k.

[0068] In some embodiments, preferably, the dialysis time is 12 to 24 hours, such as 12 hours.

[0069] In the present invention, the white flocculent precipitate formed after the dialysis is the sugar-responsive insulin. The sugar-responsive insulin is soluble or easily soluble in weakly alkaline water.

[0070] The sugar-responsive insulin prepared by the present invention is an amorphous flocculent precipitate, and subcutaneous injection can form an insulin reservoir. Sugar response is achieved by competitive reversible binding of glucose with 1,3-diol and phenylboronic acid under physiological conditions ( Figure 1 ). Under the condition of hyperglycemia, high concentration of glucose and 1,3-diol structure compete with phenylboronic acid, resulting in the rupture of phenylboronic acid ester bond, which increases the solubility of insulin and promotes the release of insulin, making insulin have good glucose response release performance, and can quickly restore the body to normal blood sugar; under the condition of normal blood sugar, only a small part of insulin is in a free state, reducing the occurrence of hypoglycemia.

[0071] 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.

[0072] In still another aspect, the present invention provides a composition for delivering insulin, comprising the compound as described above and insulin.

[0073] In another aspect, the present invention provides use of the glucose-responsive insulin as described above in the preparation of a drug for treating diabetes.

[0074] Furthermore, the diabetes may be type 1 diabetes or late stage type 2 diabetes.

[0075] In some embodiments, the glucose-responsive insulin can be resuspended in PBS or physiological saline and administered by subcutaneous injection for treatment.

[0076] The present invention has the following beneficial effects:

[0077] 1. For the first time, a new sugar-responsive molecular connector compound was constructed. This compound can be directly biolinked with insulin through amidation to form sugar-responsive insulin, and has very good glucose response function. It has a simple structure and is easy to prepare at a low cost.

[0078] 2. Using the principle that glucose can compete with 1,3-diol structure to bind to phenylboronic acid, a new mechanism is proposed to achieve insulin sugar response. At high concentrations, glucose can compete with 1,3-diol structure, and glucose preferentially binds to phenylboronic acid, resulting in the cleavage of phenylboronic acid ester bond, increasing insulin solubility and promoting insulin release. At low concentrations, only a small amount of insulin is in a free state, reducing the occurrence of hypoglycemia. This mechanism makes the sugar response effect more sensitive, prolongs the treatment time, and more effectively controls blood sugar.

[0079] 3. Sugar-responsive molecular linker compounds are 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 will occur. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Schematic diagram of the formation of glucose-responsive insulin and the mechanism of glucose-responsive insulin release;

[0081] Figure 2 The hydrogen nuclear magnetic resonance spectrum of 2-fluoro-4-(5-propyl-1,3,2-dioxaborohex-2-yl)benzoic acid synthesized in Example 1 (the deuterated reagent is DMSO-d6);

[0082] Figure 3 is the MALDI-TOF mass spectrum of the sugar-responsive insulin FPRDBA-insulin synthesized in Example 2;

[0083] Figure 4 The hydrogen nuclear magnetic resonance spectrum of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborohexan-2-yl)benzoic acid synthesized in Example 3 (the deuterated reagent is DMSO-d6);

[0084] Figure 5 is the MALDI-TOF mass spectrum of the sugar-responsive insulin FPTDBA-insulin synthesized in Example 4;

[0085] Figure 6 This is the hydrogen nuclear magnetic resonance spectrum of 4-(dihydroxyboryl)-N-dodecyl-2-fluorobenzamide synthesized in Example 5 (the deuterated reagent is DMSO-d6);

[0086] Figure 7 The 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);

[0087] Figure 8 is the MALDI-TOF mass spectrum of the recombinant human insulin conjugate (DFPMDCA-insulin) synthesized in Example 6;

[0088] Fig. 9 is the hydrogen nuclear magnetic resonance spectrum of C5M synthesized in Example 7;

[0089] Fig.10 This is the HPLC purification spectrum of C5M-INS synthesized in Example 8;

[0090] Fig.11 is the hydrogen nuclear magnetic resonance spectrum of C5P synthesized in Example 9;

[0091] Fig.12 This is the HPLC purification spectrum of C5P-INS synthesized in Example 10;

[0092] Fig.13 is the insulin concentration-absorbance standard curve in Example 11;

[0093] Fig.14 The glucose-responsive insulin release curves of the glucose-responsive recombinant human insulin (FPTDBA-insulin) and the glucose-responsive recombinant human insulin (DFPMDCA-insulin) in Example 11;

[0094] Fig.15 The blood glucose level of type 1 diabetic mice treated with recombinant human insulin and commercial long-acting insulin preparation glargine insulin and phosphate buffer in Example 12;

[0095] Fig.16 The spectrum of pure C5F-INS obtained by HPLC purification in Example 13;

[0096] Fig.17 is the ESI mass spectrum in Example 13;

[0097] Fig.18 is the circular dichroism spectrum in Example 13;

[0098] Fig.19 is the scanning electron microscope spectrum in Example 13;

[0099] Fig. 20 is the MALDI-TOF mass spectrum in Example 13;

[0100] Fig.21 is the solid-state NMR boron spectrum in Example 13;

[0101] Fig. 22This is a comparison chart of the blood sugar control effects of the sugar-responsive insulin prepared by using three groups of sugar-responsive molecular linker compounds in Example 14;

[0102] Fig.23 Schematic diagram of the results of hypoglycemic effect in diabetic mice using different concentrations of glucose-responsive insulin in Example 15;

[0103] Fig.24 The results of blood glucose control in mice after single-injection and multiple-injection injections of glargine insulin in Example 15;

[0104] Fig.25 is the survival rate of mice after injection of different insulins in Example 15;

[0105] Fig.26 The figure is a graph showing the results of the intraperitoneal glucose tolerance test of type I diabetic mice in Example 15, wherein the left figure is the result of the glucose tolerance test after 1 day of glucose stimulation, and the right figure is the result of the glucose tolerance test after two glucose stimulations on day 1 and day 5;

[0106] Fig. 27 This is a diagram showing the verification results of the in vivo sugar response insulin release in type I diabetic mice in Example 15;

[0107] Fig.28 The pharmacokinetic results of type I diabetic mice treated with the sugar-responsive insulin analog (C5F-INS) in Example 15, wherein the left figure is the result of intravenous injection, and the right figure is the result of subcutaneous injection;

[0108] Fig.29 These are the results of the biosafety evaluation of type 1 diabetic mice treated with the sugar-responsive insulin analog (C5F-INS) in Example 15. DETAILED DESCRIPTION

[0109] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0110] The manufacturers or models of the reagents or instruments used in the following examples are shown in Table 1.

[0111] Table 1. Manufacturer or type of reagents or instruments

[0112]

[0113] Example 1. Preparation of Sugar-Responsive Molecular Linker Compound FPRDBA

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

[0115]

[0116] The preparation process is as follows:

[0117] 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. Cool and filter, 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-dioxaborohex-2-yl)benzoic acid (FPRDBA), and 1 H-NMR characterization (see Figure 2 ).

[0118] Example 2. Preparation of glucose-responsive insulin (FPRDBA-insulin)

[0119] 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:

[0120] (1) Take 6.9 mg of the sugar-responsive molecular linker compound FPRDBA prepared in Example 1 and 7.8 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, add 1.5 mL of anhydrous N,N-dimethylformamide, and stir until clear. Add 5.4 μL of N,N-diisopropylethylamine dropwise under an ice bath and react at room temperature for 3 hours. During the reaction, the reaction progress was monitored by TLC spot plate, and the developing solvent was methanol-dichloromethane (v / v=1 / 4).

[0121] (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. 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.

[0122] (3) Place the prepared recombinant human insulin solution in an ice bath, quickly add the fully reacted FPRDBA solution, and adjust the pH to > 8 with 1 M sodium hydroxide solution during the reaction. Allow to react overnight in an ice bath.

[0123] (4) After the reaction is completed, take a regenerated cellulose dialysis bag (3500) of appropriate length and add the reaction solution. Dialyze with deionized water for 12 hours (3×4L). After the dialysis is completed, take out the dialysate and freeze-dry it to obtain the obtained product. Characterization by MALDI-TOF MS ( Figure 3 ).

[0124] Example 3. Preparation of Sugar-Responsive Molecular Linker Compound FPTDBA

[0125] The reaction formula of the sugar-responsive molecular linker compound FPTDBA provided in this example is:

[0126]

[0127] The preparation process is as follows:

[0128] 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, take the filtrate, remove ethyl acetate by vacuum rotary evaporation at 25°C, and dry it in a vacuum drying oven at 40°C for 24 hours to obtain the product 2-fluoro-4-(5-pentyl-1,3,2-dioxaborohex-2-yl)benzoic acid (FPTDBA), through 1 H-NMR characterization (see Figure 4 ).

[0129] Example 4. Preparation of glucose-responsive insulin (FPTDBA-insulin)

[0130] This example uses the sugar-responsive molecular linker compound FPTDBA prepared in Example 3 to prepare sugar-responsive insulin FPTDBA-insulin. The preparation process is as follows:

[0131] (1) Take 9.1 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborin-2-yl)benzoic acid and 9.3 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, add 1.5 mL of anhydrous N,N-dimethylformamide, and stir until clear. Add 6.2 μL of N,N-diisopropylethylamine dropwise under ice bath, and react at room temperature for 3 hours. During the reaction, the reaction progress was monitored by TLC spot plate, and the developing solvent was methanol-dichloromethane (v / v=1 / 4).

[0132] (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. Then, 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.

[0133] (3) Place the prepared recombinant human insulin solution in an ice bath, quickly add the fully reacted FPTDBA solution, and adjust the pH to > 8 with 1 M sodium hydroxide solution during the reaction. Allow to react overnight in an ice bath.

[0134] (4) After the reaction is completed, take a regenerated cellulose dialysis bag (3500) of appropriate length and add the reaction solution. Dialyze with deionized water for 12 hours (3×4L). After the dialysis is completed, take out the dialysate and freeze-dry it to obtain the obtained product. Characterize by MALDI-TOF MS (see Figure 5 ).

[0135] Example 5. Preparation of Sugar-Responsive Molecular Linker Compound DFPMDCA

[0136] The reaction formula of the sugar-responsive molecular linker compound DFPMDCA provided in this example is:

[0137]

[0138] The preparation process is as follows:

[0139] (1) 10.00 g of 4-carboxyl-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 and 100 mL of DMF was added. After stirring until the mixture was clear, the reaction system was placed in an ice bath and 16.65 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was slowly added under stirring. The mixture was reacted at room temperature overnight. After the reaction was allowed to proceed overnight, DMF was removed by rotary evaporation under reduced pressure, 150 mL of ethyl acetate was added and then washed with a 1 M hydrochloric acid solution (150 mL × 3). The organic phase was removed by rotary evaporation under reduced pressure at 25° C. and then washed with a 1 M hydrochloric acid solution (30 mL × 3), filtered, and the precipitate was dried in a vacuum oven at 40° C. for 24 hours to obtain 1-({[4-(dihydroxyboryl)-2-fluorophenyl]carbonyl}oxy)tetrahydropyrrole-2,5-dione.

[0140] (2) 2.81 g of 1-({[4-(dihydroxyboryl)-2-fluorophenyl]carbonyl}oxy)pyrrole-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 mixture was reacted at room temperature overnight.

[0141] 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 H-NMR characterization (see Figure 6 ).

[0142] (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, take the filtrate, remove acetone by vacuum rotary evaporation at 30°C, and dry it 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), and then 1 H-NMR characterization (see Figure 7 ).

[0143] Example 6. Preparation of glucose-responsive insulin (DFPMDCA-insulin)

[0144] This example uses the sugar-responsive molecular linker compound DFPMDCA prepared in Example 5 to prepare sugar-responsive insulin DFPMDCA-insulin. The preparation process is as follows:

[0145] (1) Take 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, place them in a 5 mL sample bottle, add 1.5 mL of anhydrous DMF, and stir until clear. Add 4.0 μL of N,N-diisopropylethylamine dropwise under ice bath, and react at room temperature for 6 hours. During the reaction, the reaction progress was monitored by TLC spot plate, and the developing solvent was methanol-dichloromethane (v / v=1 / 4).

[0146] (2) Take 60.0 mg of recombinant human insulin and place it in a 10 mL sample bottle. Add 1.00 mL of 1× PBS and 5.00 mL of dimethyl sulfoxide in an ice bath and stir until dissolved.

[0147] (3) Place the prepared recombinant human insulin solution in an ice bath, quickly add the DFPMDCA solution that has reacted completely, and adjust the pH to > 8 with 1 M sodium hydroxide solution during the reaction. Allow to react overnight in an ice bath.

[0148] (4) After the reaction is completed, take a regenerated cellulose dialysis bag (3500) of appropriate length and add the reaction solution. Dialyze with deionized water for 12 hours (3×4L). After the dialysis is completed, take out the dialysate and freeze-dry it to obtain the obtained product. Characterize by MALDI-TOF MS (see Figure 8 ).

[0149] Example 7. Preparation of Sugar-Responsive Molecular Linker Compound C5M

[0150] The reaction formula of the sugar-responsive molecular linker compound C5M provided in this example is:

[0151]

[0152] The preparation process is as follows:

[0153] 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 taken. Ethyl acetate was removed by vacuum rotary evaporation at 25°C, and then the mixture was dried in a vacuum drying oven at 40°C for 24 hours to obtain the product C5M. 1 H-NMR characterization (see Fig. 9 ).

[0154] Example 8. Preparation of Sugar Responsive Insulin (C5M-INS)

[0155] 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:

[0156] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborin-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, 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.

[0157] 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.

[0158] The C5F solution that has reacted completely was added dropwise to the recombinant human insulin solution and reacted for 1 hour.

[0159] 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 out and dried to obtain a crude product.

[0160] Then, pure C5M-INS was obtained by HPLC purification (see Fig.10 ).

[0161] Example 9. Preparation of Sugar-Responsive Molecular Linker Compound C5P

[0162] The reaction formula of the sugar-responsive molecular linker compound C5P provided in this embodiment is:

[0163]

[0164] The preparation process is as follows:

[0165] 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 taken. The ethyl acetate was removed by vacuum rotary evaporation at 25°C, and then the mixture was dried in a vacuum drying oven at 40°C for 24 hours to obtain the product C5P. 1 H-NMR characterization (see Fig.11 ).

[0166] Example 10. Preparation of Sugar Responsive Insulin (C5P-INS)

[0167] In this example, the sugar-responsive molecular linker compound C5P prepared in Example 8 was used to prepare sugar-responsive insulin C5P-INS. The preparation process is as follows:

[0168] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborin-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, 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.

[0169] 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.

[0170] The C5F solution that has reacted completely was added dropwise to the recombinant human insulin solution and reacted for 1 hour.

[0171] 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 out and dried to obtain a crude product.

[0172] Then pure C5P-INS was obtained by HPLC purification (see Fig.12 ).

[0173] Example 11: In vitro glucose response verification of glucose-responsive insulin

[0174] 1. In vitro glucose response verification of glucose-responsive insulin (FPTDBA-insulin)

[0175] Using PBS (10 mM, pH = 7.4) as solvent, a 1 mg / mL sugar-responsive insulin (FPTDBA-insulin) (prepared in Example 4) solution was prepared, and 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, 1600 mg / dL). At 0h, 0.25h, 0.5h, 1h, 2h and 4h, 30μL of the complex was aspirated and centrifuged. Then, 4μL of the supernatant was mixed with 200μL of Coomassie Brilliant Blue. The absorbance at 595 nm was measured by an ELISA instrument, and the insulin concentration in the supernatant was calculated using an established standard curve (the insulin concentration-absorbance standard curve is shown in Fig.13 ).

[0176] In a phosphate buffer solution at pH 7.4, the concentration of insulin in the supernatant was determined by the Coomassie brilliant blue method, and the glucose-responsive insulin release performance of the recombinant insulin conjugate (FPTDBA-insulin) was evaluated at different glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, 1600 mg / dL). When glucose was added to the PBS solution, the insulin release rate and the insulin concentration at equilibrium increased significantly. Under high glucose concentrations, free glucose competes for the binding sites of the 1,3-diol structure and the phenylboronic acid analog, enhancing the solubility of insulin and resulting in a faster insulin release rate (see Fig.14 -left picture).

[0177] 2. In vitro glucose response verification of glucose-responsive insulin (DFPMDCA-insulin)

[0178] Using PBS (10 mM, pH = 7.4) as solvent, a 1 mg / mL solution of recombinant insulin conjugate (DFPMDCA-insulin) (prepared in Example 5) was prepared, and 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, 1600 mg / dL). At 0h, 0.25h, 0.5h, 1h, 2h and 4h, 30μL of the complex was aspirated and centrifuged. Then, 4μL of the supernatant was mixed with 200μL of Coomassie Brilliant Blue. The absorbance at 595 nm was measured by an ELISA instrument, and the insulin concentration in the supernatant was calculated using the established standard curve ( Fig.13 ).

[0179] In a phosphate buffer solution at pH 7.4, the concentration of insulin in the supernatant was determined by the Coomassie brilliant blue method, and the glucose-responsive insulin release performance of the recombinant insulin conjugate (DFPMDCA-insulin) was evaluated at different glucose concentrations (0, 30, 50, 70, 100, 200, 400, 800, 1600 mg / dL). When glucose was added to the PBS solution, the insulin release rate and the insulin concentration at equilibrium increased significantly. Under high glucose concentrations, free glucose will compete with the 1,3-diol structure for the binding sites of the phenylboronic acid analog, enhance the solubility of insulin, and have a faster insulin release rate (see Fig.14 -right picture).

[0180] Example 12: Animal efficacy study

[0181] 1. Construction of type 1 diabetes mouse model

[0182] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected, and after fasting overnight, STZ solution was intraperitoneally injected in a dark environment at a dose of 120 mg / kg. Blood glucose of mice was measured one week and two weeks after injection. If blood glucose>300 mg / dL, the model was successfully established. Otherwise, STZ solution was intraperitoneally injected again after two weeks at a dose of 50 mg / kg, and blood glucose of mice was measured again after one week and two weeks.

[0183] 2. In vivo hypoglycemic study in type 1 diabetic mice

[0184] 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 were selected for evaluation of the therapeutic effect. After subcutaneous injection of the sugar-responsive recombinant insulin FPTDBA-insulin (2.4 mg / kg) prepared in Example 4, the blood glucose 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), sugar-responsive recombinant insulin showed a faster blood glucose lowering effect and a significantly more stable blood glucose control ability. No diabetic mice showed hypoglycemia (below 50 mg / dL) (see Fig.15 ).

[0185] Example 13. Preparation of Sugar Responsive Insulin (C5F-INS)

[0186] 1. Preparation of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborohex-2-yl)benzoic acid (sugar-responsive molecular linker compound FPTDBA)

[0187] 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, 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. 1 H-NMR characterization (see Figure 4 ).

[0188] 2. Preparation of insulin analog (C5F-INS) (C5F-INS is the purified product of FPTDBA-INS)

[0189] Take 101 mg of 2-fluoro-4-(5-pentyl-1,3,2-dioxaborin-2-yl)benzoic acid and 90 mg of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate, place them in a 5 mL sample bottle, 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.

[0190] 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.

[0191] The C5F solution that has been reacted completely was added dropwise to the recombinant human insulin solution and reacted for 1 hour. After the reaction was completed, the solution was added dropwise to a precipitant of isopropyl acetate: methyl tert-butyl ether = 4:1 and stirred for one hour. The precipitate was filtered out and dried to obtain a crude product. Then, pure C5F-INS ( Fig.16 ).

[0192] By ESI mass spectrometry ( Fig.17 ), circular dichroism ( Fig.18 )、Scanning electron microscope( Fig.19 )、MALDI-TOF mass spectrometry( Fig. 20 ), solid-state boron NMR spectroscopy ( Fig.21 ) to characterize its structure. It proved that sugar-responsive insulin was successfully prepared.

[0193] Example 14 Comparison of therapeutic effects of sugar-responsive insulin prepared from sugar-responsive molecular linker compounds with different structures

[0194] In the previous study, this example compared the sugar-responsive insulins prepared by sugar-responsive molecular linker compounds with different structures, and found that the sugar-responsive molecular linker compounds with different structures had different effects on the therapeutic effects of sugar-responsive insulin, and there were certain differences in the therapeutic effects. The three most typical ones were screened out, namely, sugar-responsive insulin C5M-INS prepared in Example 8, sugar-responsive insulin C5P-INS prepared in Example 10, and sugar-responsive insulin C5F-INS prepared in Example 13, and animal experiments were carried out. The experimental process is as follows:

[0195] According to Example 12, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose of 400-500 mg / dL were selected for evaluation of the therapeutic effect. After subcutaneous injection of the sugar-responsive insulin C5M-INS prepared in Example 8, the sugar-responsive insulin C5P-INS prepared in Example 10, and the sugar-responsive insulin C5F-INS prepared in Example 13 (12 mg / kg), the blood glucose levels of the mice were detected to examine the blood glucose control effects of the three groups. Fig. 22 shown.

[0196] according to Fig. 22 It can be seen that the three groups of sugar-responsive insulin have good blood sugar control effects, but there are also 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 150h, 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 phenylboronic acid is not conducive to the later therapeutic effect.

[0197] 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 the purified product of FPTDBA-INS) has better blood sugar control effect and more therapeutic advantages.

[0198] Example 15: Rodent efficacy study

[0199] 1. Construction of type 1 diabetes mouse model

[0200] C57BL / 6J mice (purchased from Hangzhou Medical College) were selected, and after fasting overnight, STZ solution was intraperitoneally injected in a dark environment at a dose of 120 mg / kg. Blood glucose of mice was measured one week and two weeks after injection. If blood glucose>300 mg / dL, the model was successfully established. Otherwise, STZ solution was intraperitoneally injected again after two weeks at a dose of 50 mg / kg, and blood glucose of mice was measured again after one week and two weeks.

[0201] 2. In vivo hypoglycemic study in type 1 diabetic mice

[0202] The type I diabetic mouse model was established according to the above method, and type I diabetic mice with blood glucose of 400-500 mg / dL were selected for evaluation of the therapeutic effect. After subcutaneous injection of the glucose-responsive insulin analog (C5F-INS), the blood glucose level of the diabetic mice remained in the normal range of less than 200 mg / dL for more than 214 hours ( Fig.23 ), significantly longer than glargine insulin ( Fig.24 Compared with the commercially available long-acting insulin (insulin glargine), the glucose-responsive insulin analog (C5F-INS) showed a faster blood sugar lowering effect and more stable blood sugar control ability, and no diabetic mice showed hypoglycemia (less than 50 mg / dL).

[0203] 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 (about 13.8 mg / kg in mass units), 40% of the mice developed hypoglycemia, and when glargine insulin controlled blood sugar for a 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 ( Fig.25 ).

[0204] 3. Intraperitoneal glucose tolerance test in type 1 diabetic mice

[0205] A type 1 diabetic mouse model was established according to the above method, and type 1 diabetic mice (n=5) with blood sugar of 400-500 mg / dL were selected to evaluate the glucose control effect. On the first and fifth days after subcutaneous injection of a sugar-responsive insulin analog (C5F-INS) (12 mg / kg), mice were intraperitoneally injected with 1.5 g / kg of glucose, and the blood sugar of the mice was detected and recorded every 15 minutes, and monitored continuously for 2 hours. At the same time, a group of healthy C57BL / 6J mice were selected, and 1.5 g / kg of glucose was intraperitoneally administered, and the blood sugar of the mice was detected and recorded every 15 minutes, and monitored continuously for 2 hours as a control.

[0206] The results showed that when the mice were stimulated with sugar on the first day after administration, their blood sugar levels could be maintained in the normal blood sugar range (50–200 mg / dL), and the blood sugar 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 stimulated with sugar on the fifth day after administration, their blood sugar levels showed a more obvious peak at 15 minutes, but returned to normal (below 200 mg / dL) after 1 hour, and remained stable for 2 hours ( Fig.26 ), the sugar control effect is still better than that of normal mice.

[0207] 4. Verification of in vivo glucose-responsive insulin release in type 1 diabetic mice

[0208] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose of 400-500 mg / dL (n=5) were selected for in vivo glucose-responsive insulin release evaluation. On the first and fifth days 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, and the blood glucose of the mice was detected and recorded every 15 minutes for 2 hours, and blood samples were taken at 0, 15, 30, 60, and 120 minutes (orbital blood, 20 μL, and plasma was obtained after centrifugation).

[0209] The insulin content in plasma was detected by recombinant human insulin ELISA kit.

[0210] The results showed that after the sugar stimulation, the blood glucose of mice showed an obvious peak (higher than 300mg / kg), and the insulin concentration in the plasma also changed synchronously with the change of blood glucose, and both peaked at 30 minutes. On the first day after administration, the peak insulin value increased by 4-5 times compared with the initial value; on the fifth day after administration, the peak insulin value increased by 2-3 times. At the same time, when the blood glucose gradually dropped to the initial value, the insulin concentration in the plasma also decreased and returned to the initial level, verifying the sugar-responsive insulin release (see Fig. 27 ).

[0211] 5. Pharmacokinetics of glucose-responsive insulin analog (C5F-INS) in type 1 diabetic mice

[0212] According to the above method, a type I diabetic mouse model was established, and type I diabetic mice with blood glucose of 400-500 mg / dL (n=5) were selected for pharmacokinetic evaluation. After subcutaneous injection of a glucose-responsive insulin analog (C5F-INS) (12 mg / kg), blood samples were collected at predetermined time points (orbital blood, 20 μL, and plasma was collected after centrifugation). The blood samples were continuously monitored for 17 days (see Fig.28 ).

[0213] At the same time, a group of healthy C57BL / 6J mice were selected and intravenously administered with 1 nmol / kg of glucose-responsive insulin analog (C5F-INS). Blood samples were collected at predetermined time points (orbital blood, 20 μL, and plasma was collected after centrifugation). The mice were monitored continuously for 21 days (see Fig.28 ).

[0214] The plasma insulin concentration-time curve data were analyzed by non-compartmental model using Winnonlin. The half-life of C5F-INS in diabetic mice was 79.0±17.7 hours after subcutaneous injection, and 93.7.0±9.5 hours after intravenous injection in healthy mice, which is 1000 times that of normal human insulin (half-life of about 5 minutes).

[0215] 6. Biosafety evaluation of glucose-responsive insulin analog (C5F-INS) in type 1 diabetic mice

[0216] A type 1 diabetic mouse model was established according to the above method, and type 1 diabetic mice (n=5) with blood glucose of 400-500 mg / dL were selected for biosafety evaluation. Blood samples were collected from mice before administration. Blood samples were collected on the 7th day after subcutaneous injection of a sugar-responsive insulin analog (C5F-INS) (12 mg / kg). Blood-related biochemical indices and routine blood tests were performed. 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 Fig.29 ).

[0217] The embodiments described in the present invention are all preferred embodiments and features. Any person skilled in the art can make some changes and modifications based on the essence of the description of the present invention. These changes and modifications are also considered to be 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; the sugar-responsive molecular linker 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.

2. 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.

3. The compound according to claim 1, characterized in that The phenylboronic acid or phenylboronic acid analogue is modified with a carboxyl group; including one or more of the following:

4. The compound according to claim 3, 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.

5. The compound according to claim 4, characterized in that The alcohol compound is a compound with a diol structure, including one or more of the following:

6. The compound according to claim 5, characterized in that The alcohol compound is a 1,3-diol compound.

7. The method for preparing the sugar-responsive molecular linker compound according to any one of claims 1 to 6, 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.

8. 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 6 and insulin.

9. 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 6; (2) Add insulin solution and react to obtain the product.

10. Use of the sugar-responsive molecular linker compound according to any one of claims 1 to 6 for preparing a reagent for linking and releasing insulin in a glucose-responsive manner.

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