JP2002542386A - Releasable bonds and compositions containing the same - Google Patents

Abstract

(57) Abstract A compound composed of a hydrophilic polymer conjugated and reversibly linked to an amine-containing ligand via a dithiobenzyl bond is described.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to hydrophilic polymer compounds, such as polyethylene glycol, cleavably bound to an amine-containing ligand (in a preferred embodiment, an amine-containing lipid, drug or protein). The compound can be cleaved under mild thiol cleavage conditions to regenerate the amine-containing ligand in its original form.

BACKGROUND OF THE INVENTION [0002] Various substrates, such as polypeptides, drugs and liposomes, are modified to reduce the immunogenicity of a substrate, improve its blood circulation lifetime, or fulfill both functions. For this reason, hydrophilic polymers such as polyethylene glycol (PEG) have been used.

[0003] For example, parenterally administered proteins are immunogenic and may have a short pharmaceutical half-life. The protein is relatively water-insoluble, and as a result, achieving therapeutically useful blood levels of the protein in the patient's body may be difficult. As a method of overcoming this problem, PEG and protein conjugation have already been reported. Davis et al., U.S. Pat.
Methods have been disclosed for conjugating PEG to proteins such as enzymes and insulin to reduce immunogenicity and form PEG-protein conjugates that still retain biological activity. Veronese et al. (Applied Biochem. And Biotech. 11 , 141-152, 198
5) reports on a method for modifying ribonuclease and superoxide dismutase by activating polyethylene glycol with phenyl chloroformate. Katre et al., U.S. Pat. Nos. 4,766,106 and 4,917,888 disclose methods of solubilizing proteins by conjugating polymers. PEG and other polymers are conjugated to recombinant proteins, reducing immunogenicity and increasing half-life (Nitecki et al., US Patent No. 4,902,502, Enzon Inc., PCT / US90 / 02133).
). Garman (US Pat. No. 4,935,465) describes proteins modified with a water-soluble polymer linked to the protein by a reversible linking group.

[0004] However, the PEG-protein conjugates reported so far have the disadvantage that the protein is often inactivated and the biological activity of the resulting conjugate is reduced. In the prior art, the PEG usually forms a stable bond with the protein, trying to retain the favorable properties provided by the PEG. For some protein-PEG conjugates, there is also the problem that the conjugate degrades to form undesirable products.

With respect to PEG, a method has been described for using it to improve the circulating life of liposomes (US Pat. No. 5,103,556). Here, PEG is conjugated to a polar lipid head group to mask or protect the liposome from being recognized and removed by the reticuloendothelial system. Liposomes with releasable PEG chains have also been described, where the PEG chains are released from the liposomes upon exposure to an appropriate stimulus such as a pH change (PCT / US97 / 18813). However, the release of the PEG chains from the liposomes has the disadvantage that the degradation products are chemically modified in vivo and may exhibit unpredictable and potentially negative effects.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a compound in which a ligand is conjugated to a hydrophilic polymer in a conjugated and reversible manner. When the bond is cleaved, the ligand is regenerated in its original natural form.

In one embodiment, the present invention includes a compound represented by the following general formula: That is, Here, R ¹ is a hydrophilic polymer constituting a bond for addition to the dithiobenzyl moiety, R ² is selected from H, an alkyl group and an aryl group, and R ³ is O (
Selected from C ＝ O) R ⁴ , S (CＣO) R ⁴ , and O (C ＝ S) R ⁴ , wherein R ⁴ is comprised of an amine-containing ligand, and R ⁵ is H, an alkyl group and an aryl And the configuration of CH ₂ —R ³ is selected from the ortho and para positions.

In one embodiment, R ⁵ is H and R ² is CH ₃ , C ₂ H ₅ and C ₃ H ₈
Selected from In another embodiment, R ² and R ⁵ is an alkyl group.

In another embodiment, the amine-containing ligand R ⁴ is selected from a polypeptide, an amine-containing drug, and an amine-containing lipid. The amine-containing ligand R ⁴
In one embodiment, where is an amine-containing lipid, the lipid comprises a single hydrocarbon tail or a double hydrocarbon tail. In one preferred embodiment, the lipid is a hydrocarbon double tailed phospholipid.

In yet another embodiment, the lipophilic polymer R ¹ is polyvinylpyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethyl. Acrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, copolymers thereof, and polyethylene oxide-polypropylene oxide.

[0011] In one preferred embodiment, the hydrophilic polymer R ¹ is polyethylene glycol. In another embodiment, when R ¹ is polyethylene glycol, R ⁵ is H and R ² is CH ₃ or C ₂ H ₅ .

[0012] In yet another embodiment, the amine-containing ligand R ⁴ is a polypeptide. In other embodiments, the polypeptide may be a recombinant polypeptide. Preferred polypeptides to recommend include cytokins such as interferons, interleukins, growth factors, and enzymes.

In another embodiment, the present invention provides a compound of the general structure A composition comprising a conjugate obtained by reaction with a compound having the following formula: Here, R ¹ is a hydrophilic polymer constituting a bond for addition to the dithiobenzyl moiety, R ² is selected from H, an alkyl group and an aryl group, and R ³ is O (
Selected from among CＲO) R ⁴ , S (C ＝ O) R ⁴ , and O (C ＝ S) R ⁴ , wherein R ⁴ is constituted by a group to be removed, and R ⁵ is H, an alkyl group and an aryl group. The coordination of CH ₂ —R ³ is selected from the ortho and para positions. The composition also includes pharmaceutical carriers such as physiological saline and buffers.

In one embodiment of this aspect, R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ .

In another embodiment, R ³ is O (C ＝ O) R ⁴ , wherein R ⁴ is a hydroxy or oxy group-containing removed group. In another embodiment, the group to be removed is chloride, paranitrophenol, orthonitrophenol, N-hydroxy-.
Tetrahydrophthalimide, N-hydroxysuccinimide, N-hydroxy-
Glutarimide, N-hydroxynorbornene-2,3-dicarboximide, 1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine, 2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, It is derived from a compound selected from imidazole, triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol and trichlorophenol.

In one embodiment, the claimed compound is reacted with an amine-containing ligand that replaces R ⁴ to form a conjugate containing the amine-containing ligand. For example, the amine-containing ligand may be a phospholipid.

In one preferred embodiment, the hydrophilic polymer R ¹ is polyethylene glycol, R ⁵ is H, and R ² is CH ₃ or C ₂ H ₅ .

In another embodiment of the present embodiment, the composition containing the conjugate is composed of a liposome. The liposome can be further comprised of a capture therapeutic.

In another embodiment, the amine-containing ligand comprises a polypeptide.

[0020] In yet another aspect of the invention, a liposome composition comprising a liposome comprising a surface coating of a hydrophilic polymer chain is included. Here, at least a part of the hydrophilic polymer chain has a general structure have. Here, R ¹ is a hydrophilic polymer constituting a bond for addition to the dithiobenzyl moiety, R ² is selected from H, an alkyl group and an aryl group, and R ³ is O (
Selected from C ＝ O) R ⁴ , S (CＣO) R ⁴ , and O (C ＝ S) R ⁴ , wherein R ⁴ is comprised of an amine-containing ligand, and R ⁵ is H, an alkyl group and an aryl Selected from among the groups, wherein the coordination of CH ₂ —R ³ is selected from the ortho and para positions. The liposome has a longer circulation life in blood than liposomes having a hydrophilic polymer chain attached to the liposome via an aliphatic disulfide bond.

[0021] In one embodiment, the liposome is further comprised of a entrapped therapeutic agent.

In yet another aspect, the invention includes a method for improving the circulating longevity of liposomes having a surface coating of releasable hydrophilic polymer chains.
The method includes preparing liposomes having about 1% to about 20% of a compound having the general structure: Here, R ¹ , R ² , R ³ and R ⁵ are as described above, and R ⁴ is composed of an amine-containing lipid.

In one preferred embodiment of this aspect, R ⁵ is H and R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ .

In one embodiment, the amine-containing lipid is comprised of a phospholipid and R ¹ is polyethylene glycol.

In this embodiment, the liposome may be further composed of a capture therapeutic.

These and other objects and features of the present invention will become more fully understood from the following detailed description of the invention when read in conjunction with the accompanying drawings.

(Detailed Description of the Invention) Definitions As used herein, the term "polypeptide" refers to polymers of amino acids in general, and does not specifically mean amino acid polymers of a particular length. Therefore,
For example, peptides, oligopeptides, proteins, enzymes and the like are included within the definition of "polypeptide". The term also includes post-expression modifications of the polypeptide, such as glycosylated, acetylated, phosphorylated, and the like.

The term “amine-containing” refers to RNH ₂ (primary amine) and R ₂ NH obtained by replacing one or two hydrogen atoms of ammonia with an alkyl or aryl group.
All compounds having a derived moiety having the general structure of (secondary amine) are meant. Here, R may be any hydrocarbyl group.

“Hydrophilic polymer” as used herein refers to a polymer having a water-soluble moiety. The water soluble moiety confers some water solubility to the polymer at room temperature. Typical hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethyl-acrylamide, polyhydroxypropylmethacrylate, Examples include polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, polyaspartamide, copolymers of the above polymers, and polyethylene oxide-polypropylene oxide copolymers. The properties of these polymers and their reaction with many of these polymers are described in U.S. Patent Nos. 5,395,619 and 5,631,018.

The “polymer composed of a reactive functional group” or the “polymer composed of a bonding group for addition” is mainly a terminal portion (but not necessarily a terminal portion) which reacts with another compound to form a conjugated bond. (Not necessarily a moiety). For those skilled in the art, it is easy to choose a reaction mechanism for functionalizing the polymer by introducing such a reactive functional group moiety, for example, US Pat.
No. 3,018 or the report of Zalipsky et al., For example, Eur. Polymer J., 19 (12), 1177-1183 (
1983); Bioconj. Chem., 4 (4), 296-299 (1993).

The term “recombinant,” as used in “recombinant polypeptides” and the like, refers to the attachment of an amino acid into a desired sequence by laboratory procedures.

As used herein, the term “alkyl group” refers to a group of the form “C _n H _{2n + 1} ” _where one hydrogen atom has been removed from any carbon atom of an alkane group. is there. By removal of one hydrogen atom from a terminal carbon atom of straight-chain alkane group, a subclass of positive alkyl (n- alkyl) group H [CH _2] n groups are formed.
RCH ₂ - group and R ₂ CH- group (R is not H), and R ₃ C-group (R is not hydrogen), the primary, respectively, are called secondary and tertiary alkyl groups.

The term “aryl group” is intended to mean a substituted or unsubstituted monovalent aromatic group having one ring (eg, a benzene ring) or two fused rings (eg, a naphthyl group). The term includes heteroaryl groups, which are aromatic ring groups containing one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole. included. The term "substituted" means that one or more ring hydrogens in the aryl group is a halogen such as fluorine, chlorine or bromine; a lower alkyl group containing one or more carbon atoms; a nitro group; Group; methylamino group; dimethylamino group; methoxy group; halomethoxy group; halomethyl group; or haloethyl group.

The term “aliphatic disulfide” bond means a bond in the form of R′—S—S—R ″, wherein R ′ and R ″ are straight or branched alkyl chains. Yes, the linear or branched alkyl chain may be further substituted with another element or group.

The following abbreviations have been used herein: That is, PEG is poly (ethylene glycol), mPEG is methoxy PEG, DTB is dithiobenzyl group, M
eDTB is methyldithiobenzyl group, EtDBP is ethyldithiobenzyl group, DSPE is distearoyl phosphatidylethanolamine, DOPE is dioleoyl phosphatidylethanolamine, PHPC is partially hydrogenated phosphatidylcholine, and MALDI-TOFMS is flight mass spectrometry. Matrix-assisted laser desorption / ionization time
desorption / ionization time-of-flight mass spectrometry).

II. Compounds According to the Invention In one embodiment, the invention consists of compounds having the following structure: Here, R ¹ is composed of a hydrophilic polymer containing a functional group suitable for conjugation to the dithiobenzyl moiety of the polymer. R ² and R ⁵ are H, an alkyl group or an aryl group, which can be independently selected so that the rate of disulfide cleavage can be controlled, as will be apparent from the description of the present specification. For example, in order to accelerate the cleavage rate, hydrogen is preferably selected as R ² and R ⁵ . In order to decrease the cleavage rate, it is preferable to select an alkyl group or an aryl group for R ² and R ⁵ or any one of them to give steric hindrance to disulfide. R ³ is comprised of a binding moiety that joins R ⁴ , wherein R ⁴ is comprised of an amine-containing ligand. In a preferred embodiment, the binding moiety is O (C = O), S (C = O), or O (C = O). The amine-containing ligand R ⁴ is a primary or secondary amine, which can be selected from any number of substrates. Such substrates include, but are not limited to, lipids, pharmaceuticals, polypeptides, viruses, biological material surfaces, and aminoglycosides.
In a preferred embodiment, R ⁴ is a lipid containing primary or secondary amine, pharmaceutical or polypeptide. In the compounds of the present invention, the coordination of CH ₂ —R ³ is either ortho or para.

FIG. 1A shows the structure of a representative compound according to the present invention. Where R ¹
Is a hydrophilic polymer methoxy-polyethylene glycol, mPEG = CH ₃
O (CH ₂ CH ₂ O) n, where n is an integer from about 10 to about 2300. The integer range corresponds to a molecular weight from about 440 daltons to about 100,000 daltons. The molecular weight of the polymer is dependent on some degree the choice of R ^3. In embodiments, such as R ³ is a amine-containing lipids used in the liposomes, the preferred molecular weight of the PEG is from about 750 to about 10,000 daltons, more preferably in the range of about 2,000 to about 5,000 daltons. The mPEG in this embodiment includes a urethane binding moiety. In embodiments where R ³ is an amine-containing polypeptide,
A preferred range for the PEG molecular weight is from about 2,000 to about 40,000 daltons, more preferably from about 2,000 to about 20,000 daltons. R ¹ may be selected from a variety of hydrophilic polymers, and it will be understood that representative polymers are as described above. It will also be appreciated that for certain ligands, such as polypeptides, the molecular weight of the polymer will vary depending on the number of polymer chains attached to the ligand. That is, when the number of bonding polymer chains is small, a polymer having a molecular weight as large as possible is selected.

Still referring to FIG. 1 a, R ² and R ⁵ in this representative compound are H. However, either or both of R ² and R ⁵ may be a linear or branched alkyl or aryl group. In a preferred embodiment R ⁵ is H, R ² is an alkyl group. In this regard, some examples are given below. In the compound shown in FIG. 1A, R ³ takes the general form of O (C ＝ O)-(NH ₂ -ligand). Where NH ₂
The ligand may be an amine-containing polypeptide, a pharmaceutical or a lipid. Specific examples of each embodiment are shown below. R ³ is O (C = S) - ( NH 2 - ligand) or S (C = O) - can also take the form of - (NH ₂ ligand).

FIG. 1B shows the thiol cleavage mechanism of the mPEG-DTB- (NH ₂ -ligand) compound of FIG. 1A. The ortho- or para-dithiobenzyl carbamate moiety is
It can be cleaved under mild thiol cleavage conditions, such as in the presence of cysteine or other naturally occurring reducing agents. When cleavage occurs, the amine-containing ligand is regenerated in its native, unmodified form. The following research results supporting the present invention are:
This indicates that the cleavage of the DTB bond is sufficiently initiated under natural physiological conditions in vivo, and that this can be achieved. From this fact, it will be understood that thiol cleavage conditions sufficient for cleavage and degradation of the compound can be artificially induced by administering a reducing agent.

As noted above, R ³ takes the general form of a linking moiety, for example, O (C = O), S (C = O) or O (C = S) attached to an amine-containing ligand. In a preferred embodiment, the amine-containing ligand comprises an amine-containing polypeptide, pharmaceutical or lipid. Here, these embodiments will be described.

A. Amine-Containing Lipids In one embodiment, the amine-containing ligand is an amine-containing lipid.
As used herein, the term "lipid" refers to an aqueous solution having at least one acyl chain containing at least about 8 carbon atoms, and more preferably having an acyl chain containing about 8-24 carbon atoms. Means a sex molecule. Preferred lipids include amine-containing polar head groups and acyl chain-containing lipids. Representative lipids include:
Examples include phospholipids having one acyl chain, such as stearoylamine, or two acyl chains. Preferred phospholipids having an amine-containing head group include phosphatidylethanolamine and phosphatidylserine.
The tail of the lipid includes groups also having from about 12 to about 24 carbon atoms, which may be either fully saturated or unsaturated. One preferred lipid is distearoyl phosphatidylethanolamine (DSPE). However, those skilled in the art will appreciate that a wide variety of other lipids fall within this description. It will also be appreciated that the lipids naturally contain or can be derived to contain amine groups. It is also possible to use other lipid moieties that do not have an acyl tail, such as cholesterolamine.

The synthesis of the polymer-DTB-lipid compound is shown schematically in FIG. By reacting 2- (methoxycarbonyldithio) ethanamine with mPEG chloroformate, an mPEG derivative having a methoxycarbonyldithioalkyl end group (MW2000
And 5000 daltons). Chloroformic acid mPEG could be easily prepared by phosgenating a dry mPEG-OH solution (Zalipsky,
S. et al., Biotechnol. Appl. Biochem. 15 , 100-114, 1992). 2- (Methoxycarbonyldithio) ethanamine was prepared by reacting 2-aminoethanethiol hydrochloride with an equivalent amount of methoxycarbonylsulfenyl chloride according to published procedures (Brois, SJ et al., J. Amer. Chem. Soc. 92 , 7629-7631, 1970;
Koneko, T. et al., Bioconjugate Chem. 2 , 133-141, 1991). Precise coupling of the resulting PEG-linked acyl disulfide with the para and ortho isomers of mercaptobenzyl alcohol (Grice, R. et al., J. Chem. Soc. 1947-1954, 1963) provides dithiobenzyl alcohol MPEG having a terminal group was obtained. As with uninduced mPEG-OH, the introduction of active carbonate proceeded to give paranitrophenyl carbonate. By adding DSPE to ethanolamine, the desired mP
An EG-DTB-DSPE product was obtained. Ortho DTB-lipid compounds and para-DTB-lipid compounds were prepared, purified by silica gel chromatography, and characterized by NMR and MALDI-TOFMS. The details of the results are shown in Example 1.

FIG. 3 shows the thiol cleavage mechanism of the mPEG-DTB-DSPE conjugate. When cleavage occurs, the phosphatidylethanolamine lipid is regenerated in its native, unmodified form.

4A-4B show an mPEG-DTB-DSP having an alkyl group adjacent to a disulfide bond, for example, having a disulfide bond having relatively large steric hindrance.
The reaction mechanism for the synthesis of the E conjugate is shown. As provided a complete description in Example 2A,
MPEG-OH in dichloromethane was reacted with p-nitrophenyl chloroformate in the presence of triethylamine (TEA) to give mPEG-nitrophenyl carbonate. 1-amino-2-propanol or 1 in dimethylformamide (DMF)
-An amino alcohol such as amino-2-butanol is converted to methylene carbonate in the presence of TEA.
Reaction with PEG-nitrophenyl gave a PEG conjugate of a secondary alcohol. The secondary alcohol was then converted to the desired mPEG-DTB-DS as shown in FIG. 4A.
Converted to PE compound. The details are described in Example 2A.

In this reaction mechanism, mPEG-methyl-dithiobenzyl-nitrophenyl chloroformate was reacted with DSPE to obtain a desired compound. The chloroformic acid mP
The nitrophenyl chloroformate moiety of the EG-methyl-dithiobenzyl-nitrophenyl compound acts as a leaving group and reacts with the selected lipid to yield the desired product. In another aspect, the present invention is directed to a composition comprising a compound obtained by reacting with a compound such as mPEG-methyl-dithiobenzyl-R ³ . Here, R ³ is a removing group bonded to a benzene ring via a bonding moiety. The scavenging group reacts with and is displaced by an amine-containing ligand, such as a DSPE, a polypeptide or an amine-containing drug. The scavenging group is selected based on the reactivity of the amine in the ligand, and is preferably derived from a hydroxy group containing scavenging group or various acidic alcohols having an oxy group containing scavenging group. These include chloride, p-nitrophenol, o-nitrophenol, N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide, N-hydroxyglutarimide, N-hydroxynorbornene-2,3-dicarboximide. , 1-
Hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine, 2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, imidazole, triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol and trichlor Phenol and the like are included.

Example 2B describes a method for preparing an mPEG-EtDTB-lipid conjugate.
Here, the disulfide bond is sterically hindered by the ethyl moiety.

FIG. 5 shows a synthesis reaction mechanism of another mPEG-DTB-ligand compound in the present invention. Examples 3A-3B provide details of the reaction procedure. Briefly, 1-amino-2-propanol was reacted at low temperature with sulfuric acid to produce 2-amino-1-methylethyl hydrogen sulfate. This product was reacted with carbon disulfide and sodium hydroxide in ethanol water to obtain 5-methylthiazolidine-2-thione. An aqueous solution of hydrochloric acid was added to the 5-methylthiazolidine-2-thione and heated. After refluxing for one week, the product, 1-mercapto (methyl) ethylammonium chloride, crystallized and was recovered. This product was reacted with methoxycarbonylsulfenyl chloride to give 2- (methoxycarbonyldithio) ethanamine. Using the procedure described above with respect to FIG. 2, reacting the 2- (methoxycarbonyldithio) ethanamine with mPEG chloroformate and further reacting with a selected amine-containing ligand to provide a compound of the present invention, Desired m
A PEG-DTB-nitrophenyl compound was obtained.

Example 3B illustrates the synthesis reaction of mPEG- (ethyl) DTB-nitrophenyl.

FIG. 6A shows a reaction mechanism for preparing another mPEG-DTB-lipid compound according to the present invention. The details of the reaction are shown in Example 4. The lipid 1,2-distearoyl-sn-glycerol is activated, and FIG. 4A or FIG.
With mPEG-DTB-nitrophenyl prepared by the method described in (1). The resulting mPEG-DTB-lipid compound differs from the above compounds by the absence of a phosphate head group. The mPEG-DTB-lipid compound of FIG. 6A is neutral before cleavage. As shown in FIG. 6B, by thiol cleavage reduction of disulfide bonds, the compound is decomposed to generate a cationic lipid. The positively charged lipid causes an electrostatic interaction in vivo, and a corresponding in vivo targeting effect can be obtained.

In the above reaction scheme, R ⁵ in the claimed compound is H. However, in other embodiments, R ⁵ is an alkyl group or an aryl group. In this method, for example, when both R ² and R ³ are CH ₃ , α, β-unsaturated acyl chloride (
R'R "C = CHCOCl; R 'is, for example CH _3, R" is a CH _3, other alkyl or aryl groups also are subject) is reacted with the terminal amine of the PEG, the corresponding N To obtain PEG-substituted α, β-unsaturated amides. This compound is reacted with thiolacetic acid and the corresponding N-PEG substituted β via conjugate addition to the C へ C bond.
(Acetylthio) amide is obtained. Hydrolyzing acetylthio based (-SCOCH ₃₎ to a thiol group (-SH), then the thiol group is reacted with methyl formate (chlorosulfonated phenyl) (ClSCOOCH ₃₎ with methoxycarbonyl dithio group (-
SSCOOCH ₃ ). This intermediate is then reacted with p- mercapto benzyl alcohol and N-PEG-substituted beta (dithiobenzyl alcohol) amide (PEG-NH-CO-CH 2 CR'R "-SS-p- phenyl -CH ₂ OH The benzyl alcohol moiety is then reacted with nitrophenyl chloroformate to give the nitrophenyl carbonate removing group as described above.

1. ortho-mPEG-DTB-DSPE and para-m in mPEG-DTB-DSPE compound in vitro cleavage buffer (pH = 7.2)
By preparing a micelle solution of PEG-DTB-DSPE (prepared by the method described in Example 1), the in vitro cleavage rate of the compound was examined. The progress of thiol cleavage of the compound was monitored by analyzing the disappearance of the compound by HPLC as described in Example 5 in the presence and absence of cysteine 150 μM. The results are shown in FIG. 7A. Here, in the absence of cysteine, the ortho and para compounds (represented by the symbols * and +, respectively) did not undergo a cleavage reaction. That is, it was found that these compounds were stable under the condition that cysteine was not present. In contrast, as shown in FIG. 7A, in the presence of 150 μM cysteine, it was revealed that the ortho and para compounds (indicated by open circles and open squares, respectively) undergo cleavage. The decomposition rate of the ortho compound was slightly faster than that of the para compound (T _1/2 was about 12 minutes and about 18 minutes, respectively).

[0052] 2. Liposome composition composed of mPEG-DTB-lipid compound a. In Vivo Characterization In one embodiment, the mPEG-DTB-lipid compound is formulated into liposomes. Liposomes are closed lipid vesicles. By systemically administering the liposome, it can be used for various therapeutic purposes, particularly for delivering therapeutic agents to target areas or cells. In particular, polyethylene glycol (PEG)
A liposome coated with a hydrophilic polymer chain such as) is suitable as a drug carrier. This is because these liposomes often exert the effect of extending the blood circulation life as compared to the liposomes not coated with the polymer. The polymer chains in the polymer coating protect the liposomes and form a "hard brush" of water-solvated polymer chains around the liposomes. In this way, the polymer acts as a barrier to blood proteins, preventing them from binding and being taken up by macrophages and other cells of the reticuloendothelial system that recognize the liposomes.

Typically, liposomes having a surface coating of polymer chains are prepared by including from about 1 mol% to about 20 mol% of the lipid coated with the polymer in the lipid mixture. The actual amount of polymer-coated lipid may vary depending on the molecular weight of the polymer. In the present invention, the liposome contains from about 1 mol% to about 20 mol%.
It is prepared by adding up to mol% of the polymer-DTB-lipid conjugate to the other liposome lipid bilayer component. As shown by the following tests, liposomes containing the polymer-DTB-lipid conjugate of the present invention have longer circulation in the blood than liposomes containing the polymer-lipid conjugate in which the polymer and the lipid are linked by cleavable aliphatic disulfide bonds. Have a lifetime.

In a study supporting the present invention, liposomes were composed of vesicle-forming lipids of partially hydrogenated phosphatidylcholine along with cholesterol, and ortho-mPEG-DTB-
DSPE or para-mPEG-DTB-DSPE compounds were prepared as described in Example 6. The cysteine-mediated cleavage reaction of the mPEG-DTB-DSPE compound in an aqueous buffer solution was monitored in the presence and absence of 150 μM cysteine. The result is shown in FIG. 7B. FIG. 7B also shows the data of FIG. 7A for comparison. In FIG. 7B, the ortho compound and the para compound in the micelle form (* each)
(Indicated by + and +) did not cleave in the absence of cysteine. This fact indicates that the conjugate is stable in the absence of thiol. As in the case of FIG. 7A, open circles and open squares correspond to the micelle-shaped ortho compound and para compound, respectively, in the presence of cysteine. In the presence of cysteine, the filled circles and filled squares correspond to the liposome-shaped ortho and para compounds, respectively.

The data in FIG. 7B shows that both the ortho and para compounds have somewhat higher thiol cleavage resistance when incorporated into liposomes. TCL (silica gel G, chloroform / methanol / water = 90: 18: 2) (Dittmer, JC et al., J. Biol.
Lipid Res. 5 , 126-127, 1964) indicates that DSPE is the only lipid component and that the other spot is a lipid-free mPEG derivative with thiol. Is shown.

In other studies performed to support the present invention, lipid dioleoyl
Liposomes were prepared from phosphatidylethanolamine (DOPE) to prepare either ortho-mPEG-DTB-DSPE compounds or para-mPEG-DTB-DSPE compounds. DOPE is a hexagonal phase lipi
d), which alone does not form lipid vesicles. However, liposomes are DO
Formed when PE is combined with several mole% of mPEG-DTB-DSPE compound. When the mPEG-DTB-DSPE compound is cleaved, this causes degradation of the liposome and release of the liposome-entrapped contents. Thus, the content release characteristics of such liposomes provide a convenient and quantitative method of evaluating cleavable PEG-loaded liposomes.

DOPE and ortho-mPEG-DTB-DSPE compound or para-mPEG
Liposomes composed of -DTB-DSPE compounds and capturing fluorophore, p-xylene-bis-pyridinium bromide and trisodium 8-hydroxypyrene trisulfonate were prepared by the method described in Example 7A. The release status of the fluorophore from liposomes incubated in the presence of various concentrations of cysteine was monitored by the method described in Example 7B.

The results for liposomes composed of ortho-compounds are shown in FIG. 8A. In this figure, the concentrations are 15 μM (black diamond), 150 μM (black inverted triangle), 300 μM (black triangle).
And the percentage of captured fluorophores released from liposomes incubated in the presence of 1.5 mM (closed circles) cysteine. FIG. 8B is a similar plot for a liposome composed of para-compounds. Here, the liposome has a concentration of 15 μM (solid diamond), 300 μM (solid triangle), 1 μM (solid square) and 1.5 mM (solid square).
The cells were incubated in cysteine (solid circles).

FIGS. 8A-8B show that when the ortho compound or para compound is introduced into the liposome, both the ortho compound and the para compound are cleaved at a rate depending on the concentration of cysteine, as is apparent from the release state of the capture dye. It shows that. Non-cleavable mPEG
In a comparative control test using liposomes containing -DSPE, no release of the contents was observed (the results are not particularly shown). These results also suggest that ortho-conjugates tend to be slightly thiol-cleavable. For example, 300 μM cysteine releases most of the contents of DOPE liposomes within 20 minutes. Under the same conditions, for liposomes with para mPEG-DTB-DSPE,
Only part of it decomposed. Similarly, in the case of liposomes containing the ortho-type compound, half of the captured content was released after incubation for 20 minutes at a cysteine concentration of 150 μM. In contrast, only about 10% of the liposomes containing the para-compound were released. Both the ortho and para compounds have a half-life of less than 20 minutes at a cysteine concentration of 150 μM (FIG. 7B).
See data at). This fact suggests that more than half of the original 3 mol% mPEG-DTB-lipid has been cleaved and release of the contents from the liposomes must be observed.

15 μM cysteine (mean plasma concentration in humans and rodents; Lash, LH
Et al., Arch. Biochem. Biophys. 240 , 583-592, 1985).
Degradation of B-DSPE / DOPE liposomes was at the lowest level within the time frame (60 minutes) of these experiments. This fact indicates that the longevity of the mPEG-DTB-lipid compound in plasma is long enough that the PEG-grafted vesicles can be distributed systemically in vivo, or that specific sites targeted passively or ligand-mediated. Suggests that it can accumulate. A local or short-term increase in cysteine concentration
It may be achieved by its intravenous or intraarterial administration. The results shown in FIGS. 8A-8B also suggest that prolonged exposure to natural plasma cysteine concentrations (約 15 μM) largely degrades these compounds. These possibilities were further investigated by the following in vivo experiments.

In another supporting test in the present invention, DOPE and three different mPE
Liposomes composed of G-DTB-lipid compounds were prepared. The liposome was prepared by the method described in Example 7. The liposome trapped the fluorophore inside. The three types of mPEG-DTB-lipid compounds are mPEG-DTB-DSPE shown in FIG. 1A; mPEG-MeDTB-DSP shown in FIG. 4B.
E (R is CH ₃ ); and mPEG-MeDTB-distearoyl- shown in FIG. 6A.
It was glycerin. The liposomes were 97 mol% DOPE and 3 mol% mP
It was composed of one of the EG-DTB-lipid compounds. The rate of cysteine-mediated cleavage of the compound was determined by monitoring the release of the captured fluorophore as a function of time in the presence of various concentrations of cysteine. The results are shown in FIGS. 9A-9C. Here, the release rate (%) of the captured fluorophore was normalized to the release rate from liposomes incubated in buffer only.

FIG. 9A shows the release rate (%) of trapped fluorophores for liposomes composed of DOPE and para-mPEG-DTB-DSPE (compound of FIG. 1A) as a function of time. In this figure, the conjugate is included, and the concentration is 15 μM (solid square), 75 μM (open triangle), 150 μM (marked X), 300 μM (open circle), 1500 μM (solid circle)
The release rates from liposomes incubated in the presence of cysteine at 3000, 3000 μM (+), and 15000 μM (open diamonds) are shown.

FIG. 9B shows the release rate of captured fluorophores as a function of time for liposomes composed of DOPE and para-mPEG-MeDTB-DSPE (the compound of FIG. 4B). Concentrations of 15 μM (closed square), 75 μM (open triangle), 150 μM (
X mark), 300 μM (open circle), 1500 μM (black circle), 3000 μM (+ mark), and 15000
The figure shows the rate at which the fluorophore is released from the liposome by incubating the liposome in the presence of μM (open diamond) cysteine.

FIG. 9C is a similar plot determined above for liposomes formed with DOPE and mPEG-MeDTB-distearoyl glycerin (the compound of FIG. 6A). Concentrations of 15 μM (black square), 75 μM (open triangle), 150 μM (X mark), 300
This shows the rate of release of dye from liposomes when liposomes were incubated in the presence of cysteine at μM (open circle), 1500 μM (black circle), 3000 μM (+ mark), and 15000 μM (open diamond). .

FIGS. 9A-9C show that the cleavage rate of mPEG-MeDTB-lipid is dependent on cysteine concentration. That is, cleavage proceeds slowly, as can be seen from the release rates of the captured fluorophores at cysteine concentrations of 15-75 μM. By comparing the data in FIG. 9A with the data in FIG. B, it can be seen that the mPEG-MeDTB-DSPE compound (FIG. 9B) cleaves at about one-tenth the rate of the mPEG-DTB-DSPE compound (FIG. 9A). . Therefore, the cleavage rate is dependent on the R portion of the DTB bond (FIG. 2).
Can be designed.

B. Liposome prepared by the method described in Example 8 for evaluation of characteristics in vivo and polymer-DTB-
The circulating lifetime of the liposomes containing the lipid conjugate was measured in mice. In ¹¹¹ was captured in the liposome, and the liposome was intravenously administered. One group of test mice received an additional injection of cysteine and a control group received additional saline. By taking blood samples at various times, the presence of the liposomes were analyzed by the presence of an In ^111.

[0067] Figure 10, after injection of the liposomes and saline (open circles) or 200 mM cysteine (closed circles), an illustration counts per minute In ¹¹¹ the (CPM) as a function of time is there. This figure shows that cleavage of mPEG-DTB-DSPE occurred in a group of mice to which physiological saline was administered after administration of liposome, indicating that cleavage occurs by exposure to natural physiological conditions. mPEG
Administration of an exogenous reducing agent (cysteine) to mice was effective in increasing the rate of cleavage of the -DTB-lipid compound within a time frame of about 2 to about 8 hours.

It is important that the cleavage of the polymer-DTB-lipid compound according to the invention regenerates the original lipid in an unmodified state. It is desirable that the original lipid be regenerated in its original form, as modified lipids that do not exist in nature may have undesirable in vivo effects. At the same time, the compound can stably store it in the absence of a reducing agent.

Although not specifically shown herein, in other studies, the circulating lifetime of liposomes containing mPEG-DTB-lipids was measured using a polymer-lipid conjugate (where the polymer and lipid are cleavable aliphatic disulfide bonds). Conjugated liposomes). Aliphatic disulfide bonds are easily cleaved in vivo. Therefore, as observed with liposomes having polymer chains stably bound, the circulating lifetime of liposomes with polymer chains grafted to the surface by aliphatic disulfide bonds is rarely extended. The dithiolbenzyl bond of the present invention, particularly the DTB bond having a relatively high degree of steric hindrance, is stable in vivo, and therefore can achieve a longer blood circulation lifetime than liposomes having a polymer chain linked by an aliphatic disulfide bond. it can.

B. Amine-Containing Polypeptides In other embodiments, the present invention also includes the compounds described in FIG. 1A, wherein the amine-containing ligand is a polypeptide. The synthetic reaction mechanism showing the method for preparing the polymer-DTB-polypeptide is shown in FIG. 11A using mPEG as an example. Generally, an mPEG-DTB-removing compound is prepared according to one of the synthetic routes described in FIGS. 2, 4a and 5 above. As the removing group, nitrophenyl carbonate or any one of the above can be selected. The mPEG-DTB-nitrophenyl carbonate compound is coupled to the amine moiety in the polypeptide via a urethane bond. The R group adjacent to the disulfide in the compound can be selected from H, CH ₃ , C ₂ H _5, etc., based on the desired disulfide cleavage rate.

FIG. 11B shows the degradation products of the compound in cysteine-mediated cleavage. From this figure, it can be seen that a natural protein without any modification to the amine group of the protein is regenerated by a cleavage reaction.

When a polymer chain such as PEG is bound to a polypeptide, the enzymatic activity or other biological activity of the polypeptide, such as receptor binding activity, is often reduced.
However, by modifying the polypeptide with a polymer, the circulating life of the polypeptide in blood is increased. In the present invention, the polymer-modified polypeptide is administered to a subject. As the polymer-modified polypeptide circulates, exposure to physiological reducing conditions, such as blood cysteine and other in vivo thiols, causes the polymer chain to begin to cleave from the polypeptide. As the polymer chain is detached from the polypeptide, the biological activity of the polypeptide gradually recovers. In this way, the polypeptide initially has a sufficient circulating lifetime in the blood to achieve sufficient biodistribution, but regains its full biological activity over time as cleavage of the polymer chain proceeds.

In a test supporting the present invention, mPEG-MeDTB-lysozyme conjugate was prepared using lysozyme as a model polypeptide and using a synthetic route similar to that described above. Lysozyme was incubated with a 0.1 M borate solution of mPEG-MeDTB-nitrophenyl carbonate (pH = 9; nitrophenyl carbonate: amino group of lysozyme = 2: 1). After reacting for 15 minutes and 3 hours, the sample characteristics were
-Evaluated by the PAGE method. Comparative compounds were prepared by reacting lysozyme with mPEG-nitrophenyl carbonate conjugate under the same conditions for 60 minutes.
This reaction produces a stable mPEG-lysozyme conjugate.

FIG. 12 is a view for explaining the test results using SDS-PAGE gel. Lane 1 corresponds to the compound formed 15 minutes after the start of the reaction between lysozyme and mPEG-MeDTB-nitrophenyl carbonate. Lane 2 shows the compound formed one hour after the start of the reaction of the same compound. Lane 3 represents native lysozyme and lane 4 corresponds to lysozyme after reacting with mPEG-nitrophenyl carbonate for 1 hour.
The molecular weight markers in lane 5 are as follows (from top to bottom).

By comparing Lane 1 and Lane 2, it can be seen that the conjugation of the mPEG chain to the polypeptide gradually proceeds, and the molecular weight of the conjugated mPEG chain compound increases with the reaction time.

Lanes 6-9 of the SDS-PAGE profile correspond to the samples of lanes 1-4 after treatment with 2% β-mercaptoethanol at 70 ° C. for 10 minutes. The mP
The EG-MeDTB-lysozyme conjugate was decomposed by exposing it to a reducing agent to regenerate natural lysozyme. The regeneration reaction was performed in lanes 6 and 7 at 14
It is evident from the .4 kDa band. In contrast, stable mPEG-lysozyme compounds were not affected when incubated with reducing agents.
This is apparent from the fact that the profiles of lane 9 and lane 4 match.

A conjugation bond is formed between mPEG-MeDTB and the protein, and various mPE
Conjugate mixtures containing G-protein ratios can also be obtained, also by SDS-PAGE.
It is clear from the profile. This ratio varies depending on the reaction time and reaction conditions. This is clear from the bands in lanes 1 and 2. Here, lane 1 shows lysozyme coated with about 1-6 PEG chains. In lane 2, the longer the reaction time, the higher the mPEG-protein ratio m
A PEG-MeDTB-lysozyme conjugate has been formed. All cleavable conjugates were easily cleaved to regenerate the native protein. This is apparent from the bands in lanes 6 and 7.

It will be readily understood that any of the above hydrophilic polymers can be used in the present invention. The molecular weight of the polymer is chosen according to the type of polypeptide used, the number of reactive amines on the polypeptide, and the desired size of the polymer modifying compound.

The polypeptides to be used are endless and various, such as naturally occurring polypeptides or recombinantly produced polypeptides. The use of small human recombinant polypeptides is preferred, with a molecular weight range of 10-30 kDa. Typically, polypeptides include cytokins such as tumor necrosis factor (NTF), interleukins and interferons, erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), enzymes and the like. Viral polypeptides are also of interest. In this case, the surface of the virus is modified to introduce one or more polymer chains bound by reversible DTB bonds. By modifying the virus containing the gene and transfecting the cells, the circulation time of the virus can be increased and immunogenicity reduced, thereby improving the delivery of the exogenous gene.

C. Amine-Containing Drugs In yet another embodiment of the present invention, compounds in the form of polymer-DTB-amine-containing drugs are of interest. The compound has the above structure, especially the structure of FIG. 1A. In the structure of FIG. 1A, the amine-containing ligand is an amine-containing drug. Modification of a therapeutic agent with PEG can effectively improve the blood circulation lifetime of the agent and reduce the immunogenicity of the agent if it is present.

The polymer-DTB-amine-containing drug can be prepared by making necessary modifications to the specific drug according to any of the above reaction mechanisms. A wide range of therapeutic agents, such as mitomycin C, bleomycin, doxorubicin, and ciprofloxacin, have a reactive amine moiety, and in the present invention, these agents can be added to a subject without any restrictions. it can. It will be appreciated that the present invention is also useful for drugs containing an alcohol or carboxyl moiety. If the drug contains a hydroxyl or carboxyl moiety suitable for reaction, the polymer-DTB moiety can be attached to the drug via a urethane, ester, ether, thioether or thioester linkage. In all these embodiments, the polymer-DTB-drug compound is thiolyzed after its in vivo administration to regenerate the amine-containing drug in its native, active form. Thus, it is not necessary that the compound have therapeutic activity after modification and before administration. Thus, even when the therapeutic activity is reduced or eliminated by modifying the drug with the DBT-polymer, the D prepared from the drug may be used.
After administration and cleavage of the TB-polymer, the activity of the drug is regenerated.

In a test supporting the present invention, the nitroanilide drug was added to mPEG-MeDTB-
After reacting with nitrophenyl carbonate, mPEG-MeDTB-para-
A nitroanilide compound was obtained. Decomposition of the compound upon exposure to a reducing agent yields the product of FIG. 13 and regenerates the para-nitroanilide drug in an unmodified state.

The mPEG-MeDTB-para-nitroanilide compound was added in vitro at 5 mM
The samples were incubated in a buffer solution containing cysteine, and the absorbance of samples collected at various times was measured. The results are shown in FIG. 14A. As can be seen in FIG. 14A, the measurement times for the samples were 0 minutes (black diamond), 2 minutes (black square), 5 minutes (X mark), 10 minutes (white square), 20 minutes (white triangle), 40 minutes (White diamond) and 80 minutes (black circle). It is clear that the UV spectrum is changing as a function of the incubation time in cysteine, indicating that para-nitroanilide is released from the mPEG-MeDTB-para-nitroanilide compound via cysteine. ing.

FIG. 14B shows mPE incubated in vitro in the presence of 5 mM cysteine (closed circle), 1 mM cysteine (closed square) and 0.15 mM cysteine (closed diamond).
The amount (mol / L) of para-nitroanilide released from the G-MeDTB-para-nitroanilide conjugate is shown. The rate of drug release from the conjugate varied depending on the concentration of reducing agent present.

From the above facts, it is clear how many such objects and features of the present invention are compatible with the present invention. The compounds of the present invention are comprised of an amine-containing ligand reversibly linked to a hydrophilic polymer via an ortho- or para-disulfide group of a benzylurethane linkage. This linkage undergoes cleavage and regenerates the original amine-containing ligand in its unmodified form when mild thiol degradation conditions are applied thereto. The cleavage rate can be controlled by the steric hindrance of the disulfides in the bond and by controlling the thiol degradation conditions in vivo. The compound before cleavage of the dithiobenzyl bond has high circulating life and stability and low immunogenicity.

III. Examples The following examples are provided to illustrate the invention in more detail and are not intended to limit the scope of the invention in any way.

(Materials) All materials were obtained from commercially suitable suppliers such as Aldrich.

Example 1 Synthesis of mPEG-DTB-DSPE mPEG-MeDTB-nitrophenyl carbonate (300 mg, 0.12 mmol, 1.29 eq)
Was dissolved in CHCl ₃ (3 ml). DSPE (70 mg, 0.093 mol) and TEA
(58.5 μl, 0.42 mmol, 4.5 eq) was added to the PEG solution and stirred at 50 ° C. (oil bath temperature). After 15 minutes, TLC indicated that the reaction was incomplete. Then T
EA was added twice (10 μl and 20 μl) and mPEG-MeDTB-nitrophenyl carbonate several times (50 mg, 30 mg, 10 mg) at 10 minute intervals until the reaction was completed, and the solvent was evaporated. And the resulting mixture was dissolved in MeOH and 1 g of C8 silica was added. The solvent was evaporated again and the product containing C8 silica was added to the top of the column and eluted by MeOH: H ₂ O (pressure) gradient. The eluent used was MeOH: H ₂ O = 30: 70, 60 ml; MeOH: H ₂ O = 50: 50, 60 ml;
_{MeOH: H 2 O = 70:} 30, 140 ml ( starting material _{eluted); MeOH: H 2 O =} 75:
25, 40 ml; MeOH: H 2 O = 80: 20, 80 ml ( product _{eluted); MeOH: H 2 O =} 85: 15, 40 ml; MeOH: H 2 O = 90: 10, 40 ml; MeOH = 40 ml; CH
Cl ₃ : MeOH: H ₂ O = 90: 18: 10, 40 ml. Fractions containing the pure product were collected and evaporated to give the product as a colorless viscous liquid. was added t- butanol (5 ml) thereto, it was lyophilized, vacuum dried over P ₂ O _5, fluffy white solid product (
252 mg, 89% yield).

The ortho- and para-DTB-DSPE compounds were purified by silica gel chromatography (concentration gradient method including 0-10% of methanol in chloroform, isolated yield: about 70%), and the structures were analyzed by NMR and Confirmed by MALDI-TOFMS. ¹ H NMR of paraconjugate: (d ₆ -DMSO, 360 MHz) δ 0.86 (t, CH ₃
, 6H), 1.22 (s, CH 2 of lipid, 56H), 1.57 (m, CH 2 CH 2 CO 2, 4H
), 2.50 ( ₂ × t, CH ₂ CO ₂ , 4H), 2.82 (t, CH ₂ S, 2H), 3.32 (
s, OCH ₃ , 3H), 3.51 (m, PEG, about 180 H), 4.07 (t, PEG-C
H ₂ OCONH, 2H), 4.11 and 4.28 (CH ₂ CH, 2H of ₂ × dd glycerin)
), 4.98 (s, benzyl _{-CH 2, 2H), 5.09 (} m, lipid CHCH _2), 7.35
And 7.53 (2xd, aromatic, 4H) ppm]. The ortho conjugate is 5.11 (s, CH ₂ , 2
H), and 7.31 (d, 1H), 7.39 (m, 2H), 7.75 (d, 1H).

MALDI-TOFMS created a 44 Da equally spaced ion distribution. The ion distribution corresponds to a repeating unit of ethylene oxide. The average molecular weight of the compound was 3127 Da and 3139 Da, corresponding to the para and ortho isomers, respectively (theoretical molecular weight about 3100 Da).

FIG. 2 shows this reaction mechanism.

Example 2: Synthesis of mPEG-DTB-DSPE mPEG-MeDTB-DSPE The mechanism of this reaction is shown in FIGS. 4A-4B.

MPEG (5K) -OH (40 g, 8 mmol) was azeotropically dried with toluene (total volume 270 ml, 250 ml distilled off by Dean-Stark method). Dichloromethane (100 ml) was added to mPEG-OH. P-nitrophenyl chloroformate (2.42 g, 12 mmol, 1.5 eq) and TEA (3.3 ml, 24 mmol, 3 eq) were added to the PEG solution at 4 ° C. (ice-water bath) while being careful not to mix moisture. Was added. A pale yellow TEA hydrochloride was obtained. After 15 minutes, the cooling bath was removed and the reaction mixture was stirred at room temperature overnight. TLC (CHCl ₃ : MeOH: H ₂ O = 90: 18: 2) indicated the completion of the reaction. The solvent was evaporated and the residue was dissolved in ethyl acetate (50 ° C.). The TEA hydrochloride was removed by filtration and washed with warm ethyl acetate. The solvent was evaporated and the product was recrystallized with isopropanol (3 times). Yield: 38.2 g (92
%); ¹ H NMR (DMSO-d ₆ , 360 MHz) δ 3.55 (s, PEG, 450H)
; 4.37 (t, PEG-CH 2, 2H); 7.55 (d, C 6 H 5, 2H); 8.31 (d,
C ₆ H _5, 2H).

1-amino-2-propanol (1.1 ml, 14.52 mmol, 3 eq) and TEA (2
.02 ml, 14.52 mmol, 3 eq) was added to mPEG (5K) -nitrophenyl carbonate (25 g,
(4.84 mmol) in DMF (60 ml) and CH ₂ Cl ₂ (40 ml). The solution was a yellow clear solution. The reaction mixture was stirred at room temperature for 30 minutes. TL
C (CHCl ₃ : MeOH = 90: 10) indicated that the reaction was complete. solvent(
Dichloromethane) was evaporated. Isopropanol (250 ml) was added to a DMF solution (60 ml) of the product mixture, whereupon the product precipitated. The precipitate was recrystallized with iPrOH (3 times). Yield: 22.12 g (90%), ¹ H NMR
_{(DMSO-d 6, 360 MHz} ) δ .98 (d, CH 3 CH (OH) CH 2, 3H); 3.
50 (s, PEG, 180H) ; 4.03 (t, PEG-CH 2, 2H); 4.50 (d, CH 3 CHOH, 1H); 7.0 (t, mPEG-OCONH).

MPEG (5K) -urethane-2-methyl propanol (22.12 g, 4.34 mm
ol) was azeotropically dried with toluene (45 ml). To this was added dichloromethane (60 ml). Methanesulfonyl chloride (604.6 μl, 7.81 mmol, 1.8 eq) and TEA
(3.93 ml, 28.21 mmol, 6.5 eq) with care to avoid moisture contamination.
The solution was added to the mPEG solution at 0 ° C. with stirring. After 30 minutes, the cooling bath was removed and the reaction mixture was stirred at room temperature for 16 hours. Evaporate the solvent, add ethyl acetate and add TE
The A salt was removed. The product was recrystallized from isopropanol (3 times). Yield: 20
.27 g (90%); ¹ H NMR (DMSO-d ₆ , 360 MHz) δ 1.27 (d, CH ₃ C
HOSO ₂ CH ₃ , 3H); 3.162 (s, CH ₃ O ₂ SOCH, 3H); 3.50 (s,
PEG, 180H); 4.07 (t , PEG-CH 2, 2H); 4.64 (q, CH 3 CHO
H, 1H); 7.43 (t, mPEG-OCONH).

MPEG (5K) -urethane-2-methylmethanesulfone (10.27 g, 1.98 mm
ol) was azeotropically dried with toluene (20 ml each). Sodium hydride (377 mg, 9
.4 mmol, 4.75 eq) was added to anhydrous toluene (60 ml) at 0 ° C. (ice-water bath). 5
After minutes, triphenylmethanethiol (3.92 g, 14.6 mmol, 7.15 eq) was added to the solution. After 10 minutes, mPEG-urethane-2-methylmethanesulfone (10.27 g
, 1.98 mmol) was added to the reaction mixture. The reaction mixture turned into a yellow solution. After 45 minutes, TLC (CHCl ₃ : MeOH: H ₂ O = 90: 18: 2) indicated that the reaction was complete. Acetic acid (445.57 μl, 7.42 mmol, 3.75 eq) was added to the reaction mixture to neutralize excess sodium hydride. The solution turned into a viscous white solution. The solvent was evaporated and the solid was recrystallized with ethyl acetate (30 ml) and isopropanol (70 ml). Since the product mixture did not completely dissolve, the precipitate was removed by filtration. The product mixture was then recrystallized from isopropanol / t-butyl alcohol (100 ml / 20 ml). Yield: 8.87 g (84%); ¹ H
NMR (DMSO-d ₆ , 360 MHz) δ .74 (d, CH ₃ CHSC (C ₆ H ₅ ) 3,3
H), 3.50 (s, PEG , 180H), 4.0 (t, PEG-CH 2, 2H), 4.64 (
_{q, CH 3 CHOH, 1H)} ; 7.49 (t, mPEG-OCONH); 7.20-7.41
_{(M, SC (C 6 H} 5) 3, 15H).

MPEG (5K) -urethane-2-methyl-triphenylmethanethiol (8.
87 g, 1.65 mmol) was dissolved in TFA / CH ₂ Cl ₂ (10 ml / 10 ml) at 0 ° C.
While stirring vigorously, methoxycarbonylsulfenyl chloride (185.5 μl, 1.99
mmol, 1.2 eq) was added to the solution. The reaction mixture was stirred at room temperature for 15 minutes. TLC (CHCl ₃ : MeOH = 90: 10) indicated the reaction was complete.
The solvent is evaporated and the product mixture is treated with isopropanol: t-butyl alcohol (
(80 ml: 20 ml) twice. To the product was added t-butanol (5 ml), lyophilized and vacuum dried over P ₂ O ₅ to give a white fluffy solid product (8.32 g, yield: 97%). . ¹ H NMR (DMSO-d ₆ , 360 MHz) δ 1.17 (d
, CH ₃ CHSSCOOCH ₃ , 3H); 3.42 (s, PEG, 180H); 3.84 (s
_{, CH 3 OCOSSCH, 3H);} 4.05 (t, mPEG-CH 2, 2H); 7.38 (
t, mPEG-OCONH, 1H).

MPEG (5K) -urethane ethyl (methyl) dithiocarbonyl methoxide (8.32 g, 1.6 mmol) was added to dry methanol (20 ml) and chloroform (2.5 ml).
). Mercaptobenzyl alcohol (592 mg, 4 mmol, 2.5 eq
) In dry methanol (2 ml) was added to the PEG solution. The reaction mixture was stirred at room temperature for 18 hours. The solvent was evaporated and the product mixture was recrystallized with ethyl acetate / isopropanol (30 ml / 100 ml) (3 times). NMR indicated that only about 16% product was obtained. Therefore, a solution of mercaptobenzyl alcohol (322 mg, 2.18 mmol, 1.8 eq) in MeOH (2 ml) was further added dropwise to the resulting mixture in MeOH / CHCl ₃ (24 ml / 1 ml) at 0 ° C. (ice-water bath). . After completion of the dropwise addition (about 10 minutes), the ice bath was removed and the reaction mixture was stirred at room temperature for 24 hours. TLC (CHCl ₃ : MeOH: H ₂ O = 90: 18: 2) indicated completion of the reaction. The solvent was evaporated, then the product mixture was recrystallized with ethyl acetate / isopropanol (30 ml / 100 ml). Yield: 7.25 g (94%), ¹ H NM
R (DMSO-d ₆ , 360 MHz) δ 1.56 (d, CH ₃ CHSSC ₆ H ₅ CH ₂ OH,
_{3H); 3.29 (CH 3 O} -PEG, 3H); 3.50 (s, PEG, 450H); 4.03 (
_{t, mPEG-CH 2, 2H} ); 4.46 (d, HOCH 2 C 6 H 5, 2H); 5.16 (t
_{, HOCH 2 C 6 H 5,} 1H); 7.30 (d, C 6 H 5, 2H); 7.40 (br t, mPE
G-OCONH, 1H); 7.50 (d, C 6 H 5, 2H).

MPEG (5K) -urethane-ethyl (methyl) -dithiobenzyl alcohol (6.75 g, 1.27 mmol) was dissolved in CHCl ₃ (30 ml) and P-nitrophenyl chloroformate (513 mg, 2.54 mmol, 2 eq) at 0 ° C. (ice-water bath). After 5 minutes, triethylamine (531 μl, 3.81 mmol, 3 eq) was added. After 30 minutes, the ice bath was removed and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated and the product mixture was dissolved in ethyl acetate. The TEA salt was filtered off and the solvent was evaporated.
The product mixture is washed with ethyl acetate / isopropanol (30 ml / 100 ml) (3 times)
Recrystallized. Yield: 6.55 g (94%); ¹ H NMR (DMSO-d ₆ , 360 MH)
z) δ 1.17 (d, CH ₃ CHSSC ₆ H ₅ , 3H); 3.24 (CH ₃ O-PEG, 3H
); 3.40 (s, PEG, 180H); 4.03 (br t, mPEG-CH 2, 2H); 5.28
_{(S, C 6 H 5 CH} 2 OCO, 2H); 7.45-8.35 (m, (C 6 H 5) 2,8H).

MPEG-MeDTB-nitrophenyl carbonate (766 mg, 0.14 mmol, 1.29 eq)
To CHCl_Three(5 ml), and DSPE (70 mg, 0.093 mol) and TEA (
58.5 μl, 0.42 mmol, 4.5 eq) was added to the PEG solution and stirred at 50 ° C (oil bath temperature).
Stirred. After 20 minutes, TLC indicated that the reaction was incomplete. Further mPE
G-MeDTB-nitrophenyl carbonate (total amount: 1239 mg, 0.23 mmol, 2.47 eq)
) And 1-hydroxybenztriazole (HOBt) (25 mg, 0.19 mmol, 2
eq) was added. After 20 minutes, TLC (CHCl_Three: MeOH: H_TwoO = 90: 18: 2+
Molybdenum and ninhydrin) indicated that the reaction was complete. Evaporate the solvent,
The resulting mixture was dissolved in warm (42 ° C) ethyl acetate. This solution was cloudy
(TEA salt precipitated), which was filtered and the solvent was evaporated. MeOH, and
And 2 g of C8 silica were added to the product mixture. The solvent was again evaporated. C8
The product containing silica is added to the top of the column and MeOH: H_TwoO (Pressure) tilt method
Was eluted. That is, MeOH: H_TwoO = 30: 70, 100 ml; M
eOH: H_TwoO = 50: 50, 100 ml; MeOH: H_TwoO = 70: 30, 250 ml (starting material
Is eluted); MeOH: H_TwoO = 75: 25, 40 ml; MeOH: H_TwoO = 80: 20, 200 m
l (product eluted); MeOH = 100 ml; CHCl_Three: MeOH: H_TwoO = 90: 18
: 2,100 ml; CHCl_Three: MeOH: H_TwoO = 75: 36: 6,100 ml was used.
Fractions containing the pure product were collected and evaporated to give a colorless viscous liquid product. t-pig
Then, the product was freeze-dried, and P_TwoO_FiveVacuum dried on white cotton
A product as a hairy solid (467 mg; yield: 83%) was obtained.¹1 H NMR (DMSO-d₆ , 360 MHz) δ 0.83 (d, 2 (CH_Three), 3H); 1.16 (d, CH_ThreeCHSSC₆
H_Five, 3H); 1.21 (s, 28 (CH_Two, 56H); 1.47 (br m, CH_TwoCH_TwoCO, 4
H); 2.23 (2 x t, CH_TwoCH_TwoCO, 4H); 3.50 (s, PEG, 180H); 4
.04 (br t, mPEGCH_Two, 2H); 4.05 (transd, PO_FourCH_TwoCHCH_Two, 1
H); 4.24 (cisd, PO_FourCH_TwoCHCH_Two, 1H); 4.97 (s, C₆H_FiveCH_TwoO
CO-DSPE, 2H); 5.03 (brs, PO_FourCH_TwoCH, 1H); 7.32 (d, C ₆ H_Five, 2H); 7.35 (d, C₆H_Five, 2H); 7.52 (brs, mPEG-OCONH)
, 1H). MALDI-TOFMS has a bell-shaped ion component that is arranged at equal intervals of 44 Da.
Produced a cloth. This distribution corresponds to the repeating units of ethylene oxide. The conjugate and
And the average molecular weight of mPEG-thiol (mostly cleaved disulfide bonds)
, 6376 Da and 5368 Da (theoretical molecular weights: about 6053 daltons and 5305 daltons)
).

B. mPEG-ethyl DTB-DSPE mPEG-urethane ethyl (ethyl) dithiocarbonyl methoxide (2 g
, 0.90 mmol) was dissolved in dry methanol (8 ml). Initially, the solution was cloudy, but after 5 minutes the cloudiness had disappeared and a clear solution was obtained. Mercaptobenzyl alcohol (265.2 mg, 1.79 mmol, 2 eq) was added to the PEG solution. The reaction mixture was stirred at room temperature for 30 hours. Ether (70 ml) was added to the reaction to precipitate the product and left at 4 ° C. overnight. The white solid was filtered and recrystallized from ethyl acetate / ether (30 ml / 70 ml). Yield: 1.69 g (94%); ¹ H
_{NMR (DMSO-d 6, 360} MHz) δ 0.86 (d, CH 3 CH 2 CHSSC 6 H 5 C
_{H 2 OH, 3H); 1.42} (p, CH 3 CH 2 CHSSC 6 H 5 CH 2 OH, 1H); 1.
_{64 (p, CH 3 CH 2} CHSSC 6 H 5 CH 2 OH, 1H); 3.51 (s, PEG, 180
H); 4.03 (t, mPEG-CH ₂ , 2H); 4.47 (d, HOCH ₂ C ₆ H ₅ , 2H)
_{); 5.20 (t, HOCH 2} C 6 H 5, 1H); 7.31 (d, C 6 H 5, 2H); 7.42 (b
rt, mPEG-OCONH, 1H) ; 7.49 (d, C 6 H 5, 2H).

N-hydroxy-s-norbornene-2,3-dicarboxylic imide (HONB
) (48 mg, 0.269 mmol) was added to a solution of DSPE (55 mg, 0.073 mmol) in CHCl _3.
Added at 0 ° C. (oil bath temperature). After 3-4 minutes, the solution became cloudy and clear. Next, mPEG-EtDTB-nitrophenyl chloroformate (334 mg, 0.134 mm
ol), and triethylamine (TEA, 45 μl, 0.329 mmol) was further added. After 20 minutes, TLC (CHCl ₃ : MeOH: H ₂ O = 90: 18: 2) showed the reaction was complete (molybdenum and ninhydrin spray). The solvent was evaporated, the resulting mixture was dissolved in methanol, mixed with C8 silica (1 g) and the solvent was removed by rotary evaporation. Add the solid residue to the top of the C8 column and add MeOH: H ₂ O (pressurized)
Elution was performed by the concentration gradient method. The eluate used was MeOH: H ₂ O = 30: 70,6
0 ml, MeOH: H ₂ O = 50: 50, 60 ml, MeOH: H ₂ O = 70: 30, 140 ml,
MeOH: H ₂ O = 75: 25, 140 ml (starting material eluted), MeOH: H ₂ O = 80:
20, 80 ml, MeOH: H ₂ O = 90: 10, 140 ml (product eluted), MeOH = 40
ml; CHCl ₃ : MeOH: H ₂ O = 90: 18: 10,40 ml. Fractions containing the pure product were collected and evaporated to give a colorless viscous liquid product. t-butanol (5 ml
) Was added, lyophilized and dried in vacuo over P ₂ O ₅ to give a white fluffy solid product (175 mg, yield: 78%). ¹ H NMR (DMSO-d ₆ , 360 MHz) δ
0.85 (d, 2 (CH ₃ ), 6H; d, CH ₃ CHSSC ₆ H ₅ , 3H); 1.22 (s
_{, 28 (CH 2), 56H} ); 1.49 (br m, CH 2 CH 2 CO, 4H); 2.24 (2 xt,
_{CH 2 CH 2 CO, 4H)} ; 3.50 (s, PEG, 180H); 4.04 (br t, mPEG
_{-CH 2, 2H); 4.08 (} transd, PO 4 CH 2 CHCH 2, 1H); 4.27 (cisd
, PO ₄ CH ₂ CHCH ₂ , 1H); 4.98 (s, C ₆ H ₅ CH ₂ OCO-DSPE, 2
H); 5.06 (br s, PO ₄ CH ₂ CH, 1H); 7.34 (d, C ₆ H ₅ , 2H); 7.53
_{(D, C 6 H 5,} 2H); 7.55 (br s, mPEG-OCONH, 1H).

Example 3 Synthesis of mPEG-DTB-Nitrophenyl chloroformate Synthesis procedure of 1- (mercaptomethyl) ethylammonium chloride 2-amino-1-methylethyl hydrogen sulfate . 1-Amino-2-propanol (22.53 g, 0.3 mol) was stirred vigorously in an ice bath. Sulfuric acid (16.10 ml, 0.3 mol)
Was added very slowly over 1 hour. A thick vapor and a very viscous solution formed in the flask. After the addition was complete, the reaction mixture was connected to a private vacuum line and heated under reduced pressure at 170-180 ° C. The heating changed the color of the reaction mixture to light brown. After all water was removed (about 1 hour), the reaction mixture was allowed to cool to room temperature. Upon cooling, a brown glassy solid formed. The product crystallized on addition of methanol. 60 ° C
Was dissolved in water (50 ml). A sufficient amount of warm methanol was added until the methanol concentration of the solution reached 80%. The resulting solution was cooled to produce crystals, which were filtered and dried over P ₂ O ₅ . Yield: 17.17 g (37%)
¹ H NMR (D ₆ -DMSO): δ 1.16 (d, CH ₃ , 3H); δ 2.78 (dd
_{, NH3-CH 2, 1H)} ; δ 2.97 (dd, NH 3 -CH 2, 1H); δ 4.41 (m,
_{CH-OSO 3, 1H);} δ 7.69 (s, H 3 N, 3H). Melting point: 248-250 ° C (literature value: 250 ° C).

2.5-Methylthiazolidine-2-thione. Hydrogen 2-amino-1-methylethyl sulfate (23.03 g, 148 mmol) and carbon disulfide (10.71 ml, 178 mmol, 1.2 eq) were placed in a 250 ml round bottom flask and a 50% aqueous ethanol solution (40 ml) was added. And stirred. To this solution was added sodium hydroxide (13.06 g, 327 mmol, 2.2 eq) in 50% aqueous ethanol (50 ml) very slowly dropwise. The addition of sodium hydroxide dissolved all starting materials and the solution turned orange. The reaction mixture was refluxed (85 ° C.) for 40 minutes and turned bright yellow, producing a viscous precipitate. The ethanol was evaporated, then the resulting aqueous solution was warmed and filtered on a Buchner funnel to remove any water-soluble impurities. Dissolve the remaining crystals in warm ethanol,
Then warm water was added until the amount of water in the solution reached 80%. The mixture was allowed to cool and then cooled in a refrigerator to yield long needle-like crystals. Yield: 14.64 g (75%). ¹ H NMR (D ₆ -DMSO): δ 1.33 (d, CH ₃ , 3
H); δ 3.50 (m, R ₃ CH, 1H); δ 3.95 (dd, N-CH ₂ , 1H); δ 4
_{.05 (m, N-CH 2} , 1H); δ 10.05 (s, NH, 1H). Melting point: 92.5-93.5
° C (literature value: 94-95 ° C)

[0105] 3. 1- (mercaptomethyl) ethylammonium chloride . 5-Methylthiazolidine-2-thione (6.5 g, 49 mmol) was placed in a 250 ml round bottom flask. An aqueous hydrochloric acid solution (40 ml, 18% aqueous solution) was added and the flask was heated in an oil bath.
The reaction mixture was refluxed (120 ° C.) for one week. Three times during the week, 1 ml of concentrated hydrochloric acid was added. The reaction was monitored by TLC (using ethyl acetate as eluent). The progress of the reaction was monitored using UV, ninhydrin, and iodine vapors. For most of the week in which the reaction was performed, the reaction mixture was in a heterogeneous state. That is, the starting material was oily and had a higher density than water.
The oily starting material disappeared after one week, but was still present by TLC. The heating of the reaction mixture was stopped, allowed to cool to room temperature, and cooled in a refrigerator to crystallize the starting material. The crystallized starting material was filtered, the filtrate was evaporated and dried over P ₂ O ₅ and NaOH to completely remove water and HCl. The resulting crude product was washed twice with diethyl ether (50 ml each) to completely remove the starting material. Dry again over P ₂ O ₅ . Yield: 2.83 g (45%). ¹ H NMR (D ₆ -D
MSO); δ 1.33 (d, CH ₃ , 3H); δ 2.92 (m, N-CH ₂ , 2H); δ
3.12 (m, SH, 1H); δ 3.18 (m, R ₃ —CH, 1H); δ 8.23 (bs, N
H _3, 3H). Melting point: 80-82 ° C (literature: 92-94 ° C).

FIG. 5 shows the reaction mechanism.

B. Synthesis of mPEG-ethyl DTB-nitrophenyl chloroformate 2-amino-1-ethylethyl hydrogen sulfate . 1-amino-2-butanol (
15 ml, 158 mmol) were stirred vigorously in a 100 ml round bottom flask immersed in an ice bath. Sulfuric acid (8.43 ml, 158 mmol) was added very slowly over 1 hour. A thick vapor and a very viscous solution formed in the flask. After the addition was completed, the reaction mixture was connected to an in-house vacuum line and
Heated at between 180C and 180C. Due to the heating, the reaction mixture turned pale brown.
After the water was completely removed (about 1 hour), the reaction mixture was allowed to cool to room temperature. After cooling, a brown, glassy solid formed. Add the solid product to hot water (50 ml)
And stored in the refrigerator overnight. After cooling, crystals formed. The crystals were collected by filtration and dried over P ₂ O ₅ . Yield: 9.98 g (37%). ¹ H NMR (D ₆ -D
MSO): δ 0.87 (t, CH ₃ , 3H); δ 1.51 (q, CH ₃ —CH ₂ , 2H)
Δ 2.82 (dd, NH ₃ —CH ₂ , 1H); δ 3.00 (dd, NH ₃ —CH ₂ , 1H);
δ 4.21 (m, CH-OSO ₃ , 1H); δ 7.70 (s, H ₃ N, 3H).

[0108] 2. 5-ethylthiazolidine-2-thione . 2-amino-1-ethyl-ethyl hydrogen sulfate (9.98 g, 59 mmol) and carbon disulfide (4.26 ml, 71 mmol, 1.2 eq) were combined with 1
Stir in a 50% aqueous ethanol solution (15 ml) in a 00 ml round bottom flask.
Sodium hydroxide (5.20 g, 130 mmol, 2.2 eq) dissolved in 50% aqueous ethanol (20 ml) was added dropwise very slowly to the mixture. After the addition of sodium hydroxide, the starting material was completely dissolved and the solution turned orange. When the reaction mixture was refluxed (85 ° C.) for 40 minutes, the color of the solution turned bright yellow and a thick precipitate layer was formed. The ethanol was evaporated and the resulting aqueous solution was warmed and filtered on a Buchner funnel to remove any water-soluble impurities. The remaining crystals were dissolved in warm ethanol, then warm water was added until the water in the solution reached 80%.
The mixture was allowed to cool, then placed in a refrigerator and cooled to obtain needle-like crystals. Yield: 7.28 g (86%). ¹ H NMR (D ₆ -DMSO): δ 0.88 (t, C
_{H 3, 3H); δ 1.66} (in, CH 3 -CH 2, 2H); δ 3.58 (m, R 3 CH, 1
H); δ 3.93 (m, N-CH ₂ , 2H); δ 10.06 (s, NH, 1H). Melting point:
76-78 ° C (literature value: 76.6-76.9 ° C).

[0109] 3. 1- (mercaptoethyl) ethylammonium chloride . 5-Ethylthiazolidine-2-thione (7.24 g, 50 mmol) was placed in a 250 ml round bottom flask. Hydrochloric acid aqueous solution (45 ml, 18% aqueous solution) was added and the flask was heated in an oil bath. The heating melted the starting material to form a completely heterogeneous mixture. The reaction mixture was refluxed (120 ° C.) for one week. Four times a week, 1 ml of concentrated hydrochloric acid was added. The reaction was monitored by TLC (using ethyl acetate as eluent). The reaction was observed using UV, ninhydrin, and iodine vapors.
Over the past week, the reaction mixture was heterogeneous and the starting material was an oil denser than water. The reaction mixture was turned off and allowed to cool to room temperature, then placed in a refrigerator and cooled to crystallize the starting material. The crystallized starting material was filtered. The filtrate was evaporated and dried over P ₂ O ₅ and NaOH to completely remove water and HCl. The resulting crude product was washed twice with diethyl ether (50 ml each) to completely remove the starting material. Dry again over P ₂ O ₅ . Yield: 3.66 g (52%). ¹
1 H NMR (D ₆ -DMSO).

[0110] The reaction mechanism is shown in FIG.

Example 4: Synthesis of mPEG-DTB-lipid 1,2-Distereoyl-sn-glycerin (500 mg, 0.8 mmol) was azeotropically dried with benzene (3 times). P-Nitrophenyl chloroformate (242 mg, 1.2 mmol,
1.5 eq), dimethylaminopyridine (DMAP) (10 mg, 0.08 mmol, 0.1 eq)
, And TEA (334.5 μl, 2.4 mmol, 3 eq) were added to a solution of 1,2-disasteryl glycerin in CHCl ₃ (5 ml). The reaction mixture was stirred at room temperature for 2 hours. TLC (toluene: ethyl acetate = 7: 3) indicated that the reaction was complete. The resulting mixture was then extracted with 10% citric acid to remove dimethylaminopyridine (DMAP) and washed with acetonitrile (3 ml, 4 times) to remove excess p-nitrophenyl chloroformate. The pure product was dried over P ₂ O ₅ in vacuo.
Yield: 557 mg (88%). ¹ H NMR (CHCl ₃ , 360 MHz) δ 0.88 (t, end
_{CH 3, 6H); 1.25 (} s, 28xCH 2, 56H); 1.58 (m, CH 2 CH 2 CO, 4
H); 2.34 (2xt, CH 2 CO, 4H); 4.22 (transd, CH 2 OCOC 17 H 35,
1H); 4.35 (m, OCOOCH 2 CH, 2H); 4.51 (cisd, CH 2 OCOC 1 7 H 35, 1H); 5.37 (m, OCOOCH 2 CH, 1H); 7.39 (d, C 6 H 5, 2
H); 8.28 (d, C 6 H 5, 2H).

Ethylenediamine (42 μl, 0.63 mmol, 5-fold excess) and pyridine (200 μl
) To CHCl_Three(1 ml). 2-disteroyl-sn-p-nitrocarbonate
Phenyl (100 mg, 0.13 mmol) in CHCl_Three(1 ml) and dissolve in ethylene dia
Using a Pasteur pipette, add dropwise to the min solution at 0 ° C (ice water temperature).
Continued overnight (16 hours). TLC (CHCl_Three: MeOH; H_TwoO = 90: 18: 2
And CHCl_Three: MeOH = 90: 10) indicated that the reaction was complete.
. The solvent was evaporated to remove pyridine. The resulting mixture is then_ThreeDissolved in
Disassemble and put on a column (Aldrich, silica gel, 60 degree A, 200-400 mesh)
Load CHCl_Three: CH_ThreeCOCH_Three, And CHCl_Three: Dissolved by MeOH concentration gradient method
Issued. The eluent composition used was CHCl_Three: CH_ThreeCOCH_Three= 90:10, 60 ml (
CHCl is eluted._Three: MeOH = 90: 10, 60 ml (product eluted)
Met. Fractions containing the pure product were collected and evaporated. t-butanol is added,
P_TwoO_FiveVacuum dried on top. Yield: 64 mg (75%).¹1 H NMR (DMSO-d₆,
 360 MHz) δ.83 (t, endCH_Three, 6H); 1.22 (s, 28xCH_Two, 56H); 1.51
(M, CH_TwoCH_TwoCO, 4H); 2.25 (2xt, CH_TwoCO, 4H); 2.83 (m, H _Two NCH_TwoCH2NH, 2H); 3.21 (m, H_TwoNCH_TwoCH_TwoNH, 2H); 4.10
−4.14 (m & cisd, COOCH_TwoCHCH_Two, 4H); 5.17 (m, OCOOCH_Two CH, 1H); 7.78 (m, H_TwoNCH_TwoCH_TwoNH, 2H).

MPEG-MeDTB-nitrophenyl chloroformate (400 mg, 0.162 mmol, 2.
2 eq) to CHCl_Three(2 ml). 1,2-Steroyl-sn-ethyl
Amine (51 mg, 0.075 mmol) and TEA (37 μl, 0.264 mmol, 3.52 eq)
Was added to the solution. Next, the reaction mixture was stirred at 45 ° C. for 20 minutes. TLC
(CHCl_Three: MeOH: H_TwoO = 90: 18: 2 and CHCl_Three: MeOH = 90: 1
0) indicated that the reaction was completed. Evaporate the solvent and mix
Was dissolved in methanol. 2 g of C8 silica were added and then the solvent was evaporated
. The C8 silica containing the product mixture was applied to a C8 column (Spelco, Spelclean,
No. SP0824), and this is added to MeOH: H_TwoO (Pressure) concentration gradient
Was eluted. The composition of the eluate used was MeOH: H_TwoO = 60: 40, 40 ml;
MeOH: H_TwoO = 70: 30, 80 ml (starting material eluted); MeOH: H_TwoO = 80: 2
0, 40 ml; MeOH: H_TwoO = 90: 10, 20 ml; CHCl_Three: MeOH: H_TwoO = 5
: 80: 15, 20 ml; CHCl_Three: MeOH: H_TwoO = 90: 18: 10, 40 ml (product is
Elution). The fractions containing the pure product are collected and evaporated to a colorless, viscous liquid
Was obtained. t-Butanol (5 ml) was added and the solution was lyophilized,
Then P_TwoO_FiveVacuum drying on the above gave the product as a white solid (200 mg, yield: 89%). ¹ 1 H NMR (DMSO-d₆, 360 MHz) δδ .83 (t, endCH_Three, 6H); 1.2
2 (s, 28xCH_Two, 56H); 1.48 (m, CH_TwoCH_TwoCO, 4H); 2.25 (2xt, C
H_TwoCO, 4H); 3.10 (m, HNCH_TwoCH_TwoNH, 4H); 3.50 (s, PEG
, 180H); 4.04 (t, mPEG-CH_Two, 2H); 4.09 (transd, COOCH_Two CHCH_Two, 1H); 4.25 (cisd, COOCH_TwoCHCH_Two, 1H); 4.98 (s,
C₆H_FiveCH_TwoOCO, 2H); 5.23 (m, COOCH_TwoCHCH_Two, 1H); 7.18
(M, NHCH_TwoCH_TwoNH, 2H); 7.33 (d, C₆H_Five, 2H); 7.38 (m, m
PEG-OCONH, 1H); 7.52 (d, C₆H_Five, 2H).

FIG. 6A shows the reaction mechanism.

Example 5: In vitro cleavage of mPEG-DTB-DSPE compounds Ortho-mPEG-DTB-DSPE and para-mPEG-DTB-DSPE
(Prepared by the method described in Example 1) was added to an aqueous buffer solution (pH = 7.2) in the presence and absence of 150 μM cysteine. HPLC of conjugate disappearance
(Phenomenex C8 Prodigy, 4.6 x 50 mm column, detected at 277 nm,
(Mobile phase: methanol: water = 95: 5 + 0.1% trifluoroacetic acid, 1 mL / min). The results are shown in FIG. 7A. In FIG. 7A, the ortho conjugate is represented by a white circle,
Paraconjugates are represented by open squares.

Example 6: o-mPEG-DTB-DSPE compound and p-mPEG-DTB-
In vitro cleavage of DSPE compounds in liposomes Preparation of liposomes Partially hydrogenated phosphatidylcholine (PHPC) lipids, cholesterol and ortho- or para-mPEG-DTB-DSPE (prepared by the method described in Example 1, mPE
G in MW = 1980 daltons) in a suitable organic solvent (chloroform: methanol = 1: 1 or 1: 3 can be used as a representative solvent) at a molar ratio of 95: 5: Dissolved at a ratio of 3. The solvent was removed by rotary evaporation to form a dry lipid film. The film was hydrated with a buffered aqueous solution to form liposomes, which were extruded to an average diameter of 120 nm.

B. Evaluation of in vitro properties The liposome was treated with phosphate buffered saline (pH 7.2) containing 5 mM EDTA.
And incubated at 37 ° C. in the presence of 150 μM cysteine. The conjugate of disappearance of HPLC (Phenomenex Inc. C ₈ Purodijii, 4.6 x 50 mm column, detection at 277 nm, mobile phase = methanol: water = 95: 5 + 0.1% trifluoroacetic acid,
(1 mL / min). The results are shown in FIG. 7B. In FIG. 7B, the liposome composed of the ortho conjugate is indicated by a black circle, and the conjugate composed of the para conjugate is indicated by a black square. Open circles and open squares indicate micelle-shaped ortho-mPEG-DTB-DS.
It corresponds to PE and para-mPEG-DTB-DSPE (described in Example 5 above and FIG. 7A).

Example 7: o-mPEG-DTB-DSPE and p-mPEG-DTB-DSP
In vitro cleavage of E compounds in liposomes Preparation of liposomes Dioleoyl phosphatidylethanolamine (DOPE) lipids and ortho- or para-mPEG-DTB-DSPE (prepared by the method described in Example 1, mP
EG of EG = 1980 dalton) in a 97: 3 molar ratio of chloroform: methanol = 1.
: 1 dissolved in a mixed solvent. The solvent was removed by rotary evaporation to form a dry lipid film. The lipid film was hydrated with an aqueous solution containing 30 mM each of the fluorophores P-xylene-pis-pyridinium bromide and trilidium 8-hydroxypyrene trisulfonate to form liposomes. The liposome was formed into an average diameter of 100 nm by an extrusion method.

B. Characterization of the liposome in vitro In a HEPES buffer (pH 7.2) at 37 ° C, a concentration of 15 μM, 15
Incubation was performed in the presence of 0, 300, and 1.5 mM cysteine. The ratio of the release dye to the fluorescence (λem = 512 nm, λex = 413 nm-isobestic point independent of pH) emitted from the sample was increased by the amount before the incubation (release amount = 0) (sample before incubation) 0.2% of Triton X-100
(100% release), normalized to the amount of fluorescence increase obtained after lysis (K
irpotin, D. et al., FEBS Letters 388 , 115-118, 1996). FIG. 8A shows the results obtained for liposomes composed of ortho compounds at various cysteine concentrations.
FIG. 8B shows the results obtained for the para compound.

Example 8 In Vivo Characterization of Liposomes Composed of mPEG-DTB-DSPE Compounds Preparation of liposomes Partially hydrogenated phosphatidylcholine (PHPC) lipid, cholesterol and para-mPEG-DTB-DSPE (prepared by the method described in Example 1, MW of mPEG =
1980 daltons) were dissolved in organic solvents at a molar ratio of 55: 40: 5, respectively, and the solvents were removed by rotary evaporation to form dry lipid films. The film was hydrated with a buffered aqueous solution containing diethylenetriaminepentaacetic acid (DTPA) to form liposomes. After the liposome mean diameter is miniaturized to a 120 nm, to remove non-trapped DTPA, it was added In ¹¹¹ the external medium. The liposomes for a time sufficient In ¹¹¹ is chelated with DTPA across through the bilayer of the lipid, and incubated.

B. In vivo administered mice were divided into two test groups, and the liposome composition was injected into all test mice. One of the test groups included 1,3 liposomal injections.
And 5 hours later, 200 μL of 200 mM cysteine was injected. Another test group was injected with saline at the same time as above. The liposome content in the blood was determined by monitoring the In ¹¹¹ content in the blood sample. The results are shown in FIG.

Although the present invention has been described above with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the present invention. It will be obvious.

[Brief description of the drawings]

FIG. 1A is a diagram showing one embodiment of the present invention. In this embodiment, dithiobenzyl (DTB) links the methoxy-polyethylene glycol (mPEG) moiety to the amine-containing ligand. B shows the product after thiol cleavage of the compound of FIG. 1A.

FIG. 2 is a diagram showing a synthesis reaction mechanism when synthesizing mPEG-DTB-amine lipid. Here, the amine-ligand is distearoyl phosphatidylethanolamine (DSPE) lipid.

FIG. 3 shows the thiol cleavage mechanism of para-dithiobenzyl urethane (DTB) -conjugated mPEG-DSPE conjugate.

FIG. 4 is a diagram showing a synthetic reaction mechanism of an mPEG-DTB-DSPE compound when it is prepared according to the present invention. Here, the DTB bond is sterically hindered by the alkyl group.

FIG. 5 is a diagram showing another synthetic reaction mechanism when an mPEG-DTB-ligand compound is prepared according to the present invention.

FIG. 6A is a diagram showing a synthesis reaction mechanism when synthesizing an mPEG-DTB-lipid compound. Upon thiol cleavage of the compound, a cationic lipid is formed. B shows the product after thiol cleavage of the compound of FIG. 6A.

FIG. 7A shows the results when A is the buffer alone (ortho-conjugate (*), para-conjugate (+)) and when 150 μM cysteine is present (ortho-conjugate (open circle), para-conjugate) (
White square)], ortho-mPEG-DTB-DSPE and para-mPEG
It is a figure which shows the cleavage rate when a -DTB-DSPE conjugate cleaves in a solution and forms a micelle. B, micellar mPEG-DTB-DSPE conjugate as described in FIG. 7A, and ortho-mPEG-DTB-DSPE (filled circles) and para-mPEG-DTB formulated in liposomes and incubated in the presence of 150 μM cysteine. -DSPE
It is a figure which shows the cleavage rate of a (black square) conjugate.

FIG. 8: DOPE incubated in the presence of cysteine (concentrations indicated in the figure):
Ortho-mPEG-DTB-DSPE (FIG. 8A) or DOPE: para-mPEG
FIG. 9 is a diagram showing the ratio of trapped fluorophores released from liposomes composed of -DTB-DSPE (FIG. 8B).

FIG. 9A shows the normalized value of the capture fluorophore release ratio as a function of time for liposomes composed of DOPE and para-mPEG-DTB-DSPE. The percentage of capture fluorophore released was normalized to the percentage of fluorophore released from liposomes incubated in the absence of cysteine. Presence of cysteine 15 μM (black square), 75 μM (white triangle), 150 μm (X mark), 300 μM (white circle), 1500 μM (black circle), 3000 μM (+ mark), and 15000 μM (white diamond) The release rate from liposomes incubated below was indicated. B shows the normalized release ratio (%) of the captured fluorophore as a function of time for liposomes composed of DOPE and para-mPEG-MeDTB-DSPE. Percent release of capture fluorophore was normalized to the percentage of fluorophore released from liposomes incubated in the absence of cysteine. Cysteine 15 μM (solid square), 75 μM (open triangle), 150 μM (marked X), 300
μM (open circle), 1500 μM (black circle), 3000 μM (+ mark), and 15000 μM (open diamond)
The release rate for liposomes incubated in the presence of. C shows the normalized release rate of the captured fluorophore for the liposomes composed of DOPE and mPEG-meDTB-distearoyl-glycerin compound of FIG. 6A (
%) As a function of time. The percentage release of the captured fluorophore was normalized to the percentage release of the fluorophore released from the liposomes incubated in the absence of cysteine. Cysteine 15 μM (solid square), 75 μM (open triangle), 150 μM (marked X), 300 μM (open circle), 1500 μM (solid circle), 3000 μM (+
) And the release rate of the dye released by cleavage of the compound from the liposomes incubated in the presence of 15,000 μM (open diamonds).

FIG. 10: PHPC: cholesterol: mPEG-DTB-DSPE (molar ratio: 55: 40: 5)
The amount of liposomes in mouse blood samples taken at various times after injection of liposomes composed in), is a plot in units of counts / min / mL of the liposomes comprising capturing an In ^111. One group of mice was injected with 200 μL of 200 mM cysteine at time zero (closed square). The control group was injected with saline at time zero (open circles).

FIG. 11A shows a reaction mechanism for synthesizing an mPEG-DTB-protein compound according to another embodiment of the present invention. B is a diagram showing a decomposition product of the compound of FIG. 11A after thiol cleavage.

FIG. 12: Lysozyme was treated with mPEG-MeDT for 15 minutes (lane 1) or 1 hour (lane 2).
M-PEG-MeDTB-lysozyme conjugate obtained by reacting with B-nitrophenyl chloroformate; natural lysozyme (lane 3), lysozyme reacted with mPEG-nitrophenyl chloroformate for 1 hour (lane 4), molecular weight marker (lane 5) ),
And samples of lanes 1-4 treated with 2% β-mercaptoethanol at 70 ° C. for 10 minutes (lanes 6-9) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), FIG. 3 shows a perspective view of a photograph.

FIG. 13 is a diagram showing a degradation product after thiol cleavage of an mPEG-DTB-p-nitroanilide conjugate.

FIG. 14A shows the absorbance of mPEG-MeDTB-para-nitroanilide (filled diamond); and the addition of 5 mM cysteine for 2 minutes (filled squares), 5 minutes (marked X), 10 minutes.
FIG. 2 shows the absorbance as a function of wavelength (nm) after in vitro incubation for minutes (open squares), 20 minutes (closed triangles), 40 minutes (open diamonds) and 80 minutes (closed circles) for 80 minutes. B is para-nitroanilide released in vitro from mPEG-MeDTB-para-nitroanilide conjugate incubated in the presence of 5 mM (closed circle), 1 mM (closed square) and 0.15 mM (closed diamond) cysteine Amount (mol / L), time (minute)
FIG.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] June 5, 2001 (2001.6.5)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 30

[Correction method] Change

[Contents of correction]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Contents of correction]

In another embodiment, the compound is reacted with an amine-containing ligand that replaces R ⁴ to form a conjugate containing the amine-containing ligand. For example, the amine-containing ligand may be a phospholipid.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Contents of correction]

In another embodiment of the present embodiment, the composition containing the conjugate is composed of a liposome. The liposome can be further comprised of a capture therapeutic.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Contents of correction]

Typically, liposomes having a surface coating of polymer chains are prepared by including from about 1 mol% to about 20 mol% of the lipid coated with the polymer in the lipid mixture. The actual amount of polymer-coated lipid may vary depending on the molecular weight of the polymer. In the present invention, the liposome contains from about 1 mol% to about 20 mol%.
It is prepared by adding up to mol% of the polymer-DTB-lipid conjugate to the other liposome lipid bilayer component. As shown by the following tests, liposomes containing the polymer-DTB-lipid conjugate of the present invention have longer circulation in the blood than liposomes containing the polymer-lipid conjugate in which the polymer and the lipid are linked by cleavable aliphatic disulfide bonds. Have a lifetime.

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Example 8 In Vivo Characterization of Liposomes Composed of mPEG-DTB-DSPE Compounds Preparation of liposomes Partially hydrogenated phosphatidylcholine (PHPC) lipid, cholesterol and para-mPEG-DTB-DSPE (prepared by the method described in Example 1, MW of mPEG =
1980 daltons) were dissolved in organic solvents at a molar ratio of 55: 40: 5, respectively, and the solvents were removed by rotary evaporation to form dry lipid films. The film was hydrated with a buffered aqueous solution containing diethylenetriaminepentaacetic acid (EDTA) to form liposomes. After the liposome was miniaturized to an average diameter of 120 nm, uncaptured EDTA was removed, and ^In111 was added to the external medium. The liposomes were incubated for a time sufficient for In ¹¹¹ to traverse the lipid bilayer and be chelated with EDTA.

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[Submission date] October 25, 2001 (2001.10.25)

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FIG. 9

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) C08F 8/34 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, D , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZWF terms (reference) 4C076 AA19 FF63 4C084 AA02 AA03 AA07 BA44 CA53 DA12 DA21 DB52 DC01 MA24 NA03 NA05 4H045 AA10 AA20 DA89 DA89 FA51 4J005 AA04 BD02 BD04 BD05 BD08 4J100 AE03P AL09P AM15P AM19P AM21P AQ08P BA03P BA22H BA38H BA41H BA50H BA51H BC43H CA31 HA00 HA11 HA55 HG00 JA51 JA53

Claims (41)

[Claims] 1. General structure Wherein R ¹ is a hydrophilic polymer comprising a bond for addition to a dithiobenzyl moiety, R ² is selected from H, alkyl and aryl groups, and R ³ is O (C =
O) selected from among ^{R 4, S (C = O} ) R 4, and ^{O (C = S) R 4} , R 4 is composed of an amine-containing ligand, R ⁵ is H, alkyl group and aryl group Chosen, C
A compound in which the coordination of H ₂ -R ³ is selected from the ortho position and the para position. 2. The compound according to claim 1, wherein R ⁵ is H and R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ . 3. The compound according to claim 1, wherein said amine-containing ligand R ⁴ is selected from a polypeptide, an amine-containing drug and an amine-containing lipid. 4. The compound according to claim 1, wherein the amine-containing ligand R ⁴ is an amine-containing lipid composed of either a single hydrocarbon tail or a double hydrocarbon tail. 5. The amine-containing lipid is a phospholipid having a hydrocarbon double tail.
A compound according to claim 4. 6. The compound according to claim 1, wherein R ² and R ⁵ are an alkyl group. 7. R ¹ is polyvinylpyrrolidone, polyvinyl methyl ether,
Polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethyl-acrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspal The compound according to any one of claims 1 to 6, wherein the compound is selected from toamide, a copolymer thereof, and polyethylene oxide-polypropylene oxide. 8. The compound according to claim 1, wherein R ¹ is polyethylene glycol. 9. The compound according to claim 8, wherein R ⁵ is H and R ² is CH ₃ or C ₂ H ₅ . 10. A liposome composed of the compound according to any one of claims 1 to 9. 11. The compound according to any one of claims 1, 2, 7, 8 or 9, wherein said amine-containing ligand R ⁴ is a polypeptide. 12. The method of claim 1, wherein said polypeptide is a recombinant polypeptide.
2. The compound according to 1. 13. The compound according to claim 11, wherein said polypeptide is cytokin. 14. The compound of claim 11, wherein said polypeptide is selected from interferons, interleukins, growth factors, and enzymes. 15. General structural formulaAnd R¹Polymer comprising a bond for attaching to a dithiobenzyl moiety
And R^TwoIs selected from H, an alkyl group and an aryl group;^ThreeIs O (C =
O) R^Four, S (C = O) R^Four, And O (C = S) R^FourSelected from among R^FourIs removed
A group represented by R^FiveIs selected from H, an alkyl group and an aryl group;_Two-R ^Three Is obtained by reaction with a compound selected from the ortho and para positions.
A composition comprising a conjugate and a pharmaceutical carrier. 16. The composition according to claim 15, wherein R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ . 17. The composition according to claim 15, wherein R ³ is O (C ＝ O) R ⁴ and R ⁴ is a hydroxy group or a group to be removed containing an oxy group. 18. The method according to claim 18, wherein the group to be removed is chloride, paranitrophenol, orthonitrophenol, N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide, N-hydroxy-glutalimide, N-hydroxynorbornene-2,3-. Dicarboximide, 1-hydroxybenzotriazole, 3-
Hydroxypyridine, 4-hydroxypyridine, 2-hydroxypyridine, 1-
16. The composition according to claim 15, which is hydroxy-6-trifluoromethylbenzotriazole, imidazole, triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol and trichlorophenol. 19. The composition of claim 15, wherein said compound is reacted with an amine-containing ligand substituting R ⁴ to form a conjugate containing said amine-containing ligand. 20. The method of claim 1, wherein said amine-containing ligand is comprised of a phospholipid.
9. The composition according to 9. 21. The composition of claim 19, wherein said amine-containing ligand is comprised of a polypeptide. 22. When R ¹ is polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxypropylmethacrylate 22. The composition according to claim 20, wherein the composition is selected from hydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, polyaspartamide, a copolymer thereof, and polyethylene oxide-polypropylene oxide. 23. The method of claim 20, wherein R ¹ is comprised of polyethylene glycol.
Or the composition according to 21. 24. The composition according to claim 23, wherein R ² is CH ₃ or C ₂ H ₅ . 25. The composition according to claim 20, wherein the composition containing the conjugate is composed of a liposome. 26. The composition of claim 25, wherein said liposome further comprises a capture therapeutic. 27. The composition of claim 21, wherein said polypeptide is comprised of a recombinant polypeptide. 28. The composition of claim 21, wherein said polypeptide is comprised of cytokin. 29. The composition of claim 21, wherein said polypeptide is selected from interferons, interleukins, growth factors, and enzymes. 30. A liposome comprising a surface coating of a hydrophilic polymer chain, wherein at least a part of the hydrophilic polymer chain has a general structure. Wherein R ¹ is a hydrophilic polymer composed of a bond for addition to a dithiobenzyl moiety, R ² is selected from H, an alkyl group and an aryl group, and R ³ is O (
Selected from C ＝ O) R ⁴ , S (C ＝ O) R ⁴ , and O (C ＝ S) R ⁴ , wherein R ⁴ is comprised of an amine-containing ligand, R ⁵ is H, an alkyl group and an aryl selected from the group, coordination of CH ₂ -R ³ is selected from ortho and para positions, the liposome is longer than liposomes having a hydrophilic polymer chain attached to a liposome via an aliphatic disulfide bond A liposome composition comprising a liposome having a blood circulation lifetime. 31. The composition according to claim 30, wherein R ⁵ is H and R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ . 32. The composition according to claim 30, wherein the amine-containing lipid is composed of a phospholipid. 33. R ¹ is polyvinylpyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl - methacrylamide, poly methacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, poly The composition according to any one of claims 30 to 32, wherein the composition is selected from hydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, polyaspartamide, a copolymer thereof, and polyethylene oxide-polypropylene oxide. object. 34. The composition according to claim 30, wherein said R ¹ is comprised of polyethylene glycol. 35. The composition according to any one of claims 30 to 34, wherein said liposome further comprises a capture therapeutic. 36. General Structure About 1 percent compound having a - about Liposomes were prepared containing 20%, a hydrophilic polymer composed of binding to R ¹ is bonded to the dithiobenzyl moiety, R ² is H, alkyl group and aryl group R ³ is O (C = O) R ⁴ , S (C = O)
R ⁴ , and O (C = S) R ⁴ , wherein R ⁴ comprises an amine-containing lipid;
A liposome having a surface coating of a releasable hydrophilic polymer chain, wherein R ⁵ is selected from H, an alkyl group and an aryl group, and the coordination of CH ₂ —R ³ is selected from an ortho position and a para position. How to improve blood circulation life. 37. The method of claim 36, wherein R ⁵ is H and R ² is selected from CH ₃ , C ₂ H ₅ and C ₃ H ₈ . 38. The method according to claim 36, wherein the amine-containing lipid is composed of a phospholipid. 39. R ¹ is polyvinylpyrrolidone, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl - methacrylamide, poly methacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, poly The method according to any one of claims 36 to 38, wherein the method is selected from hydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyethylene glycol, polyaspartamide, a copolymer thereof, and polyethylene oxide-polypropylene oxide. . 40. The method of claim 36, wherein R ¹ is comprised of polyethylene glycol.
39. The method according to any one of -38. 41. The method of any one of claims 36 to 40, wherein the liposome is further comprised of a capture therapeutic.