US20050058620A1 - Modified bio-related substance, process for producing the same, and intermediate - Google Patents

Modified bio-related substance, process for producing the same, and intermediate Download PDF

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US20050058620A1
US20050058620A1 US10/716,432 US71643203A US2005058620A1 US 20050058620 A1 US20050058620 A1 US 20050058620A1 US 71643203 A US71643203 A US 71643203A US 2005058620 A1 US2005058620 A1 US 2005058620A1
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group
formula
compound
related substance
bio
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Ken-ichiro Nakamoto
Syunsuke Ohashi
Yuji Yamamoto
Kenji Sakanoue
Chika Itoh
Tohru Yasukohchi
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NOF Corp
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NOF Corp
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Assigned to NOF CORPORATION reassignment NOF CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YASUKOHCHI, TOHRU, ITOH, CHIKA, NAKAMOTO, KEN-ICHIRO, OHASHI, SYUNSUKE, SAKANOUE, KENJI, YAMAMOTO, YUJI
Publication of US20050058620A1 publication Critical patent/US20050058620A1/en
Priority to US11/142,255 priority Critical patent/US7524875B2/en
Priority to US11/142,683 priority patent/US8828373B2/en
Priority to US11/142,678 priority patent/US8003117B2/en
Priority to US12/399,249 priority patent/US7851491B2/en
Priority to US12/942,554 priority patent/US8034981B2/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/26—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/26—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds
    • C08G65/2603—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds the other compounds containing oxygen
    • C08G65/2606—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds the other compounds containing oxygen containing hydroxyl groups
    • C08G65/2609—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds the other compounds containing oxygen containing hydroxyl groups containing aliphatic hydroxyl groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment

Definitions

  • the present invention relates to a bio-related substance modified by the bonding to a polyalkylene glycol derivative, a process for producing the same, and a reactive polyalkylene glycol derivative which is an intermediate thereof.
  • Polyethylene glycol exhibits a low interaction with the other bio-components owing to its steric repulsion effect and as a result, proteins and polypeptides such as enzymes modified with polyethylene glycol exhibit an effect of avoiding the filtration through glomeruli in the kidney and bio-reactions such as immunoreaction, so that they achieve half-lives in blood longer than those of unmodified substances. Moreover, they also have decreased toxicity and antigenicity and further exhibit an effect of enhancing the solubility of a sparingly water-soluble compound having a high hydrophobicity.
  • JP-B-61-42558 proposes a polyethylene glycol-modified L-asparaginase.
  • cyanuric chloride as a starting material for a reactive polyethylene glycol derivative has three reactive sites and hence it is difficult to introduce two polyethylene glycol chains thereinto selectively. Accordingly, it is difficult to synthesize a highly pure polyethylene glycol-modified L-asparaginase.
  • JP-A-10-67800 proposes a polyethylene glycol-modified interferon a.
  • this substance has three urethane and amide bonds including the linkage between interferon ⁇ and the poly(ethylene glycol)oxy group. These bonds are labile to hydrolysis during storage or during the reaction under an alkaline condition and as a result, there arises a problem that the branched polyethylene glycol moiety is decomposed to a single chain.
  • the polyethylene glycol derivative which is the intermediate material has been produced by a method wherein two monomethoxypolyethylene glycols and amino groups at ⁇ - and ⁇ -positions of lysine are combined through urethane bonds and then the carboxyl residue of lysine is converted into a succinimide ester.
  • the polyethylene glycol-modified interferon ⁇ there arises a problem that increased impurities are produced owing to the multi-step process, such as the activation of the terminal hydroxyl groups of two monomethoxypolyethylene glycols, the combination with lysine, the activation of the carboxyl residue of lysine, and the combination with interferon ⁇ .
  • a first object of the invention is to provide a bio-related substance having a branched poly(alkylene glycol)oxy group which is formed by stable bonds and is hardly decomposed to a single chain, and a process for producing the same.
  • a second object of the invention is to provide a polyalkylene glycol derivative having a reactive group, which can be combined with a bio-related substance, at the primary carbon at the 1-position of the glycerin skeleton and having polyalkylene glycol chains at the 2- and 3-positions.
  • the invention relates to a modified bio-related substance, wherein at least one poly(alkylene glycol)oxy group represented by the following formula (1): wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA 1 and OA 2 are each an oxyalkylene group having 2 to 4 carbon atoms, R and OA 2 are the same or different from each other in one molecule, n and m are each average number of moles of the oxyalkylene group added, n represents 0 to 1000, and m represents 10 to 1000, is combined in a molecule.
  • R is a hydrocarbon group having 1 to 24 carbon atoms
  • OA 1 and OA 2 are each an oxyalkylene group having 2 to 4 carbon atoms
  • R and OA 2 are the same or different from each other in one molecule
  • n and m are each average number of moles of the oxyalkylene group added
  • n represents 0 to 1000
  • m represents 10 to 1000
  • the invention relates to an intermediate for the modified bio-related substance, which is represented by the following formula (2): wherein R, OA 1 , OA 2 , n, and m are the same as above, and X represents a functional group capable of chemically reacting with a bio-related substance.
  • the invention relates to a process for producing a modified bio-related substance wherein at least one poly(alkylene glycol)oxy group represented by the formula (1) is combined in a molecule,
  • the invention relates to a compound of the formula (p) as a starting material of the compound of the formula (2) and a process for producing the same.
  • the modified bio-related substance of the invention is formed by stable bonds and is hardly decomposed to a single chain.
  • the invention can provide a polyalkylene glycol derivative having a reactive group, which can be combined with a bio-related substance, at the primary carbon at the 1-position of the glycerin skeleton and having polyalkylene glycol chains at the 2- and 3-positions.
  • FIG. 1 illustrates an experimental result by polyacrylamide gel electrophoresis of OVA and modified OVA.
  • FIG. 2 is a chart illustrating a result of GPC measurement before an accelerated aging test of the compound p-8.
  • FIG. 3 is a chart illustrating a result of GPC measurement after an accelerated aging test of the compound p-8.
  • FIG. 4 is a chart illustrating a result of GPC measurement before an accelerated aging test of the compound p-10.
  • FIG. 5 is a chart illustrating a result of GPC measurement after an accelerated aging test of the compound p-10.
  • FIG. 6 is a result of electrophoresis of the compound obtained by modifying Humanin with the compound (p31).
  • FIG. 7 is a result of electrophoresis of the compound obtained by modifying insulin with the compound (p32) or (p35).
  • the modified bio-related substance of the invention is a substance wherein a bio-related substance is combined with at least one poly(alkylene glycol)oxy group represented by the above formula (1).
  • R in the poly(alkylene glycol)oxy group of the formula (1) is a hydrocarbon group having 1 to 24 carbon atoms and specific hydrocarbon groups include hydrocarbon groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a hexyl group, a heptyl group, a 2-ethylhexyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, an oleyl group, a nona
  • OA 1 and OA 2 represent each an oxyalkylene group having 2 to 4 carbon atoms. Specifically, they include an oxyethylene group, an oxypropylene group, an oxytrimethylene group, an oxy-1-ethylethylene group, an oxy-1,2-dimethylethylene group, and an oxytetramethylene group.
  • the oxyalkylene groups may be the same or different from each other and may be added randomly or block-wise. In general, the fewer the carbon atoms are, the higher the hydrophilicity is.
  • the group is preferably an oxyethylene group or an oxypropylene group, more preferably an oxyethylene group.
  • m and n are each average number of moles of the oxyalkylene group added.
  • n represents 10 to 1000, preferably 20 to 800, more preferably 50 to 800, most preferably 100 to 800.
  • n represents 0 to 1000, preferably 0 to 500, more preferably 0 to 200, most preferably 0 to 50. In a preferable embodiment, n is 0. In another preferable embodiment, n is 1 to 50. In the latter case, n is particularly preferably 1 to 3.
  • the number of modifications with the poly(alkylene glycol)oxy group to the bio-related substance is not particularly limited but is preferably 1 to 100, more preferably 1 to 20.
  • the “bio-related substance” means a substance relating to a body.
  • the substances relating to a body include the following.
  • the animal cell-constituting materials are components constituting cell membranes and the kind is not particularly limited but examples thereof include phospholipids, glycolipides, and glycoproteins. Examples of more specific phospholipids include phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, cardiolipin, phosphatidylserine, and phosphatidylinositol. In addition, lyso isomers thereof are also included. These phospholipids may be those derived from natural products such as egg yolk or soybean or may be synthesized products.
  • the composition of fatty acids is not particularly limited but may include fatty acids having 12 to 22 carbon atoms. These fatty acids may be saturated fatty acids or may be those containing an unsaturated bond.
  • glycolipids examples include ceramides, cerebrosides, sphingosines, gangliosides, and glyceroglycolipids.
  • fatty acids, monoglycerides, diglycerides, cholesterols, and bile acid are also included.
  • the body fluid-constituting substances mean fluid components existing inside and outside cells and the kind is not particularly limited but examples thereof include blood, lymph, and bone marrow liquid. Examples of more specific body fluid-constituting components include hemoglobin, albumin, and blood coagulation factors.
  • the physiologically active substances mean components controlling body functions and the kind is not particularly limited but examples thereof include vitamins, neurotransmitters, proteins, polypeptides, and drugs.
  • vitamins examples include vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.
  • neurotransmitters examples include adrenalin, noradrenalin, dopamine, acetylcholine, GABA, glutamic acid, and aspartic acid.
  • proteins and polypeptides include the following. Hormones such as neurohypophysial hormone, thyroid hormone, male sex hormone, female sex hormone, and adrenal cortex hormone. Serum proteins such as hemoglobin and blood factors. Immunoglobulins such as IgG, IgE, IgM, IgA, and IgD.
  • Cytokines and fragments thereof such as interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12 subtypes), interferons (- ⁇ , - ⁇ , - ⁇ ), granulocyte-colony stimulating factors ( ⁇ and ⁇ types), macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, platelet-derived growth factor, phospholipase-activating protein, insulin, glucagon, lectin, ricin, tumor necrosis factor, epidermal growth factor, transforming growth factors (- ⁇ , - ⁇ ), fibroblast growth factor, hepatocyte growth factor, vascular endothelial growth factor, nerve growth factor, bone growth factor, insulin-like growth factor, heparin binding growth factor, tumor growth factor, glial cell line-derived neurotrophic factor, macrophage differentiating factor, differentiation-inducing
  • Enzymes such as proteolytic enzymes, oxidoreductases, transferases, hydrases, lyases, isomerases, ligases, asparaginases, arginases, arginine deaminases, adenosine deaminases, superoxide dismutases, endotoxinases, catalases, chymotrypsin, lipases, uricases, elastases, streptokinases, urokinases, prourokinases, adenosine diphosphatases, tyrosinases, bilirubin oxidases, glucose oxidases, glucodases, glactosidases, glucocerebrosidases, and glucouronidases.
  • Monoclonal and polyclonal antibodies and fragments thereof Monoclonal and polyclonal antibodies and fragments thereof.
  • Polyamino acids such as poly-L-lysine, poly-D-lysine.
  • Vaccines such as hepatitis B vaccine, malaria vaccine, melanoma vaccine, and HIV-1 vaccine, and antigens.
  • glycoproteins are also included.
  • structurally similar substances having physiological activity similar to that of these physiologically active substances.
  • proteins and polypeptides may be isolated from natural sources thereof or cells subjected to genetic engineering or may be produced via various synthetic processes.
  • the drugs are not particularly limited but more preferably include anticancer drugs and antifungal drugs.
  • More specific anticancer drugs are not particularly limited but, for example, include paclitaxel, adriamycin, doxorubicin, cisplatin, daunomycin, mitomycin, vincristine, epirubicin, methotrexate, 5-fluorouracil, aclacinomycin, idamycin, bleomycin, pirarubicin, peplomycin, vancomycin, and camptothecine.
  • Specific antifungal drugs are not particularly limited but, for example, include amphotericin B, nystatin, flucytosine, miconazole, fluconazole, itraconazole, ketoconazole, and peptide antifungal drugs.
  • these physiologically active substances also include flavonoids, terpenoids, carotenoids, saponins, steroids, quinones, anthraquinones, xanthones, coumarins, alkaloids, porphyrins, and polyphenols.
  • the intermediate for the bio-related substance of the invention is represented by the following formula (2).
  • X is not particularly limited as far as it is a functional group or an unsaturated bond capable of forming a chemical bond with a bio-related substance.
  • X is a group represented by the group (I) or (II).
  • the groups represented by (a), (b), (d), (f), (h), (i), and (k) are preferable.
  • the groups represented by (a), (b), (c), (d), (e), (f), (h), (i), and (k) are preferable.
  • the groups represented by (c) is preferable.
  • the groups represented by (c), (g), and (j) are preferable.
  • Z in the group (I) or (II) is a linker between the poly(alkylene glycol)oxy group and the reactive functional group and is not particularly limited as far as it is a covalent bond, but preferably includes an alkylene group and an alkylene group containing an ester bond, a urethane bond, an amide bond, an ether bond, a carbonate bond, or a secondary amino group.
  • Preferable alkylene group includes a methylene group, an ethylene group, a trimethylene group, a propylene group, an isopropylene group, a tetramethylene group, a butylene group, an isobutylene group, a pentamethylene group, and a hexamethylene group.
  • More preferable is a structure of the following (z1). More preferable as an alkylene group containing an ester bond is a structure of the following (z2). More preferable as an alkylene group containing an amide bond is a structure of the following (z3).
  • a group of the following (z4) is more preferable as an alkylene group containing an ether bond. More preferable as an alkylene group containing a urethane bond is a structure of the following (z5).
  • the following (z6) is a structure more preferable as an alkylene group containing a secondary amino group.
  • s is an integer of 1 to 6, preferably an integer of 1 to 3, more preferably an integer of 2 to 3.
  • Y is a hydrocarbon group having 1 to 10 carbon atoms which may contain fluorine atom(s).
  • Y includes a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a tert-butyl group, a hexyl group, a nonyl group, a vinyl group, a phenyl group, a benzyl group, a 4-methylphenyl group, a trifluoromethyl group, a 2,2,2-trifluoroethyl group, and a 4-(trifluoromethoxy)phenyl group, and is preferably a methyl group, a vinyl group, a 4-methylphenyl group, and a 2,2,2-trifluoroethyl group.
  • R, A 1 O, A 2 O, n, and m are the same as above.
  • Tables 1 and 2 show relation between a residual group T of the above bio-related substance and a functional group X of the poly(alkylene glycol)oxy group side which forms a chemical bond with the residual group T.
  • Tables 1 and 2 also show types of the chemical bonds between the poly(alkylene glycol)oxy groups and bio-related substances, which are formed by the reaction of the bio-related substances with X.
  • the poly(alkylene glycol)oxy group and the bio-related substance are combined by, for example, an amide bond, a secondary amino group, a urethane bond, a thioester bond, a sulfide bond, a disulfide bond, or a thiocarbonate bond.
  • the modified bio-related substances of the invention can be produced as follows.
  • the intermediates (a), (b), (d), (f), (h), (i), and (k) of the invention are used. More preferably, (a), (b), (d), and (f) are used.
  • the intermediates (a), (b), (d), (f), (h), (i), and (k) of the invention may be reacted in a ratio of equimolar or more to the bio-related substance.
  • the reaction solvent is not particularly limited as far as it does not participate in the reaction, but in the case of reacting a protein or polypeptide, preferable solvents include buffer solutions such as phosphate buffer solutions, borate buffer solutions, Tris-acid buffer solutions, acetate buffer solutions, and carbonate buffer solutions. Furthermore, an organic solvent which does not deactivate the protein or polypeptide and does not participate in the reaction, such as acetonitrile, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, may be added.
  • buffer solutions such as phosphate buffer solutions, borate buffer solutions, Tris-acid buffer solutions, acetate buffer solutions, and carbonate buffer solutions.
  • an organic solvent which does not deactivate the protein or polypeptide and does not participate in the reaction such as acetonitrile, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, may be added.
  • preferable solvents include, in addition to the above buffer solutions, toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, methylene dichloride, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, water, methanol, ethanol, n-propanol, 2-propanol, and n-butanol. Also, the solvent need not be used. The order of adding the intermediate and the bio-related substance is optional.
  • the reaction temperature is not particularly limited as far as it does not deactivate the bio-related substance, but the temperature is preferably 0 to 40° C. in the case of reacting a protein or polypeptide and is preferably ⁇ 20 to 150° C. in the case of reacting an anticancer drug, antifungal drug, or phospholipid.
  • the reaction time is preferably 0.5 to 72 hours, more preferably 1 to 24 hours.
  • a condensing agent such as N,N′-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) may be used.
  • DCC N,N′-dicyclohexylcarbodiimide
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • a covalent bond is formed between the bio-related substance and the intermediate of the invention by carrying out the reaction.
  • An amide bond is formed in the case of using (a) or (k), a secondary amino group in the case of using (b), a urethane bond in the case of using (d), (h), or (i), and a Schiff base in the case of using (f).
  • a Schiff base When a Schiff base is formed, it may be subjected to a reduction treatment using a reducing agent such as sodium cyanoborohydride to form a secondary amino group.
  • the product may be purified by a purifying means such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, electrophoresis, extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • a purifying means such as dialysis, salting-out, ultrafiltration, ion-exchange chromatography, electrophoresis, extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • the intermediates (a), (b), (c), (d), (e), (f), (h), (i), and (k) of the invention are used. More preferably, (e) is used.
  • the reaction solvent, reaction conditions, and the like are the same as in the case of using an amino group.
  • a radical initiator such as iodine or AIBN may be used.
  • a covalent bond is formed between the bio-related substance and the intermediate of the invention by carrying out the reaction, and a thioether bond is formed in the case of using (a) or (k), a thiocarbonate bond in the case of using (d), (h), or (i), a disulfide bond in the case of using (c), and a sulfide bond in the case of using (b), (e), or (f).
  • the intermediate (c) of the invention is used.
  • the reaction solvent, reaction conditions, and the like are the same as in the case of using an amino group.
  • a radical initiator such as iodine or AIBN may be used.
  • a sulfide bond is formed between the bio-related substance and the intermediate of the invention by carrying out the reaction.
  • the intermediate (c), (g), or (j) of the invention is used.
  • the reaction solvent, reaction conditions, and the like are the same as in the case of using an amino group.
  • a condensing agent such as DCC. or EDC. may be optionally used.
  • a covalent bond is formed between the bio-related substance and the intermediate of the invention by carrying out the reaction, and a thioester bond is formed in the case of using (c) and an amide bond in the case of using (g) or (j).
  • a bio-related substance does not have any of an amino group, a mercapto group, an unsaturated bond, and a carboxyl group
  • the substance can be modified by introducing a reactive group suitably into the bio-related substance and using an intermediate of the invention.
  • the intermediates of the invention can be, for example, produced as follows.
  • An alkylene oxide is polymerized in an amount of 0 to 1000 mol to the primary hydroxyl group residue of 2,2-dimethyl-1,3-dioxolane-4-methanol and the terminal hydroxyl group is protected with a benzyl group or a t-Bu group.
  • the cyclic acetal structure is deprotected under an acidic condition and an alkylene oxide is polymerized in an amount of 10 to 1000 mol to the newly formed two hydroxyl groups, followed by alkyl-etherification of the terminal ends.
  • the protective group such as the benzyl group or t-Bu group is deprotected and thereby, the compound of the general formula (p) can be obtained.
  • n is 0, the primary hydroxyl group residue of 2,2-dimethyl-1,3-dioxolane-4-methanol is protected with a benzyl group or t-Bu group and then an alkylene oxide is polymerized in an amount of 10 to 1000 mol to the newly formed two hydroxyl groups, followed by alkyl-etherification of the terminal ends. Then, the protective group such as the benzyl group or t-Bu group is deprotected and thereby, the compound of the general formula (p) can be obtained.
  • the compound (p) can be also produced by the following method.
  • the primary hydroxyl group of 2,2-dimethyl-1,3-dioxolane-4-methanol is protected by a benzyl group or t-Bu group.
  • the cyclic acetal structure is deprotected under an acidic condition and an alkylene oxide is polymerized in an amount of 10 to 1000 mol to the newly formed two hydroxyl groups, followed by alkyl-etherification of the terminal ends.
  • the protective group such as the benzyl group or t-Bu group is deprotected and thereby, the compound of the general formula (p) wherein n is 0 can be obtained.
  • the compound may be also produced by polymerizing an alkylene oxide in an amount of 0 to 1000 mol to the newly formed hydroxyl group.
  • n 1 to 3
  • the cyclic acetal structure is deprotected under an acidic condition and an alkylene oxide is polymerized in an amount of 10 to 1000 mol to the newly formed two hydroxyl groups, followed by alkyl-etherification of the terminal ends.
  • the protective group such as the benzyl group or t-Bu group is deprotected and thereby, the compound of the general formula (p) can be obtained.
  • a highly pure branched polyalkylene glycol derivative can be produced in high yields in an industrially suitable manner by using the alkylene oxide-addition polymerization reaction, without column purification.
  • the intermediates of the invention can be produced by modifying hydroxy group into various reactive groups shown in the groups (I) and (II). Furthermore, using the formed reactive groups, various bio-related substances can be reacted and modified to produce modified bio-related substances of the invention.
  • the intermediate having each functional group of the groups (I) and (II) can be reacted with a bio-related substance but in some cases, the intermediate can be further reacted with the other compound to produce other intermediate and the other intermediate can be then reacted with a bio-related substance.
  • the intermediate having a functional group (g), (j), or (k) belonging to the group (II) as an starting material the intermediate having (a), (e), or (f) of the group (I) can be synthesized.
  • the addition polymerization of an alkylene oxide to the primary hydroxyl group residue of 2,2-dimethyl-1,3-dioxolane-4-methanol can be carried out in the following manner.
  • the addition polymerization of an oxyalkylene can be achieved in toluene or without solvent under an alkaline condition such as sodium, potassium, sodium hydride, potassium hydride, sodium methoxide, or potassium t-butoxide.
  • n is 1 to 3
  • the subsequent benzyl etherification can be carried out in the following manner.
  • the addition polymerization of an alkylene oxide to the compound of the following formula (9) having two hydroxyl groups newly formed by the deprotection of the cyclic acetal is not particularly limited but can be achieved via the following steps (C1) and (C2).
  • Step (C1) The alcoholation of the compound of the formula (9) is achieved by using sodium or potassium, preferably sodium as a catalyst in an catalyst amount of 5 to 50 mol %, followed by dissolution at 10 to 50° C.
  • Step (C2) An alkylene oxide addition polymerization is carried out at a reaction temperature of 50 to 130° C.
  • the use of the catalyst in an amount of 5 mol % or more is advantageous in the production of a high quality high-molecular-weight compound.
  • the catalyst amount exceeds 50 mol %, the viscosity of the reaction liquid increases or the liquid solidifies at the alcoholation reaction and thus there is a tendency that the stirring efficiency decreases and the alcoholation is not accelerated.
  • the liquid solidifies handling thereof tends to be difficult, which causes water absorption.
  • an alkylene glycol compound derived from water is formed and is contained as an impurity undesirable in medical use.
  • benzyl alcohol When the temperature at the dissolution is higher than 50° C., a decomposition reaction may occur to form benzyl alcohol and glycerin.
  • benzyl alcohol When benzyl alcohol is formed, it initiates addition polymerization with the alkylene oxide, whereby a low-molecular-weight impurity having a molecular weight 0.5 time the molecular weight of the target compound.
  • the low-molecular-weight impurity derived from benzyl alcohol When the low-molecular-weight impurity derived from benzyl alcohol is formed, a functional group is introduced via alkyl-etherification of the hydroxyl group and deprotection in the subsequent steps as in the case of the target compound, so that the impurity is converted into a low-molecular-weight impurity which is reactive with a bio-related substance.
  • the reaction solvent is not particularly limited as far as it is an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, methylene dichloride, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, but preferable is toluene or no solvent.
  • the reaction time is preferably 1 to 24 hours. When the time is less than 1 hour, there is the possibility that the catalyst does not completely dissolved. When the time is longer than 24 hours, there is the possibility that the above decomposition reaction may occur.
  • reaction temperature in the step (C2) when the temperature is lower than 50° C., the polymerization rate is low and heat history increases to result in a tendency to decrease the quality of the compound of the formula (5). Moreover, when the temperature is higher than 130° C., side reactions such as vinyl etherification of the terminal end occur during the polymerization and thus the quality of the target compound tends to decrease.
  • an aprotic solvent preferably toluene may be optionally added.
  • step (C3) As another production process in the step of alcoholation, the following step (C3) may be mentioned.
  • the catalyst amount is preferably 5 to 50 mol % for the reason mentioned above.
  • the reaction temperature when the temperature is lower than 60° C., the conversion of the exchange reaction decreases and alcohols such as methanol remain, which leads to the formation of impurities having a molecular weight 0.5 time that of the target compound.
  • the temperature is higher than 80° C., a degradation reaction occurs.
  • the alcoholation reaction requires elevation of the temperature and the reaction time is desirably 1 to 3 hours since the degradation reaction is apt to occur.
  • the time is shorter than 1 hour, there is the possibility that the conversion into the alcoholate decreases.
  • the time is longer than 3 hours, the decomposition may occur.
  • the reaction solvent is not particularly limited as far as it is an aprotic solvent, but preferable is toluene or no solvent.
  • the subsequent alkyl-etherification of the terminal end may be achieved by either of the following (1) or (2):
  • each charged molar ratio satisfies the following relationship: 20Va ⁇ Vc ⁇ 3Va 4Vc>Vb>Vc.
  • Vc When Vc is smaller than 3Va, the conversion decreases and thus some part of the hydroxyl groups in the oxyalkylene chain terminal ends remain unchanged.
  • a functional group is introduced to the remaining hydroxyl group to form a polyfunctional impurity having a molecular weight the same as that of the target compound.
  • polyfunctional impurity When such polyfunctional impurity is present, it acts as a crosslinking agent at the combination with a bio-related substance to result in a tendency to decrease the purity of the resulting modified bio-related substance.
  • Vb is not larger than Vc, the conversion decreases owing to inefficient trapping of an acid which is produced as a by-product with the progress of the reaction, so that some part of the hydroxyl groups in the oxyalkylene chain terminal ends remain unchanged.
  • Vc is larger than 20Va or Vb is not smaller than 4Vc, an excess of each reagent or compound may be contained to cause side reactions in the subsequent processes.
  • the dehalogenating agent to be used includes organic bases such as triethylamine, pyridine, and 4-dimethylaminopyridine, and inorganic bases such as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium carbonate, and potassium hydroxide.
  • organic bases such as triethylamine, pyridine, and 4-dimethylaminopyridine
  • inorganic bases such as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium carbonate, and potassium hydroxide.
  • Preferable dehydrochlorinating agent is an organic base such as triethylamine, pyridine, or 4-dimethylaminopyridine.
  • W is preferably Cl or Br
  • R 1 is preferably a methyl group, a phenyl group, or a p-methylphenyl group. More suitably, methanesulfonyl chloride where W is Cl and R 1 is a methyl group is most preferable.
  • the solvent to be used at that time is not particularly limited as far as it is an aprotic solvent and preferably includes toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, methylene dichloride, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, but more preferable is toluene which enables azeotropic removal of water in the system.
  • the amount of the solvent to be used at the reaction is preferably 0.5 equivalent weight to 10 equivalent weight to the compound of the formula (5). In the case that the compound of the formula (5) has a large molecular weight, the viscosity of the reaction liquid increases and the conversion decreases, so that it is preferable to dilute the reaction liquid with the solvent.
  • the reaction temperature is not particularly limited but is preferably 60° C. or lower for the purpose of inhibiting side reactions and is preferably 20° C. or higher for the purpose of inhibiting increase of the viscosity of the reaction liquid.
  • the reaction time is preferably 1 to 24 hours. When the time is less than 1 hour, there is the possibility that the conversion is low. When the time is longer than 24 hours, there is the possibility that a side reaction may occur.
  • the operation of removing water from the starting materials such as azeotropic removal of water may be carried out prior to the reaction.
  • an antioxidant such as 2,6-di-tert-butyl-p-cresol may be added.
  • a salt is formed with the progress of the reaction and the formation of the compound of the formula (7), but the reaction mixture may be used in the subsequent step as it is, or the salt may be removed by filtration, or after the filtration, the compound of the formula (7) may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • Step (B2) A step of adding a compound represented by the formula (8) to the compound of the formula (7) and reacting them at 20 to 80° C. to obtain the compound of the formula (4). At that time, each charged molar ratio satisfies the following relationship: Vd>Vc Vd: number of moles of the compound represented by the formula (8).
  • R is as mentioned above and M is sodium or potassium, preferably sodium.
  • Vd is not larger than Vc
  • the alkyl-etherification does not sufficiently proceed and a reactive group such as a mesylate group remains unchanged at the oxyalkylene chain terminal end.
  • a reactive group remains at the oxyalkylene chain terminal end, as mentioned above, a polyfunctional compound is formed and a serious side reaction is caused at the combination with a bio-related substance.
  • Vd is not smaller than 10Vc
  • an excess of the alcoholate may be contained to cause side reactions in the subsequent process.
  • the solvent to be used in the reaction is not particularly limited as far as it is an aprotic solvent and is preferably toluene.
  • The.amount of the solvent to be used at the reaction is preferably an amount of 0.5 equivalent to 10 equivalent to the compound of the formula (7).
  • the viscosity of the reaction liquid increases, so that it is preferable to dilute the reaction liquid with the solvent.
  • the reaction temperature is not particularly limited but is preferably 80° C. or lower for the purpose of inhibiting side reactions and is preferably 20° C. or higher for the purpose of inhibiting increase of the viscosity of the reaction liquid.
  • the reaction time is preferably 1 to 24 hours. When the time is less than 1 hour, there is the possibility that the conversion is low. When the time is longer than 24 hours, there is the possibility that a side reaction occurs.
  • an operation of removing water from the starting materials such as azeotropic removal of water may be carried out prior to the reaction.
  • Step (B3) A step of filtrating the reaction liquid or washing the reaction liquid with an aqueous inorganic salt solution having a concentration of 10 wt % or more.
  • the inorganic salt is not particularly limited but is preferably sodium chloride.
  • concentration is less than 10 wt %, the target compound migrates into an aqueous layer to decrease the process yield remarkably.
  • the operation of washing with water may be repeated several times.
  • the step (B3) is carried out for removing starting materials excessively added and salts produced as by-products. The omission of the step may cause side reactions in the case that the steps (B1) to (B3) are again carried out in the next place. In the case that a debenzylation step is carried out as a next step, these impurities act as catalyst poisons and thus the conversion may be affected.
  • the compound of the formula (4) thus obtained may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • the production of the compound (p) by successive debenzylation is not particularly limited but it can be produced by hydrogenation of the following step (A) using a hydrogenative reduction catalyst and a hydrogen donor.
  • the decomposition reaction of the polyoxyalkylene chain occurs. Since polyalkylene glycol formed by the decomposition has a hydroxyl group, it is functionalized in the next step to form a reactive low-molecular-weight impurity.
  • Such reactive low-molecular-weight impurity reacts with a bio-related substance as mentioned above and thus tends to change the properties of the resulting preparation.
  • the hydrogenative reduction catalyst is preferably palladium.
  • the support is not particularly limited but is preferably alumina or carbon, more preferably carbon.
  • the amount of palladium is preferably 1 to 20 wt % based on the compound of the formula (4). When the amount is less than 1 wt %, the conversion of deprotection decreases and thus there is the possibility that the ratio of functionalization in the next step decreases. Moreover, when the amount is more than 20 wt %, the decomposition reaction of the polyalkylene glycol chain may occur and there is the possibility that the above reactive low-molecular-weight compound is produced as a by-product.
  • the reaction solvent is not particularly limited as far as the water content in the reaction system is less than 1%, but preferably includes methanol, ethanol, 2-propanol, and the like and more preferable is methanol.
  • the hydrogen donor is not particularly limited but include hydrogen gas, cyclohexene, 2-propanol, and the like.
  • the reaction temperature is preferably 40° C. or lower. When the temperature is higher than 40° C., the decomposition reaction of the polyalkylene glycol chain may occur and there is the possibility that the reactive low-molecular-weight compound is produced as a by-product.
  • the reaction time is not particularly limited. When large amount of the catalyst is used, the reaction is completed within a short period of time.
  • the reaction time is preferably 1 to 5 hours.
  • the time. is shorter than 1 hour, there is the possibility that the conversion is low.
  • the decomposition reaction of the poly(alkylene glycol) may occur.
  • the resulting compound of the formula (p) may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • the thus obtained compound is a polyalkylene glycol derivative represented by the following formula (p) and containing substantially no secondary hydroxyl group: wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA 1 and OA 2 are each an oxyalkylene group having 2 to 4 carbon atoms, R and OA 2 are the same or different from each other in one molecule, n and m are each average number of moles of the oxyalkylene group added, n represents 0 to 1000, and m represents 10 to 1000.
  • the conversion of the subsequent functional group-introducing reaction is high and a highly pure polyalkylene glycol derivative can be obtained.
  • a secondary hydroxyl group is present, the conversion of the subsequent functional group-introducing reaction is low and the purity of intermediate of the modified bio-related substance decreases, so that there may arise the problem of contamination of the drug or the like with an impurity.
  • the compound of the formula (p) of the invention satisfies the relationship: Hrd/Mpx1000000 ⁇ 3 wherein Mp is a molecular weight corresponding to the peak top obtained from gel-permeation chromatography of the polyalkylene glycol derivative of the formula (p), and Hrd is a ratio of remaining hydroxyl group contained in the alkyl group R at the polyoxyalkylene chain terminal end in the 2- and 3-positions.
  • Mp means a weight-average molecular weight at the point of the maximum refractive index among peaks excluding the peaks caused by a developing solvent used in gel-permeation chromatography and false peaks derived from base line fluctuation caused by the column and apparatus used.
  • gel permeation chromatography is carried out using SHODEX GPC SYSTEM-11 as a GPC system and measurement was conducted under the following conditions:
  • the ratio Hrd of the remaining hydroxyl group contained in the alkyl group R is measured after mesylation of the compound of the formula (4) which is a precursor before deprotection. The following will illustrate the case that R is a methyl group.
  • Hrd can be determined by suitably identifying a peak position where the alkyl group is detected and applying a similar equation in consideration of the proton number.
  • Hrd thus determined satisfies the following relationship: Hrd/Mpx1000000>3, the case means that a large amount of impurities where hydroxyl groups remain at 2- and 3-positions of the polyoxyalkylene chain terminal end are contained.
  • impurities When such impurities are present, the hydroxyl group of the polyoxyalkylene chain terminal end are also functionalized in the subsequent step to form polyfunctional impurities.
  • Such impurities may act as crosslinking agents at the combination with a bio-related substance as mentioned above to cause side reactions.
  • polydispersity Mw/Mn in all the peaks from the starting point of elution to the end point of elution satisfies the relationship: Mw/Mn ⁇ 1.07 at the measurement of gel permeation chromatography. More preferable is the case that the relationship: Mw/Mn ⁇ 1.05 is satisfied.
  • Mw/Mn is larger than 1.07, it means the presence of a large amount of the above-mentioned high-molecular-weight impurities and low-molecular weight impurity and when the compound is combined with a bio-related substance, there is the possibility that the formation of by products increases to result in an insufficient purity. Moreover, when the purity is insufficient, the product may cause an adverse effect when used as an medical product.
  • the compound of the formula (p) of the invention satisfies the relationship: M2/(M1+M2) ⁇ 100 ⁇ 10 wherein M1 is an integral value of the methyl group detected at around 3.13 ppm, which is originated from the mesyl group derived from the hydroxyl group at the 1-position directly bonded to the glycerin skeleton in the case that n is 0 when the compound is reacted with methanesulfonyl chloride to obtain a mesylated compound and a nuclear magnetic resonance spectrum thereof is measured as a deuterated methanol solution, and M2 is an integral value of the methyl group detected at around 3.12 ppm, which is originated from the mesyl group derived from the hydroxyl group of the polyalkylene glycol chain. More preferably, the relationship: M2/(M1+M2) ⁇ 100 ⁇ 8 is satisfied.
  • M1 is determined as an integral value of the methyl group detected at around 3.13 ppm, which is originated from the mesyl group derived from the hydroxyl group at the 1-position directly bonded to the glycerin skeleton in the case that n is 0, a TMS base peak being 0 ppm.
  • M2 is determined as an integral value of the methyl group detected at around 3.12 ppm, which is originated from the mesyl group derived from the polyalkylene glycol chain terminal end or polyalkylene glycol chain formed by the decomposition reaction.
  • the case means that a large amount of impurities having a hydroxyl group at the polyoxyalkylene chain terminal end, which are originated from the impurities:
  • the debenzylation reaction of the invention is widely applicable to other derivatives.
  • X 1 is an amino group, a carboxyl group, or a protected group thereof; and g1, g2, and g3 represent each an integer and satisfy the following relational equations: 1 ⁇ g1 ⁇ 3, 0 ⁇ g2, 0 ⁇ g3, 2 ⁇ g1+g2+g3 ⁇ 4.
  • More specific residual group of a compound having 2 to 4 hydroxyl groups in G includes ethylene glycol, glycerin, pentaerythritol, diglycerin, and the like, and more preferable is ethylene glycol or glycerin.
  • R 2 includes a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, and the like, and preferable is a methyl group.
  • m1, m2, and m3 they are not particularly limited as far as the relationships: 0 ⁇ m1 ⁇ 1000, 0 ⁇ m2 ⁇ 1000, 0 ⁇ m3 ⁇ 1000, 10 ⁇ m1+m2+m3 ⁇ 1000 are satisfied, but preferable is the case of 20 ⁇ m1+m2+m3 ⁇ 1000, more preferably is the case of 40 ⁇ m1+m2+m3 ⁇ 1000, and most preferable is the case of 100 ⁇ m1+m2+m3 ⁇ 1000.
  • Specific X 1 includes an amino group, a Boc amino group, an Fmoc amino group, a carboxyl group, and the like, and more preferable is a Boc amino group, wherein Boc means a t-butoxycarbonyl group and Fmoc means a 9-fluorenylmethoxycarbonyl group.
  • the water content in the reaction system, catalyst amount, reaction time, solvent, and the like are the same as those in the aforementioned step (A).
  • the hydrogenative reduction reaction can be carried out using a hydrogenative reduction catalyst.
  • the hydrogenative reduction catalyst is preferably palladium.
  • the alkyl-etherification of the invention is widely applicable to other derivatives.
  • Step (BB1) A step of adding a dehalogenating agent and a compound represented by the formula (14) to a compound represented by the formula (12) and reacting them at 20 to 60° C. to obtain a compound of the formula (13). At that time, each charged molar ratio satisfies the following relationship: Vj ⁇ 1.5 ⁇ Vh ⁇ g5 Vi>Vj
  • Step (BB2) A step of adding a compound represented by the formula (15) to a compound of the formula (13) and reacting them at 20 to 80° C. to obtain a compound of the formula (16). At that time, each charged molar ratio satisfies the following relationship: Vk>Vj
  • Step (BB3) A step of filtrating the reaction liquid or washing the reaction liquid with an aqueous inorganic salt solution having a concentration of 10 wt % or more.
  • W is preferably Cl or Br
  • R 3 is preferably a methyl group, a phenyl group, or a p-methylphenyl group, and most preferable is methanesulfonyl chloride where W is Cl and R 3 is a methyl group.
  • the inorganic salt is not particularly limited but is preferably sodium chloride.
  • an organic base such as triethylamine, pyridine, or 4-dimethylaminopyridine or an inorganic base such as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium carbonate, or potassium hydroxide and any one of the compounds represented by the following general formulae (b1), (d1), (h1), and (i1) in an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butyl methyl ether, tetrahydrofuran, chloroform, methylene dichloride, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide or without any solvent, (b), (d), (h), and (i) can be introduced, respectively.
  • an organic base such as triethylamine, pyridine, or 4-dimethylaminopyridine
  • an inorganic base such as sodium carbonate,
  • the ratio of the organic base or inorganic base to be used is not particularly limited but is preferably equimolar or more to the compound (p). Furthermore, an organic base may be used as a solvent.
  • W in (b1) or (d1) is a halogen atom selected from Cl, Br and I, and is preferably Cl.
  • the ratio of the compounds represented by the general formulae (b1), (d1), (h1), and (i1) to be used is not particularly limited but is preferably equimolar or more, more preferably equimolar to 50 molar to the compound (p).
  • the reaction temperature is preferably 0 to 300° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • the compound formed may be purified by a purification means such as extraction, recrystallization, adsorption treatment, reprecipitation, column chromatography, or supercritical extraction.
  • W is a halogen atom selected from Cl, Br and I.
  • the succinimide compound (a) can be obtained by reacting the compound (p) with a dicarboxylic acid anhydride such as succinic anhydride or glutaric anhydride to obtain a carboxyl compound (k), followed by condensation with N-hydroxysuccinimide in the presence of a condensing agent such as DCC. or EDC.
  • a dicarboxylic acid anhydride such as succinic anhydride or glutaric anhydride
  • the reaction of the compound (p) with a dicarboxylic acid anhydride is carried out in the aforementioned aprotic solvent or without any solvent.
  • the ratio of the dicarboxylic acid anhydride to be used is not particularly limited but is preferably equimolar or more, more preferably equimolar to 5 molar to the compound (p).
  • the reaction temperature is preferably 0 to 200° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 12 hours.
  • an organic base such as triethylamine, pyridine, or dimethylaminopyridine or an inorganic base such as sodium carbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassium carbonate, or potassium hydroxide may be used as a catalyst.
  • the ratio of the catalyst to be used is preferably 0.1 to 50 wt %, more preferably 0.5 to 20 wt %.
  • the carboxyl compound (k) thus formed may be purified by the aforementioned purification means or may be used as it is in the next condensation reaction.
  • the subsequent condensation reaction is also carried out in the aforementioned aprotic solvent or without any solvent.
  • the condensing agent is not particularly limited but is preferably DCC.
  • the ratio of DCC to be used is preferably equimolar or more, more preferably equimolar to 5 molar to the compound (p).
  • the ratio of N-hydroxysuccinimide to be used is preferably equimolar or more, more preferably equimolar to 5 molar to the compound (p).
  • the reaction temperature is preferably 0 to 100° C., more preferably 20 to 80° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 12 hours.
  • the compound formed may be purified by the aforementioned purification means.
  • the amine compound (g) can be obtained by reacting the compound (p) to acrylonitrile or the like using an inorganic base such as sodium hydroxide or potassium hydroxide in a solvent such as water and acetonitrile to obtain a nitrile compound and then subjecting it to hydrogenation of the nitrile group in the presence of a nickel or palladium catalyst in an autoclave.
  • the ratio of the inorganic base to be used for obtaining the nitrile compound is not particularly limited but is preferably 0.01 to 50 wt % to the compound (p).
  • the ratio of acrylonitrile or the like to be used is not particularly limited but is preferably 0.5 to 5 equivalent weight, more preferably 1 to 4 equivalent weight to the weight of the compound (p).
  • acrylonitrile may be used as a solvent.
  • the reaction temperature is preferably ⁇ 50 to 100° C., more preferably ⁇ 20 to 60° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • the reaction solvent in the subsequent hydrogenation reaction is not particularly limited as far as it does not participate in the reaction, but is preferably toluene.
  • the ratio of the nickel or palladium catalyst to be used is not particularly limited but is 0.05 to 30 wt %, preferably 0.5 to 20 wt % to the nitrile compound.
  • the reaction temperature is preferably 20 to 200° C., more preferably 50 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • the hydrogen pressure is preferably 2 to 10 MPa, more preferably 3 to 8 MPa.
  • ammonia may be added to the reaction system.
  • the ammonia pressure is not particularly limited but is 0.1 to 10 MPa, more preferably 0.3 to 2 MPa.
  • the compound formed may be purified by the aforementioned purification means.
  • the above amine compound (g) or (j) can be also obtained by reacting (b) with aqueous ammonia.
  • the reaction is carried out in aqueous ammonia and the concentration of ammonia is not particularly limited but is preferably in the range of 10 to 40%.
  • the ratio of aqueous ammonia to be used is preferably 1 to 300 times the weight of (b).
  • the reaction temperature is preferably 0 to 100° C., more preferably 20 to 80° C.
  • the reaction time is preferably 10 minutes to 72 hours, more preferably 1 hour to 36 hours.
  • the amine compound (g) or (j) can be also obtained by reacting (b) with ammonia in an autoclave.
  • the reaction solvent is not particularly limited but preferably includes methanol and ethanol.
  • the amount of ammonia is preferably 10 to 300 wt %, more preferably 20 to 200 wt %.
  • the reaction temperature is preferably 50 to 200° C., more preferably 80 to 150° C.
  • the reaction time is preferably 10 minutes to 24 hours, more preferably 30 minutes to 12 hours.
  • the compound formed may be purified by the aforementioned purification means.
  • the maleimide compound (e) can be obtained by reacting the resulting amine (g) with maleic anhydride in the aforementioned aprotic solvent or without any solvent to obtain an maleamide compound and then subjecting it to a ring closure reaction using acetic anhydride or sodium acetate.
  • the ratio of maleic anhydride to be used in the maleamidation reaction is not particularly limited but is preferably equimolar or more, more preferably equimolar to 5 molar to the compound (p).
  • the reaction temperature is preferably 0 to 200° C., more preferably 20 to 120° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 12 hours.
  • the maleamide compound formed may be purified by the aforementioned purification means or may be used as it is in the next ring closure reaction.
  • the reaction solvent in the subsequent ring closure reaction is not particularly limited but is preferably aprotic solvent or acetic anhydride.
  • the ratio of sodium acetate to be used is not particularly limited but is preferably equimolar or more, more preferably equimolar to 50 molar to the maleamide compound.
  • the reaction temperature is preferably 0 to 200° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 12 hours.
  • the compound formed may be purified by the aforementioned purification means.
  • the above maleimide compound can be also obtained by reacting the compound of the following formula (e1) with the aforementioned amine (g) or (j).
  • the reaction is carried out in the aforementioned aprotic solvent or without any solvent and the compound (e1) is added in an amount of equimolar or more to the amine (g) or (j).
  • the ratio of the compound (e1) to be used is preferably equimolar or more, more preferably equimolar to 5 molar to the amine (g) or (j).
  • the reaction temperature is preferably 0 to 200° C., more preferably 20 to 80° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 12 hours. During the reaction, light shielding may be conducted.
  • the compound formed may be purified by the aforementioned purification means. wherein Q represents a hydrocarbon group having 1 to 7 carbon atoms.
  • the aldehyde compound (f) can be obtained by reacting the compound (b) with an acetal compound (f1) to obtain an acetal compound and then subjecting it to hydrolysis under an acidic condition.
  • the compound (b) is produced as mentioned above.
  • the acetalization reaction can be achieved by reacting the compound (b) with an equimolar or more amount, preferably an equimolar to 50 molar amount of the compound (f1) in the aforementioned aprotic solvent or without any solvent.
  • the compound (f1) can be prepared from the corresponding alcohol using sodium, potassium, sodium hydride, sodium methoxide, potassium t-butoxide, or the like.
  • the reaction temperature is preferably 0 to 300° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • an acetal compound can be obtained by converting the hydroxyl group of the compound (p) into an alcoholate by the aforementioned method and then reacting it with an equimolar or more amount, preferably an equimolar to 100 molar amount of the compound (f2) in the aforementioned aprotic solvent or without any solvent.
  • the reaction temperature is preferably 0 to 300° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • an acetal compound can be obtained by reacting the compound (f3) with the compound (a), (b), (d), (h), (i), or (k).
  • the compound (a), (b), (d), (h), (i), or (k) is produced as mentioned above.
  • the solvent is not particularly limited but the reaction is preferably carried out in the aforementioned aprotic solvent.
  • the charging ratio of the compound (f3) is preferably equimolar or more, more preferably equimolar to 10 molar to the compound (a), (b), (d), (h), (i), or (k).
  • the reaction temperature is preferably ⁇ 30 to 200° C., more preferably 0 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • a condensing agent such as DCC or EDC may be optionally used. Any acetalization reaction may be carried out under light shielding.
  • the acetal compound thus obtained may be purified by the aforementioned purification means or may be used as it is in the next aldehyde-formation reaction.
  • the aldehyde compound can be produced by hydrolyzing the acetal compound in a 0.1 to 50% aqueous solution which is adjusted to pH 1 to 4 with an acid such as acetic acid, phosphoric acid, sulfuric acid, or hydrochloric acid.
  • the reaction temperature is preferably ⁇ 20 to 100° C., more preferably 0 to 80° C.
  • the reaction time is preferably 10 minutes to 24 hours, more preferably 30 minutes to 10 hours.
  • the reaction may be carried out under light shielding.
  • the compound formed may be purified by the aforementioned purification means.
  • R 1 and R 2 are each a hydrocarbon group having 1 to 3 carbon atoms and may be the same or different from each other, and they may together form a ring;
  • M is sodium or potassium;
  • W is a halogen atom selected from Cl, Br, and I; and
  • t is an integer of 1 to 5.
  • the mercapto compound (c) can be obtained by reacting the compound (b) with a thiol-forming agent such as thiourea.
  • the compound (b) is produced as mentioned above.
  • the thio-formation reaction is carried out in a solvent such as water, an alcohol, or acetonitrile or without any solvent.
  • the ratio of thiourea to be used is equimolar or more, more preferably equimolar to 50 molar to the compound (b).
  • the reaction temperature is preferably 0 to 300° C., more preferably 20 to 150° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • the mercapto compound can be obtained by subjecting the resulting thiazolium salt to alkali hydrolysis.
  • the compound formed may be purified by the aforementioned purification means.
  • the above mercapto compound can be also obtained by reacting the compound (b) with the following compound (c1), followed by decomposition with a primary amine.
  • the reaction of the compound (b) with the compound (c1) is carried out in the aforementioned aprotic solvent or without any solvent.
  • the ratio of the compound (c1) to be used is equimolar or more, more preferably equimolar to 50 molar to the compound (b).
  • the reaction temperature is preferably 0 to 300° C., more preferably 20 to 80° C.
  • the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours.
  • the subsequent alkali decomposition with a primary amine is carried out in the aforementioned aprotic solvent or without any solvent.
  • the primary amine to be used is not particularly limited but preferably includes ammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, cyclohexylamine, ethanolamine, propanolamine, and butanolamine.
  • the primary amine may be used as a solvent.
  • the compound formed may be purified by the aforementioned purification means.
  • a bio-related substance modified with a branched poly(alkylene glycol)oxy group can be obtained.
  • the bio-related substance is formed by ether bonds only except for the linker part with the poly(alkylene glycol)oxy group, so that a high stability can be expected with no decomposition to a single chain. Therefore, by modifying a bio-related substance with a branched polyalkylene glycol, a bio-related substance exhibiting an improved behavior in a body can be provided.
  • the intermediate of the bio-related substance of the invention is a novel compound having a reactive group, which can be combined with a bio-related substance, at the primary carbon at the 1-position of the glycerin skeleton and having polyalkylene glycol chains at the 2- and 3-positions.
  • Mn represents a number average molecular weight
  • Mw a weight average molecular weight
  • Mp a peak top molecular weight
  • a Karl Fisher's moisture meter 7S8/3-20 type manufactured by Metrome-Shibata
  • “HYDRANAL-composite 2” manufactured by Sigma Aldrich was employed as a Karl Fisher's reagent.
  • GPC analysis ⁇ main peak>number average molecular weight (Mn): 9978, weight average molecular weight (Mw): 10171, polydispersity (Mw/Mn): 1.019, peak top molecular weight (Mp): 10044; ⁇ whole peak>number average molecular weight (Mn): 9865, weight average molecular weight (Mw): 10114, polydispersity (Mw/Mn): 1.025, peak top molecular weight (Mp): 10044.
  • the reaction liquid was filtered and the filtrate was transferred into a 500 ml round-bottom flask fitted with a thermometer, a nitrogen-introducing tube, a stirrer, and a condenser tube. Then, 19.3 g (100 mmol) of 28% methanol solution of sodium methoxide was added thereto, followed by 6 hours of reaction at 70° C. Subsequently, 27 g of an adsorbent “KYOWAAD 700” (manufactured by Kyowa Chemical Industry Co., Ltd.) was added to the reaction liquid and the whole was further stirred at 70° C. for 1 hour to adsorb excessive sodium methoxide.
  • KYOWAAD 700 manufactured by Kyowa Chemical Industry Co., Ltd.
  • the filtrate was charged into a 1 L beaker and crystallization was carried out by adding 300 g of ethyl acetate and 350 g of hexane.
  • the precipitated crystals were collected into a 1 L beaker by filtration and dissolved under heating at 40° C. with adding 400 g of ethyl acetate. Thereafter, 300 g of hexane was added and crystallization was again carried out.
  • the precipitated crystals were collected by filtration and dried to obtain the following compound (p2).
  • the reaction liquid was filtered and 1.0 g of an adsorbent “KYOWAAD 1000” (manufactured by Kyowa Chemical Industry Co., Ltd.) was added to the filtrate and the whole was further stirred at 60° C. for 1 hour to adsorb triethylamine salt of methanesulfonic acid as a by-product.
  • the filtrate was charged into a 500 ml beaker and crystallization was carried out by adding 100 ml of ethyl acetate and 150 ml of hexane.
  • the precipitated crystals were collected into a 300 ml beaker by filtration and dissolved under heating at 40° C.
  • reaction liquid was poured into the round-bottom flask containing the compound (p4) from which water had been removed as above, followed by 12 hours of reaction at 110° C. After cooling of the reaction liquid to 40° C., 0.36 g (20 mmol) of ion-exchange water was added and the whole was stirred for 30 minutes. Then, 50 ml of 20% aqueous sodium chloride solution was added thereto and the aqueous layer was adjusted to pH 7.0 with 85% phosphoric acid. After the upper toluene layer was separated, the aqueous layer was extracted twice with chloroform. The toluene layer and the chloroform layer were combined and dried over sodium sulfate.
  • the concentrate was dissolved under heating by adding 30 ml of toluene and 30 ml of ethyl acetate and then 60 ml of hexane was added to precipitate crystals, which was collected by filtration.
  • the resulting crystals were weighed into a 200 ml beaker and dissolved under heating by adding 30 ml of toluene and 30 ml of ethyl acetate and then 60 ml of hexane was added to precipitate crystals again, which was collected by filtration and dried to obtain the following aldehyde compound (p7).
  • the treated liquid was analyzed by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 20%). The gel was stained by CBB staining. The results were shown in FIG. 1 .
  • (A) is a lane of OVA+aldehyde compound,
  • (B) a lane of OVA alone, and
  • (C) a lane of a marker (Bio-rad Broad range SDS-PAGE standards) which shows bands of molecular weights of 201000, 130000, 94000, 48600, 36400, 29800, 20600, and 600 from the top.
  • the following model compound was synthesized and the stability was compared.
  • the resulting concentrate was dissolved by adding 20 ml of ethyl acetate and then 30 ml of hexane was added to precipitate crystals, which was collected by filtration.
  • the resulting crystals were weighed into a 100 ml beaker and dissolved under heating with adding 20 ml of ethyl acetate and then 20 ml of hexane was added to precipitate crystals again, which was collected by filtration and dried to obtain the following compound (p8).
  • FIG. 2 is a GPC chart of the sample before starting and FIG. 3 is a GPC chart of the sample of (p8) after heating.
  • FIG. 4 is a GPC chart of the sample of (p10) before starting and FIG. 5 is a GPC chart of the sample of (p10) after heating.
  • the reaction liquid was filtered and, after addition of 100 ml of ethyl acetate to the filtrate, 200 ml of hexane was added to precipitate crystals.
  • the precipitated crystals were collected by filtration and dissolved under heating with adding 100 ml of ethyl acetate. Then, 100 ml of hexane was added thereto to crystallize the product again. The crystallization operation was repeated five times in total.
  • the crystals collected by filtration were dried to obtain the following p-nitrophenyl carbonate compound (p15).
  • the reaction liquid was filtered and 0.5 g of an adsorbent “KYOWAAD 1000” was added to the filtrate and the whole was stirred at 60° C. for 1 hour to adsorb triethylamine salt of methanesulfonic acid as a by-product.
  • the filtrate was charged into a 300 ml beaker and crystallization was carried out with adding 50 ml of ethyl acetate and 70 ml of hexane.
  • the precipitated crystals were collected into a 300 ml beaker by filtration and dissolved under heating at 40° C. with adding 50 ml of ethyl acetate. Thereafter, 50 ml of hexane was added and crystallization was again carried out.
  • the precipitated crystals were collected by filtration and dried to obtain the following mesylate compound (p17).
  • reaction liquid was poured into the round-bottom flask containing the compound (p17) from which water had been removed as above, followed by 4 hours of reaction at 70° C. After cooling of the reaction liquid to 40° C., 0.18 g (10 mmol) of ion-exchange water was added and the whole was stirred for 30 minutes. Then, 30 ml of 20% aqueous sodium chloride solution was added thereto and the aqueous layer was adjusted to pH 7.0 with 85% phosphoric acid. After the upper toluene layer was separated, the aqueous layer was extracted twice with chloroform. The toluene layer and the chloroform layer were combined and dried over sodium sulfate.
  • the concentrate was dissolved under heating with adding 30 ml of toluene and 30 ml of ethyl acetate and then 60 ml of hexane was added to precipitate crystals, which was collected by filtration.
  • the resulting crystals were weighed into a 200 ml beaker and dissolved under heating with adding 30 ml of toluene and 30 ml of ethyl acetate and then 60 ml of hexane was added to precipitate crystals again, which was collected by filtration and dried to obtain the following aldehyde compound (p19).
  • the reaction liquid was filtrated and the water content of the filtrate was measured by means of Karl Fisher's moisture meter and found to be 1259 ppm.
  • the filtrate was concentrated and 1 L of ethyl acetate was added to the concentrate, followed by addition of hexane until crystals were precipitated. The resulting crystals were collected by filtration and dried to obtain the following compound (p25).
  • Example 16-3 2.0 kg of dry toluene was added to about 1 kg of the reaction liquid which remained in the autoclave. After 1.0 kg of toluene was removed by evaporation at an autoclave temperature of 95° C. under slight reduced pressure and then the atmosphere of the autoclave was replaced by nitrogen. After heating to 120° C., 1260 g of ethylene oxide was introduced under pressure thereto at 100 to 150° C. under a pressure of 1 MPa or lower, followed by continuation of the reaction for another 4 hours. After completion of the reaction, the whole was cooled to 70° C. and the reaction liquid was adjusted to pH 7.5 with 85% aqueous phosphoric acid solution to obtain the following compound (p26).
  • the reaction liquid was filtrated and the water content of the filtrate was measured by means of Karl Fisher's moisture meter and found to be 2215 ppm.
  • the filtrate was concentrated and 1 L of ethyl acetate was added to the concentrate, followed by addition of hexane until crystals were precipitated. The resulting crystals were collected by filtration and dried to obtain the following compound (p28).
  • the resulting chloroform layer was dried over magnesium sulfate, filtrated, and then concentrated. Thereafter, 700 ml of ethyl acetate was added to the concentrate, which was dissolved therein. Then, hexane was added thereto until crystals were precipitated. The crystals were collected by filtration and again dissolved in 700 ml of ethyl acetate under heating. After cooling to room temperature, hexane was added thereto until crystals were precipitated. The crystals were collected by filtration and dried to obtain the following nitrile compound (p29).
  • reaction liquid was charged into a 5 L autoclave thoroughly dried beforehand and the same operations as in Examples 16-3, 16-6, 16-7, and 16-8 were conducted to obtain the compound (p33) having the same structure as that of (p28).
  • GC system HP6890, column: HP-5 (0.25 ⁇ m ⁇ 30 cm), detector: FID, injection temperature: 320° C., injection: splitless, injection amount: 0.2 ⁇ l, carrier gas: helium, flow rate: 23 cm/sec, column temperature 80° C. (0 min) ⁇ 15° C./min ⁇ 320° C. 24 min), detector temperature: 320° C. TABLE 3 Benzyl alcohol Glycerin Example 1-3 0% 0.4% Example 16-3 0% 0.3% Example 20 2.2% 1.3%
  • Example 7-2 Using 10 g (0.5 mmol) of the compound (p12) obtained in Example 7-2, 1.01 g (10 mmol) of triethylamine, and 0.344 g (3 mmol) of methanesulfonyl chloride, the same operations as in Example 22 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, Mme was 6 and Mms was 0.102.
  • Example 16-4 Using 10 g (0.5 mmol) of the compound (p24) which had been twice alkyl-etherified in Example 16-4, 1.01 g (10 mmol) of triethylamine, and 0.344 g (3 mmol) of methanesulfonyl chloride, the same operations as in Example 22 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, Mme was 6 and Mms was 0.019.
  • Example 16-7 Using 11.3 g (0.25 mmol) of the compound (p27) which had been twice alkyl-etherified in Example 16-7, 0.506 g (5 mmol) of triethylamine, and 0.172 g (1.5 mmol) of methanesulfonyl chloride, the same operations as in Example 22 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, Mme was 6 and Mms was 0.026.
  • Example 16-5 Using 10 g (0.5 mmol) of the compound (p25) obtained in Example 16-5, 1.01 g (10 mmol) of triethylamine, and 0.344 g (3 mmol) of methanesulfonyl chloride, the same operations as in Example 27 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, M1 was 3 and M2 was 0.091.
  • Example 16-8 Using 11.3 g (0.25 mmol) of the compound (p28) obtained in Example 16-8, 0.51 g (5 mmol) of triethylamine, and 0.172 g (1.5 mmol) of methanesulfonyl chloride, the same operations as in Example 27 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, M1 was 3 and M2 was 0.112.
  • Example 20 Using 11.3 g (0.25 mmol) of the compound (p33) obtained in Example 20, 0.51 g (5 mmol) of triethylamine, and 0.172 g (1.5 mmol) of methanesulfonyl chloride, the same operations as in Example 27 were conducted. Then, 1 H-nuclear magnetic resonance measurement was conducted (integration: 256 times) to obtain a spectrum. At that time, M1 was 3 and M2 was 0.212.
  • SP-Sepharose FF manufactured by Amersham
  • insulin recombinant human insulin, Mw 5800, manufactured by SEROLOGICALS CORPORATION
  • a 10 mg/ml buffer solution of the insulin was prepared.
  • Into 100 ⁇ l of the solution was added 6.8 mg of the compound of the formula (p32), followed by 20 hours of reaction at 4° C.
  • a solution obtained by adding NaCl to the buffer so as to be 1N was passed through the column and a fraction of the peptide modified with (p32) was obtained with monitoring the elute by UV.
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