阅读说明：本技术 基于Kdo的糖脂衍生物及其制备和抗菌应用 (Kdo-based glycolipid derivatives, their preparation and their antibacterial applications ) 是由 冯颖乐 柴永海 刘爱云 张琦 于 2021-01-08 设计创作，主要内容包括：本发明公开了一种基于Kdo的糖脂衍生物及其制备和抗菌应用,所述衍生物的结构式为：式中R～1为脂肪族氨基(即CH-3(CH-2)-nNH-,n＝1～17),R～2＝-NH-3～+Cl～-；或者R～1为-OH或-O～-NH-4～+,R～2为RCONH-(R为C-(1～18)的饱和或者不饱和烷基)。本发明中的糖脂与已报道的糖脂相比,是基于革兰式阴性菌来源的重要单糖Kdo(3-脱氧-D-甘露-2-辛酮糖酸)制备的非天然糖脂类化合物,该类化合物可作为新型的抗菌物质。(The invention discloses a Kdo-based glycolipid derivative, a preparation method and an antibacterial application thereof, wherein the structural formula of the derivative is as follows: in the formula R 1 Is an aliphatic amino group (i.e. CH) 3 (CH 2 ) n NH‑，n＝1～17)，R 2 ＝‑NH 3 + Cl ‑ (ii) a Or R 1 is-OH or-O ‑ NH 4 + ，R 2 Is RCONH- (R is C) 1～18 Saturated or unsaturated alkyl groups). Compared with the reported glycolipids, the glycolipid is an unnatural glycolipid compound prepared based on an important monosaccharide Kdo (3-deoxy-D-manno-2-octulosonic acid) from gram-negative bacteria, and the compound can be used as a novel antibacterial substance.)

1. A Kdo-based glycolipid derivative characterized in that it has the following structural formula:

in the formula, R¹Is an aliphatic amino group, R²is-NH₃ ⁺Cl^-Wherein said aliphatic amino group is CH₃(CH₂)_nNH-, and n is an integer of 1 to 17; or R¹is-OH or-O^-NH₄ ⁺，R²Is RCONH-, wherein R is C_1～18Saturated or unsaturated alkyl groups.

2. A method of preparing a Kdo-based glycolipid derivative according to claim 1, in which R is¹Is an aliphatic amino group, R²is-NH₃ ⁺Cl^-The method is characterized in that:

(1) taking methanol and water as reaction media, reacting the compound 1 and alkali for 1-24 hours at 0-50 ℃ according to the molar ratio of 1: 1-10, and separating and purifying the product to obtain a compound 2;

(2) adding the compound 2, aliphatic amine, a condensing agent and N, N-diisopropylethylamine into N, N-dimethylformamide according to the molar ratio of 1: 1-2: 1-3: 1.5-2.5, reacting at 0-40 ℃ for 3-24 hours, and separating and purifying a product to obtain a compound 3; wherein, the structural formula of the aliphatic amine is CH₃(CH₂)_nNH₂In the formula, n is an integer of 1-17;

(3) dissolving a compound 3 in a polar solvent, adding palladium carbon, introducing hydrogen, stirring at room temperature for 1-12 hours to obtain a reduction product, filtering to remove the palladium carbon, and acidifying the obtained solution with a hydrogen chloride aqueous solution to obtain a target compound I;

3. the method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (1), the alkali is sodium hydroxide or lithium hydroxide.

4. The method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (2), the condensing agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate or a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1-2: 1.

5. The method of preparing Kdo-based glycolipid derivatives according to claim 2, characterized in that: in the step (3), the polar solvent is ethanol or methanol.

6. A method of preparing a Kdo-based glycolipid derivative according to claim 1, in which R is¹is-OH or-O^-NH₄ ⁺，R²Is RCONH-, R is C_1～18Is characterized in that:

(1) dissolving the compound 1 and organic phosphine in a molar ratio of 1: 1-5 in a mixed solution of tetrahydrofuran and water, reacting at 40-90 ℃ for 3-24 hours, cooling the reaction solution to room temperature, concentrating to remove the solvent, and performing column chromatography to obtain a compound 4;

(2) adding the compound 4, aliphatic carboxylic acid, a condensing agent and N, N-diisopropylethylamine into N, N-dimethylformamide according to the molar ratio of 1: 1-2: 1-3: 1-2, reacting for 3-24 hours at 0-40 ℃, and separating and purifying a product to obtain a compound 5;

or adding the compound 4, pentafluorophenol ester and N, N-diisopropylethylamine into N, N-dimethylformamide according to the molar ratio of 1: 1-2, reacting for 3-24 hours at 0-60 ℃, and separating and purifying the product to obtain a compound 5;

the structural formulas RCOOH and pentafluorophenol ester of the above aliphatic carboxylic acidIn which R is C_1～18Saturated or unsaturated alkyl groups of (a);

(3) dissolving a compound 5 in a mixed solution of dichloromethane and methanol, adding an alkaline aqueous solution into the mixed solution under stirring at room temperature, wherein the molar ratio of the compound 5 to alkali is 1: 1-8, reacting for 3-6 hours under stirring at room temperature, neutralizing a product with acidic ion exchange resin after the reaction is finished, and filtering and concentrating to obtain a target compound II; further treating the compound II with ammonia water, and concentrating to obtain a target compound III;

7. the method of preparing Kdo-based glycolipid derivatives according to claim 6, characterized in that: in the step (1), the organic phosphine is triphenylphosphine or trimethylphosphine.

8. The method of preparing Kdo-based glycolipid derivatives according to claim 6, characterized in that: in the step (2), the condensing agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate or a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole in a molar ratio of 1-2: 1.

9. The method of preparing Kdo-based glycolipid derivatives according to claim 6, characterized in that: in the step (3), the alkali is sodium hydroxide or lithium hydroxide.

10. Use of a Kdo-based glycolipid derivative according to claim 1, in the preparation of an antibacterial material, said bacterium being a gram-positive or gram-negative bacterium.

Technical Field

The invention belongs to the technical field of synthesis of glycolipid compounds, and particularly relates to a glycolipid derivative taking a scarce monosaccharide Kdo (3-deoxy-D-manno-2-octulosonic acid) in organisms such as gram-negative bacteria, plants and algae as a framework. The compounds can be used as potential novel antibacterial compounds, and are used for inhibiting the growth of gram-positive bacteria and gram-negative bacteria.

Background

Natural glycolipids have the advantages of low toxicity, degradability, reproducibility, environmental friendliness and the like, and natural glycolipids represented by sophorolipid, trehalose glycolipids and rhamnolipids can reduce surface and interface tension, promote the formation of detergent foam and wetting ability and are widely applied to cosmetics, medicines, environmental protection and energy-saving technologies. Importantly, glycolipids also induce the formation of channels or ion channels in cell membranes, destroying membrane integrity and permeability. Thus, glycolipids also have antibacterial, antifungal, viral and mycoplasma action. In addition, glycolipids can also be used to modulate the activity of enzymes and to increase or inhibit the activity of enzymes (carbohydrate res.2015,416, 59.).

Nevertheless, the natural glycolipids have microscopic heterogeneity, and the research on physicochemical properties and functions of natural glycolipids with definite structures has great limitation, and the research is mainly focused on the research on some common glycolipids such as sophorolipids. Studies based on chemically synthesized glycolipids have also been performed only on the basis of simple glucose, mannose, lactose, etc. (chem. rev.2016,116, 1693.).

Kdo, 3-deoxy-D-manno-2-octulosonic acid, is an important component of the outermost lipopolysaccharide layer of gram-negative bacteria. Introduction of Kdo is a critical step in lipopolysaccharide synthesis during lipopolysaccharide synthesis. Meanwhile, a large number of studies have shown that two derivatives of Kdo (2-deoxy- β -Kdo and 8-amino-2, 8-dideoxy- β -Kdo) have very good inhibitory activity on enzymes critical for lipopolysaccharide synthesis in vitro assays. However, due to the strong water solubility, the compounds are difficult to penetrate cell membranes to reach the cells for inhibitory activity. To solve this problem, there are several groups that hopefully screen compounds with good inhibitory effect on gram-negative bacteria by derivatization with Kdo (nat. prod. rep.2010,27, 1618-. For example, Claesson et al introduced amino acids and dipeptides at position 8 of 8-amino-2, 8-dideoxy-. beta. -Kdo, wherein the dipeptide derivatives introduced through the N-terminus showed a better inhibitory effect against several gram-negative bacteria (J.Med.chem.1987,30, 2309-. In addition, other terminal Kdo derivatives (Carbohydr. Res.1987,166,233-251) or 8-position Kdo derivatives have been used for inhibition assays of key enzymes in lipopolysaccharide synthesis. However, even compounds having inhibitory activity against enzymes are not necessarily able to achieve growth inhibition of bacteria (J.Med.chem.1989,32, 1069-1074.).

Disclosure of Invention

The invention aims to provide a kind of glycolipid derivative based on Kdo, and a preparation method and application of the glycolipid derivative.

For the above purpose, the structural formula of the Kdo-based glycolipid derivative employed in the present invention is shown below:

in the formula, R¹Is an aliphatic amino group, R²is-NH₃ ⁺Cl^-The aliphatic amino group is CH₃(CH₂)_nNH-, and n is an integer of 1 to 17; or R¹is-OH or-O^-NH₄ ⁺，R²Is RCONH-, wherein R is C_1～18Saturated or unsaturated alkyl groups.

When R in the above formula is¹Is an aliphatic amino group, R²is-NH₃ ⁺Cl^-The glycolipid derivative can be prepared by the following method:

1. taking methanol or water as a reaction medium, reacting the compound 1 and alkali at a molar ratio of 1: 1-10 at 0-50 ℃ for 1-24 hours, and separating and purifying a product to obtain a compound 2, wherein the reaction equation is as follows:

2. reacting compound 2 with an aliphatic amine (CH)₃(CH₂)_nNH₂Wherein N is an integer of 1 to 17), a condensing agent, and N, N-Diisopropylethylamine (DIPEA) in a molar ratio of 1:1 to 2:1 to 3:1.5 to 2.5, adding to N, N-Dimethylformamide (DMF), and adding to DMF at 0Reacting at the temperature of-40 ℃ for 3-24 hours, separating and purifying the product to obtain a compound 3, wherein the reaction equation is as follows:

3. dissolving a compound 3 in a polar solvent, adding palladium carbon, introducing hydrogen, stirring at room temperature for 1-12 hours to obtain a reduction product, filtering to remove the palladium carbon, and acidifying the obtained solution with an aqueous solution of hydrogen chloride to obtain a target compound I, namely the glycolipid derivative, wherein the reaction equation is as follows:

when R in the above formula is¹is-OH or-O^-NH₄ ⁺，R²Is RCONH-, wherein R is C_1～18The glycolipid derivative is prepared by the following method:

1. dissolving the compound 1 and organic phosphine in a molar ratio of 1: 1-5 in a mixed solution of tetrahydrofuran and water, reacting at 40-90 ℃ for 3-24 hours, cooling the reaction solution to room temperature, concentrating to remove the solvent, and performing column chromatography to obtain a compound 4, wherein the reaction equation is as follows:

2. adding the compound 4, aliphatic carboxylic acid, a condensing agent and N, N-Diisopropylethylamine (DIPEA) into DMF according to the molar ratio of 1: 1-2: 1-3: 1.5-2.5, reacting for 3-24 hours at 0-40 ℃, and separating and purifying a product to obtain a compound 5; or adding the compound 4, pentafluorophenol ester and N, N-Diisopropylethylamine (DIPEA) into DMF according to the mol ratio of 1: 1-2, reacting for 3-24 hours at 0-60 ℃, and separating and purifying the product to obtain a compound 5. The reaction equation is as follows:

the structural formulas RCOOH and pentafluorophenol ester of the above aliphatic carboxylic acidIn which R is C_1～18Saturated or unsaturated alkyl groups.

3. Dissolving a compound 5 in a mixed solution of dichloromethane and methanol, adding an alkaline aqueous solution into the mixed solution under stirring at room temperature, wherein the molar ratio of the compound 5 to the alkali is 1: 1-8, reacting for 3-6 hours under stirring at room temperature, neutralizing a product with acidic ion exchange resin after the reaction is finished, and filtering and concentrating to obtain a target compound II. The target compound II can be further treated by ammonia water and concentrated to obtain the ammonium salt thereof, namely the target compound III, and the specific reaction equation is as follows:

in the above preparation method, the alkali is specifically sodium hydroxide, lithium hydroxide, etc.; the condensing agent is benzotriazole-1-yl-oxy tripyrrolidinyl phosphorus hexafluorophosphate (PyBOP) or a mixture of 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) in a molar ratio of 1-2: 1; the organic phosphine is specifically trimethyl phosphine or triphenyl phosphine; the polar solvent is specifically alcohol solvent such as ethanol and methanol.

The glycolipid derivative can be prepared into an antibacterial material for inhibiting the growth of gram-positive bacteria and gram-negative bacteria.

The invention has the following beneficial effects:

the invention is based on that the glycolipid compound can realize the antibacterial effect by physically destroying the structure of a bacterial membrane, the Kdo derivative has the inhibitory activity on the key enzyme of gram-negative bacteria, the membrane penetrating capacity of the Kdo derivative is improved by introducing an alkyl chain on an 8-amino-2, 8-di-deoxy-beta-Kdo skeleton, and the obtained glycolipid derivative, particularly a cationic compound, has good to moderate inhibitory effect on gram-positive bacteria or gram-negative bacteria.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the final product in example 1.

FIG. 2 is a nuclear magnetic hydrogen spectrum of the final product in example 2.

FIG. 3 is a nuclear magnetic hydrogen spectrum of the final product in example 3.

FIG. 4 is a nuclear magnetic hydrogen spectrum of the final product in example 4.

FIG. 5 is a nuclear magnetic hydrogen spectrum of the final product in example 5.

FIG. 6 is a graph comparing the bacteriostatic effects of the final product of example 3 on E.coli and P.aeruginosa.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

1. 100mg (0.36mmol) of Compound 1 was dissolved in 1mL of methanol, 3.6mL of a 0.5mol/L aqueous solution of lithium hydroxide was added dropwise thereto with stirring at room temperature, the resulting mixture was stirred at room temperature for 2 hours, neutralized to neutrality with an acidic ion exchange resin, filtered and concentrated to give Compound 2(87mg, 0.36mmol, yield 100%) which was used as a crude product for the subsequent reaction.

2. 87mg (0.36mmol) of Compound 2, 116mg (0.43mmol) of stearyl amine were dissolved in 4mL of DMF, 89mg (0.46mmol) of EDCI, 63mg (0.47mmol) of HOBt and 108. mu.L (0.62mmol) of DIPEA were added to the solution successively with stirring at room temperature, and after stirring at room temperature for 8.5 hours, the solvent was removed by concentration, and the resulting syrup was taken up in CH₂Cl₂Dissolving and then adding H₂O washing, collecting organic phase and adding Na₂SO₄Drying, filtering, concentrating, and performing column Chromatography (CH)₂Cl₂MeOH ═ 10:1, v/v) gave compound 3-1(80mg, 0.16mmol, yield 44%)。

The structural characterization data of the obtained compound 3-1 are:¹H NMR(400MHz,DMSO-d₆)δ7.41(t,J＝5.8Hz,1H),5.28(d,J＝6.2Hz,1H),4.69(d,J＝5.4Hz,1H),4.34(d,J＝4.7Hz,1H),4.25(d,J＝6.2Hz,1H),3.87(d,J＝7.0Hz,1H),3.71(d,J＝4.3Hz,1H),3.57(dd,J＝12.8,2.3Hz,1H),3.28–3.14(m,3H),3.00(dd,J＝12.8,6.4Hz,1H),2.01(dd,J＝12.4,4.6Hz,1H),1.81(td,J＝12.1,6.5Hz,1H),1.42(s,2H),1.23(s,30H),0.85(t,J＝6.6Hz,3H)；¹³C NMR(100MHz,DMSO)δ170.01,73.08,67.97,65.89,65.80,54.05,38.49,31.27,29.08,29.00,28.93,28.74,28.68,27.03,26.39,22.08,13.94；MALDI-TOF MS:[M+Na]⁺theoretical value 521.3679, calculated value 521.3854.

3. 80mg (0.16mmol) of Compound 3-1 was dissolved in 5mL of methanol, 17mg of 10% Pd/C was added to the solution with stirring, and after deoxygenation under reduced pressure, hydrogen gas was introduced into the reaction flask and the reaction was stirred at room temperature for 12 hours. After completion of the reaction, Pd/C was removed by filtration, and 0.5mL of 1mol/L aqueous HCl solution was added to the filtrate, followed by concentration to give Compound I-1 as a white solid (31mg, 0.06mmol, yield 32%).

The structural characterization data of the obtained compound I-1 are:¹H NMR(400MHz,DMSO-d₆) δ 8.05(t, J ═ 5.8Hz,2H),7.88(t, J ═ 5.8Hz,1H),4.30(dd, J ═ 6.0,1.5Hz,1H),3.86(td, J ═ 7.8,3.0Hz,1H),3.72(d, J ═ 2.8Hz,1H),3.44(ddd, J ═ 11.7,4.8,2.8Hz,1H),3.23(dd, J ═ 7.7,1.0Hz,1H), 3.15-3.06 (m,1H), 3.06-2.97 (m,1H), 2.82-2.69 (m,1H), 2.03-1.81 (m,2H),1.39(t, J ═ 6.9, 2.81 (m,1H), 1.81(m,2H), 1.87 (m,1H), see fig. 1.87 (t, J ═ 6.9, 2H), 1H), 1.81(m, 1H);¹³C NMR(100MHz,DMSO-d₆)δ170.84,76.40,72.61,65.97,65.81,65.59,42.81,38.62,31.33,29.15,29.10,29.08,29.05,28.81,28.75,28.15,26.47,22.12,13.95；MALDI-TOF MS:[M+H]⁺theoretical value 473.3949, found:473.3700.

example 2

1. Dissolving 200mg (0.73mmol) of compound 1 in 7mL of a mixed solution of tetrahydrofuran and deionized water in a volume ratio of 6:1, adding 526mg (1.78mmol) of triphenylphosphine, and heating the obtained mixture to 60 ℃ for reaction for 7 h; after the reaction solution was cooled to room temperature, the solvent was removed by concentration, the resulting syrup was dissolved in deionized water and washed three times with ethyl acetate, and the aqueous phase was concentrated to give compound 4(172mg, 0.68mmol, yield 96%) as a white solid.

The structural characterization data for compound 4 obtained are:¹H NMR(400MHz,Methanol-d₄)δ4.61–4.53(m,1H),4.30–4.18(m,2H),3.94(d,J＝2.5Hz,1H),3.93–3.87(m,1H),3.64–3.54(m,1H),3.49(dd,J＝8.5,0.8Hz,1H),3.01(dd,J＝13.4,3.4Hz,1H),2.93(dd,J＝13.4,5.7Hz,1H),2.20–2.09(m,2H),1.30(t,J＝7.1Hz,3H).

2. 132mg (0.53mmol) of Compound 4, 305mg (0.68mmol) of pentafluorophenol octadecanecarboxylic acid ester, and 111. mu.L (0.64mmol) of DIPEA were added to 3mL of DMF, and reacted at 50 ℃ for 8 hours; cooling the solution to room temperature, concentrating under reduced pressure to remove the solvent, and separating by column Chromatography (CH)₂Cl₂MeOH ═ 10:1, v/v) gave compound 5-1(113mg, 0.22mmol, yield 32%).

The structural characterization data of the obtained compound 5-1 are:¹H NMR(400MHz,Chloroform-d)δ7.09(dd,J＝7.8,4.5Hz,1H),4.53(d,J＝6.24Hz,1H),4.22(q,J＝7.1Hz,2H),4.05(s,1H),4.00–3.85(m,2H),3.62(ddt,J＝12.2,5.4,2.5Hz,1H),3.44(d,J＝8.9Hz,1H),3.26(dt,J＝14.5,4.2Hz,1H),2.36–2.12(m,4H),1.69–1.54(m,2H),1.36–1.12(m,33H),0.93–0.83(m,3H)；¹³C NMR(100MHz,CDCl₃)δ176.55,172.11,74.81,72.42,68.83,66.62,66.20,61.57,42.92,36.49,31.90,29.70,29.69,29.68,29.64,29.55,29.36,29.34,29.32,29.01,25.70,22.67,14.18,14.10；MALDI-TOF MS:[M+Na]⁺theoretical value 538.3720, found value 538.3184.

3. 75mg (0.14mmol) of compound 5-1 was dissolved in 3mL of a mixed solution of dichloromethane and methanol at a volume ratio of 1:1, 0.65mL of a 0.5mol/L aqueous solution of sodium hydroxide was added thereto with stirring at room temperature, and after stirring at room temperature for 3.5 hours, the product was neutralized with an acidic ion exchange resin Dowex 50WX8, and concentrated by filtration to give the objective compound II-1(59mg, 0.12mmol, yield 86%).

The structural characterization data of the obtained compound II-1 are:¹h NMR (400MHz, DMSO-d6) δ 4.36(d, J ═ 5.3Hz,1H),3.72(d, J ═ 2.8Hz,1H), 3.68-3.60 (m,3H),3.38(ddd, J ═ 11.2,5.8,2.7Hz,1H),3.26(d, J ═ 8.7Hz,1H), 2.88-2.78 (m,1H),2.08(t, J ═ 7.4Hz,2H), 2.00-1.84 (m,2H),1.46(p, J ═ 7.2Hz,2H),1.21(s,26H), 0.89-0.78 (m,3H), see fig. 2;¹³C NMR(100MHz,DMSO)δ172.50,75.62,72.01,67.26,66.61,65.93,42.77,35.53,31.35,29.11,29.06,28.95,28.77,25.40,22.15,14.01；MALDI-TOF MS:[M-H]^-theoretical value 486.3436, found value 486.8383.

And adding excessive ammonia water/MeOH solution into the compound II-1, stirring for 10min, and spin-drying to obtain ammonium carboxylate, namely the target compound III-1.

Example 3

1. 396mg (1.44mmol) of the compound 1 was dissolved in 6mL of methanol, 13.34mL of a 0.5mol/L aqueous lithium hydroxide solution was added dropwise thereto with stirring at room temperature, and after the resulting mixture was stirred at room temperature for 4 hours, it was neutralized to neutrality with an acidic ion exchange resin, and concentrated by filtration to obtain a compound 2(340mg, 1.38mmol, yield 96%) as a crude product which was used directly for the subsequent reaction.

2. 184mg (0.73mmol) of Compound 2, 173mg (0.81mmol) of saturated decatetramine were dissolved in 4mL of DMF, 172mg (0.90mmol) of EDCI, 118mg (0.56mmol) of HOBt and 300. mu.L (1.82mmol) of DIPEA were added to the solution in this order with stirring at room temperature, and after stirring at room temperature for 12 hours, the solvent was removed by concentration, and the resulting syrup was taken up in CH₂Cl₂Dissolving and then adding H₂O washing, collecting organic phase and adding Na₂SO₄Drying, filtering, concentrating, and performing column Chromatography (CH)₂Cl₂MeOH ═ 10:1, v/v) gave compound 3-2(206mg, 0.47mmol, yield 64%).

The structural characterization data of the obtained compound 3-2 are:¹H NMR(400MHz,DMSO-d₆)δ7.41(t,J＝5.7Hz,1H),5.29(d,J＝6.1Hz,1H),4.70(d,J＝5.4Hz,1H),4.35(d,J＝4.6Hz,1H),4.25(d,J＝6.1Hz,1H),3.93–3.83(m,1H),3.71(s,1H),3.56(dd,J＝12.8,2.2Hz,1H),3.46–3.37(m,1H),3.30–3.14(m,2H),3.00(dq,J＝12.5,6.7Hz,1H),2.02(dt,J＝12.4,4.4Hz,1H),1.81(td,J＝12.3,6.5Hz,1H),1.48–1.34(m,2H),1.23(s,22H),0.89–0.81(m,3H)；¹³C NMR(100MHz,DMSO-d₆)δ170.05,75.12,73.12,67.99,65.92,65.83,54.06,38.53,31.32,29.11,29.08,29.05,29.02,28.97,28.78,28.73,27.04,26.43,22.11,13.95；MALDI-TOF MS:[M+Na]⁺theoretical value 465.3053, found value 465.2897.

3. 192mg (0.38mmol) of Compound 3-2 was dissolved in 10mL of methanol, 158mg of 10% Pd/C was added to the solution with stirring, and after deoxygenation under reduced pressure, hydrogen gas was introduced and the mixture was stirred at room temperature for 12 hours. After completion of the reaction, Pd/C was removed by filtration, and 1mL of 1mol/L aqueous HCl solution was added to the filtrate, followed by concentration to give Compound I-2(170mg, 0.038mmol, yield 100%) as a white solid.

The structural characterization data of the obtained compound I-2 are:¹H NMR(400MHz,DMSO-d₆) δ 7.99(s,2H),7.87(t, J ═ 5.7Hz,1H),4.32(d, J ═ 5.3Hz,1H), 4.18-4.04 (m,1H),3.87(td, J ═ 7.7,2.9Hz,1H),3.73(s,1H),3.45(ddd, J ═ 11.8,4.8,2.8Hz,1H),3.23(d, J ═ 7.9Hz,1H), 3.19-2.97 (m,2H), 2.83-2.72 (m,1H), 2.06-1.83 (m,2H), 1.45-1.33 (m,2H),1.23(s,22H), 0.87-0.83 (t, J ═ 6.9,3H), see fig. 3;¹³C NMR(100MHz,DMSO-d₆)δ170.87,76.45,72.62,65.97,65.83,65.60,42.81,38.62,31.33,29.15,29.10,29.06,28.80,28.75,28.18,26.47,22.13,13.98；MALDI-TOF MS:[M+H]⁺theoretical value 417.3323, found value 417.3350.

Example 4

1. Dissolving 200mg (0.73mmol) of compound 1 in 14mL of a mixed solution of tetrahydrofuran and deionized water in a volume ratio of 6:1, adding 300mg (1.14mmol) of triphenylphosphine, and heating the obtained mixture to 60 ℃ for reaction for 12 h; after the reaction solution was cooled to room temperature, the solvent was removed by concentration, the resulting syrup was dissolved in deionized water and washed three times with ethyl acetate, and the aqueous phase was concentrated to give compound 4(152mg, 0.61mmol, yield 84%) as a white solid.

2. 100mg (0.40mmol) of Compound 4, 271mg (0.69mmol) of pentafluorophenol tetradecylcarboxylate, and 111. mu.L (0.64mmol) of DIPEA were added to 3mL of DMF and reacted at 50 ℃ for 8 hours; cooling the solution to room temperature, concentrating under reduced pressure to remove the solvent, and separating by column Chromatography (CH)₂Cl₂MeOH ═ 10:1, v/v) gave compound 5-2(120mg, 0.26mmol, yield 49%).

The structural characterization data of the obtained compound 5-2 are:¹H NMR(400MHz,Chloroform-d)δ6.91(t,J＝6.1Hz,1H),4.52(d,J＝6.7Hz,1H),4.23(q,J＝7.2Hz,2H),4.08(s,1H),4.00–3.86(m,2H),3.71–3.57(m,2H),3.46(d,J＝9.0Hz,2H),3.37–3.25(m,1H),2.36–2.22(m,2H),2.15(td,J＝12.9,6.7Hz,1H),1.70–1.58(m,2H),1.38–1.18(m,25H),0.88(t,J＝6.7Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.94,171.98,77.32,77.00,76.68,74.61,72.36,69.44,66.56,66.30,61.58,43.05,36.40,31.90,30.12,29.68,29.65,29.52,29.34,29.28,25.66,22.67,14.20,14.10；MALDI-TOF MS:[M+Na]⁺theoretical value 482.3094, found value 482.2968.

3. 80mg (0.17mmol) of compound 5-2 was dissolved in 3mL of a mixed solution of dichloromethane and methanol at a volume ratio of 1:1, 0.65mL of a 0.5mol/L aqueous solution of sodium hydroxide was added thereto with stirring at room temperature, and after stirring at room temperature for 3.5 hours, the product was neutralized with an acidic ion exchange resin Dowex 50WX8, and concentrated by filtration to give compound II-2(68mg, 0.16mmol, yield 94%).

The structural characterization data of the obtained compound II-2 are:¹H NMR(400MHz,DMSO-d₆) δ 4.71(s,1H),4.64(s,1H),4.33(s,1H),4.23(s,1H),3.73(s,1H), 3.70-3.62 (m,2H), 3.43-3.26 (m,2H),2.83(ddd, J ═ 13.7,6.7,3.4Hz,1H),2.09(t, J ═ 7.5Hz,2H), 1.97-1.89 (m,2H),1.52(t, J ═ 7.1Hz,3H),1.24(s,22H), 0.90-0.82 (t,3H), see fig. 4;¹³C NMR(100MHz,DMSO)δ172.52,75.51,72.11,67.23,66.60,65.95,42.74,35.51,31.30,29.08,29.03,29.00,28.95,28.90,28.72,25.36,22.10,13.95；MALDI-TOF MS:[M-H]^-theoretical value 430.2810, found value 430.6582.

Example 5

1. This step is the same as step 1 of example 2.

2. 62.5mg (0.25mmol) of Compound 4 and 90mg (0.32mmol) of linoleic acid were dissolved in 2mL of DMF, and 198mg (0.38mmol) of PyBOP and 66. mu.L (0.40mmol) of DIPEA were added to the mixture solution with stirring at room temperature, and after stirring at room temperature for 16 hours, the mixture was concentrated and subjected to silica gel column chromatography to obtain Compound 5-3(71mg, 0.14mmol, 56%).

3. 71mg (0.14mmol) of Compound 5-3 was dissolved in 1.8mL of methanol, 0.45mL of 0.5mol/L aqueous sodium hydroxide solution was added thereto with stirring at room temperature, and after stirring at room temperature for 1.5 hours, it was neutralized with acidic ion exchange resin Dowex 50WX8, and concentrated by filtration to give Compound II-3(44mg, 0.091mmol, yield 65%).

The structural characterization data of the obtained compound II-3 are:¹h NMR (400MHz, MeOD) δ 5.41 to 5.31(m,2H),4.47(d, J ═ 6.1Hz,1H),4.00 to 3.91(m,2H),3.85(dd, J ═ 14.0,2.7Hz,1H),3.66(ddd, J ═ 11.8,4.6,2.9Hz,1H),3.55(d, J ═ 8.7Hz,1H),3.24(dd, J ═ 13.9,4.7Hz,1H),2.35 to 2.09(m,4H),2.09 to 1.97(m,4H),1.74 to 1.55(m,2H),1.47 to 1.21(m,20H),0.92 to 0.88(t, J ═ 6.7, 3H), see fig. 5 Hz.

Example 6

In vitro antibacterial Activity test of glycolipid derivatives prepared in examples 1 and 3

The testing process comprises the following steps: two gram-positive bacteria (enterococcus faecalis E.faecium and staphylococcus aureus S.aureus) and two gram-negative bacteria (escherichia coli E.coli and pseudomonas aeruginosa) are respectively selected, and the inhibition effect of the glycolipid derivative on the bacterial growth (24h) is determined by adopting a multiple dilution method. Two commercially available antibiotics vancomycin and gentamicin were used as positive controls. The MIC value was measured by the Constant broth dilution method (Constant broth dilution method) in the following specific procedures: 384. mu.L of 4mg/mL sample solution was added to 1.808mL of medium, after mixing, 1mL was aspirated into tube 2, after mixing, 1mL was aspirated into tube 3, and this was repeated, and 1mL was discarded from the last 10 th tube, tube 11 being a drug-free growth control. The sample concentrations were formulated to 768, 384, 192, 96, 48, 24, 12, 6, 3, 1.5. mu.g/mL. Diluting newly shake strain 0.2mL by 100 times to 20mL, respectively adding 1mL into the above samples to make the concentration of the bacteria liquid about5 x 105 CFU/mL. The final concentration of the sample was 384, 192, 96, 48, 24, 12, 6, 3, 1.5, 0.75. mu.g/mL, and after incubation in an incubator at 37 ℃ for 24h, the solution was visually checked for turbidity and OD₆₀₀The measured value is equivalent to the negative control and is the MIC value, and the final MIC value is determined by repeating the steps for three times.

And (3) testing results: experimental results show that the compound I-1 has good inhibition effect on gram-positive bacteria, and the inhibition concentrations of the compound I-1 on staphylococcus aureus and enterococcus faecalis are 12 mu g/mL and 6 mu g/mL respectively. And example 3 contains C₁₄The alkyl chain compound I-2 shows good inhibitory activity to gram-positive bacteria and gram-negative bacteria, and the inhibitory concentrations to escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and enterococcus faecalis are as follows in sequence: 24. 24, 24 and 12. mu.g/mL. The transmission electron microscope can observe that after the bacteria and the glycolipid I-2 are incubated for 24h, the membrane structure of the bacteria is destroyed, so that the bacteria die, and the bacterial state before and after partial antibiosis is shown in figure 6.