WO2023147789A1

WO2023147789A1 - Chemical synthesis method of chitosan oligosaccharide with controllable polymerization degree

Info

Publication number: WO2023147789A1
Application number: PCT/CN2023/079441
Authority: WO
Inventors: 尹健; 成道泉; 邹小鹏; 王祥传; 胡静; 刘海玉; 朋小军; 王成; 田光宗; 王殿海
Original assignee: 山东京博农化科技股份有限公司; 江南大学
Priority date: 2022-03-15
Filing date: 2023-03-03
Publication date: 2023-08-10
Also published as: CN115215910A

Abstract

Disclosed in the present invention is a chemical synthesis method of chitosan oligosaccharide with a controllable polymerization degree, which belongs to the field of sugar chemistry. The method of the present invention comprises the following steps: (1) using a monosaccharide building block which is connected with an activatable leaving group on an anomeric carbon and contains a C-4 exposed hydroxyl as a substrate, so that the monosaccharide building block is used as both a donor and a receptor; adding an accelerant to carry out a glycosylation reaction, so as to generate chitosan oligosaccharide derivatives with different polymerization degrees; and changing the protection groups of the saccharide building block and the glycosylation reaction conditions to generate fully-protected chitosan oligosaccharide derivatives with different polymerization degrees; and (2) performing deprotection to obtain corresponding chitosan oligosaccharides. The method of the present invention has the advantages of a controllable polymerization degree, simple and convenient steps and a high conversion rate.

Description

A kind of chemical synthesis method of chitosan oligosaccharide with controllable degree of polymerization

technical field

The invention relates to a method for chemically synthesizing chitosan oligosaccharides with a controllable degree of polymerization, belonging to the field of sugar chemistry.

Background technique

Oligochitosan is an oligosaccharide composed of 2 to 20 glucosamine or acetylglucosamine connected by β-1,4 glycosidic bonds. It is widely used in biomedicine, daily chemical industry, agriculture, food and other fields. Compared with macromolecular chitosan, oligochitosan has attracted attention because of its good properties such as low viscosity, high water solubility, high biocompatibility and biodegradability. Oligochitosan has many physiological activities, including antibacterial, antitumor, antioxidation, immune regulation, blood sugar regulation, etc. Oligochitosan has low cytotoxicity and is easily absorbed and utilized by the human small intestine. Different degrees of polymerization and acetylation directly affect their biological activity.

In recent years, chitosan oligosaccharides are mostly prepared by extracting chitin from the exoskeleton of crustaceans, followed by deacetylation and hydrolysis. The extraction of chitin can be summarized as "three removals", which are deproteinization, desalination, and decolorization. Chitin needs to be treated with acid, alkali and reducing agent for a long time. Usually, deacetylation is completed by reacting with high-concentration lye, and the degradation of sugar chains includes acid hydrolysis, oxidative degradation, sodium borate degradation, ultrasonic method, microwave method, photodegradation method and enzymatic method. However, chitosan oligosaccharides prepared by degrading chitosan will have residues of heterogeneous sources, such as proteins, salts, etc. to a certain extent. This caused certain difficulties for subsequent purification.

With the rise of sugar chemistry in recent years, the chemical synthesis of oligosaccharides has become a hotspot. The degree of polymerization of chitosan oligosaccharide synthesis can be controlled by chemical methods to meet its requirements in different fields.

Contents of the invention

Technical problem: The technical problem to be solved by the present invention is to synthesize chitosan oligosaccharides (β-1,4-oligo-glucosamine) with different degrees of polymerization, and realize polymerization by changing the protecting group strategy and glycosylation reaction conditions of sugar building blocks The degree is controllable. Prior to this, it was only necessary to design and synthesize a sugar building block with an activatable leaving group attached to the anomeric carbon and a C-4 exposed hydroxyl group.

Technical solutions:

The invention provides a chemical synthesis method of oligochitosan with a controllable degree of polymerization. The oligochitosan is formed by connecting 2 to 20 glucosamines through β-1,4 glycosidic bonds. The synthesis method includes the following process:

(1) Using the monosaccharide block shown in formula I as a substrate, a self-assembled glycosylation reaction occurs under the action of an accelerator: after the leaving group of the anomeric carbon of a sugar block is activated, another sugar is linked The C-4 hydroxyl of the building block generates fully protected chitosan oligosaccharides with different degrees of polymerization;

Among them, R is selected from ethylthio (SEt), p-tolylthio (STol), phenylthio (SPh), trifluoroacetimide ester (OCNPhCF ₃ ), dibenzyloxyphosphate (OP (OBn) ₂ ) and other leaving groups that can be activated; R ₁ is selected from phthaloyl (Phth), trichloroethoxycarbonyl (Troc), benzylmethoxycarbonyl (Cbz) and other amino protecting groups; R ₂ , R ₃ are independently selected from protecting groups such as acetyl (Ac), benzoyl (Bz), levulinyl (Lev), benzyl (Bn) or naphthylidene (Nap); R ₂ and R ₃ are different is benzyl;

(2) Carry out full deprotection reaction, obtain chitosan oligosaccharide product.

In one embodiment of the present invention, in the monosaccharide building block shown in formula I, it contains a leaving group that can be activated at the anomeric carbon, and C-4 contains an exposed hydroxyl group, making it both as a donor body, and as an acceptor; for the anomeric carbon, some leaving groups (R) that can be activated can be selected, such as common thioglucosides, phosphoglycosides, glycosyl sulfoxides, imidoglycosides, etc. ; For the amino protecting group (R ₁ ), optional phthaloyl (Phth), trichloroethoxycarbonyl (Troc) or benzyloxycarbonyl (Cbz); for C-3, C-6 hydroxyl protection The groups (R ₂ , R ₃ ) can be protected with acetyl, benzyl, 2-naphthylidene, and p-methoxybenzyl, respectively.

In one embodiment of the present invention, R ₃ is preferably acetyl (Ac), benzoyl (Bz), levulinyl (Lev), naphthylidene (Nap).

In one embodiment of the present invention, the degree of polymerization of oligosaccharides is changed by changing the protecting groups of sugar building blocks, reaction time, concentration and different accelerators. The different protecting groups of the sugar building block will affect the glycosylation reactivity of the sugar building block, and then affect the degree of polymerization of the link. Moreover, when the electron-donating ability of the sugar block protecting group is strong, the reactivity will be too high and a large number of by-products will be produced, and chitosan oligosaccharides with a required degree of polymerization cannot be obtained.

In one embodiment of the present invention, the accelerators are NIS and TfOH. In the present invention, the accelerator can be one or both of NIS, TfOH, and MeOTf. It has been proved by experiments that when NIS and TfOH are used as accelerators together, the self-assembly glycosylation conversion rate is the highest, which is higher than that of using any one alone. has significant advantages.

In one embodiment of the present invention, the self-assembly glycosylation reaction is carried out in an organic solvent, and the substrate concentration in the self-assembly glycosylation reaction is 0.02-0.5M. The organic solvent is preferably dichloromethane (DCM).

Further, in one embodiment of the present invention, in the self-assembly glycosylation reaction, the amount of organic solvent used is: 12-15 mL/mmol monosaccharide block.

In one embodiment of the present invention, the self-assembly glycosylation reaction also includes adding Molecular sieve. The addition of molecular sieves is to remove the water in the reaction solvent as much as possible and reduce the occurrence of side reactions.

In one embodiment of the present invention, in the self-assembly glycosylation reaction, the molar ratio of NIS to monosaccharide building blocks is 1.5-3.0:1; the molar ratio of TfOH to monosaccharide building blocks is 0.1-0.3:1 .

Further, in one embodiment of the present invention, in the self-assembly glycosylation reaction, the molar ratio of NIS to monosaccharide building blocks is 1.8-2.5:1; the molar ratio of TfOH to monosaccharide building blocks is 0.15- 0.25:1.

In one embodiment of the present invention, in the self-assembly glycosylation reaction, the temperature is controlled at -78° C., and the time is 1-5 hours.

In one embodiment of the present invention, the self-assembled glycosylation reaction comprises the following process: dissolving the sugar block in DCM, adding Molecular sieves, the mixture was cooled to -78°C; NIS and TfOH were added and reacted for 4 hours; after the reaction was completed, triethylamine was added to quench the reaction, the reaction solution was diluted with DCM and washed with saturated sodium bicarbonate, extracted three times with DCM, and the organic phase It was dried with anhydrous Na ₂ SO ₄ , concentrated, and purified by column chromatography.

In one embodiment of the present invention, when R is ethylthio (SEt), _R is phthaloyl (Phth) _, _R is acetyl (Ac), R is acetyl (Ac) , the corresponding monosaccharide building block is prepared by the following synthetic route:

Using compound 1 glucosamine hydrochloride as the starting material, remove the hydrochloric acid under the action of a base, and add a weak base to protect the amino group at the C-2 position, and the obtained compound is fully acetylated under the action of pyridine and acetic anhydride to obtain the compound 2;

Compound 2 generates glucosinolate compound 3 with ethanethiol under the action of Lewis acid;

The obtained glucosinolate compound 3 is deacetylated under alkaline conditions and then combined with benzaldehyde dimethyl acetal under the catalysis of p-toluenesulfonic acid to generate 4,6-benzylidene protected compound 4;

Then 3-OH is protected with acetyl to obtain fully protected compound 5;

Compound 5 removes the benzylidene group in acetic acid aqueous solution to obtain compound 6;

Compound 6 reacted with acetic anhydride under the action of pyridine to generate monosaccharide building block 7.

In one embodiment of the present invention, when R is ethylthio (SEt), _R is phthaloyl (Phth), _R is benzyl (Bn), _R is acetyl (Ac) , the corresponding monosaccharide building block is prepared by the following synthetic route:

Compound 4 was benzylated at the third position to obtain 8, opened the 4,6-benzylidene group under acidic conditions to obtain 9, and finally selectively acetylated at the sixth position to obtain the monosaccharide building block 10.

In one embodiment of the present invention, the process of self-assembly glycosylation reaction synthesis controllable polymerization degree chitosan oligosaccharide is as follows:

n is 2-20.

In one embodiment of the present invention, the structure of the obtained chitosan oligosaccharide product after full deprotection is as follows:

n is 2-20.

In one embodiment of the present invention, n may specifically be 2-6, or 2-3.

In one embodiment of the present invention, chitosan oligosaccharides with different degrees of polymerization are obtained by performing a full deprotection reaction on the fully protected chitosan oligosaccharide.

Among them, the radical and phthaloyl group are removed under the action of hydrazine hydrate; the benzyl group is removed under the action of palladium carbon; other protecting groups can be removed by existing methods.

Beneficial effect:

The invention can prepare chitosan oligosaccharides with different polymerization degrees through chemical synthesis, and realize the controllable polymerization degree by changing the protecting group strategy of the sugar building blocks and the glycosylation reaction conditions.

Description of drawings

Figure 1 is the mass spectrometry characterization of chitosan oligosaccharide A.

Fig. 2 is the HPLC analysis of chitosan oligosaccharide A.

Figure 3 is the mass spectrum characterization of chitosan oligosaccharide B.

Fig. 4 is the HPLC analysis of chitosan oligosaccharide B.

Figure 5 is the mass spectrum characterization of chitosan oligosaccharide C.

Fig. 6 is the HPLC analysis of chitosan oligosaccharide C.

Figure 7 is the mass spectrum characterization of chitosan oligosaccharide D.

Figure 8 is the mass spectrum characterization of chitosan oligosaccharide E.

Figure 9 is the mass spectrum characterization of chitosan oligosaccharide F.

Detailed ways

The beneficial effect of the present invention is further described through examples below, it should be understood that these examples are only for the purpose of illustration, and in no way limit the scope of the present invention. Those who do not indicate specific conditions in the embodiments, carry out according to conventional conditions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

The calculation method of the yield in the present invention is "product (mol)/reaction substrate (mol)*100%". The nuclear magnetic data of the compound in the present invention is measured by Bruker Ascend 400M/600M nuclear magnetic resonance instrument at 25 ℃; Mass spectrum data is measured by TSQ quantum Ultra EMR; The unit of concentration (c) is g/100mL.

Example 1:

The synthesis route of sugar block 7 is as follows:

Using glucosamine hydrochloride 1 as the starting material, the hydrochloric acid is removed under the action of a base, and a weak base is added to protect the amino group at the C-2 position. The resulting compound is fully acetylated under the action of pyridine and acetic anhydride to obtain compound 2 . Compound 2 reacted with ethanethiol under the action of Lewis acid to generate glucosinolate compound 3; the obtained glucosinolate compound was deacetylated under alkaline conditions and then reacted with benzaldehyde dimethyl acetal to generate 4 under the catalysis of p-toluenesulfonic acid, 6-benzylidene protected compound 4; then 3-OH was protected with acetyl to obtain fully protected compound 5, and compound 5 was removed in acetic acid aqueous solution to obtain compound 6. Compound 6 reacted with acetic anhydride under the action of pyridine to generate the desired sugar building block 7. The sugar building block 7 self-assembles and connects under the activation of iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) to form a chitosan oligosaccharide derivative mixture.

Specific experimental operations and steps:

Compound 2: Glucosamine hydrochloride 1 (40.0 g, 185.5 mmol) was dissolved in methanol (640 mL). add methanol Sodium (10.0g, 186mmol), after stirring at room temperature for 1h, the mixture was cooled to 0°C, phthalic acid (55.0g, 370mmol) was added to the reaction solution, stirred at 0°C for 3h, concentrated and co- Steam twice. The mixture was dissolved in pyridine (180 mL), acetic anhydride (128 mL, 1.36 mol) was added at 0° C., and the reaction was stirred at room temperature for 8 h. After the reactants disappeared, the solution was cooled to 0°C, and washed with saturated aqueous sodium bicarbonate solution. The mixture _was extracted 3 times with DCM, the organic phase was dried over anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (petroleum ether/ethyl acetate, 4:1) gave compound 2 (68.3 g, 77%). ¹ H NMR (400MHz, Chloroform-d) δ: 7.87-7.75(m, 4H, Ar-H), 6.54(d, 1H, H-1, J=8.9Hz), 5.89(t, 1H, H-4 ),5.22(t,1H,H-4),4.49(t,1H,H-2),4.37,4.16(m,2H,H-6a,H-6b),4.03(m,1H,H-5 ),2.13(s,3H,OAc),2.05(s,3H,OAc),2.01(s,3H,OAc),0.88(s,3H,OAc).

Compound 3: Compound 2 (95.0 g, 200 mmol) was dissolved in DCM (400 mL), and Molecular sieves, and to the mixture was added ethanethiol (34.0 mL, 452 mmol). Boron trifluoride diethyl ether (50 mL, 0.4 mol) was added dropwise under . The mixture was stirred overnight at room temperature. After the reactants disappeared, the mixture was cooled to 0 °C, and triethylamine was added to quench the reaction. The reaction solution was washed with saturated aqueous sodium bicarbonate solution. DCM was extracted ₃ times, and the organic phase was dried over anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (petroleum ether/ethyl acetate, 4:1) gave compound 3 (90 g, 95%). ¹ H NMR (400MHz, Chloroform-d) δ7.94–7.64 (m, 4H, Ar), 5.84 (dd, J＝10.2, 9.1Hz, 1H, H-3), 5.49 (d, J＝10.6Hz, 1H,H-1),5.19(dd,J=10.2,9.1Hz,1H,H-4),4.40(t,J=10.4Hz,1H,H-2),4.32(dd,J=12.3,4.9 Hz,1H,H-6a),4.18(dd,J=12.3,2.3Hz,1H,H-6b),3.95–3.86(m,1H,H-5),2.79–2.57(m,2H,SEt) ,2.11(s,3H,OAc),2.04(s,3H,OAc),1.87(s,3H,OAc),1.22(t,J=7.4Hz,3H,SEt).

Compound 4: Compound 3 (34g, 68mmol) was dissolved in methanol (140mL), sodium methoxide (1.8g, 34mmol) was added, and stirred at room temperature for 1h. H ⁺ ) neutralization. After filtration and concentration, the crude product was azeotroped twice with toluene. The obtained crude product was dissolved in DMF (140mL), benzaldehyde dimethyl acetal (12.24mL, 81.6mmol) and p-toluenesulfonic acid monohydrate (1.6g, 8.16mmol) were added, and the reaction was stirred at 65°C for 10h. The reaction was quenched with triethylamine. After concentration, it was separated and purified by column chromatography (petroleum ether/ethyl acetate, 3:1) to obtain compound 4 (25.0 g, 82%). ¹ H NMR (400MHz, Chloroform-d) δ8.00–7.32 (m, 9H, Ar), 5.57 (s, 1H, ArCH), 5.41 (d, J=10.6Hz, 1H, H-1), 4.66 ( dd,J=10.0,8.9Hz,1H,H-3),4.40(dd,J=10.3,4.8Hz,1H,H-6a),4.33(t,J=10.3Hz,1H,H-2), 3.81(t,J=10.1Hz,1H,H-6b),3.70(td,J=9.5,4.8Hz,1H,H-5),3.61(t,J=9.1Hz,1H,H-4), 2.69(ttd,J=12.5,7.5,5.0Hz,2H.SEt),1.25(t,J=7.2Hz,3H,SEt).

Compound 5: Dissolve compound 4 (5.5g, 12.5mmol) in pyridine (25mL), add acetic anhydride (2.4mL, 25.0mmol) at 0°C, and stir at room temperature for 5h. After the reactant is completely consumed, The reaction solution was washed with saturated sodium bicarbonate solution. DCM was extracted three times, the organic phase was dried over _anhydrous _Na2SO4 and concentrated. Column chromatography separation and purification (stone Oil ether/ethyl acetate, 4:1), to obtain compound 5 (5.89g, 88%). [α] ²² _D ＝-1.72(c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.98–7.70(m,4H,Ar),7.53–7.33(m,5H,Ar), 5.92(t, J=9.4Hz, 1H, H-3), 5.58(d, J=10.6Hz, 1H, H-1), 5.55(s, 1H, ArCH), 4.45–4.41(m, 1H, H -5), 4.37(dd, J=10.6, 9.9Hz, 1H, H-2), 3.86–3.75(m, 3H, H-4, H-6a, H-6b), 2.69(ttd, J=12.5 ,7.4,5.1Hz,2H,SEt), 1.90(s,3H,OAc),1.21(t,J=7.4Hz,3H,SEt). ¹³ C NMR(151MHz,Chloroform-d)δ170.12,167.80,167.42, 136.94, 134.43, 134.18, 131.76, 131.25, 129.17, 128.26, 126.26, 123.71, 123.65, 101.68, 81.78, 79.31, 70.60, 70.54, 68.68, 54.33, 24.44, 20 .57, 14.93.

Compound 6: Compound 5 (3.26 g, 6.74 mmol) was dissolved in 80% acetic acid aqueous solution (60.0 mL), and the reaction solution was stirred at 65° C. for 3 h. After concentration, it was purified by column chromatography (dichloromethane/methanol, 50:1) to obtain compound 6 (2.55 g, 96%). [α] ²² _D = 4.27 (c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.97–7.66 (m, 4H, Ar), 5.72 (dd, J = 10.2, 8.9Hz, 1H ,H-3),5.55(d,J=10.5Hz,1H,H-1),4.30(t,J=10.4Hz,1H,H-2),4.01(dd,J=12.1,3.3Hz,1H ,H-6a), 3.90(dd,J=12.0,4.7Hz,1H,H-6b),3.81(t,J=9.4Hz,1H,H-4),3.71(ddd,J=9.8,4.6, 3.2Hz,1H,H-5),2.69(qq,J=12.6,7.4Hz,2H,SEt),1.94(s,3H,OAc),1.21(t,J=7.4Hz,3H,SEt). ¹³ C NMR (151MHz, Chloroform-d) δ171.42, 167.96, 167.42, 134.47, 134.27, 131.68, 131.24, 123.69, 81.10, 79.76, 74.55, 70.21, 62.51, 53.82, 24.41, 20.70, 1 4.97.

Compound 7: Dissolve compound 6 (2.35g, 5.94mmol) in pyridine (15.0mL), add acetic anhydride (0.84mL, 8.91mmol) at 0°C, stir at 0°C for 12h, and wash the reaction solution with saturated bicarbonate Sodium solution for washing. DCM was extracted three times, the organic phase was dried over _anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (petroleum ether/ethyl acetate, 2:1) gave compound 7 (1.65 g, 64%). [α] ²² _D =-7.34 (c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.81 (ddd, 4H, Ar), 5.70 (dd, J = 10.3, 8.9Hz, 1H, H-3), 5.50(d, J=10.5Hz, 1H, H-1), 4.53–4.45(m, 1H, H-6a), 4.41(dd, J=12.2, 2.3Hz, 1H, H-6b ), 4.31(t, J=10.4Hz, 1H, H-2), 3.78(ddd, J=10.0, 4.7, 2.3Hz, 1H, H-5), 3.66(dd, J=10.0, 8.9Hz, 1H ¹³ C NMR (101MHz, Chloroform-d) δ171.70, 171.23, 167.87, 167.42, 134.45, 134.28, 131.65, 131.23, 123.69, 81.20, 77.36, 74.08, 69.63, 63.31, 53.67, 24.52, 20.91, 20.69, 14.94.

Example 2

The synthesis of sugar block 10 is as follows:

In compound 4, the benzyl group at the third position was obtained to obtain 8, the 4,6-benzylidene group was opened under acidic conditions to obtain 9, and finally the compound 10 was obtained by selective acetylation at the sixth position.

Specific experimental operations and steps:

Compound 8: Compound 4 (5.27g, 11.5mmol) was dissolved in DMF (40.0mL), at 0°C, 60% sodium hydride (688mg, 17.2mmol) was added and stirred for 0.5h, and benzyl bromide was added to the reaction (2.04 mL, 17.2 mmol). React at room temperature for 4 h, until the reactant completely disappears, add water at 0°C to quench the reaction, extract three times with dichloromethane, dry the organic phase with anhydrous Na ₂ SO ₄ , and concentrate. Separation and purification by column chromatography (petroleum ether/ethyl acetate, 10:1) gave compound 8 (5.3 g, 85%). [α] ²² _D ＝43.18(c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.91–7.65(m,4H,Ar),7.61–7.37(m,5H,Ar),7.05 –6.89(m,5H,Ar),5.66(s,1H,ArCH),5.38(d,J=10.6Hz,1H,H-1),4.83(d,J=12.3Hz,1H,ArCH),4.54 (d,J=12.3Hz,1H,ArCH),4.49(dd,J=10.0,8.9Hz,1H,H-3),4.44(dd,J=10.3,4.8Hz,1H,H-6a),4.38 -4.28(m,1H,H-2),3.87(dd,J=9.7,5.3Hz,1H,H-6b),3.83(d,J=3.6Hz,1H,H-4),3.73(td, J＝9.8, 4.9Hz, 1H, H-5), 2.68(qq, J＝12.5, 7.5Hz, 2H, SEt), 1.19(t, J＝7.4Hz, 3H, SEt).

Compound 9: Compound 8 (5.00 g, 9.10 mmol) was dissolved in 80% acetic acid aqueous solution (90.0 mL), and the reaction solution was stirred at 65° C. for 5 h. After concentration, it was purified by column chromatography (dichloromethane/methanol, 50:1) to obtain compound 9 (3.7 g, 100%). [α] ²² _D ＝34.67(c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.86–7.67(m,4H,Ar),7.13–6.94(m,5H,Ar),5.31 (d,J=10.3Hz,1H,H-1),4.71(d,J=12.2Hz,1H,ArCH),4.55(d,J=12.1Hz,1H,ArCH),4.31(dd,J=10.2 ,8.5Hz,1H,H-3),4.23(t,J=10.2Hz,1H,H-2),3.96(dd,J=11.9,3.5Hz,1H,H-6a),3.87(dd,J =12.0,4.6Hz,1H,H-6b),3.80(dd,J=9.8,8.5Hz,1H,H-4),3.58(ddd,J=9.6,4.5,3.4Hz,1H,H-5) , 2.64(ttd, J＝12.6, 7.5, 5.1Hz, 2H, SEt), 1.17(t, J＝7.4Hz, 3H, SEt). ¹³ C NMR (101MHz, Chloroform-d) δ168.24, 167.50, 137.93, 134.05 ,133.97,131.60,128.34,128.27,127.85,127.68,123.65,123.36,81.42,80.31,79.37,74.65,72.23,62.67,54.61,24.22,14.93.

Compound 10: Dissolve compound 9 (2.58g, 5.60mmol) in pyridine (18.6mL), add acetic anhydride (0.53mL, 5.60mmol) at 0°C, stir at 0°C for 10h, and wash the reaction solution with saturated bicarbonate Sodium solution for washing. DCM was extracted three times, the organic phase was dried over _anhydrous _Na2SO4 and concentrated. Column chromatography separation and purification (petroleum ether/ethyl acetate, 4:1), Compound 10 (1.4 g, 51%) was obtained. [α] ²² _D ＝17.01(c 1.0, CHCl ₃ ). ¹ H NMR (400MHz, Chloroform-d) δ7.87–7.65(m,4H,Ar),7.08–6.93(m,5H,Ar),5.28 (d,J=10.1Hz,1H,H-1),4.74(d,J=12.1Hz,1H,ArCH),4.55(d,J=12.1Hz,2H,ArCH,H-6a),4.32(dd ,J=12.2,1.8Hz,1H,H-6b),4.30–4.26(m,1H,H-3),4.22(t,J=10.1Hz,1H,H-2),3.64(d,J= 2.1Hz, 2H, H-4, H-5), 2.74–2.53(m, 2H, SEt), 2.14(s, 3H, OAc), 1.18(t, J＝7.4Hz, 3H, SEt). ¹³ C NMR (151MHz, Chloroform-d) δ172.01, 168.14, 167.51, 137.93, 134.02, 133.92, 131.60, 128.25, 127.92, 127.59, 123.61, 123.33, 81.41, 79.51, 78.05, 74.7 2,71.68,63.37,54.54,24.23,20.91, 14.89.

Embodiment 3: the preparation of chitosan oligosaccharide derivative

A mixture of two different chitooligosaccharide derivatives was formed by self-assembled glycosylation of two different sugar building blocks. In this example, two different glucosinolates are used, the amino protecting group is phthaloyl, and C-4 and C-6 are respectively protected by acetyl or benzyl. The synthesized chitosan derivatives were characterized by MALDI-TOF and HPLC.

The general steps of glycosidation are shown in the following formula:

Specific experimental operations and steps:

Sugar block 7 (108 mg, 0.25 mmol) was dissolved in DCM (3.10 mL), added Molecular sieves, the mixture was cooled to -78°C. NIS (111 mg, 0.49 mmol) and TfOH (4 μL, 0.05 mmol) were added to the reaction solution. After 4 h of reaction, triethylamine was added to quench the reaction. The reaction solution was diluted with DCM, washed with saturated sodium bicarbonate, and extracted three times with DCM. The organic phase was dried over _anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (dichloromethane/methanol, 50:1) gave mixture A (86 mg); the mass spectrum characterization data of chitooligosaccharide A are shown in Figure 1, and the HPLC analysis data are shown in Figure 2.

Sugar block 10 (98 mg, 0.2 mmol) was dissolved in DCM (2.5 mL). join in Molecular sieves, the mixture was cooled to -78°C, NIS (90.8mg, 0.40mmol) and TfOH (3.2μL, 0.04mmol) were added to the reaction solution, and after 4h of reaction, triethylamine was added to quench the reaction, diluted with DCM and saturated Washed with sodium bicarbonate, extracted three times with DCM, the organic phase was dried over anhydrous Na ₂ SO ₄ and concentrated. Separation and purification by column chromatography (dichloromethane/methanol, 50:1) yielded mixture B (67 mg); the mass spectrum characterization data of chitooligosaccharide B are shown in Figure 3, and the HPLC analysis data are shown in Figure 4.

Sugar block 7 (113 mg, 0.26 mmol) was dissolved in DCM (3.20 mL), added Molecular sieves, the mixture was cooled to -15°C, MeOTf (80 μL, 0.78 mmol) was added to the reaction solution, after 48 hours of reaction, triethylamine was added to quench the reaction, diluted with DCM, washed with saturated sodium bicarbonate, extracted three times with DCM, organic The phase was dried over anhydrous Na ₂ SO ₄ , concentrated shrink. Separation and purification by column chromatography (dichloromethane/methanol, 50:1) gave mixture C (66 mg); the mass spectrum characterization data of chitooligosaccharide C are shown in Figure 5, and the HPLC analysis data are shown in Figure 6.

Sugar block 7 (54 mg, 0.12 mmol) was dissolved in DCM (1.50 mL), added Molecular sieves, the mixture was cooled to -78°C. NIS (55mg, 0.25mmol) and TMSOTf (4.8μL, 0.02mmol) were added to the reaction solution. After 4h of reaction, triethylamine was added to quench the reaction. The reaction solution was diluted with DCM, washed with saturated sodium bicarbonate, and extracted three times with DCM. , the organic phase was dried over _anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (dichloromethane/methanol, 50:1) gave mixture D (44 mg); the mass spectrometry data of chitooligosaccharide D are shown in Figure 7.

Composite result:

The mass spectrometry and HPLC analysis of the oligosaccharide product A obtained by the self-assembly connection of the sugar block 7 under the action of NIS and TfOH are shown in Figure 1 and Figure 2 (it should be noted that the chitosan oligosaccharide derived after self-assembly glycosylation The anomeric carbon at the reducing end will be hydrolyzed, because the glucosinolate cannot return to the original state after being activated, but it does not affect the experiment and subsequent deprotection). Through HPLC and mass spectrometry analysis, disaccharide 4%, trisaccharide 11%, tetrasaccharide 30%, pentasaccharide 24%, hexasaccharide 19%, heptasaccharide 7%, octasaccharide 2%, nonasaccharide 1%.

The mass spectrometry analysis and HPLC analysis of the oligosaccharide product B obtained by the self-assembly and connection of the sugar block 10 under the action of NIS and TfOH are shown in Figure 3 and Figure 4 . Through HPLC and mass spectrometry analysis, disaccharides were 44%, trisaccharides 20%, tetrasaccharides 9%, pentasaccharides 19%, hexasaccharides 5%.

The mass spectrometry analysis and HPLC analysis of the oligosaccharide product C obtained by the self-assembly and connection of sugar block 7 under the action of MeOTf are shown in Figure 5 and Figure 6 . According to HPLC and mass spectrometry analysis, 11% of disaccharides, 16% of trisaccharides, 56% of tetrasaccharides, 12% of pentasaccharides, 1% of hexasaccharides.

The mass spectrometry analysis of the oligosaccharide product D obtained by the self-assembly and connection of sugar block 7 under the action of NIS and TMSOTf is shown in Figure 7. After purifying the product, 60% of monosaccharide, 35% of disaccharide and 5% of trisaccharide are obtained.

The specific results are shown in Table 1.

Table 1 The influence of different sugar building blocks and different conditions on the degree of polymerization

Embodiment 4: full deprotection prepares chitosan oligosaccharide

Dissolve chitosan oligosaccharide A in methanol (21mg) Dissolve chitosan oligosaccharide A (21mg) in methanol (1.0mL), add hydrazine hydrate (1.0mL), reflux at 65°C for two days, concentrate and filter , Purified with C18 reverse column to obtain full chitosan oligosaccharide A' (6.5 mg). (Chitooligosaccharide A can remove acetyl and phthaloyl groups simultaneously under the condition of hydrazine hydrate).

Chitooligosaccharide B (28 mg) was dissolved in DCM/t-Butanol (1:1, v/v, 1.0 mL), and palladium carbon (Pd/C) was added and stirred, and the mixture was heated under 1 atmosphere of H ₂ React under pressure for three days, concentrate and filter to obtain the semi-deprotected product. The semi-deprotected product was dissolved in methanol (1.0mL), added hydrazine hydrate (1.0mL), refluxed at 65°C for two days, concentrated and filtered, and purified with a C18 reverse column to obtain a fully deprotected chitosan oligosaccharide B' (5.9 mg)

Comparative example 1

The synthetic route of sugar block 11 is as follows:

The specific operation is as follows:

Under the protection of argon, compound 8 (3.55g, 6.68mmol) was dissolved in DCM (70mL), and at 0°C, triethylsilane (12.8mL, 80mmol) and boron trifluoride diethyl ether (1.68ml , 13.3mmol), stirred at room temperature for 3h, until the reactant completely disappeared, adding triethylamine to quench the reaction, the resulting reaction solution was washed with saturated aqueous sodium bicarbonate, extracted three times with dichloromethane, and the organic phase was washed with anhydrous Na ₂ Dry over SO ₄ and concentrate. Separation and purification by column chromatography (petroleum ether/ethyl acetate, 5:1) gave Compound 11 (2.9 g, 88%). [α] ²² _D ＝+32.38(c 1.0,CH ₃ Cl). ¹ H NMR(400MHz,Chloroform-d)δ7.87–7.64(m,4H,Ar),7.43–7.31(m,5H,Ar) ,7.13–6.92(m,5H,Ar),5.27(d,J=10.0Hz,1H,H-1),4.75(d,J=12.2Hz,1H,ArCH),4.64(d,J=11.9Hz ,1H,ArCH),4.60(s,1H,ArCH),4.54(d, J=12.1Hz,1H,ArCH),4.29(d,J=10.2Hz,1H,H-2),4.28–4.18(m,1H,H-3),4.03–3.83(m,1H,H-6a ),3.84(d,J=4.9Hz,1H,H-4),3.77(dd,J=10.1,5.2Hz,1H,H-6b),3.68(dt,J=9.8,5.0Hz,1H,H -5),2.73–2.52(m,2H,SEt),1.16(t,J＝7.4Hz,3H,SEt). ¹³ C NMR(151MHz,Chloroform-d)δ168.10,167.55,138.14,137.60,133.94,133.85 ,131.68,128.55,128.19,127.93,127.84,127.47,123.56,123.30,81.20,79.60,74.57,74.49,73.83,70.94,54.42,23.99,14.92.

Dissolve sugar block 11 (134 mg, 0.25 mmol) in DCM (3.10 mL), add Molecular sieves, the mixture was cooled to -78°C. NIS (111 mg, 0.49 mmol) and TfOH (4 μL, 0.05 mmol) were added to the reaction solution. After 4 h of reaction, triethylamine was added to quench the reaction. The reaction solution was diluted with DCM, washed with saturated sodium bicarbonate, and extracted three times with DCM. The organic phase was dried over _anhydrous _Na2SO4 and concentrated. Column chromatography separation and purification (dichloromethane/methanol, 50:1) to obtain mixture E; the mass spectrum characterization data of chitosan oligosaccharide E are shown in Figure 8, and the molecular weight of the obtained product components does not match the expected molecular weight, indicating that 3,6- Dibenzylglucose building blocks are not suitable for glycosylation in this method.

Dissolve sugar block 11 (220 mg, 0.41 mmol) in DCM (5.10 mL), add Molecular sieves, the mixture was cooled to -15°C, MeOTf (128 μL, 1.24 mmol) was added to the reaction solution, after 48 h of reaction, triethylamine was added to quench the reaction, diluted with DCM, washed with saturated sodium bicarbonate, extracted three times with DCM, organic The phase was dried over _anhydrous _Na2SO4 and concentrated. Separation and purification by column chromatography (dichloromethane/methanol, 50:1) gave mixture F (188mg). The results of mass spectrometry analysis are shown in Figure 9. From Figure 9, it can be seen that the 3,6-dibenzylglucose building block also cannot effectively carry out the glycosylation reaction to synthesize chitosan products.

Through the comparison of Comparative Example 1 and Example 4 (Table 1), it is illustrated that due to the strong electron-donating effect of benzyl, the activity of the 4,6-dibenzyl-protected sugar building block 11 is improved, resulting in the The assembly reaction process is too active and produces a large number of by-products.

Claims

A chemical synthesis method of oligochitosan with controllable degree of polymerization is characterized in that it comprises the following process:

(1) Using the monosaccharide block shown in formula I as a substrate, a self-assembled glycosylation reaction occurs under the action of an accelerator: after the leaving group of the anomeric carbon of a sugar block is activated, another sugar is linked The C-4 hydroxyl of the building block generates fully protected chitosan oligosaccharides with different degrees of polymerization;

Wherein, R is selected from ethylthio, p-tolylthio, phenylthio, trifluoroacetimide ester, dibenzyloxyphosphate; R is selected from phthaloyl, trichloroethoxycarbonyl, Benzylmethoxycarbonyl; R 2 and R 3 are independently selected from acetyl, benzoyl, levulinyl, benzyl or naphthylidene; R 2 and R 3 are not benzyl at the same time;

(2) Carry out full deprotection reaction, obtain chitosan oligosaccharide product.
The chemical synthesis method according to claim 1, characterized in that, the accelerator is NIS and TfOH.
The chemical synthesis method according to claim 1, characterized in that the self-assembly glycosylation reaction is carried out in an organic solvent, and the concentration of the substrate in the self-assembly glycosylation reaction is 0.02-0.5M.
The chemical synthesis method according to claim 3, characterized in that, in the self-assembled glycosylation reaction, the amount of the organic solvent is: 12-15mL/mmol monosaccharide block.
The chemical synthesis method according to claim 1, characterized in that, in the self-assembled glycosylation reaction, also includes adding Molecular sieve.
The chemical synthesis method according to claim 2, characterized in that, in the self-assembled glycosylation reaction, the mol ratio of NIS to the monosaccharide building block is 1.5-3.0:1; the mol ratio of TfOH to the monosaccharide building block is 0.1-0.3:1.
The chemical synthesis method according to claim 6, characterized in that, in the self-assembled glycosylation reaction, the mol ratio of NIS to the monosaccharide building block is 1.8-2.5:1; the mol ratio of TfOH to the monosaccharide building block is 0.15-0.25:1.
The chemical synthesis method according to claim 1, characterized in that, in the self-assembly glycosylation reaction, the temperature is controlled at -78°C and the time is 1-5h.
According to the chemical synthesis method described in any one of claims 1-8, the process of self-assembled glycosylation reaction synthesis controllable polymerization degree chitosan oligosaccharide is:

n is 2-20.
The chemical synthesis method according to claim 9, characterized in that: n is 2-6.