Stearic acid derivatives and oleic acid derivatives of multi-arm polyethylene glycol

Abstract

Disclosed are multi-arm polyethylene glycol derivatives having structure of formula I or formula VI. Compared with straight chain polyethylene glycol, multi-arm polyethylene glycol has a plurality of terminal groups, thus has a plurality of introducing points of functional groups and can support a plurality of reactive terminal groups, thereby enabling multi-arm polyethylene glycol to have more flexibility and wider range of application. ##STR00001##

Claims

1. A multi-arm polyethylene glycol derivative, wherein the multi-arm polyethylene glycol derivative has a structure of general formula I or general formula VI ##STR00047## wherein: PEG is the same or different and is (CH.sub.2CH.sub.2O).sub.m, m is an integer of 3-250; l is an integer greater than or equal to 1 and less than or equal to 10; X.sub.1, X.sub.2, X.sub.3, and X.sub.4 are independently selected from the group consisting of the following groups: ##STR00048## wherein i is an integer of 0-10; F.sub.1, F.sub.2, F.sub.3, and F.sub.4 are independently selected from the group consisting of the following groups: ##STR00049## and, in general formula I at least one of F.sub.1, F.sub.2, F.sub.3, and F.sub.4 is ##STR00050## or in general formula VI at least one of F.sub.1, F.sub.2, and F.sub.4 is ##STR00051##

2. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula I at least two of F.sub.1, F.sub.2, F.sub.3, and F.sub.4 are ##STR00052## or in general formula VI at least two of F.sub.1, F.sub.2, and F.sub.4 are ##STR00053##

3. The multi-arm polyethylene glycol derivative according to claim 1, wherein the said F.sub.2 and/or F.sub.4 are COOH or ##STR00054##

4. The multi-arm polyethylene glycol derivative according to claim 1, wherein the said X.sub.2 and X.sub.4 are selected from (CH.sub.2).sub.iNH, wherein i is an integer of 1-5.

5. The multi-arm polyethylene glycol derivative according to claim 1, wherein the said X.sub.2 and X.sub.4 are selected from ##STR00055## wherein i is an integer of 1-5.

6. The multi-arm polyethylene glycol derivative according to claim 1, wherein the said X.sub.1 and/or X.sub.3 are (CH.sub.2).sub.iNH, wherein i is an integer of 1-5.

7. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula I of multi-arm polyethylene glycol derivative, at least one of F.sub.1 and F.sub.3 is ##STR00056##

8. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula I of multi-arm polyethylene glycol derivative, both F.sub.1 and F.sub.3 are ##STR00057##

9. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula VI of multi-arm polyethylene glycol derivative, F.sub.1 is ##STR00058##

10. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula I of multi-arm polyethylene glycol derivative, the said F.sub.1 and F.sub.3 are ##STR00059## and F.sub.2 and F.sub.4 are COOH or ##STR00060##

11. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula VI of multi-arm polyethylene glycol derivative, the said F.sub.1 is ##STR00061## and F.sub.2 and F.sub.4 are COOH or ##STR00062##

12. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula I of multi-arm polyethylene glycol derivative, the said F.sub.1, F.sub.2 and F.sub.3 are selected from ##STR00063## and F.sub.4 is selected from COOH and ##STR00064##

13. The multi-arm polyethylene glycol derivative according to claim 1, wherein in general formula VI of multi-arm polyethylene glycol derivative, the said F.sub.1 and F.sub.2 are selected from ##STR00065## and F.sub.4 is selected from COOH and ##STR00066##

14. The multi-arm polyethylene glycol derivative according to claim 1, having a structure of general formula II-X: ##STR00067## ##STR00068##

15. A method for preparation of a multi-arm polyethylene glycol derivative according to claim 1 comprising the following steps: (1) dissolving a multi-arm polyethylene glycol amino-monoacetic acid or multi-arm polyethylene glycol amino-diacetic acid in a solvent, adding thionyl chloride and reacting for 1-10 h to give a multi-arm polyethylene glycol amino-monoacetic acid methyl ester or multi-arm polyethylene glycol amino-diacetic acid methyl ester; (2) dissolving multi-arm polyethylene glycol amino-monoacetic acid methyl ester or multi-arm polyethylene glycol amino-diacetic acid methyl ester in a solvent, adding stearic acid and triethylamine for reaction to give a multi-arm polyethylene glycol stearic acid-monoacetic acid methyl ester or multi-arm polyethylene glycol stearic acid-diacetic acid methyl ester; (3) to multi-arm polyethylene glycol stearic acid-monoacetic acid methyl ester or multi-arm polyethylene glycol stearic acid-diacetic acid methyl ester, adding NaOH to adjust pH value to 10-12, reacting for 1-8 h, filtering, adjusting pH value to 2-3 with hydrochloric acid, extracting, and separating to obtain a multi-arm polyethylene glycol stearic acid-monoacetic acid or multi-arm polyethylene glycol stearic acid-diacetic acid; and optionally, (4) dissolving multi-arm polyethylene glycol stearic acid-monoacetic acid or multi-arm polyethylene glycol stearic acid-diacetic acid in a solvent, adding N-hydroxysuccinimide (NHS) and N,N-dicyclohexylacarbodiimide for reaction to give a multi-arm polyethylene glycol stearic acid-monoacetic acid NHS ester or multi-arm polyethylene glycol stearic acid-diacetic acid NHS ester.

16. A method for preparation of a multi-arm polyethylene glycol derivative according to claim 1 comprising the following steps: (1) dissolving a multi-arm polyethylene glycol amino-monoacetic acid or multi-arm polyethylene glycol amino-diacetic acid in a solvent, adding thionyl chloride and reacting for 1-10 h to give a multi-arm polyethylene glycol amino-monoacetic acid methyl ester or multi-arm polyethylene glycol amino-diacetic acid methyl ester; (2) dissolving multi-arm polyethylene glycol amino-monoacetic acid methyl ester or multi-arm polyethylene glycol amino-diacetic acid methyl ester in a solvent, adding oleoyl chloride and triethylamine for reaction to give a multi-arm polyethylene glycol oleic acid-monoacetic acid methyl ester or multi-arm polyethylene glycol oleic acid-diacetic acid methyl ester; (3) to multi-arm polyethylene glycol oleic acid-monoacetic acid methyl ester or multi-arm polyethylene glycol oleic acid-diacetic acid methyl ester, adding NaOH to adjust pH value to 10-12, reacting for 1-8 h, filtering, adjusting pH value to 2-3 by using hydrochloric acid, extracting, and separating to obtain a multi-arm polyethylene glycol oleic acid-monoacetic acid or multi-arm polyethylene glycol oleic acid-diacetic acid; and optionally, (4) dissolving multi-arm polyethylene glycol oleic acid-monoacetic acid or multi-arm polyethylene glycol oleic acid-diacetic acid in a solvent, adding N-hydroxysuccinimide (NHS) and N,N-dicyclohexylacarbodiimide for reaction to give a multi-arm polyethylene glycol oleic acid-monoacetic acid NHS ester or multi-arm polyethylene glycol oleic acid-diacetic acid NHS ester.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) 4-arm polyethylene glycol amino-monoacetic acid, 4-arm polyethylene glycol amino-diacetic acid used in examples are provided by Beijing JenKem Technology Co., Ltd., other reagents used in the following examples are purchased from Beijing Chemical Reagent Company or other similar public chemical sales companies.

Example 1: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Stearic Acid-Monoacetic Acid

(2) ##STR00035##

(3) 10 g of 4-arm polyethylene glycol (3,000 Da) amino-monoacetic acid was dissolved in 100 mL of anhydrous methanol, placed in an ice-water bath, then 1 mL of thionyl chloride was added dropwise, the reaction was kept for 5 h at room temperature, evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 150 mL of diethyl ether, filtered, the product was dried under vacuum to give 8.5 g of 4-arm polyethylene glycol (3,000 Da) amino-monoacetic acid methyl ester.

(4) .sup.1H NMR (DMSO): 3.51 (m, 272H), 2.95 (m, 6H), 4.13 (s, 2H).

(5) 4.91 g of 4-arm polyethylene glycol (3,000 Da) amino-monoacetic acid methyl ester was dissolved in 100 mL of chloroform, then 0.89 mL of triethylamine and 6.98 g of stearic acid were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue was dissolved by heating after addition of 80 mL of isopropanol and then frozen for precipitation, filtered, evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 80 mL of diethyl ether, filtered, the product was dried under vacuum to give 5.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid methyl ester.

(6) .sup.1H NMR (DMSO): 0.82-0.87 (t, 9H), 3.51 (m, 272H), 4.13 (s, 2H), 7.82 (m, 3H).

(7) To 5.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid methyl ester, 50 mL of deionized water was added, then the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, then the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 100 mL of dichloromethane, and the organic phases were combined, washed twice with 100 mL of saturated saline water with a pH value of 2-3, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 2.5 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid.

(8) .sup.1H NMR (DMSO): 0.82-0.87 (t, 9H), 3.51 (m, 272H), 4.08 (s, 2H), 7.77 (m, 3H).

Example 2: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Stearic Acid-Monoacetic Acid Active NHS Ester

(9) ##STR00036##

(10) 3.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid was dissolved in 30 mL of dichloromethane, then 0.15 g of N-hydroxysuccinimide (NHS) and 0.29 g of N, N-dicyclohexylcarbodiimide (DCC) were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue was dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 2.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid active NHS ester.

(11) .sup.1H NMR (DMSO): 0.82-0.87 (t, 9H), 2.81 (s, 4H), 3.51 (m, 272H), 4.60 (s, 2H), 7.77 (m, 3H).

Example 3: Synthesis of 8-Arm Polyethylene Glycol (20,000 Da) Stearic Acid-Monoacetic Acid

(12) ##STR00037##

(13) 10 g of 8-arm polyethylene glycol (20,000 Da) amino-monoacetic acid was dissolved in 100 mL of anhydrous methanol, placed in an ice-water bath, then 0.3 mL of thionyl chloride was added dropwise, the reaction was kept for 5 h at room temperature, evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 150 mL of diethyl ether, filtered, the product was dried under vacuum to give 9.8 g of 8-arm polyethylene glycol (20,000 Da) amino-monoacetic acid methyl ester.

(14) .sup.1H NMR (DMSO): 3.51 (m, 1818H), 2.95 (m, 14H), 4.13 (s, 2H).

(15) 5 g of 8-arm polyethylene glycol (20,000 Da) amino-monoacetic acid methyl ester was dissolved in 100 mL of chloroform, then 0.27 mL of triethylamine and 2.1 g of stearic acid were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 80 mL of isopropanol and then frozen for precipitation, filtered, evaporated by rotary to remove solvent, the residue obtained was frozen for precipitation after addition of 80 mL of diethyl ether, filtered, the product was dried under vacuum to give 4.8 g of 8-arm polyethylene glycol (20,000 Da) stearic acid-monoacetic acid methyl ester.

(16) .sup.1H NMR (DMSO): 0.82-0.87 (t, 21H), 3.51 (m, 1818H), 4.13 (s, 2H), 7.82 (m, 7H).

(17) To 4.0 g of 8-arm polyethylene glycol (20,000 Da) stearic acid-monoacetic acid methyl ester, then 40 mL of deionized water was added, the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 80 mL of dichloromethane, and the organic phases were combined, washed twice with 80 mL of saturated saline water with a pH value of 2-3, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 80 mL of diethyl ether, filtered, the product was dried under vacuum to give 2.5 g of 8-arm polyethylene glycol (20,000 Da) stearic acid-monoacetic acid

(18) .sup.1H NMR (DMSO): 0.82-0.87 (t, 21H), 3.51 (m, 1818H), 4.08 (s, 2H), 7.77 (m, 7H).

Example 4: Synthesis of 8-Arm Polyethylene Glycol (20,000 Da) Stearic Acid-Monoacetic Acid Active NHS Ester

(19) ##STR00038##

(20) 3.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid was dissolved in 30 mL of dichloromethane, then 0.15 g of N-hydroxysuccinimide (NHS) and 0.29 g of N, N-dicyclohexylcarbodiimide (DCC) were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 2.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-monoacetic acid active NHS ester.

(21) .sup.1H NMR (DMSO): 0.82-0.87 (t, 27H), 2.81 (s, 4H), 3.51 (m, 1818H), 4.60 (s, 2H), 7.77 (m, 7H).

Example 5: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Stearic Acid-Diacetic Acid

(22) ##STR00039##

(23) 10 g of 4-arm polyethylene glycol (3,000 Da) amino-diacetic acid was dissolved in 100 mL of anhydrous methanol, placed in an ice-water bath, then 2 mL of thionyl chloride was added dropwise, the reaction was kept for 5 h at room temperature, evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 150 mL of diethyl ether, filtered, the product was dried under vacuum to give 8.8 g of 4-arm polyethylene glycol (3,000 Da) amino-diacetic acid methyl ester.

(24) .sup.1H NMR (DMSO): 3.51 (m, 272H), 2.95 (m, 4H), 4.13 (s, 4H).

(25) 5 g of 4-arm polyethylene glycol (3,000 Da) amino-diacetic acid methyl ester was dissolved in 100 mL of chloroform, then 0.6 mL of triethylamine and 4.74 g of stearic acid were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 100 mL of isopropanol and then frozen for precipitation, filtered, evaporated by rotary to remove solvent, the residue obtained was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 5.5 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-diacetic acid methyl ester.

(26) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 3.51 (m, 272H), 4.13 (s, 4H), 7.82 (m, 2H).

(27) To 5.0 g of 4-arm polyethylene glycol stearic acid-diacetic acid methyl ester, 50 mL of water was added, then the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 100 mL of dichloromethane, and the organic phases were combined, washed twice with 100 mL saturated saline water with a pH value of 2-3, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue obtained was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 3.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-diacetic acid.

(28) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 3.51 (m, 272H), 4.08 (s, 4H), 7.77 (m, 2H).

Example 6: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Stearic Acid-Diacetic Acid Active NHS Ester

(29) ##STR00040##

(30) 3.0 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-diacetic acid was dissolved in 30 mL of dichloromethane, then 0.30 g of N-hydroxysuccinimide (NHS) and 0.58 g of N, N-dicyclohexylcarbodiimide (DCC) were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 2.2 g of 4-arm polyethylene glycol (3,000 Da) stearic acid-diacetic acid active NHS ester.

(31) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 2.81 (s, 8H), 3.51 (m, 272H), 4.15 (s, 4H), 7.77 (m, 2H).

Example 7: Synthesis of 4-Arm Polyethylene Glycol (10,000 Da) Oleic Acid-Monoacetic Acid

(32) ##STR00041##

(33) 10 g of 4-arm polyethylene glycol (10,000 Da) amino-monoacetic acid methyl ester was dissolved in 120 mL of toluene, the mixture obtained was heated to remove water, then cooled to room temperature, then 0.19 mL of triethylamine was added, and 0.45 mL of oleoyl chloride was added dropwise, the reaction was stirred overnight, then evaporated by rotary to remove solvent, the residue was dissolved by heating after addition of 200 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 7.5 g of 4-arm polyethylene glycol (10,000 Da) oleic acid-monoacetic acid methyl ester.

(34) .sup.1H NMR (DMSO): 0.82-0.87 (t, 9H), 3.51 (m, 909H), 4.15 (s, 2H), 5.32 (t, 6H), 7.82 (m, 3H).

(35) To 5.0 g of 4-arm polyethylene glycol (10,000 Da) oleic acid-monoacetic acid methyl ester, 50 mL of water was added, then the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 100 mL of dichloromethane, and the organic phases were combined, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 3.1 g of 4-arm polyethylene glycol (10,000 Da) oleic acid-monoacetic acid.

(36) .sup.1H NMR (DMSO): 0.82-0.87 (t, 9H), 3.51 (m, 909H), 4.01 (s, 2H), 5.32 (t, 6H), 7.78 (m, 3H).

Example 8: Synthesis of 4-Arm Polyethylene Glycol (10,000 Da) Oleic Acid-Monoacetic Acid Active NHS Ester

(37) ##STR00042##

(38) 3.0 g of 4-arm polyethylene glycol (10,000 Da) oleic acid-monoacetic acid was dissolved in 30 mL of dichloromethane, then 0.90 g of N-hydroxysuccinimide (NHS) and 0.174 g of N, N-dicyclohexylcarbodiimide (DCC) were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 2.2 g of 4-arm polyethylene glycol (10,000 Da) oleic acid-monoacetic acid active NHS ester.

(39) .sup.1H NMR (DMSO): 0.82-0.87 (OH), 3.51 (m, 909H), 2.83 (s, 4H), 4.60 (s, 2H), 5.32 (t, 6H), 7.78 (m, 3H).

Example 9: Synthesis of 8-Arm Polyethylene Glycol (Hexamer Glycerol, 10,000 Da) Oleic Acid-Monoacetic Acid

(40) ##STR00043##

(41) 10 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) amino-monoacetic acid methyl ester was dissolved in 120 mL of toluene, the mixture obtained was heated to remove water, then cooled to room temperature, then 0.44 mL of triethylamine was added, and 1.05 mL of oleoyl chloride was added dropwise, the reaction was stirred overnight, then evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 200 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 8.7 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) oleic acid-monoacetic acid methyl ester.

(42) .sup.1H NMR (DMSO): 0.82-0.87 (t, 21H), 3.51 (m, 909H), 4.15 (s, 2H), 5.32 (t, 14H), 7.82 (m, 7H).

(43) To 5.0 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) oleic acid-monoacetic acid methyl ester, 50 mL of water was added, then the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 100 mL of dichloromethane, and the organic phases were combined, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue obtained was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 3.1 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) oleic acid-monoacetic acid.

(44) .sup.1H NMR (DMSO): 0.82-0.87 (t, 21H), 3.51 (m, 909H), 4.01 (s, 2H), 5.32 (t, 14H), 7.78 (m, 7H).

Example 10: Synthesis of 8-Arm Polyethylene Glycol (Hexamer Glycerol, 10,000 Da) Oleic Acid-Monoacetic Acid Active NHS Ester

(45) ##STR00044##

(46) 3.0 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) oleic acid-monoacetic acid was dissolved in 30 mL of dichloromethane, then 0.90 g of N-hydroxysuccinimide (NHS) and 0.174 g of N, N-dicyclohexylcarbodiimide (DCC) were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 2.7 g of 8-arm polyethylene glycol (hexamer glycerol, 10,000 Da) oleic acid-monoacetic acid active NHS ester.

(47) 1H NMR (DMSO): 0.82-0.87 (t, 21H), 3.51 (m, 909H), 2.83 (s, 4H), 4.60 (s, 2H), 5.32 (t, 14H), 7.78 (m, 7H).

Example 11: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Oleic Acid-Diacetic Acid

(48) ##STR00045##

(49) 10 g of 4-arm polyethylene glycol (3,000 Da) amino-diacetic acid methyl ester was dissolved in 120 mL of toluene, the mixture obtained was heated to remove water, then cooled to room temperature, then 0.46 mL of triethylamine was added, and 1.0 mL of oleoyl chloride was added dropwise, the reaction was stirred overnight, then evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 200 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 8.9 g of 4-arm polyethylene glycol (3,000 Da) oleic acid-diacetic acid methyl ester.

(50) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 3.51 (m, 272H), 4.15 (s, 4H), 5.32 (t, 4H), 7.82 (m, 2H).

(51) To 5.0 g of 4-arm polyethylene glycol (3,000 Da) oleic acid-diacetic acid methyl ester, 50 mL of water was added, then the pH value of the system was adjusted to 12 with 0.5 mol/L of NaOH, the reaction was kept for 2 h at room temperature, filtered, the pH value was adjusted to 2-3 with 1 mol/L of hydrochloric acid, 15% NaCl was added, the reactive liquid was extracted three times with 100 mL of dichloromethane, and the organic phases were combined, dried with anhydrous sodium sulfate, filtered, the filtrate was evaporated by rotary to remove solvent, the residue was frozen for precipitation after addition of 100 mL of diethyl ether, filtered, the product was dried under vacuum to give 2.4 g of 4-arm polyethylene glycol (3,000 Da) oleic acid-diacetic acid.

(52) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 3.51 (m, 272H), 4.01 (s, 4H), 5.32 (t, 4H), 7.78 (m, 2H).

Example 12: Synthesis of 4-Arm Polyethylene Glycol (3,000 Da) Oleic Acid-Diacetic Acid Active NHS Ester

(53) ##STR00046##

(54) 2.0 g of 4-arm polyethylene glycol (3,000 Da) oleic acid-diacetic acid was dissolved in 20 mL of dichloromethane, then 0.20 g of NHS and 0.174 g of DCC were added, the reaction was stirred overnight at room temperature, filtered, evaporated by rotary to remove solvent, the residue obtained was dissolved by heating after addition of 60 mL of isopropanol and then frozen for precipitation, filtered, the product was dried under vacuum to give 1.6 g of 4-arm polyethylene glycol (3,000 Da) oleic acid-diacetic acid active NHS ester.

(55) .sup.1H NMR (DMSO): 0.82-0.87 (t, 6H), 3.51 (m, 272H), 2.83 (s, 8H), 4.60 (s, 4H), 5.32 (t, 4H), 7.78 (m, 2H).