Branched hetero polyethylene glycol and intermediate

Abstract

A branched hetero polyethylene glycol according to the present invention is represented by the formula [1]: ##STR00001##
wherein X and Y represent each an atomic group containing at least a functional group which reacts with a functional group present in a bio-functional molecule to form a covalent bond and the functional group contained in the atomic group X and the functional group contained in the atomic group Y are different from each other; s is an integer of 2 to 8, which represents the number of polyethylene glycol chains; n is the number of average added moles for the polyethylene glycol chain and 20n2000; and E is a branching linker moiety having s-valent bonding valency for the polyethylene glycol chains and having monovalent bonding valency for the functional group Y.

Claims

1. A branched hetero polyethylene glycol represented by the formula [1]: ##STR00032## wherein X represents an atomic group containing at least a functional group which reacts with a functional group present in a bio-functional molecule to form a covalent bond and Y represents an atomic group consisting essentially of a functional group which reacts with a functional group present in a bio-functional molecule to form a covalent bond and a bonding moiety to be bonded to a branching linker moiety E selected from the group consisting of an ester bond, a urethane bond, an amide bond, an ether bond, a carbonate bond, a saturated hydrocarbon group containing a secondary amino group, a single bond, and a saturated hydrocarbon group, and the functional group contained in the atomic group X and the functional group contained in the atomic group Y are different from each other; wherein as a combination of the functional group X and the functional group Y: Y is a group selected from a maleimide group, a vinylsulfone group, an iodoacetamide group, an active carbonate group, an amino group, and an aminoxy group when X is an acetal group, Y is a group selected from a maleimide group, a vinylsulfone group, an acetylene group, and an azide group when X is an aldehyde group, Y is a group selected from an acetal group, an active carbonate group, an acetylene group, and an azide group when X is a maleimide group, a vinylsulfone group, or an iodoacetamide group, Y is a group selected from an acetal group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an acetylene group, and an azide group when X is an active ester group or an active carbonate group, Y is a group selected from an acetal group, an acetylene group, and an azide group when X is an amino group or an aminoxy group, Y is a group selected from an acetal group, an aldehyde group, a maleimide group, a vinylsulfone group, an iodoacetamide group, an active ester group, an active carbonate group, an amino group, and an aminoxy group when X is an acetylene group or an azide group, wherein the active ester group is a group represented by the following formula [9] which is reactive with nucleophilic groups: ##STR00033## wherein R.sub.2 is a phenyl group, a 3-pyridyl group, a succinimide group, a 2-benzothiazole group, or a 1-benzotriazole group and W and W are each independently a linker comprising a covalent bond selected from the group consisting of an ester bond, a urethane bond, an amide bond, an ether bond, a carbonate bond, a saturated hydrocarbon group containing a secondary amino group, a single bond, and a saturated hydrocarbon group; s is an integer of 2 or 4, which represents a number of polyethylene glycol chains; n is a number of average added moles for the polyethylene glycol chain and 20n2000; and E is a branching linker moiety represented by the formula [A] or [B]: ##STR00034## wherein each D is (CH.sub.2).sub.aO(CH.sub.2).sub.b, (CH.sub.2).sub.aOC(O)NH(CH.sub.2).sub.b, OC(O)NH(CH.sub.2).sub.b, (CH.sub.2).sub.aNHC(O)O(CH.sub.2).sub.b, (CH.sub.2).sub.aC(O)NH(CH.sub.2).sub.b, or (CH.sub.2).sub.aNHC(O)(CH.sub.2).sub.b, or a single bond, a=1-12, b=1-12, v=0 or 2, and w=0 or 1.

2. The branched hetero polyethylene glycol according to claim 1, which is represented by the formula [3] or [4]: ##STR00035## wherein v=0 or 2 and w=0 or 1.

3. The branched hetero polyethylene glycol according to claim 1, wherein as the combination of the functional group X and the functional group Y: Y is a group selected from a maleimide group, an active carbonate group, an amino group, and an aminoxy group when X is an acetal group, Y is a group selected from an acetal group, an active carbonate group, an acetylene group, and an azide group when X is a maleimide group, Y is a group selected from an acetal group, a maleimide group, an acetylene group, and an azide group when X is an active ester group or an active carbonate group, Y is a group selected from an acetal group, an acetylene group, and an azide group when X is an amino group or an aminoxy group, Y is a group selected from an acetal group, a maleimide group, an active ester group, an active carbonate group, an amino group, and an aminoxy group when X is an acetylene group or an azide group.

Description

EXAMPLES

(1) The following will describe the invention in further detail with reference to Examples.

(2) The molecular weight and molecular weight distribution of polyethylene glycol compounds including the branched hetero polyethylene glycol or intermediates thereof were determined by analysis on gel permeation chromatography (GPC). In the invention, measurement was performed using SHODEX GPC SYSTEM-11 as a GPC system and SHODEX RIX8 as a differential refractometer that was a detector, connecting three columns of SHODEX KF801L, KF803L, and KF804L ( 8 nm300 mm) serially, setting the temperature of the column oven to 40 C., using tetrahydrofuran as an eluent, setting the flow rate to 1 ml/minute, setting the concentration of a sample to 0.1% by mass, and setting an injection volume to 0.1 ml. As a calibration curve, a curve prepared using ethylene glycol, diethylene glycol, and triethylene glycol manufactured by Kanto Chemical Co., Inc. and Polymer Standards for GPC of polyethylene glycols or polyethylene oxides each having a molecular weight of 600 to 70,000 manufactured by Polymer Laboratory was used. For data analysis, BORWIN GPC calculation program was used. Mn indicates number-average molecular weight, Mw indicates weight-average molecular weight, and Mp indicates peak-top molecular weight. As for the molecular weight distribution, a calculated value thereof was shown as Mw/Mn.

(3) For .sup.1H-NMR analysis, JNM-ECP400 or JNM-ECA600 manufactured by JEOL DATUM Ltd. was used. Measurement was performed using CDCl.sub.3 as a deuterated solvent, using a 5 mm tube, and using TMS as an internal standard substance.

Example 1

(4) To a 300 ml four-neck flask equipped with a thermometer, a nitrogen-inlet tube, and a stirrer were added 18.2 g (0.1 mol) of 3-benzyloxy-1,2-propanediol, 150 g of anhydrous toluene, 0.9 g (39 mmol:26% by mol) of metal sodium, and the whole was stirred at room temperature with introducing nitrogen until metal sodium was dissolved. The solution was charged into a 5 L autoclave and, after inside of the system was replaced with nitrogen, the temperature was raised to 100 C. After 1,982 g (45 mol) of ethylene oxide was added thereto at 100 to 130 C. under a pressure of 1 MPa or less, the reaction was further continued for 2 hours. After unreacted ethylene oxide gas was removed under reduced pressure, the whole was cooled to 60 C. and pH was adjusted to 7.5 with an 85% aqueous phosphoric acid solution to obtain the following compound (a1).

(5) .sup.1H-NMR; (ppm):

(6) 3.40-3.90 (1785H, m, HO(CH.sub.2CH.sub.2O).sub.nCH.sub.2, HO(CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OCH.sub.2Ph), 4.54 (2H, s, CH.sub.2Ph), 7.27-7.38 (5H, m, CH.sub.2Ph)

(7) GPC analysis;

(8) number-average molecular weight (Mn): 19,654, weight-average molecular weight (Mw): 20,285, polydispersity (Mw/Mn): 1.032

(9) ##STR00020##

Example 2

(10) Into a 3 L four-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 372 g (18.6 mmol) of the above compound (a1) and 1,860 g of toluene. The whole was heated under reflux and water was removed as an azeotrope. After cooling to room temperature, 6.02 g (59.5 mmol) of triethylamine and 5.54 g (48.4 mmol) of methanesulfonyl chloride were added thereto and allowed to react at 40 C. for 3 hours. Subsequently, a toluene (256.8 g) solution of 85.6 g (465 mmol) of 3,3-diethoxy-1-propanol containing 2.58 g (112 mmol) of sodium dissolved therein was added thereto, followed by reaction at 70 C. for 5 hours. After filtration of the reaction solution, the filtrate was transferred to a 10 L stainless steel pot and crystallization was performed with adding 1.488 g of ethyl acetate, 1,488 g of ethanol, and 2,976 g of hexane. After the precipitated crystals were filtrated to remove the solvents, the crystals were transferred to a 10 L stainless steel pot. After 1,488 g of ethyl acetate and 1,488 g of ethanol were added thereto and heated to dissolve the crystals at 40 C., the solution was cooled to 20 C. and crystallization was performed with adding 2.976 g of hexane. Thereafter, similar crystallization was repeated three times and, after washing with hexane, the crystals were collected by filtration and dried to obtain the following compound (a2).

(11) .sup.1H-NMR; (ppm):

(12) 1.16-1.24 (12H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 1.85-1.95 (4H, q, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 3.40-3.90 (1725H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OCH.sub.2Ph, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 4.54 (2H, s, CH.sub.2Ph), 4.60-4.68 (2H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 7.27-7.38 (5H, m, CH.sub.2Ph)

(13) GPC analysis;

(14) number-average molecular weight (Mn): 19,190, weight-average molecular weight (Mw): 19,496, polydispersity (Mw/Mn): 1.016

(15) ##STR00021##

Example 3

(16) Into a 1 L four-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, and a cooling tube were charged 50 g of the above compound (a2) and 25 g of 5% palladium-carbon (a 50% hydrous product). After replacement with nitrogen, 400 g of methanol and 67 g of cyclohexene were added thereto, the temperature was raised, and gentle refluxing was performed at 52 to 55 C. to effect reaction for 2 hours. After cooling of the reaction solution to room temperature, palladium-carbon was filtrated off and the filtrate was concentrated. To the concentrate were added 400 g of toluene and 200 g of hexane, and crystallization was performed. The resulting crystals were collected by filtration and dried to obtain the following compound (a3).

(17) .sup.1H-NMR; (ppm):

(18) 1.16-1.24 (12H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 1.85-1.95 (4H, q, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 3.40-3.90 (1673H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 4.60-4.68 (2H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2)

(19) GPC analysis;

(20) number-average molecular weight (Mn): 18,573, weight-average molecular weight (Mw): 19,123, polydispersity (Mw/Mn): 1.030

(21) ##STR00022##

Example 4

(22) Into a 200 mL three-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 13.1 g (0.66 mmol) of the above compound (a3) and 79 g of toluene. The whole was heated under reflux and water was removed as an azeotrope. After cooling to room temperature, 0.33 g (3.3 mmol) of triethylamine and 0.30 g (2.6 mmol) of methanesulfonyl chloride were added thereto and allowed to react at 40 C. for 3 hours. After filtration of the reaction solution, the filtrate was transferred to a 300 mL beaker and crystallization was performed with adding 100 g of ethyl acetate and 50 g of hexane. After the precipitated crystals were filtrated to remove the solvents, the crystals were transferred to a 10 L stainless steel pot. After 1,488 g of ethyl acetate and 1,488 g of ethanol were added thereto and heated to dissolve the crystals at 40 C., the solution was cooled to 20 C. and crystallization was performed with adding 2,976 g of hexane. Thereafter, similar crystallization was repeated three times and, after washing with hexane, the crystals were collected by filtration and dried to obtain the following compound (a4).

(23) .sup.1H-NMR; (ppm):

(24) 1.16-1.24 (12H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 1.85-1.95 (4H, q, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 3.08 (3H, s, OSO.sub.2CH.sub.3), 3.40-3.90 (1732H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 4.24-4.44 (2H, m, CH.sub.2OOSO.sub.2CH.sub.3), 4.60-4.68 (2H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2)

(25) GPC analysis;

(26) number-average molecular weight (Mn): 19,274, weight-average molecular weight (Mw): 20,046, polydispersity (Mw/Mn): 1.040

(27) ##STR00023##

Example 5

(28) Into a 100 mL three-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 10.0 g (0.50 mmol) of the above compound (a4), 80 g of ammonia water, and 20 g of ion-exchange water, followed by reaction at 55 C. for 8 hours. Ammonia was removed at 55 C. under slightly reduced pressure for 10 hours, followed by extraction with adding 100 g of chloroform. After the chloroform solution was concentrated, 52 g of toluene was added thereto and the concentrate was dissolved at 40 C., followed by filtration. Thereafter, crystallization was performed with adding 30 g of hexane at room temperature in a 200 mL beaker. After the precipitated crystals were filtrated to remove the solvents, the crystals were washed with hexane, collected by filtration, and dried to obtain the following compound (a5).

(29) .sup.1H-NMR; (ppm):

(30) 1.16-1.24 (12H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 1.85-1.95 (4H, q, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 2.70-2.95 (2H, m, CH.sub.2NH.sub.2), 3.40-3.90 (1730H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 4.60-4.68 (2H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2)

(31) GPC analysis;

(32) number-average molecular weight (Mn): 19,217, weight-average molecular weight (Mw): 20,362, polydispersity (Mw/Mn): 1.060

(33) ##STR00024##

Example 6

(34) Into a 100 mL three-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 5.0 g (0.25 mmol) of the above compound (a5), 26 g of toluene, 4.0 g of acetonitrile, N-methylmorpholine (1.25 mmol), and N-succinimidyl 3-maleimidopropionate (0.375 mmol), followed by reaction at room temperature for 4 hours. After the solution was filtrated, crystallization was performed with adding 100 g of ethyl acetate and 50 g of hexane at room temperature in a 300 mL beaker. Then, after 100 g of ethyl acetate was added and the crystals were dissolved at 40 C., the solution was cooled to room temperature and 100 g of hexane was added to precipitate crystals. After the crystallization was repeated further twice, the crystals were washed with hexane, collected by filtration, and dried to obtain the following compound (a6).

(35) .sup.1H-NMR; (ppm):

(36) 1.16-1.24 (12H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 1.85-1.95 (4H, q, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 2.51 (2H, t, NHCOCH.sub.2CH.sub.2), 3.40-3.90 (1736H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2, CH.sub.2NHCOCH.sub.2CH.sub.2), 4.60-4.68 (2H, t, (CH.sub.3CH.sub.2O).sub.2CHCH.sub.2CH.sub.2), 6.70 (2H, s, CHCH)

(37) GPC analysis;

(38) number-average molecular weight (Mn): 19,395, weight-average molecular weight (Mw): 20,600, polydispersity (Mw/Mn): 1.062

(39) ##STR00025##

Example 7

(40) Into a 200 mL beaker fitted with a thermometer, a nitrogen-inlet tube, and a stirring bar were charged 2.0 g (0.10 mmol) of the above compound (a5) and 40 g of ion-exchange water, followed by dissolution. After the solution was prepared to be pH 2 with hydrochloric acid and stirred at room temperature for 2 hours, the solution was neutralized to pH 6 with an aqueous sodium hydroxide solution, followed by extraction with adding 100 g of chloroform. After the chloroform solution was concentrated, 40 g of toluene was added thereto and the concentrate was dissolved at 40 C., followed by filtration. Thereafter, crystallization was performed with adding 20 g of hexane at room temperature in a 200 mL beaker. After the precipitated crystals were filtrated to remove the solvents, the crystals were washed with hexane, collected by filtration, and dried to obtain the following compound (a7).

(41) .sup.1H-NMR; (ppm):

(42) 2.51 (2H, t, NHCOCH.sub.2CH.sub.2), 2.63-2.73 (4H, HCOCH.sub.2CH.sub.2), 3.40-3.90 (1735H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, HCOCH.sub.2CH.sub.2, CH.sub.2NHCOCH.sub.2CH.sub.2), 6.70 (2H, 3, CHCH), 9.80 (2H, HCOCH.sub.2CH.sub.2)

(43) GPC analysis;

(44) number-average molecular weight (Mn): 19,319, weight-average molecular weight (Mw): 20,634, polydispersity (Mw/Mn): 1.068

(45) ##STR00026##

Example 8

(46) Into a 1 L four-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 200 g (10.0 mmol) of the above compound (a1) and 600 g of toluene. The whole was heated under reflux and water was removed as an azeotrope. After cooling to 40 C., 1,000 g of anhydrous chloroform, 8.83 g (60.0 mmol) of phthalimide and 15.7 g (60.0 mmol) of triphenylphosphine were added thereto, followed by stirring and dissolution. After cooling to room temperature, 12.1 g (60.0 mmol) of diisopropylazodicarboxylate was allowed to react at 40 C. for 3 hours. Subsequently, a toluene (256.8 g) solution of 85.6 g (465 mmol) of 3,3-diethoxy-1-propanol containing 2.58 g (112 mmol) of sodium dissolved therein was added thereto and allowed to react at 70 C. for 5 hours. After filtration of the reaction solution, the filtrate was transferred to a 10 L stainless steel pot and crystallization was performed with adding 1,488 g of ethyl acetate, 1,488 g of ethanol, and 2,976 g of hexane. After the precipitated crystals were filtrated to remove the solvents, the crystals were transferred to a 10 L stainless steel pot. After 1,488 g of ethyl acetate and 1,488 g of ethanol were added thereto and heated to dissolve the crystals at 40 C., the solution was cooled to 20 C. and crystallization was performed with adding 2,976 g of hexane. Thereafter, similar crystallization was repeated three times and, after washing with hexane, the crystals were collected by filtration and dried to obtain the following compound (a8).

(47) .sup.1H-NMR; (ppm):

(48) 3.40-3.90 (1728H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OCH.sub.2Ph), 4.54 (2H, s, CH.sub.2Ph), 7.27-7.38 (5H, m, CH.sub.2Ph), 7.65-7.95 (4H, m, Ph(CO).sub.2N)

(49) GPC analysis;

(50) number-average molecular weight (Mn): 19,398, weight-average molecular weight (Mw): 19,824, polydispersity (Mw/Mn): 1.022

(51) ##STR00027##

Example 9

(52) Into a 1 L four-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, and a cooling tube were charged 50 g of the above compound (a8) and 25 g of 5% palladium-carbon (a 50% hydrous product). After replacement with nitrogen, 500 g of methanol and 68 g of cyclohexene were added thereto, the temperature was raised, and gentle refluxing was performed at 52 to 55 C. to effect reaction for 5 hours. After cooling of the reaction solution to room temperature, palladium-carbon was filtrated off and the filtrate was concentrated. To the concentrate were added 400 g of toluene and 200 g of hexane, and crystallization was performed. The resulting crystals were collected by filtration and dried to obtain the following compound (a9).

(53) .sup.1H-NMR; (ppm):

(54) 3.40-3.90 (1684H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH), 7.65-7.95 (4H, m, Ph(CO).sub.2N)

(55) GPC analysis;

(56) number-average molecular weight (Mn): 18,817, weight-average molecular weight (Mw): 19,171, polydispersity (Mw/Mn): 1.019

(57) ##STR00028##

Example 10

(58) Into a 1 L four-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 200 g (10.0 mmol) of the above compound (a1) and 600 g of toluene. The whole was heated under reflux and water was removed as an azeotrope. After cooling to 40 C., 1,000 g of anhydrous chloroform, 8.83 g (60.0 mmol) of phthalimide and 15.7 g (60.0 mmol) of triphenylphosphine were added thereto, followed by stirring and dissolution. After cooling to room temperature, 12.1 g (60.0 mmol) of diisopropylazodicarboxylate was allowed to react at 40 C. for 3 hours. Subsequently, a toluene (256.8 g) solution of 85.6 g (465 mmol) of 3,3-diethoxy-1-propanol containing 2.58 g (112 mmol) of sodium dissolved therein was added thereto and allowed to react at 70 C. for 5 hours. After filtration of the reaction solution, the filtrate was transferred to a 10 L stainless steel pot and crystallization was performed with adding 1,488 g of ethyl acetate, 1,488 g of ethanol, and 2,976 g of hexane. After the precipitated crystals were filtrated to remove the solvents, the crystals were transferred to a 10 L stainless steel pot. After 1,488 g of ethyl acetate and 1,488 g of ethanol were added thereto and heated to dissolve the crystals at 40 C., the solution was cooled to 20 C. and crystallization was performed with adding 2,976 g of hexane. Thereafter, similar crystallization was repeated three times and, after washing with hexane, the crystals were collected by filtration and dried to obtain the following compound (a10).

(59) .sup.1H-NMR; (ppm):

(60) 2.83-2.89 (4H, t, NH.sub.2CH.sub.2CH.sub.2), 3.40-3.90 (1676H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH)

(61) GPC analysis;

(62) number-average molecular weight (Mn): 18,559, weight-average molecular weight (Mw): 19,264, polydispersity (Mw/Mn): 1.038

(63) ##STR00029##

Example 11

(64) Into a 100 mL three-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 5.0 g (0.25 mmol) of the above compound (a5), 26 g of toluene, 4.0 g of acetonitrile, N-methylmorpholine (2.50 mmol), and N-succinimidyl 3-maleimidopropionate (0.75 mmol), followed by reaction at room temperature for 4 hours. After the solution was filtrated, crystallization was performed with adding 100 g of ethyl acetate and 50 g of hexane at room temperature in a 300 mL beaker. Then, after 100 g of ethyl acetate was added and the crystals were dissolved at 40 C., the solution was cooled to room temperature and 100 g of hexane was added to precipitate crystals. After the crystallization was repeated further twice, the crystals were washed with hexane, collected by filtration, and dried to obtain the following compound (a11).

(65) .sup.1H-NMR; (ppm):

(66) 2.51 (2H, t, CH.sub.2CH.sub.2CONH), 3.40-3.90 (1767H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OH, CH.sub.2CH.sub.2CONH), 6.50 (1H, s, CH.sub.2CH.sub.2CONH), 6.70 (2H, s, CHCH)

(67) GPC analysis;

(68) number-average molecular weight (Mn): 19,734, weight-average molecular weight (Mw): 21,336, polydispersity (Mw/Mn): 1.081

(69) ##STR00030##

Example 12

(70) Into a 100 mL three-neck flask fitted with a thermometer, a nitrogen-inlet tube, a stirrer, a Dean-stark tube, and a cooling tube were charged 4.0 g (0.20 mmol) of the above compound (a5), g of dichloromethane, triethylamine (2.50 mmol), and N,N-disuccinimidyl carbonate (0.75 mmol), followed by reaction at room temperature for 4 hours. After the solution was filtrated and concentrated, crystallization was performed with adding 100 g of ethyl acetate and 50 g of hexane in a 300 mL beaker. Then, after 100 g of ethyl acetate was added and the crystals were dissolved at 40 C., the solution was cooled to room temperature and 100 g of hexane was added to precipitate crystals. After the crystallization was repeated further three times, the crystals were washed with hexane, collected by filtration, and dried to obtain the following compound (a12).

(71) .sup.1H-NMR; (ppm):

(72) 2.51 (2H, t, CH.sub.2CH.sub.2CONH), 2.84 (4H, s, CH.sub.2OCOO-succinimide), 3.40-3.90 (1751H, m, (CH.sub.2CH.sub.2O).sub.nCH.sub.2, (CH.sub.2CH.sub.2O).sub.nCH, CH.sub.2OCOO-succinimide, CH.sub.2CH.sub.2CONH), 4.36-4.52 (2H, m, CH.sub.2OCOO-succinimide), 6.37 (1H, s, CH.sub.2CH.sub.2CONH), 6.70 (2H, s, CHCH)

(73) GPC analysis;

(74) number-average molecular weight (Mn): 19,659, weight-average molecular weight (Mw): 21,450, polydispersity (Mw/Mn): 1.091

(75) ##STR00031##

Example 13

(76) GRGDS (Gly-Arg-Gly-Asp-Ser, molecular weight: 490.5) was dissolved in an amount of 2.5 mg (5.1 mol) in 1 ml of 10 mM phosphoric acid buffer (pH=6.4). To 200 l of the solution was added 10 mg (0.5 mol) of the compound (a12), and reaction was carried out at room temperature for 1 hour, thereby GRGDS being modified at one active carbonate group in the compound. Subsequently, 20 mg (7.4 mol) of Humanin (Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, molecular weight: 2,687.2) was added to the solution, and reaction was carried out at room temperature for 10 hours, thereby Humanin being modified at the two maleimide groups in the compound. Then, 200 l of the reaction solution was charged onto an SP-Sepharose FF (manufactured by Amersham plc) column and equilibration was performed with 20 mM Tris-HCl buffer (pH 8.2). After equilibration, the buffer to which NaCl had been added so as to be 1N was passed through the column to obtain a fraction of a PEG compound modified with one GRGDS and two Humanin. After 20 l of the fraction and 20 l of a Tris SDS sample treating liquid were mixed, the mixture was heated in a boiled water bath for 2 minutes and 30 seconds and 20 l of the solution was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining was performed by CBB staining. As a result, through comparison with respective standard samples, it was shown that a compound where the compound (a12) was modified with one GRGDS and two Humanin.

(77) While the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

(78) The present application is based on Japanese Patent Application No. 2010-145383 filed on Jun. 25, 2010 and Japanese Patent Application No. 2011-070735 filed on Mar. 28, 2011, and the contents are incorporated herein by reference. Also, all the references cited herein are incorporated as a whole.