JPH06500077A - Conjugate materials, reagents and novel polyethers - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The terms conjugate materials, reagents and novel polyether conjugates are used to describe A common name for substances manufactured by covalently linking organic compounds, and The properties of the compound are imparted to the zygote.

The conjugation materials, reagents and novel polyethers of the present invention have a common structural feature: , consecutively linked ethoxy groups, i.e. the structure -(OCHzCHz)n- (in the formula , n = or an integer >1).

The conjugate of the present invention has the general formula A-B-A' (wherein A and A' are each an organic (residues from compounds F and F'). At least one compound is It is a polymer, and its polymer structure also exists in its residues. B is A and A' It is an organic crosslinking part that connects with each other and consists of the structure -(OCH2CIlz)n-.

A and A' are several identical residues derived from the compounds to be conjugated (F and F'). It may be composed of groups.

When manufacturing the zygote, Z-8'-Z' [where Z and Z' are is an inert (both nuclear or electronic) reactive functional group. , B' is an inert bridge]. Often used. Inert means that the bridge is stable and that Z and/or 2' It means that it does not have a group that can neutralize the reactivity of.

To produce a conjugate in which one of the compounds (F or F') is a polymer, a homogeneous It is difficult to produce a uniform conjugate material. The resulting substance is more or less , often consisting of a mixture of similar conjugate molecules. Individuals of the resulting conjugate material Common variations in molecules include variations in the number of A's attached to the same A', and variations in the bonding position. variation in position, B exists as an intermolecular and/or intramolecular crosslink, etc.

In order to impart the properties of the compound to be joined, the length and structure of the crosslinking part -B- must be adjusted. Very important. For compounds that are difficult to dissolve in water, the hydrophobic cross-linked structure makes them insoluble and and/or result in a conjugate with low activity. If the crosslink is too short, bioactivation When compounds are conjugated, their activity is often impaired. In extreme cases, there is no clue A bridge that is too short may completely degrade the activity.

The structure -(OCR2CH2)□- is present in polyethylene glycol (PEG) Ru. The molecular weight of PEG is given as an average value.

That is, PEG is usually a mixture of different molecules with different integer n. PEG is is known to have unique amphiphilic properties that can be exploited when linked to biopolymers. It is.

The structure -(OCR2CH2)n- also represents certain known amino-PEG-cal Also present in bonic acid. For example, NH2Cl'1tCHHz(O CHzCH2)no(CI’[2)2COOH(1’lowton &5o uthby, 5ynth, Commun, 19(18) (1989) 319 9-3209) has been suggested as a starting material for the production of cyclic polyetheramides. There is. The author also suggests that the same application is possible for analogs of m〉2. There is. N■2CH2C■2 (OCR2-Cu2) 20CH2COOH is a macrocycle It has been suggested as a starting material for structural ethers [Jullien et al., Tetra hedron Lett, 29 (1988) 3803-3806).

Recently (1991, 1, 20), the formula N■2CHzC[1z(OCH2CH2).

Amino-PEG-carboxylic acid represented by 0CR2COOFI (n = 1 to 10), Some of their reactive derivatives, protein conjugates prepared from them have been disclosed. (EP-A-410, 280).

Conjugates with the structure -(OCLCHz)n- in the bridge were synthesized before the priority date of the present application. has been done. 51ao+a & Rando are hydrophilic and hydrophobic image bridges [Biochea+1stry 19 (1980) 4595-4600 and Carbohydrate Re5ea rch 88 (1981) 213-221; Heteronifunctional coupling agent] . The best conjugates for those purposes are bridges: (-NHCLCHz(OCLCHz)nOcLco-)n'. In the formula, n =1, n'=1 or 2.

Slama & Rando is -0CH2C by increasing the integer n No increase in O2- structure was made. I don't know why, but instead n By setting ' to 2, the entire structure -NHCH2Cut(OCHzCHz). OCR2 CO- was doubled. Hashibe-(OCHzCH2)n- (n is various integer) Enzyme-antibody conjugates containing are known (EP-A-254,172 and F R-A-2,626,373). In the latter application, commercially available polyethylene glycol (PEG) is used as one of the starting materials for the coupling agent. this However, it is difficult to obtain a conjugate material that is uniform over the length of the bridge. Oxaa The lekylene structure, e.g. -OCH, Ctl, - represents an aldehyde group and a thiol Suggested as a heterotrifunctional reagent for selective coupling with each group (EP-^-240,200 and 10-^-89/12624).

In addition to the publications listed above, the Swedish Patent Office may also As a related publication, EP-A2-34 is included in the International Type Search Report. 5.789, WO-Al-88103412, Biochem, Biophy s, Res, Coo+m, 164 (1989-11): 3. .

The present invention shows that the spectra of individual molecules in a hydrophilic aqueous medium are Provides a conjugate material with improved uniformity and good bioavailability. this The objective is achieved by inserting a uniform, constant length amphiphilic bridge structure. It was. The conjugate of the present invention is in vivo.

and especially for diagnostic and therapeutic uses (drugs) in vitro. Are suitable.

In the joined body of the present invention, the bridge portion -B- has a structure -3rRCONI'1CH2CHz(OC[+2C[12). 0(Ctlz)m It is characterized by being composed of COY-(1).

The free valencies in formula (1) are linked to A and A', respectively. This can be done directly or even via the divalent inert structure present within the bridge -B-. -B- length, usually shorter than 180 atoms (<100 atoms, 13 atoms (preferably longer than 16 atoms).

n is an integer of >0, for example 1 to 20, and nl! , each of the conjugate (zygote substance) It is uniform so that it connects the bridges at the same position in the molecule. m is 1 or: ma2 It is. From a synthetic point of view, n is preferably 10 or <10. The zygote is P In order to exhibit the unique amphipathic nature of EG, the integer n1 must be greater than 2 , preferably: greater than +14. i.e. for various combinations of criteria. Based on this, the interval of the integer n is 1-20.1-1O11-9.2-20.2-10. 2-9.3-20.3-10.3-9.4-20.4-10.4-9.5-20 ．． 5-10, and 5-9.

Sr bonds directly to a saturated carbon atom at each of its valencies.

r=1 or 2. That is, Sr is a disl-refid group or a thioether It is the basis. When Sr is directly bonded to A, one of the sulfur atoms may be derived from F. yo0゜Y is -NH-1-NIIN[I- or -N[1N=CH-, and its The left end 1 is bonded to the Co group shown at the right end of formula I, and the right end: i is a saturated carbon atom or is bonded to a carbonyl group (only when Y is -NHNB-). Join to the right end of Y Atoms of formula Ii (not shown).

R is preferably alkylene (1 to 4 carbon atoms, often 1 or 2 carbon atoms) having one or more (1 to 3, preferably <2) hydrogen atoms It may be substituted with an xy(OH) group. In R, one identical carbon atom has , at most one oxygen atom is bonded.

From the viewpoint of availability in hydrophilic media, R is often linear or branched. and cyclic alkylene equivalently substituted with a higher alkylene selected from the group consisting of can be done. However, higher alkylenes are used to compensate for their high hydrophobicity. Must have an aqueous substituent.

As for other conditions, when r=1, B has 1-aza-2,5-dioxo- Contains a clopenkune-1,3-diyl group, and R at the 1st position to the sulfur atom at the 3rd position. or by containing a similar 1,4-diyl group and attaching it to the sulfur atom at the 4-position. Can be combined.

In a preferred embodiment, the bridge -B- must not contain aromatic rings.

The polymer may be a synthetic polymer or a biopolymer. of polymer The term refers to three or more, preferably more than ten, repeating monomer units consecutive to each other. means a compound that is bonded with The term polymer also refers to glass and other polymerized silicones. Also included are inorganic polymers such as cate.

Examples of synthetic polymers include poly[hydroxyalkyl (meth)acrylate], (meth)acrylamide, poly(vinyl alcohol), etc., and There are derivative types of polymers.

The term biopolymer refers to polymers whose basic skeleton is of biological origin. to this expression Also encompasses derivatized biopolymers. Biopolymers are generally , represents a nucleic acid or polysaccharide and/or polypeptide structure.

proteins, including polypeptides (e.g. albumin and immunoglobulins) and Polysaccharides such as soluble and insoluble polysaccharides (dextran, starch, heparin, serum (Lulose et al.) are important.

Polymers of particular interest (e.g. containing polypeptide and/or polysaccharide structures) Polymers) are usually hydroxy (-on), carboxy (-COOI'l) , amino (- or secondary) and/or mercapto (-3H) Has the ability. If a given polymer does not have suitable functional groups, one Insertion of groups is generally carried out.

Compounds F and F' are selected depending on the properties to be imparted to the conjugate. Therefore The compound is a carrier compound, a biocompatible compound, an analytically detectable compound, a water Compounds, therapeutically active compounds (drugs), enzymes, immune systems that are insoluble or soluble in the irritants, toxins, etc. Particularly important toxins exert their effects within cells. A peptide cytotoxin with a peptide segment to penetrate the cell wall and Other segments for toxicity (diphtheria toxin, ricin, pseudomonae xotoxin, etc.). Other types of peptide toxins include (e.g., T cells and class II MHC antigens) by activating cytotoxic T cells by simultaneously binding to cells with the latter An example of this type is staphylococcal enterotoxin A.

A biophilic compound, i.e. a compound that exhibits a biospecific affinity, is is of particular interest because it can be used as a target for the counterpart of a part of the zygote. be.

Examples include antibodies and antibody active fragments thereof as well as corresponding antigen/happy fragments. ten, Fc-fragments/Fc portions of immunoglobulins (Ig) and corresponding receptors (e.g., IgG binds to protein A and G), avidin/ Streptavidin and biotin, lectins and corresponding carbohydrate structures, enzymes These include elements and corresponding substrates, coenzymes, cofactors, and cosubstrates. Various classes (particularly rgG) and subclass antibodies and various specificities are useful for the conjugation of the present invention. It is a part of the body. The specificity of antibodies (and their fragments) is important for e.g. cells and/or tumor antigens/haptens, hormones, hormone receptors, etc. be.

and (insoluble polymers of interest in chromatography, immunoassays, blood A polymer used as an adsorbent in connection with liquid fractionation, etc.

In the area of in vitro and in vivo diagnostics, analytically detectable A conjugate between a compound and a compound (targeter) that exhibits biospecific affinity It is crucial for the detection and localization of the counterpart to the getter. analytically Detectable compounds include radioactivity, enzymatic activity (including enzyme substrates, cosubstrates, coenzymes, etc.) ), fluorescent, chemiluminescent, bioluminescent, particulate (e.g. latex), etc. It is a compound.

For therapeutic purposes, biocompatible compounds include drugs and other substances that exert a therapeutic effect. It can be joined to.

For the synthesis of the conjugate of the present invention, we used the general formula -Z, RCONHCH, CI'+2(OCt12CH2)nO(CH2)mZ+ We have developed a novel heterotrifunctional reagent represented by (II). During the ceremony, m and and n have the same meanings as in formula I above. Preferably m and n are uniform . That is, the reagent material is not a mixture of compounds with different m and n. R is above It is an alkylene group with the same meaning as . zl is R3-reactive electrophilic group, thiol (-SO) or a protected thiol (eg Ac5-). However, thiol group and hydroxy groups must not be bonded to one and the same carbon atom in R.

Examples of tls-reactive electrophilic groups include: (i) Halogen, saturated hydrocarbon, preferably α-haloalkylcarbonyl A halogen bonded in the form (e.g. z, cn, co-1, where z1 is preferably is bromo or iodo); (ii) activated thiols, preferably so-called anti-thiols, bonded to saturated carbon atoms; reactive disulfide (-3SI?,).

(Dan) 3.5-Dioxo-1-aza-cyclopent-3-en-1-yl. blood Regarding reactivity toward ol groups, 3,5-dioxo-1-aza-cyclopene Mixed with carbonyl group, nitro group or cyano group instead of ter-3-en-1-yl Other carbon-carbon double bonds that form a pi electron system can equally be used.

Reactive disulfides are well known in connection with synthetic chemistry (EP-A-0 63,109; see 064.040 and 128.885).

R1 is generated by a chemical reaction between -3-3-R and RS- that releases [1,-3R. In other words, the thiol-disulfide exchange reaction progresses, and R and -3R are extracted. determined to be thermodynamically stable. Many thiol compounds (R, '5 11) follows this criterion due to its natural tautomerization to the thione form in aqueous solution.

That is, the 1,000-ion form is more stable than the corresponding thiol form. This type of tautomerism One of the prerequisites for this property is that the sulfur atom of the thiol group is a carbon that forms part of the aromatic ring. bonded to an atom, the ring is (a) a heterocycle, with an odd number of atoms starting from the heteroatom in the ring. a thiol sulfur atom is located at a distance or (b) a non-heterocycle substituted with an electron-withdrawing group. This is what is being done.

Examples of R1 include 5-nitro-2-pyridyl, 5-carboxy-2-pyridyl, 2 -pyridyl, 4-pyridyl, 2-benzylthiazolyl, 4-nitro-3-cal There are N-oxides of boxyphenyl and the above-mentioned pyridyl groups.

Zl' is an activated carboxy, ie, electronic group.

Examples include carboxylic acid halides (-COC/, -COBr, and -COI) , mixed carboxylic acid anhydride (-COOOCR+), reactive ester such as N -succinimidyloxycarbonyl, -C(==NI'1)-0Rz, 4-ni Mention trophenylcarboxylate (-CO-QC, t14NO2), etc. Can be done. R1 and R2 are lower alkyl (C+~CS), R2 is also base 02-C3 alkyl or one of its valencies replaces H in NH (The 3- and/or 4-positions may be lower alkyl (C+ to C o) or a ring such as oxazolin-2-yl optionally substituted with benzyl ).

The 21' end is (i) -NI'lR+, for example in -class and secondary amines [R+ is hydrogen and lower alkyl (C+-s)], in hydrazine/hydrazide Nll, Ntl, and NH, NH- and NB2NH-CO- are aliphatic, In the compound preferably bonded to a saturated carbon atom, (if) -0■, for example, in alcohol, a compound F' having a nuclear group selected from can react selectively.

The chemical reaction of reagent ① with compound F′ at the 21′ end is as follows: [12) In the case of the group (i) above, Ill is an amide group or a hydrazide group ( -CONHNH- and -CONBN=C-), respectively, of the group (if) above In some cases, it means covalent bonding through an ester group. If necessary, Z l is then converted (reduced) into a thiol group in a thiol-disulfide exchange reaction. )can. In the latter case, F' is thiolated.

The zI terminal has a thiol group (S11) or a JIS-reactive electron-withdrawing group. Can react selectively with compound F. When F has a thiol group, the reaction can be carried out directly. be exposed. In the final product, compound F has a thioether (-3- ) or via a disulfide (-S-S-) group.

The use of reagent H takes place in a manner known per se. The reaction medium is ZI and z , 1 are chosen to avoid side reactions. F and/or F' are biopolymers - the reaction is preferably carried out in an aqueous medium to achieve selectivity. In order to So let's set it to 8. Aprotic media are often inert towards 21'. Yes, this means that they are generally preferred. Conclusion As a result, a conjugate in which F and F' are connected to each other via a bridge represented by formula I is obtained.

A reagent of formula 1 is combined with a suitable bifunctional compound at either its ZI or 21' end. By reacting, extended bridges can be introduced. For example, z ,′ for alkylene diamine, alkylene dihydrazine, alkylene dihydrazide etc., and if only one of their N[I2- groups is consumed, the remaining free The NFI2- group can be replaced with other compounds such as F or F' (preferably activated, (having a carboxy group).

Bridge lengthening can also be achieved by compound F and/or F' by use of the reagents of the present invention. Before connecting them to each other, select the appropriate type of A-B'-Z' (see page 1). This can be achieved by reacting with a difunctional reagent. Is B' the same group as R above? You can choose from. Compounds F and F' were each initially treated with the same reagent Z-8'-Z'. When reacted at the 2′ group and then linked to each other via the introduced Z group, the group B ' will appear twice in the zygote (head-to-head connection).

Useful for chain extension starting from either the reagent of formula ■ or one of compounds F and F' A large number of bifunctional reagents are known. A specific example is 4-(N-malein) imidyl)-butyric acid N-succinimidyl ester, iodoacetic acid N-succinimidyl ester Dyl ester, S-acetyl-2-mercaptoacetic acid N-succinimidyl ester 3-(pyridyl-2-dithio)propionic acid N-succinimidyl ester , alkylene diamine, alkylene diacyl hydrazide, etc. . Alkylene has the same meaning as above.

Methods known per se for the synthesis of conjugates include, for example, Oxidize the diol to two aldehydes and then -CONHNII! react with the group ,CON! Gives a lN=C- group. Important compounds F with carbohydrate structure and F' is a glycoprotein. The bifunctional reagent Z-B'-2' contains or one polymer. Even after reacting with -CONHNH2 group may be present.

In the chain extension starting from the reagent of the present invention, -8- is (2) -COR'-3r- RCONHCLCHh(OCLC■2)nO(CHz)mCONHNH-Reha structure The structure -n= means that it is a part of the structure -C=N-. This structure is an aldehyde It may be formed by a reaction between a do group and an NH2-NHCO- group.

(3) -CO((Jl,)InO(CH2CH20)nCF12CH2NHC OR'-3r-RCONH-CH2CH2 (OCFI2CHz). 0(CH,) mCOY-:C112CB, (OCH2CI'12)nO(CB2)fflCO NHNH-R'N[ln=　; Total is bonded in the same way as in case (2).

Further variations are possible. r has the same meaning as in the above case.

Construct different chain extensions starting from the Y-terminus by varying the reagents employed be able to.

The reagent (formula ■) is the formula ■ NH2CH2CH2COCHzCH2)nOccH2)mcOOFJ (m) (where m is an integer 1 or 2, and n is an integer 1 to 20, for example 2 to 20. It can be produced starting from a compound represented by

Regarding the synthesis of certain compounds represented by the formula ■, m = 1 and 2, n = 1 to 10 has been previously reported [Jullien et al.: Tetrahedron Lett , 29 (1988) 3803-3806: ■ eighty & five outhb y:5ynth, Comll1un, 19(18) (1989) 3199- 3209; and EP-^-410, 280 (public notice 1991-1-20): Rabini Slama & Rando: Carbohydrate Re5ea rch 8g (1981) 213-221 and Biochemistry 1 9 (1980) 4595-4600).

The reagent represented by the formula ■ is a compound of the formula R, as defined above, is a 2'-activated carboxy) difunctional, known per se. It can be synthesized by reacting with a chemical reagent (see page 1). After the reaction, -coon The functional group can be substituted with an activated carboxy group, e.g. 21'=activated ester, e.g. N -succinimidyloxycarbonyl, 4-nitrophenyloxycarbonyl, Convert to 2,4-dinitrophenyloxycarbonyl, etc.

In the compound represented by the formula U (m=1, n=2-20) and its derivatives, N H,- and/or -coon- groups can easily be converted into NH2- and/or The compound in which is substituted with a group that can be converted into a -COOH- group is novel and is another method of the present invention. Regarding individual aspects. This embodiment has pronounced amphiphilic properties, i.e. water as well as organic solvents. It has the property of simultaneously dissolving in lipids and lipids. offers a large number of different bifunctional substances. Ru. This is the bridge that forms the reagent used to produce the conjugate that encompasses the biomolecule. This is a desirable property for the department.

The novel compounds of formula ■ and their novel derivatives have the general formula: Polymer with XCHzC1]z(OCH2CHz-)nOcI'12Y (■) Represented by ether. n is an integer from 2 to 20, preferably from 3 to 20. X is H, N-, in its protonated form ('lImN-) or preferably in its hydrolyzed form. is a substituted H2N- which can be converted to IT, N- by reduction. For example, non-placed Converted amino (112N-); Nitro; Amide (carboxamide) such as lower acyl amide (formylamide, acetylamide 101.hexanoylamide), Acylamide group having an electron substituent on the α carbon atom of the sil residue, especially CF3 C0NH-1C113COC)I, C0NII-, etc.; even if the ring has a substituent Good phthalimidyl: carbamates, especially R,'0CONH- and (R+' 0CO)(Rz'0CO)N-1 For example, N-(t-butyloxycarbonyl) Amino (Boc), N-(benzyloxycarbohydrate which may have a substituent on the ring) nyl)amino and di(N-benzyloxycarbonyl)amino (Z and di(N-benzyloxycarbonyl)amino, respectively) and diZ); alkylar in which the carbon atom bonded to the nitrogen atom is α with respect to the aromatic system; For example, N-monobenzylamino and dibenzylamino, N-tritylamino mino (triphenylmethylamino), etc., as well as methyl carbon atoms (benzyl carbon analogous groups in which atoms (containing atoms) are substituted with silicon (St) atoms, such as N, N-di (tert-butylsilyl)amino; lower alkyl at 3- and/or 5-positions 4-oxo-1,3,5-)riazin-1-yl which may be substituted with can be done.

R1' and R2' mentioned above and below are lower alkyl, especially secondary and and a tertiary alkyl group, and 1 to 3 phenyl groups which may have substituents on the ring. means a methyl group substituted with a ru group. Lower alkyl and lower acyl groups are 1- It has 6 carbon atoms.

Y is carboxy (including -COOB, -COO-) or preferably carboxy Alternatively, it is a group that can be converted by hydrolysis or oxidation. The most important group is S In this case, the carbonyl carbon atom or the corresponding is attached to the methylene group at the right end of Formula I. Examples include Alkyl Es Ter group (-COOR,'), orthoester group [-C(ORs')s) and and reactive ester groups, such as N-succinimidyloxycarbonyl, 4-ni Trophenyloxycarbonyl, with lower alkyl in the 4- and/or 5-positions Alkylimide containing 5-membered ring type (oxazolin-2-yl) with or without There is an autogroup [-C(=NH)O-R1'). R31 is defined for R3' first It has a meaning.

Other groups of Y include -CIO1-CN, -CONR,'R,' (in this case, R, ) and R,l have the same meaning as defined above), and -CONH2.

The compounds of the invention can be prepared from known starting materials by a combination of methods known per se. Can be synthesized. A suitable synthetic route is A. Chain formation conversion of terminal functional groups Conversion of C0 symmetric polyether to asymmetric ether, bisymmetric chain cleavage of into two identical fragments It is.

Convenient starting materials having the repeat unit -0CI (2CH2-) are commercially available.

For example, there are oligoethylene glycols with 2 to 6 repeating units. same Other suitable compounds with terminal groups include the corresponding dicarboxylic acids and diamines. Ru.

A convenient starting material with different end groups is end group pure polyethylene oxide. ω-hydroxy monocarboxylic acids spaced by bridges. five Such compounds with repeating units up to (Na katsuji, Kawamura & 0kahara:5yn-thesi s (1981) p, 42].

A. Chain formation Synthesis of chains with the same or different end groups as the repeating unit -0Cf12CI'+2- For this purpose, Williamson's ether synthesis can be applied. This method uses alcohol Alkylation is (QZ″+)IOQ’-Q-0-Q’ or conversely QO1l+ Z’Q’→Q-0-Q”).

(i) Q=X'CH2(OC1'12CHz)p- (wherein, X' is XCI ’l, - or a group that can be converted into xcn 2- in one or more steps; stable under the conditions of Amson's ether synthesis) and (u　)　Q'=-(OCH2C[l2)rCH2Y' (wherein, Y' is Y or or a group that can be converted into Y in one or more steps, and is a group that can be converted into Y in one or more steps, and (stable under conditions of formation). p and r are 0 or positive integers, p+r=n.

Williamson's ether synthesis consists of a bisymmetric chain described in Section D. It can also be applied to the synthesis of

When Y' is not Y, Y' is preferably selected from groups that are stable to strong acids. and For example, Y' is preferably -CH,OR,', -CH=CR,'R,' . In this case, R4' is a lower alkyl group (C+ to C6), preferably up to 5 carbon atoms. having an elementary atom and being substituted at the α-position with up to three optionally substituted phenyl groups; is an optionally secondary or tertiary alkyl group, and R5' and IT) are hydrogen, alkyl (C+ to Cg) or an optionally substituted phenyl group.

In the above formula, 2″ is a leaving group with moderate to high reactivity, such as H B, alkanesulfonate, allenesulfonate, preferably toluenesulfone (tosylate), and perfluoroalkanesulfonates, preferably trif fluoromethanesulfonate and the like.

Williamson's ether synthesis is performed in an inert solvent, usually with a base, often a strong base. For example, it is carried out in the presence of sodium hydride or the like. Combine components of appropriate length By doing so, the elongation steps can be continued, and in principle any chain - z). can be manufactured.

Chain hamata, compound X″-C(X″)=CH2 (D HOQ or) IOQ ' can be extended by 10 CR, CH2 units by Michael addition. child , Q and Q' each have the same meaning as before. The groups X' and xm are -OQ and OQ' are respectively the 2-carbon atoms in the compound X"-C(X")=CH2 and under the conditions in which the terminal groups in Q and Q' are respectively applied. Must be selected to be stable.

X″′ is hydrogen, X′ is a group easily convertible to amino or substituted amino, e.g. It can be toro. In addition, after conversion, the group X'- A group in which C(X”)CH2- can be converted into a group HOOCCH2- or a derivative thereof can do. Preferably, X' is methanesulfinyl X nr is methyl thio, which requires hydrolysis of the product obtained in the addition step. This addition Water splitting gives aldehydes, which can then be oxidized to the corresponding carboxylic acids. can. The conditions for Michael addition are similar to those for Williamson's ether synthesis. be.

A third method of chain extension has a high affinity for Raney nickel or sulfur. reduction of the 1,000-one ester using the corresponding reagent. A suitable 1,000-one ester is , is expressed by the following equation.

X'　CHz(OCthCIh)qOCH2C3-(OC1hC1'h)s-C HzY' or X' CH2(OCH2C112)pO-C3CF12(OC H2CH2)q-CI'12Y' In the formula, X' and Y' have the same meanings as before, q and S are 0 or positive integers, q+5=n-1, where n has the previous meaning is the same as

Thione esters can be treated with alkylating agents such as methyl iodide, dimethyl sulfate or dimethyl iodide. Can be synthesized by rearrangement of the corresponding thiol ester by the action of azomethane . Thiol esters are produced by the reaction of carboxylic acids/h-rides with thiol compounds. can be synthesized by Another alternative is to add the carboxylic ester to the carboxylic ester. One method is to react the double bonded oxygen, if present, with a sulfur transfer reagent that is selective. child Examples of reagents of the type include phosphorus pentasulfide or preferably Lawesson's reagent [2,4-bisulfide]. (4-methoxyphenyl')-1,2,3,4-dithiaphosphetane-2, 4-bis sulfide]. In this rearrangement, the functional end group replaces the carboxy oxygen. Must not contain.

B. Conversion of terminal groups formula %formula% (In the formula, X'- is XC2- or XCII2- modified in one or more steps In the convertible group, Y' is Y or a group that can be converted into Y in one or more steps. ) to give the desired transformation and final product or to Depending on the conditions, reaction conditions can be applied to yield intermediate products that can be used in the coupling step. I can do it.

X' (or XCL-) +7) Examples are hydroxymethyl, -Ct120R4', -CH=CR5' R8' (R4', R2H and R, l have the same meaning as before. ). If X′ is hydroxymethyl, the conversion is e.g. reaction with a sulfonyl halide in the presence of ammonia and optionally and further reaction with a reagent that gives a suitably substituted amino group, e.g. phthalimide. This is done by responding. If X' is R4'0CH2-, then R4' is For example, by acid-catalyzed hydrolysis. The generated hydroxymethyl group is then , as described above, into a nitrogen atom-containing group. X' is CR5'R,'=C In the case of H-, the =■- of the alkylene group is converted to aldehyde by ozonation, for example. Oxidizes to hydride group. The aldehyde group is then converted to aminomethyl by reductive amination. Convert to base.

Examples of Y' (and -CH2Y) are hydroxymethyl, -CH201? ,',-C H=CR5' R6' (R4', R51 and R, l have the same meaning as before) It is. When Y' is hydroxymethyl, the reaction proceeds with the carboxy group (-CO oxidation to OH). When Y' is -CO201?4', R41 is removed, for example by acid-catalyzed hydrolysis, and R4L is at least one α If phenyl is present, removal can be accomplished by acid-catalyzed hydrolysis. Wear. The resulting hydroxymethyl group is then converted as described above. Also, that directly oxidizes the group to the corresponding carboxy group (-COOtl). Y' is -CH When =CR5'R6', =■- of the alkylene group is, for example, further acidic. carboxylic acid via an intermediate aldehyde that can be oxidizes to a group (-COOH).

The oxidation is carried out in the presence of a suitable tertiary alcohol, such as t-butanol, and the phase is directly oxidized. corresponding esters can be formed.

C1 symmetrical polyether X+CHz(OC[IzC[1z)nOcH2X+ non-pairing Conversion to ether Symmetrical ethers can be made from shorter polyethylene oxide ethers by chain extension, e.g. It can be synthesized using Williamson's ether synthesis. Two identical groups L (L is As mentioned above, the conversion of one of the groups (X' or Y') into the other group partial reaction of , then asymmetric products from unreacted starting materials and double reaction by-products This can be done by separating the

An example of such a conversion is as follows.

a) When XI is a carboxy group, convert the dicarboxylic acid into the corresponding di(acyl group) halide), which is then reacted with the missing amount of ammonia, followed by the remaining The acid nitride group is hydrolyzed. The generated amide carboxylic acid is extracted from the reaction mixture. After separation, the amide group is selectively reduced to an aminomethyl group. alternatively , the separation is carried out by a continuous ion exchange separation step after the reduction step.

b) When X is aminomethyl, the diamino compound is acylated carbon By reacting with an acid derivative such as a carboxylic acid chloride or the corresponding anhydride in insufficient amount. Ru. The monoacylated product is then separated from the starting material and the diacylated product. The aminomethyl group is then converted to a carboxylic acid group, for example via the corresponding aldehyde. The acyl group is removed by hydrolysis. A particularly useful variation of this method The method is reaction with a cyclic anhydride. In the latter case, the intermediate product is an amino acid, the starting compound Since the product is a diamine and the by-product is a dicarboxylic acid, ion exchange separation is easy. Ru.

c) X is moderately to highly lipophilic, such as trityloxymethyl or is allyloxymethyl, the difference between the product, starting material and by-product is Since the partition coefficients are significantly different, partial removal or transformation of one group is due to liquid partitioning. Easy to separate. This means that a suitable lipophilic group is a lipophilic group Xl such as trityl. Introduced into symmetrical polyethers with mono-substituted oligoethylene glycols This also applies if

If xl is hydroxymethyl, the reaction is Williamson's ether synthesis. , for example by adding an excess of haloacetic acid. After the reaction, lower the mixture to a suitable low temperature. Esterification with a grade alcohol (c+-cg), such as isopropanol. suitable By selecting the appropriate alcohol, the difference in the partition coefficients of the components in the reaction mixture becomes even more pronounced. become. This facilitates separation of the product from the starting materials and disubstituted by-products. Ru. For example, diesters migrate from the aqueous phase to organic solvents in neutral solution, whereas diesters Esters migrate with acidic p[I.

For relatively volatile compounds, most of the starting material is removed by distillation . For example, diethylene glycol or triethylene glycol.

D. Cleavage of a bisymmetric linkage into two identical fragments. formula %formula% (wherein X' and Y' each have the same meaning as before), Provide special cases. Cleavage is carried out by oxidation of the double bond. That is, In the case of A, directly to two identical carboxy groups, in the case of B, to two identical alkyl groups. It is converted to a dehyde group and then to an aminomethyl group by reductive amination.

of departure, X' CI'1z-(OCHzCH2) n-0CR2cH=cBcH20-( CHzcIIzo)n-CII2x'o and Y’　CH2-(OCH2CH2)　n-0C1f12CII=CIIC111 0-(Ct12Ctl　20)n-CH2y' are respectively Williamson's By ether synthesis, 2 equivalents (1') of F10C■2CH=CHC11,OH X'　CHz-(OCJCL)n-Z' and Y'CHz-(OCflzC[1 z) react with n-Z'' (wherein X', Y', and Z' have the same meanings as before) I like the combination of things. Alternate route Teha, Z'-CHzCH=CHCH 2Z' with 2 equivalents of X'CHz-(OCHzCH2)n-OH and A similar method can also be used to react with Y’CII, -(OCHzCH2)n-OB. Wear.

For example, the group -CH2cH=cHcH! - represents a group [in the formula, even if A is substituted] good lower alkylidene (preferably C1-C6), preferably dimethylmethylene It is. In this case, the group A is removed by acid hydrolysis.

The intermediate 1,2-diol formed is then oxidized, for example with periodic acid, and then , convert the generated aldehyde group into an aminomethyl group or a carboxy group in a manner known per se. Convert by method.

The invention is defined by the claims appended hereto, which are a part of this specification.

The example below is divided into three parts. That is, the first part is Equation IV (m=1) The synthesis of the represented amino-PEG-carboxylic acids is illustrated. The second part is represented by the formula ■ exemplifying the synthesis of a heterobifunctional reagent and a conjugate having a bridge structure of formula I; Part 3 includes T cell immune stimulants (superantigens) Staphylococcal enterotoxin (SE) The results obtained for the conjugates of A) and antibodies against tumor antigens are summarized.

Examples 5 and 6 (Part 2) illustrate the synthesis of immunoglobulin and peptide conjugates. do. In comparative experiments, it was found that the solubility of the zygote increased by having the bridge portion of the present invention. , thus clearly increasing the availability of the peptide in the aqueous medium. It was done. Comparisons are made with short, sparse samples consisting of the same peptide and antibody, but not of the invention. This test was performed on a bonded body having an aqueous bridge.

Experiment part, part 1 8-Hydroxy-3,6-thioxaoctanoic acid isopropyl ester (1) Sodium in the form of chips (23 g, l, Qmole) was added to a die in a nitrogen gas phase. Added portionwise to tyrene glycol (500*J). Sodium has completely reacted The mixture was then cooled to room temperature and bromoacetic acid (76 g, 0.5 mole) was added with stirring. Added under stirring. After reacting at 100°C for 18 hours, excess diethylene glycol was removed. was distilled off at about 4 mgHg. Next, isopropyl alcohol (400s+ 1) and acetyl chloride (51 g, 0.65 mole) were added. 65 After stirring at ℃ for 18 hours, the mixture was cooled to room temperature and treated with sodium acetate (3,59 , 0.15 mole). Filter the mixture, evaporate the filtrate to near dryness, and and dissolved in water (200++ l). The aqueous phase was separated into 1.1.1-trichloroethane (3 Extracted with X 50 l). The pooled organic phases were washed with water (20 tl). Living The product was extracted from the pooled aqueous phase with dichloromethane (5 LJ) and evaporated to an oil. A substance (559) was obtained.

11-Hydroxy-3,6,9-1-lioxaundecanoic acid isopropyl ester Tell (2) Sodium (239, l, Qwol, e) in the form of chips was treated with triethyl chloride under a nitrogen gas phase. Added little by little to lene glycol (70(1+/). Sodium reacted completely. The mixture was then cooled to room temperature and bromoacetic acid (76 g, 0.5 mole) was added with stirring. Added under stirring. After reacting at 100°C for 18 hours, excess diethylene glycol was removed. The liquid was distilled off at about 4 mmHg. Next, isopropyl alcohol (400 tl) ), and acetyl chloride (51 g, 0.65 ole) were added. 65℃ After stirring for 18 hours at room temperature, the mixture was cooled to room temperature and treated with sodium acetate (3,59, It was neutralized with 0.15 mol of oleate). The mixture was filtered, the filtrate was evaporated to near dryness, and then Dissolved in water (200mj+). The aqueous phase was 1.1.1-)dichloroethane (3X 5 (bA'). The pooled organic phases were washed with water (2To/). Living The product was extracted from the pooled aqueous phase with dichloromethane (50 mA') and evaporated. and an oily substance was obtained.

'H-n, a+, r, (CD (J'3); 1.26 (d, 6H): 3.07 (s, 2B); 3.6 to 3, 8 (m, 121); 4.11 (s, 2H); 5.09 (m, 1B)8-(N-7 talimidoyl)- 3,6-Shioxoroctanol (3) 8-chloro-3,6-thioxarooctanol (3659,2,2m+)le, ) prepared from diethylene glycol and SOC/2), dimethylformamide (400f) and potassium phthalimide (370g, 2.0 mole). Added under stirring. After stirring at 100°C for 18 hours, reduce dimethylformamide. It was distilled off under pressure. The residue was suspended in toluene (1,51) at 40-50°C and chlorinated. Potassium was filtered off. The product crystallized on cooling (-10°C). Concentrate the mother liquor By repeating the crystallization operation, a second fraction is obtained from the mother liquor.

' H-n, m, r, (CDC et al.); 2.90 (s, IH); 3.51-3.58 (m, 20): 3.60-3.68 (m, 61’l) ; 3.73-3.78 (t, 2H); 3.89-3.94 (t.

2B): 7.70-7.89 (!l, 4H) 17-(N-phthalimidoyl) -3,6,9,12,15-pentaoxa-heptadecanoic acid isopropyl ester pyridine (2.8 tl, 35 ole) in dichloromethane (30 m/) ) in dichloromethane. siroctanol (3) (8,59,36+u+ole) and trifluoromethane A solution of tansulfonic anhydride (10.2 g, 36 mmole) was stirred at approximately -5°C. It was added dropwise while stirring. After about 30 minutes, the organic phase was washed with 0.5M hydrochloric acid and water. . After drying Na25O4) and filtering, 8-hydroxy-3,6-shioxa -Octanoic acid isopropyl ester (1) (12g, 48ml1ole) and Na2P04 (6.5 g, 46.ole) was added and the mixture was stirred at room temperature for 20 hours. Stir thoroughly. The reaction mixture was filtered and the filtrate was evaporated. 1.1.1-) Partitioned between dichloroethane and water. Evaporation of the organic phase gave an oil (13 g) was gotten.

'11-n, m, r, (CDCI,); δ1.26 (d, 6■): 3.58-3.76 (m, 18H); 3.90 (t, 2■); 4 ．． 11 (s, 2H); 5.09 (Peng, 1fl): 7.70-7.8 9 (a, 4H) 17-(N-phthalimidoyl)-3,6,9,12,15-pentaoxa -Heptadecanoic acid (5) 17-(N-phthalimidoyl')-3,6,9,12,15-pentaoxa - Heptadecanoic acid isopropyl ester (4) (139) in tetrahydrofuran (50,/) and hydrochloric acid (conc., 5 parts ml). 16 hours at room temperature First, the solution was diluted with water (200++1) and the tetrahydrofuran was removed under reduced pressure. Ta. The aqueous phase was washed with toluene (1x) and extracted with dichloromethane (2x). dry Dry Na25O4) and evaporate the organic phase, leaving the product as an oil (8,59). obtained in the form.

'IT-n, ta, r, (CDCA'3); δ3.57-3.76 (+ e, 18H); 3.91 (t.

2H); 4.11 (s, 2B); 4.8 (br, IF+): 7 ．． 65-7.90 (m, 4H) 17-amino-3.6.9.12.15-pen Taoxa-heptadecanoic acid isopropyl ester (6) 17-(N-phthalimidoyl)-3,6,9,12,15-pentaoxa -Heptadecanoic acid (5) (8,59) in 150++/ethanol and 3 Dissolved in tl hydrazine hydrogen chloride.

The solution was stirred at room temperature for 16 hours and then hydrochloric acid (100 mA', 3M) was added. It was refluxed for 3 hours. After cooling to room temperature and filtering, the pH was adjusted to pH 9 and Na0. H), the filtrate was evaporated to near dryness. Add water and evaporate to near dryness again, then p The pH was adjusted to 4, MCI was adjusted, and the mixture was evaporated to dryness. The product is converted into isopropanol ( 100++ A') and acetyl chloride (2 ml) overnight at room temperature. It was then evaporated. The residue was collected in water and diluted with alkaline p[I (7-11) Extracted into dichloromethane. Evaporation gave the product (3.3g).

'H-n, m, r, (CH30D); δ1.26 (d, 6tl); 3.17 (t, 2+1); 3, 65-3.80 (m, 18H): 4 ．． 16 (s, 2H); 5.07 (m, Lll) 7.7.7-1 Rifeni Le-3,6-thioxahebutanol diethylene glycol (10017', Add triphenyl to a solution of pyridine (0.1 mole) in 0.94 mole). Nylmethyl chloride (28g, 0.In+ole) was added and the mixture was heated at 50°C. Stirred for 20 hours. The formed crystals were filtered and washed with water. Yield 31g, melting point 1 03~105℃ ' H-n, ra, r, (CFI30D); δ2.42 (br, 1 8): 3.25 (t, 211); 3.55-3.72 (i, 61’l); 7.20-7.32 (01, 9H); 7.44-7.53 (a+, 6H) 10, 10.10-triphenyl-3,6,9-)lioxadecanol (8 ) Pyridine (5 ml) in triethylene glycol (1,000 m/, 7.3 mole) 4@1.0.67a+ole), trifz:/lzmef''le chloride ( 1879,0.67 ole) was added and the mixture was stirred at 60°C for 16 hours. Living Divide the product into 4 parts, and add each part (= 250gj) to water (1,000ml) and Shake with lolomethane (250 l). The organic phases were pooled and evaporated. Yield approx. 259913.13.13-triphenyl-3,6,9,12-tetraoxa -decanol (9) Pyridine (3 To a solution of 8++/, 0.48 ole), add methyl chloride ( 1299,0,48a+ole) was added and the mixture was stirred at 80°C for 20 hours.

Water (800 mj’) was added and the mixture was extracted with dichloromethane (3X 200 m1). I put it out. The pooled organic phase was washed with water (150 mj’) and evaporated to produce The product was obtained as a syrup. The product contains small amounts of disubstituted product and unreacted raw materials. It was rare. Yield 200g Pyridine (38mCO) in pentaethylene glycol (25g, 12mmole) , 48 mole), triphni was added with methyl chloride (12 h, 0.48 m ole) was added and the mixture was stirred at 80°C for 2 hours. Add water (100mA), The mixture was extracted with dichloromethane (40 ml). The aqueous phase was extracted with toluene and ethanol. was evaporated using azeotropic distillation in the presence of alcohol. Pipet the residue as above. Treated with lysine (1 ml) and triphenylmethyl chloride (2,59).

The same operation was applied again. The three organic phases were pooled and evaporated.

The product contained small amounts of disubstituted product and unreacted starting materials. Yield 109 Pyridine (2,8 Triphenylmethyl chloride (9,79,34 mole) was added and the mixture was stirred at 80° C. for 3 hours. Add water (400m/) , the mixture was extracted with dichloromethane (150 mJ). The aqueous phase was described in the previous example. The procedure was carried out once again, but with the same amounts and reaction conditions as in this example. P The cooled organic phase was evaporated, washed with water (100ml) and dichloromethane (50ml). m1). The product contained small amounts of disubstituted and unreacted raw materials. .

Yield 41.6q 4-Methylbenzenesulfonic acid 7.7.7-)diphenyl-3,6-thioxer hebutyl ester (12) 7.7.7-) diphenyl- in pyridine (10 liters) Solution of 3,6-thioxahebutanol (7) (3,59,26m+5ole) To this, p-toluenesulfonyl chloride (59,26 ole) was added. blend After stirring at room temperature for 30 minutes, water (1 ml) was added and stirring continued for another 10 minutes. Continued. Dichloromethane (100ml) was added and the mixture was diluted with hydrochloric acid (IM). Extraction was performed until lysine was completely removed. The organic phase (NaHCOs, saturated ). After drying (Na2SO4) and evaporation, the product was dissolved in diethyl ether/hexyl Crystallized from xane. Yield 2g 4-methylbenzenesulfonic acid 10.10.1 0-triphenyl-3,6,9-dioxa-decyl ester (13) pyridine (10.10.10-)riphenyl-3,6,9-trioxade in 1(b/) To a solution of canol (8) (4.8g, 121IIlole) was added p-toluenesulfur. Honyl chloride (5g, 26a+mole) was added. Stir the mixture at room temperature for 1 hour. After stirring, water (1 mA') was added and stirring continued for an additional 10 minutes. blend diluted with dichloromethane (50 ml), 100 ml of 1M hydrochloric acid (2X), and water ( 50s+7) and NaHCO3 (saturated, 50d). evaporate the organic phase After purifying the product on silica gel (toluene:ethyl acetate, 9:1), The product was crystallized from dichloromethane/ether. Yield 4.29'H-n, m , r, (CD(Js); δ2.20(s, 311); 3.24(m, 2B): 3.60-3.80 (m, 8H); 4.20 (or, 2H); 7. 20-7.90 (m.

19H) 8-(N-phthalimidoyl)-3,6-shioxa 4-methylbenzenesulfonic acid -Octyl ester (14) 8-(N-phthalimidoyl)- Dissolve 3,6-thioxaoctanol (3) (82.2g, Q, 3 mole) and p-toluenesulfonyl chloride (56,99,0, button ole) for 1 hour. was added with stirring at 0°C. The mixture was then stirred for 16 hours at 10°C. Ta. A solid cake forms, to which ice (0.5 Ag) is added with hydrochloric acid (concentrated, 20,000 l) After stirring the mixture at 50°C for 4 hours, ethyl acetate (200g/ ) was added. The complete reaction mixture was filtered to obtain the product in solid form. Collection Amount 979 '[1-n, tr, (CDCA!s): δ2.44 (s, 3B); 3. 52-3.73 (m.

8B): 3.87 (t, 2B); 4.09 (q, 2H): 7.31 ~7.86 (m, 8H) 17-(N-phthalimidoyl)-3,6,9,12 ,15-pentaoxa-heptadecanol (15) 10.10.10-triphenylene dissolved in dimethylformamide (15@j’) Ru-3,6,9-trioxadecanol (8) (0,789,2 mmole) Sodium hydride (80%, 0.249.8 mmole.

(washed with 2X hexane). After warming the mixture to 30°C for 30 min, 4-methylene 8-(N-phthalimidoyl)-3,6-thioxaroctyl rubenzenesulfonic acid ester (14) (0.87 g, 2InlIIole) was added as a solid. −Night After stirring at room temperature, acetic anhydride (5 ml) was added and the mixture was allowed to warm to room temperature for a further 3 hours. I left it alone. Add water (2 ml), and after 30 minutes toluene liquid (50ml). The toluene phase was evaporated and approximately 1 g of the material contained was collected in dichloromethane, Q, 2 ml of trifluoroacetic acid and ca. 1 of water for 10 minutes at room temperature. The reaction mixture was then evaporated. Living The product was purified on a silica gel column (chloroform:methanol, 19:1). . Yield approximately 0.7g'B-n, m, r, (CDC/3); δ3.59-3.6 7 (m, 20H); 3.71-3.75 (i, 4H); 3.90 (t , 2■); 7.71-7.86 (s, 4H) 17-(N-phthalimidoyl ) tert-buty-3,6,9,12,15-pentaoxa-heptadecanoate ester (16) 17-(N-phthalimidoyl) in dichloromethane (51) -3,6,9,12,15-pentaoxa-heptadecanol (15) (190 m+9), pyridinium dichromate (0.64 g, 4 eq), anhydrous vinegar Acid (1 ml = 20 eq) and t-butyl alcohol (1 ml, 30 eq) added to this order. After stirring the reaction mixture for 3 hours at room temperature, ethyl acetate (2 5 ml) was added. After 10 minutes, the liquid was transferred to a column containing silica gel (5 (4X 5c++) and the column was further eluted with ethyl acetate. After evaporating, The residue was purified on a silica gel column (chloroform:methanol, 19:1). Ta. Yield approximately 0.1q 'H-n, m, r, (CDC/3); δ1.47 (s, 9H); 3.5 g ~ 3.76 (m.

18H): 3.90 (t, 2H); 4.02 (s, 2■); 7.70~ 7.87(m, 4H)14.14.14-triphenyl-64,7,10,13 -tetraoxa-tetradecene (17) Allylpromide (2 mA', 1.1 eq) and to, 10.10-triphene Nyl-3,6,9-)lioxadecanol (8) (8,59,22+mole ) was dissolved in dimethylformamide (25 liters) and hydrogenated at 0°C for 30 minutes. Dropped into sodium (1 g). After stirring at room temperature for 3 hours, methanol was dissolved. Add until the liquid becomes clear. Toluene (100g/) and water (10To/) and the reaction mixture was extracted. The toluene phase was washed with saturated saline solution and then dried. (Na2SO4). The purified product was obtained as a syrup after evaporation of the solvent (l h).

3.6.9-Trioxa-11-dodecen-1-ol (18) 14, 14.1 4-triphenyl-64,7,10,13-tetraoxa-tetradecene (17 ) (5,59,13 mmole) in dichloromethane (50 liters) and then , trifluoroacetic acid (1,2 tl.

Add 15,6 mmole) and water (IIN, 55 sa+ole) and mix the reaction. The mixture was stirred vigorously for 10 minutes. The product (1,59) was purified on a silica gel column. CCHCl3:MeOH19:1) obtained after production.

3, 6.9.12.15.18-hexaoxa-20-henicosen-1-o Le (19) 3.6.9-) Lyoxer 11-dodecen-1-ol (18) (2,079, 10,9tl1mole) and 4-methylbenzenesulfonic acid 10.10.1 0-triphenyl-3,6,9-dioxa-decyl ester (13) (5, 959,10,9 mmole) was dissolved in dimethylformamide and heated to room temperature under stirring. The mixture was added dropwise to sodium hydride over a period of 10 minutes. After 2 hours, dichloromethane and Added water. Separate the phases and add trifluoroacetic acid (1 mL 13 mmole) to the organic phase. ) and water (1 ml, 55 sa+ole) were added. Stir the mixture vigorously for 1 hour. Afterwards, it was evaporated to dryness. After adding methanol (70%), triphenylmeth Nord (2g) was filtered off and the solvent was evaporated. After purification on a silica gel column (C HCIs: MeOtl, 97:3), the purified product (2,59) was obtained and the tetrahydride 3.6.9.12.15.18-Hexaoxa-20-Hexaoxene in Lofuran Bromoacetic acid ( 0,159,1,1 mmole) and sodium hydride (0,159,5 mmole) ole) was added. After stirring the mixture for 3 hours at room temperature, the excess reagent is destroyed. Water was added to adjust the pH to 1 (11 (J). The mixture was diluted with diethyl ether. (50tl) and then extracted with dichloromethane (2X50ml). dry After drying Na25O4) and evaporating the solvent, the product (0.26 g) was obtained. Ta.

3.6.9.12.15.18.21-hebutaoki in methanol (30 mA’) Solution of Cir-23-tetradecenoic acid (20) (0.21v, Q, 55mmole) Ozone was gradually passed through the solution for 30 minutes at about -60°C. The mixture was left for 30 minutes The solution was then bubbled with air for 30 minutes and then dissolved in methanol (50+1). Dimethyl sulfide (50 μl, 0.65 mmole) was added dropwise. of this solution The temperature was allowed to rise to room temperature for 1 hour, then ammonium chloride (0.2 g) in water (5 mA’) solution and sodium cyanohydroborate (0,29,3,1 m mole) was added. After 18 hours at room temperature, the ptl of the solution was adjusted to approximately 1. HCl) and the solvent was removed by distillation. Dissolved in sodium hydroxide (IM), Extraction with dichloromethane and evaporation gives an oil (0.25 g). Pure The perfect product is to prepare amino-functional tert-butoxycarbonyl derivatives. Obtained by

That is, 0.17g of oil and sodium hydroxide (0.4v) are mixed with water (3ml). secondary acid di-tert-butyl ester dissolved in dioxane (511) (0.12g) was added. After stirring for 16 hours, the dioxane was evaporated and the aqueous phase Wash with n-hexane (2X 10 liters) and adjust the pH to about 4. HCA' ), then the mixture was extracted with dichloromethane (5X 50ml).

Dry Na25O4) and evaporate the solvent to obtain an oil, which is Purified on gel column (CBCj'3: MeOfl: AcOH, 45: 4:1). Yield was 0.15g.

'I'l-n, Il, r, (D20.500kltlz); δ 1 ．． 31 (s, H); 3.14 (t.

28); 3.48 (t, 2H): 3.59 (+e, 241): 3. 84(s, 2H) Sodium hydride (3,89,80%, 130mmole ) and bromoacetic acid (7,69,55 mole) in this order, 8-(N-phthalimidoyl)-3,6-shioxa in Ran(15(1+jl')) -Added to a solution of octanol (3) (10.1 g, 35 sa+ole). 4 o'clock After stirring at room temperature for a while, acetic anhydride (40 tl) was added and the mixture was stirred for a further 18 hours. After stirring, the mixture was filtered and evaporated in the presence of a little water. on silica gel Purification with (CHCI3:MeOH 13:2) gave the product. Yield 1 ．． 2g 11-amino-3,6,9-trioxa-undecanoic acid methyl ester (23) 11-(N-phthalimidoyl)-3,6,9-trioxa-lanticanoic acid ( 22) Solution of (1.2g, 3.5Bole) in ethanol (25all)+: , add hydrazine hittride (0.7 ml/, 14 sa + ole) and leave in room for 18 hours. After stirring at warm temperature, hydrochloric acid (3,711/, concentrated) and water were added. Refrigerate the mixture for 2 hours. After washing, and then evaporating to dryness, water was added to adjust the PTL to about 9 (Na OH).

After evaporation to dryness, methanol (200 mA') and acetyl chloride (2 mA') 1) was added and stirred at room temperature for 66 hours. The generated methanol ester is filtrated with silicage. Purified on a column.

'H-n, s, r, (D20); δ 3.17 (t, 2tl); 3.65~ 3.80 (m.

138); 4.20 (s, 2H) 3, 6.9.12.15.18.21.24.27-nonaoxa-29-trico Sodium hydride (5019,1,6 mmole) was dissolved in dimethylformamide ( Log/) medium 3.6.9.12.15.18-hexaoxa-20-hennife se ion-1-ol (19) (0.6 g, 1.9 mmole) and 4-methylbenzene 7,7.7-triphenyl-3,6-thioxerbutyl sulfonic acid It was added to a solution of Ter (12) (0.9 g, 1.8 mmole). this mixture Stir at room temperature for 56 hours, then add water (IToJ) and dichloromethane (15++4) added. The mixture was shaken, the organic phase was evaporated and purified on a silica gel column. (CH(J’3:MeOfl, 9:1). The product was purified by dichloromethane (20ml ), trifluoroacetic acid (3 drops) and water (5 drops) were added, then the mixture was dissolved in Stir for an hour and evaporate. The residue was extracted with methanol:water (70:30), The oily substance obtained by evaporating the filtrate was dissolved in 10 l of tetrahydrofuran, Add sodium hydride (about 5 eq) and bromoacetic acid (about 2 eq) to this, The mixture was stirred at room temperature for 16 hours. Decompose excess sodium hydride by adding water , the mixture was evaporated and then purified on a silica gel column (CHCA': MeOH13:1). Yield 1.14'fl-n, m, r, (CDCA'3) ; δ2.89 (s, 2H); 2.97 (s, 2H), 3.50 to 3.7 8 (Ill, 30H); 4.02 (d, 2■); 5.20 (m, 21 1), 5.913, 6.9.12.15.18.21.24.27-Nonaoxa -29-tricontinic acid (24) (0,119,0,23+*mole) Ozone was bubbled slowly through the solution in ethanol (25 ml) at about 70° C. for 30 minutes.

After 30 minutes, air was bubbled through the solution for 30 minutes and then dimethyl sulfide (50μ 165 mmole of L O) was added. The temperature of this solution was raised to room temperature for 1 hour. Then add ammonium chloride (0,19,0,23 mmole) in water (5 ml). >Solution and sodium cyanohydroborate (0,19,1,6m+*ole ) was added. After keeping it at room temperature for 16 hours, adjust the pH of the solution to about 1. l'), the solvent was distilled off. The dried material was extracted with dichloromethane and then evaporated. The residue was an oil (0.1 g). Pure product is amino was obtained by preparing a functional tert-butoxycarbonyl derivative. vinegar That is, the oil and sodium hydroxide (0.4 g) were dissolved in water (3 ml), and Secondary acid di-tert-butyl ester (0,16 g) dissolved in xane (5 ai! ) was added. After stirring for 16 hours, the dioxane was evaporated and the aqueous phase was diluted with n-hexane. Wash with a 2x10 wash and adjust the pH to about 4. Extracted with lolomethane.

Synthesized amino-PEG-carboxylic acid formula % formula % The structural formulas for the preparation of the bifunctional reagent and coupling product are shown in the appendix.

Example 1 17-Iotoacetylamino-3.6.9.12.15-pentaoxaheptade Production of N-hydroxysuccinimide ester of carnoic acid 17-Amino-3,6,9,12,15-pentaoxaheptadecanoic acid isopro Pyrester (see Experimental Section, Part 1) (1,19,3,2 mmole), 3 mg of 1M sodium hydroxide solution and left at room temperature for 30 minutes. 1.5m g of 6M hydrochloric acid were added and the mixture was evaporated to dryness. Take up the residue in dichloromethane and Filtration and evaporation of the solvent yielded 17-amino-3,6,9,12,15-pentao. 545 wq of xaheptadecanoic acid was obtained. This compound 460 times 9 (1,39 mmole) was dissolved in 10 ml of borate buffer p8.4. Add nitrogen to this solution. Air was removed through elementary gas. 4321 (1,52 mm ole) solution of N-succinimidyl 2-iodoacetate for 1 minute. dropwise, and during this time 5M Na0B was added to maintain pn at 8.4. 15% of the reaction solution The mixture was stirred for 1 minute while passing nitrogen gas. Thin phase chromatography (eluent: CB , (J,-MeOH60: 35), the reaction was completed in approximately several minutes. 15 After minutes, the pH of the reaction solution was adjusted to 3, and the solution was frozen and dried. reaction mixture Reversed phase column PEP-RPC30/26 (Pharmacia Biosyst ems AB), 0-13% acetonitrile gradient with 0.1% trifluoroacetic acid Separation with acid, followed by isolysis with 13% acetonitrile, 0.1% TFA. Fractionation was performed by cratic separation. Pool fractions from desired peaks , upon lyophilization, 17-iotoacetylamino-3.6.9.12.15-pe 351 mg of antaoxaheptadecanoic acid (A) was obtained. Yield near 6%.

The structure of the product was established by its NMR spectrum. 'II NIIRs The spectrum (020) is shown as a δ value.

Hydroxysuccinimide (4,5++v, 39μ5ole) was added to anti-3, 6.9. 12.15-pentaoxaheptadecanoic acid (A) (18,3O9.39IIm ole) was dissolved in 0.55 ml of dry dioxane and added to the reaction vial. Ba After passing nitrogen gas through the vial to remove air, add 0.15 mZ of dry dioxane. A solution of 8.0 mg (39 mole) of dicyclohexylcarbodiimide in into a vial. The vial was filled with nitrogen gas, sealed, and placed in the dark. . The reaction solution was stirred for 3.5 hours. The formed precipitate was removed by filtration. in the filtrate The yield of product B was determined to be 89% by NIIR analysis.

Example 2 (17-Iotoacetylamino-3.6.9.12.15-pentaoxahepta Production of decanoylamino) immunoglobulin (C) A, Monoclonal antibody Mab C215 immunoglobulin class IgG2a Monoclonal antibody (Mab C215) (34 genera 9.0.21811 mole ) in O, 1M borate buffer pH 8.1 containing 0.9% sodium chloride. , 17,7 mA' and this solution was added to the reaction vial. 17- Yotoa Cetyl amino-3.6.9.12.15-pentaoxaheptadecanoic acid N-h Contains 3.6 liters (6.4 μmole) of droxysuccinimide ester (B) Pour 146al of the dioxane solution into the buffer solution and stir at room temperature for 25 minutes to stir the reaction. Completed it. The reaction vial was covered with foil to protect it from light. Excess reagent (B), 5 Ephadex G25 on 26/40 column, 0.9% sodium chloride as eluent removed by fractionation using 0.1 M phosphate buffer pH 7.5 containing . Fractions containing the desired product C were pooled. Solution (22++1) was converted into Yi13θ It was passed through a filter and concentrated to 8111 in an Amlcon cell. Concentration and degree of substitution were 4.7mg/ml and 1lab C21, respectively, according to amino acid analysis. There were 18 spacers per 5.

B, Monoclonal antibody Mab C242 immunoglobulin class IgG1 mono The clonal antibodies ( ) jab C242) were prepared according to the procedure described in Example 2A. 15.20 and a 22-fold molar excess of 17-iodoacetylamino-3,6,9 , 12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide S (B) to form nona, dodeca and tetradeca (17-iodoacetyl amino)-3,6,9,12,15-pentaoxaheptadecanoylamino)- Mab C242 (C) was obtained.

C, monoclonal antibody Mab C A monoclonal antibody (Mab C) of immunoglobulin class IgG2a was prepared in Example 2. 14- and 18-fold molar excess of 17-iodoacetate, respectively, according to the procedure described in Thylamino-3,6,9,12,15-pentaoxaheptadecanoic acid N-hydro By reacting with xysuccinimide ester (B), tetra and hepta(17-io) (acetylamino) -3,6,9,12,15-pentaoxaheptadecano ylamino)-sumireabC(C)2-mercaptopropionylamino-Eu3-label Identification - Staphylococcal enterotoxin A (SEA) production A, Eu'+ labeling 5E Manufacture of A(D) from SEA (Toxin Technology Inc.) Freeze-dried product) (2■9.72nmole) in 122ul of mlli-Q water Dissolved and added to a 15 mA' polypropylene tube. 0.1M of 10hA’ Borate buffer pH 8,6 then 2160 rv in m111i-Q at 178 t/ ole's Eu''-chelating reagent (Phar+*acia Wallac Oy) added. The reaction was completed after one unit was brought to room temperature. Add the reaction solution to 5ephadexG2 On 5PD10 column (Phar+aacia Biosystems AB), Removed by fractionation using 0.1M phosphate buffer pH 8.0 as eluent. Ta. Fractions containing the desired product were pooled.

The solution (3 ml) was passed through a YM5 filter into an A1con cell with a volume of Q, 8 Concentrated to +1. According to amino acid analysis, the concentration was 1,7119/ml. . The degree of substitution is Eu (compared to J3 standard solution, Q per SEA, measured as 3Eu"") determined.

81.3-(2-pyridyldithio)propylaminoEu” labeled SE^(E) and Production of 3-mercaptopropionylamino Eu”+ labeled SEA (F) Eu””-3EA (1, 24M9.44.8nmole) was added to a 15g/polypropylene tube. Ta. 1.6me of 3-(2-pyridyldithio)-propio in 1ml of ethanol Add 35 μl of a solution of succinimidyl acid N-succinimidyl ester to the tube and add the reaction solution. was stirred at room temperature for 30 minutes. The obtained product E is not isolated and is directly used as a product. Returned to F.

To the above reaction solution, add 20 μl of 0.2M Eu”-citric acid solution and 50 μl of The pH was adjusted to 5 by adding 2M acetic acid. Then, 0.1w5l of 0.9% base 11 of dithiothreitol (Merck) in sodium chloride solution and the reaction solution was Stirred at room temperature for 20 minutes. Then add 50 μl of 0.9% sodium chloride solution. The total volume was adjusted to 1 ml. Reaction solution (1ml>5ephadexG25N Placed on AP-10 column (Pharmacia Biosystems AB) and the desired product F was added to 0.1M phosphate buffer p containing 0.9% sodium chloride. ) 17.5.1.5 mA' was added to elute. The eluted product F was 15++4 Collect in a polypropylene tube to avoid re-oxidation to disulfide compounds. , was immediately used for the synthesis of product G.

B2.2-mercaptopropionylamino-staphylococcal enterotoxin A ( SEA) (F2) manufacturing native SEA (Toxin Technology) y Inc.) or recombinantly produced SEA (rS EA) by the procedure described in Example 3B1, a two-fold molar excess of 3-(2-pyridylditi e) Propionic acid N-succinimidyl ester was reacted.

The degree of substitution was determined by Carlsson et al. (Biochem, J, 173 (197g) 72 3-737], 1.9 mercaptopropionyl per SEA. It was a group.

bile Production A of 5EA-monoclonal antibody conjugate (Gl or G2), Eu3+- Conjugate of 3EA and 1lab C215 (G1) 4-mercaptoprene described in Example 3B A solution of lopionylamino Eu”-labeled SE^(F) containing 0.9% sodium chloride was added. Octadeca (17-iotoacetylamide) in 0.1 M phosphate buffer pH 7.5 No 3.6.9.12.15-pentaoxaheptadecanoylamino) 1la b Add 1.2 ml of solution of C21C215 (C) (4). Leave at room temperature overnight. The reaction was completed. Then a solution of 20 μl of mercaptoethanol in 1 ml of water 5 μJ (1.2 μmole) was added to block unreacted iodoalkyl groups.

The reaction solution was left at room temperature for 4 hours and then filtered. Next, the filtrate is 5 uperos e 12HR16150 column (Phara+acia Biosystem II lsAB), 0.002M phosphate containing 0.9% sodium chloride as eluent Fractionation was performed using a buffer pH 7.5. pooling the fractions containing the desired product G; analyzed. The protein content by amino acid analysis was 0.22xg/ml. Place The conversion rate was determined to be 1 SEA per IgG by quantification of Eu''. The immunostimulatory effect and antibody binding ability of the products were also examined.

When the amount of Compound F relative to Compound C was increased, an increase in the degree of substitution was observed.

B, conjugate of rSEA and 1lab C215 (G2) octadeca (17-iot) Acetylamino-3.6.9.12.15-pentaoxaheptadecanoylami c) Mab C215 (C) was subjected to 1.8 times molar filtration according to the procedure described in Example 4^. It was reacted with residual 2-mercaptopropionylamino-rSEA (F2).

The composition of the conjugate was changed to sodium dodecyl sulfate on a Phast-GelT1' gradient 4-15. The binding was detected by Ph polyacrylamide gel electrophoresis (SDS-PAGE). ast IMAGE (Pharmacia Biosys-tegs AB) Scanned with. The composition of the obtained conjugate was that Mab C215 containing three SEA 6%, 15% with 2 SEAs, 28% with 1 SEA, and 28% with 1 SEA. Substitution Mab C215 was 51%.

In another experiment, when a 2.7-fold excess of F2 was used, the composition of the resulting conjugate was Mab C215 with 3 SEAs is 15%, and those with 2 SEAs are 25%. %, those with one SEA were 34%, and unsubstituted Mab C215 was 26%. Ta.

C, conjugate of rSEA and Mab C242 C1, tetradeca (17-iodoacetate) tylamino-3,6.9.12.15-pentaoxaheptadecanoylamino) Mab C242 (C) was prepared in a 3.2-fold molar excess of 2 by following the procedure described in Example 4^. -mercaptopropionylamino-rSEA (F2). set of zygotes Mab C24 with 4 SEAs was analyzed as described in Example 4B. 4% have 2 SEAs, 12% have 3 SEAs, and 28% have 2 SEAs. , 36% had one SEA, and 20% had unsubstituted 11ab C242. Ta.

The same reaction was carried out, but the product was treated with 0.2M hydroxyl chloride before fractionation on the column. MabC242 and spacer, and SEA and mercap The unstable bond between the topropionyl groups was removed. The composition of the generated zygote is 4 Mab C242 has 1% SEAs, 12% has 3 SEAs, 27% with 2 SEAs, 36% with 1 SEA, unsubstituted j ab C215 was 24%.

C2, dodeca(17-iotoacetylamino-3.6.9.12.15-penta Oxaheptadecanoylamino) l[ab C242 (C) as described in Example 4A According to the procedure, a 3-fold molar excess of 2-mercaptopropionylamino-rSEA (F 2). The resulting conjugate composition was Mab C24 with 3 SEAs. 2, 6%, 2 SEAs, 26%, 1 SEA, 36%. , unsubstituted Mab C242 was 31%.

C3, nona(17-iotoacetylamino-3.6.9.12.15-pentao xaheptadecanoylamino) 1lab C242 (C) as described in Example 4^ According to the procedure, a 3-fold molar excess of 2-mercaptopropionylamino-rSEA (F 2). The composition of the resulting conjugate was Mab C2 with two SEAs. 42 was 13%, those with one SEA were 39%, and unsubstituted Mab C242 was It was 46%.

D, conjugate of rSEA and 1lab C D1. hepta(17-iotoacetylamino-3.6.9.12.15-penta oxaheptadecanoylamino) Mab C according to the procedure described in Example 4^. React with a 5.4-fold molar excess of 2-mercaptopropionylamino-rSEA (F2). I responded. The composition of the conjugate was analyzed as described in Example 4B and found that 4 SEA 15% had 3 SEAs, 24% had 3 SEAs, and 24% had 2 SEAs. 29%, 19% with 1 SEA, 3% with unsubstituted 1lab C, Dimer type accounted for 10%.

The same reaction was carried out in the presence of 0.2M hydroxylamine, and Mab C and spacer -, and the unstable bond between SEA and the mercaptopropionyl group were removed.

The composition of the generated zygote was 11% MabC with 3 SEAs and 11% MabC with 2 SEAs. 24% have one SEA, 30% have one SEA, and unsubstituted 11abC 18% and 17% were dimeric.

D2. Tetra(17-iotoacetylamino-3.6.9.12.15-pen taoxahebtadecanoylamino) Mab C according to the procedure described in Example 4^ , with a 5.7-fold molar excess of 2-mercaptopropionylamino-rSEA (F2). Made it react. The composition of the obtained conjugate was 8% Mab C2 with 4 SEAs. , 18% had 3 SEAs, 30% had 2 SEAs, and 1 26% had SEA, 5% had unsubstituted Mab C, and 12% had dimeric type. It was.

Example 5 Peptide sequences 145-166 derived from human alloantigen HLA-^2.1, Coupling of monoclonal antibody Mab C215 (H) 1.6 mA' of 0.1 M phosphate buffer containing 0.9% sodium chloride pH 7.5 Octadeca (17-iotoacetylamino-3.6.9.12.15) dissolved in -pentaoxaheptadecanoylamino)-immunoglobulin G2a (C) (5, 4ui, 34.5 mmole) was added to a 5 mg Reacti vial. this After passing nitrogen gas through the solution to remove air, add HLA-A2 while stirring. ．． 1 peptide sequence 145-165, HisLysTrpGluAlaHisVa lAlaGlu-GlnLeuArg^1aTyrLeuG1uG1yThrC Add ysVal (2.5 y, 0.8 μmole) in solid state little by little. Ta. The pH of the reaction solution was checked to be 7.4. Before sealing the vial Then, additional nitrogen was bubbled through the solution. Cover the vial with foil and let the reaction solution cool at room temperature. Stirred overnight. 10 μ! in 1 ml of water to block unreacted iodoalkyl groups. of Add 10.5 μl (15 μl) of a solution of mercaptoethanol to the reaction solution. The liquid was stirred for 4 hours. Filter the reaction solution and add 5uperose 12■R1075 0 column (Pharmacia Biosystem+s AB), eluate and using 2111M phosphate buffer pH 7.5 containing 0.9% sodium chloride. Fractionated. Fractions containing the desired product H were pooled. Protein concentration and degree of conversion Amino acid analysis revealed that 176 μg/W protein and IgG, respectively. 11 peptides per sample were measured.

In a similar synthesis in which 5 to 9 peptides were added, 17 peptides were added per IgG. The product was obtained.

Example 6 Peptide sequence 99-113 derived from human alloantigen 1'lLA-A2.1 Coupling of monoclonal antibody Mab C215 (I) 1. In 0.1 M phosphate buffer pH 7.5 containing 6 mg of 0.9% sodium chloride. Dissolved tridecane (17-iotoacetylamino-3.6.9.12.15- Pentaoxaheptadecanoylamino) Immunoglobulin G2a (4mg, 25 ．． 6 nmole) was added to a 5 mg Reacti vial. This compound is It was prepared in the same manner as Compound C in Example 2A, except that the excess amount of Drug B was reduced.

Nitrogen gas was passed through the solution to remove air. HLA-^2.1 peptide, MetT yrGlyCysAspValGlySerAspArgPheLueArg- GlyTyr (4.7 mv, 2.1 a mole) in 0.2 mg acetonite 0.1M phosphoric acid containing 0.9% sodium chloride in Q, l++jl' Salt buffer pH 7.5 was added to dissolve. Drop this solution into the Reacti vial. I put it down. The pH of the reaction solution was checked to be 7.5. Seal the vial Further nitrogen was bubbled through the solution before the addition. Cover the vial with foil and leave the reaction solution overnight. Stirred. To block unreacted iodoalkyl groups, 10 μl of A solution of mercaptoethanol (10 μIc 1.4 μmole) was added. reaction solution was stirred for 4 hours, filtered, and then applied to a 5uperose 12HR10150 column. Top, 2allJ acid buffer pH 7 containing 0.9% sodium chloride as eluent; 5 was used for fractionation. Fractions containing the desired product (I) were pooled. Protein-rich The degree of conversion and degree of conversion were determined to be 196 μg/*l protein, respectively, by amino acid analysis. The quality and 7 peptides per IgG were determined.

sailyu 17-(3-(2-pyridyldithio)propionylamino]-3,6,9,12 , N-hydroxysuftonimide ester of 15-pentaoxaheptadecanoic acid Production of (K) A, 17-[3-(2-pyridyldithio)propionylamino] -Production of 3,6,9,12,15-pentaoxaheptadecanoic acid (J) 17-amino-3,6,9,12,15-pentaoxaheptadecanoic acid (66 , 0.2 mmole) was dissolved in 3.5 mg of IM borate buffer pH 8.4. . pFI decreased to 81. 9-(2-pyryls dissolved in 0.8 mg of dioxane) dildithio)-propionic acid succinimidyl ester (69 mg, 0.22 m mole) was added to the above solution. The pH of the reaction solution decreased to 7.6. The reaction is 1 It was completed in 0 minutes, and this was performed using thin layer chromatography (eluent: CH2CA'2- 11eOH60:35).

After 10 minutes, the pH of the reaction solution was adjusted to 4.5 with 5M hydrochloric acid, and the solution was lyophilized. . The reaction mixture was transferred to a reverse phase column PEP-RPCHR16/10 (Pharmac ia Biosystems AB) with a 0.17% acetonitrile gradient at 0 ．． Separation using 1% TFA followed by 17% acetonitrile and 0.1% TFA. Fractionation was performed by isocratic separation using F^. Fraction containing desired compound J The samples were pooled and lyophilized. Fractionation was repeated 13 times. Yield: 39 wings 9 The structure of product J was established with the aid of its NIIR spectrum.

B, 17-[3-(2-pyridyldithio)propionylamino]-3,6, 9,12,15-pentaoxaheptadecanoic acid N-hydroxysuccinimide Manufacture of Stell (K) Hydroxysuccinimide (1,815 mg, 16.3 μmole) was added to the reaction vial. It was weighed in alcohol. 17-[3-(2-pyridyldithio)-propionylamine -3,6,9,12,15-pentaoxaheptadecanoic acid (J) (6 ,0++g, 16.4μa+ole) dissolved in 0.55mm dry dioxane and added to the reaction vial. Nitrogen gas was passed through the solution. 200mA’ geoki 6.8 gg (32.8 μmole) of dicyclohexylcarbodiimide in Sun solution was added to the Reacti vial and further nitrogen gas was passed through the reaction solution. Afterwards, the vial was sealed. The reaction was allowed to wait for 24 hours, and the precipitate formed was filtered. Removed. NMR analysis of the filtrate shows that the reaction has almost completely progressed to compound K. It was revealed that.

gender Di(17-(3-(2-pyridyldithio)propionylamino)-3,6,9 , 12,15-pentaoxaheptadecanoylamino) immunoglobulin Gl (L ) Production of monoclonal antibodies of immunoglobulin class IgG l (Mab C2 42) (6g+9.88μ5ole) containing 0.9% sodium chloride 0, 1M borate buffer p[13,Q, dissolved in 2.23ml, 5mg Rea cti vial. Add this solution to 0.77 liters of the above buffer to a final concentration of 2.9 Diluted to protein/ml. 17-(3-(2-pyridyldithio)propionyl) Mino-3,6,9゜12,15-pentaoxaheptadecanoic acid N-hydroxys Dioxa of succinimide ester (K) (0,26 ole, 447 μ5 ole) N100μ! The medium solution was injected into the solution in the Reacti vial. 2 of the reaction solution After stirring for 5 minutes at room temperature, it was left in the refrigerator overnight. This reaction solution was added to 5u oerdex 76hr 10/80 column (Pharmacia Biosy stems AB), 0.1M phosphorus containing 0.9% sodium chloride as eluent. Fractionation was performed using an acid salt buffer pH 7.5. Pour the fraction containing the desired product (L). 7 proverbs), 1,5 in ^m1con cell through YII30 filter Concentrated to mg. The temperature and degree of substitution were determined to be 3.51, respectively, by amino acid analysis. It was determined to be 2 spacers per ml protein/ml and IgG.

Example 9 Cross-linking of two monoclonal antibody molecules to the product (N) A, di(17-[3-thi) opropionylamino] -3,6,9゜12,15-pentaoxabutadeca A, di(17-[3-thiopropionylamino)-3,6,9°12,15- Production of pentaoxaheptadecanoylamino)-immunoglobulin Gl (M) 0.1 M phosphate buffer containing 0.1 mg of 0.9% sodium chloride 1) 117. Di(17-[3-(2-pyridyldithio)propionylamino]- dissolved in 5 3,6,9,12,15-pentaoxaheptadecanoylamino) immunoglobulin Gl (L) (2,51 + 9.16 mmol, E11ena + an Added to Bu. The pH was adjusted to 4.7 with 1M acetic acid. Then 300 μl of 0. A solution of 9++ g dithiothreitol in 9% sodium chloride 100μ/( 2,319) was added. The reaction solution was left at room temperature for 25 minutes, then 5 epha dex g25 NAP 10 column (Pharmacia Biosyste ms AB), 0.9% sodium chloride and 2 mq EDTA as eluent Desalting was performed using 0, 1M phosphate buffer pH 7.5 containing . The product M is l , elute in a volume of 5 mg and immediately evaporate to avoid reoxidation of free mercapto groups. It was used for the synthesis of product (N).

B, (17-[3- thiopropionylamino]-3,6,9,12,15-pentaoxaheptadeca noylamino) - solution of immunoglobulin Gl (M) 0.7 genus! and birds (17 -Iotoacetylamino-3.6.9.12.15-pentaoxaheptadecano ylamino) immunoglobulin Gl (1.26 mg) was added to 5 mg of Reacti vial (the latter compound was added to a smaller excess of sample). Produced in the same manner as compound C using drug B and monoclonal antibody Mab C242. Ta).

Pass nitrogen gas through the reaction solution, then seal the vial and cover with foil to protect from light. and left at room temperature overnight. Water i++ to block unreacted iodoalkyl groups A solution of 20 μl of mercaptoethanol in 1 (2.5 μm 0.6 μa + ole) added. The reaction solution was left at room temperature for 6 hours, and then 5uperose 12H The solution was placed on an R10/30 column (Pharvacia Biosystems^B). 5mM phosphate buffer containing 0.9% sodium chloride, pH 7.5, was used as the draining solution. It was fractionated. Fractions containing dimeric product H were pooled.

Example 10 [17-(3-mercaptopropionylamino)-3,6,9°12,15- Production of pentaoxaheptadecanoylamino)-rSEA (P) A, 17-(3-(2-pyridylthio)propionylamino)-3,6,9 , 12,15-pentaoxaheptadecanoylamino)-rSEA (0) 17-[3-(2-pyridylthio)propionylamino]-3,6,9,12, 15-pentaoxaheptadecanoic acid N-hydroxysuccinimide ester (K ) (0,531+9.896 niole) in 48 μl of dioxane, ll1 in 0.1 M phosphate buffer pH 7.5 containing 0.9% sodium chloride.3. Injected into 67 mg (128 nmole) of rSEA solution. The reaction was carried out at room temperature for 30 minutes. It was completed.

For analysis of degree of substitution, 100μ! was collected. The remainder is reduced to product P. I stored it frozen.

The degree of substitution was determined by adding 100 μJ of the reaction solution to a 5 ephadex G50 NICK column ( Desalt with Pharmacia Biosystems AB) and transfer the eluate to Car lsson et al. (Biochea+, J, 175 (1976) 723-7 [37] was analyzed and measured by UV-spectroscopy. 2.7 speed on rSEA Pacer coupled.

B, [17-(3-mercaptopropionylamino)-3゜6, 9.12.15 -Production of pentaoxaheptadecanoylamino]-rSEACP> The pH of the reaction solution (0.9 m/) containing product O was adjusted to 4.4 with 2HC1, and 0. ．． Add 2.9 ng of dithiothreitol dissolved in 7.5 μl of 9% sodium chloride. I got it. The reduction was completed in 30 minutes.

The reaction solution was transferred to a 5ephadex G25 NAP 10 column (Pharmaci aBlosystem AB) plus 1.5++4 0.9% sodium chloride Product Q of Example 11 was immediately eluted with 0.1 M phosphate buffer pH 7.5 containing used for synthesis.

Interest Production of SE^-monoclonal antibody conjugate Q with double spacer Dodeca (17-Iotoacetylamino-3.6.9.12.15-Pentaoxa heptadecanoylamino) Mab C242 (C) (4,2 9.27rv ole, 10ml of 0.1M phosphate buffer containing 0.9% sodium chloride pH 7,5), (17-(3-mercaptopropionylamino)-3,6,9 ,12,15-pentaoxahebbutadecanoylamino)-rSEA(P) (1 , 17 mg, 42r+mole, in 1 ml of the above buffer) and nitrogen for 43 hours. Below, the reaction was carried out in the dark. Then, 1.14 μmole of mercaptoethanol was added. added. After another hour, the reaction solution was added to 5uperdex 200 HR16/ It was fractionated on 25 columns. The product was dissolved in 2mM phosphate containing 0.9% sodium chloride. Elution was performed with buffer pH 7.5. Fractions containing the desired product Q were pooled and Analyzed as described. The composition of the zygote is Mab C24 with two SE^ 2 is 9%, those with one SE^ are 25%, and unsubstituted Mab C242 is 66%. Met.

n=4.7.9.12.14.18 :l: --4,! Σ　z　O−〇 Experiment part, part 2 The bacterial toxin used in the following experiments was manufactured by Toxin Tech-nologie. (WI; USA) or as a recombinant protein from E. coli. This is staphylococcal enterotoxin A (SEA) produced by

Antibodies were C215, C242, and rhY-i, 2 strains^b. C215 is IgG2a Ab against human colon cancer cell lines and several human colon cell lines. Reacts with 34kD protein antigen. Literature for these mAbs is provided above. Contact The coalescence was prepared as described in Part 1.

Before the priority date, only the Eu”-tagged SE^-C215■^b conjugate was studied. I was disappointed. In the year of priority, the results are unlabeled SEA-C215, SEA-C242 and and SEA-Thy-1,2vr^b conjugate. This time it was introduced The results presented pertain to nonzygotes.

The result is a lack of MIIC class ■ or MHC class ■, which is expressed only in small, undetectable amounts. 5EA-C215m^b conjugate and unconjugated SE^ and C against intestinal cancer cells For the measurement of cytotoxic effects mediated by 215 mAb, effector cells T cell lines enriched with various human SEA were used as target cells in colon cancer cells and M. HC class II" Raji cell panel was adopted.

Colon cancer cell lines, Co1o205.5W620 and WiDr are ■L^-Dllll .

Staining with Oboro Abs for HLA-DP and HLA-DQ and FAC3 analysis All lacked expression of the operculum ■C class ■, as revealed by. SEA increase Strong T cell lines were precoated with SEA in the presence of recombinant IL-2 (20 units/ml). cells treated with mitomycin C-treated MFIC class II”BSMl nonpoma cells. was established from peripheral blood by weekly restimulation. These T cell lines are SEA Although it showed a strong cytotoxic effect on coated Raji or BSM cells, , uncoated cells, or staphylococcal enterotoxin B (SEB) showed no toxicity to cells coated with induced by this SEA Killing was achieved by blocking HLA-DR antibodies, MHC class II-Raji mutant cells and H LA-DR) transfected and labeled as SEA, as evidenced by the use of single cells. (Dohlsten et al., Im muno-1ogy 71 (1990) 96-100), these T cell lines , activated by the C2l5-SE^ conjugate, C215”MIIC class ■ - Killed colon cancer cells. On the contrary, unconjugated SEA and C215a+Ab C215”MtlC class - Very few T cells kill colon cancer cells. or could not be induced. Staphylococcal enterotoxin antibody conjugate-dependent cellular mediation Cytotoxicity is due to binding of SEA-C215-^b conjugate to C215+ tumor cells. depended on. The specificity of this binding is such that excess unconjugated C215 mAb causes lysis of colon cancer cells. This effect was absent in the unrelated C242 and v6/32 mAbs. It was clear from the facts. CD4+ and CD8'' T cells are SEA-C2 15 showed a killing effect on ff1c215+ colon cancer cells, but SEA-treated cells showed no lysis. I didn't understand. MHC class ■-SEA-C215 mAb bound to tumor cells The interaction between the zygote and T cells involves SE^-induced killing of IIHC class ■4 cells. specific VβTCR sequences are involved, as previously shown for It seems like that.

This is not an autologous SEA-specific T cell line with C215-SEA conjugate. , directed by SE^-specific T cell interactions. C242iAb and T The hy-1,2 mAb conjugate exhibits similar activity to the 0215 mAb conjugate.

Chromium labeling and incubation of target cells with SEA 0.75 x 106 target cells and ■ 50 μCi of S + chromium (^mers Has　Corp,,ArliArlln　Height,England) , in a volume of 100 μl, and incubated for 45 minutes at 37°C. The cells are 2.8% (v/v) 7.5% NaHCO3, 1% Sodium Pyruvate, 2%2 00IIML-Glutamine, 1% IMHepes, 1% 10 mq/ ml Gentamicin and 1O% fetal calf serum (FCS, Gibco, RPMI-1640 medium (Gibco , Palsley, GER). incubation After washing, cells were washed once with complete medium without FCS and incubated for 60 min at 37°C. Incubate, wash and resuspend in complete medium containing 10% FC3. U-shaped bottom 96 Well microtiter plates (Costar, Camb-ridge, US 5X103 target cells were added to each well in A).

Cytotoxicity assay Effector cells were added to the wells at various effector/target cell ratios. each The final volume of the wells was 2ooI11. Each test was performed three times. 37 plates After 4 hours of incubation at 0.degree. C., the released chromium was harvested. fit( The amount of r is measured using a gamma counter (Cobra Auto-gamma).

Packard). Cytotoxicity percentage, formula % formula %) It was calculated by where X is the chromium release (cpl) obtained for the test sample l), where M is the spontaneous release of chromium from target cells incubated with the medium. Yes, T is obtained by incubating target cells with 1% sodium dodecyl sulfate. This is a total release of romium.

result SEA-C242, SEA-C215 and 5EA-anti-Thy-1,2mA b zygotes are cells expressing the relevant epitope of that mAb, and MI IC class ■ “binds to cells. On the other hand, unconjugated SEA binds to MHC class ■” cells. Only combined. Unconjugated C215, C242 and Thy-1,2 mAbs It binds to continuous cells but not to Raji cells (Table 1).

The human T cell line is MITC class n in the presence of SEA-C215a+Ab conjugate. -3W620, Co1o205 and WiDr cell lysis Shitaka, non-conjugated SEA and remained soluble in the presence of C215 mAb (Figure 1). Colon cancer cell lysis was observed in the SEA-C215 mAb conjugate between 10 and 1100 n/ml. . For 5W620, the SEA-C215 mAb conjugate has various effector A high level of lysis was observed in the cell to target cell ratio (Figure 1). On the contrary, non-bonded SEA or C215 mAb was tested for all effector cells versus target cells. 31620 was shown to be cytotoxic (not shown). Class II - The lytic ability of Colo205 cells is limited to zygotes, non-zygotic SEA and cannot be induced by C215 mAb. SEA and SEA-C 215 mAb conjugates are MHC class II” Raji cells and Treatment with MIIC class II “Induces killing of Co1o205 cells by T cells.” However, C215 mAb did not induce this (Figure 1).

The lytic effects induced by the 5EA-C215 mAb conjugate involve C215 on target cells. To determine whether specific binding of the conjugate to the mAb molecule is involved , a blocking test was performed with excess unbound C215a+Ab and C242mAb. Ta. In this case, C242 mAb is independent of the 0215 Ab-associated antigen on colon cancer cells. Binds to relevant antigens. Although addition of C215 mAb strongly blocked cytotoxicity, , C242 mAb had no effect. Similarly, 5EA-C242mAb lysis was specifically blocked by excess unconjugated C242 mAb, but C215 Not blocked by mAb.

SEA-C215■^b conjugate, Rei HC class ll5W620 colon cancer cells The ability to induce T cell-dependent lysis was determined in both CD4 and CD8 T cell populations. (Table 2). SEA inhibits both of these T cell subsets. Although it does not activate the 1Itlc class to mediate killing of W620 cells, The lysis of Raji cells was induced (Table 2).

The SEA-C215iAb conjugate was expressed by the SEA-enhanced T cell line as 5W620 and Raji cells induced lysis, but not some SEB-enhanced T cell lines. There was no (Figure 3). Specificity of SEA and SEB systems exposed to Raji cells shown by their selective responses to SEA and SEB, respectively, when It will be done. This indicates that the SEA-C215 mAb conjugate has the same VβT as unconjugated SEA. This shows that CR specificity is maintained.

Illustration description Figure 1: 5EA-C215 mAb conjugate is effective against MHC class ■- colon cancer cells. to direct the CTL. Upper left panel shows SEA-C215 mAb conjugate, S Absence of EA, C215 and mixture of C215 and SEA (C215+5EA) Residence or IIIg/WII! In the presence of various effector cell targets at concentrations Figure 2 shows the effect of SE^-responsive CTLs on 5W620 cells at a specific cell ratio. other pa Nell is C215+1ItlC class - colon cancer cell line 5W620, Co1o20 5 and fiDr%MHC Class II’C215” Interferon-treated Co 1o205 cells and C215-11tlC class ■''''Raji cells 5EA-C215 mAb conjugate targeting SEA-responsive CTL, and SE Indicates A's ability. The effector to target cell ratio was 30:1. Several concentrations Addition of unconjugated C215 mAb at It didn't guide me at all. For HLA-DR, HLA-DP and HLA-DQ FA of 5W620 cells, Co1o205 and WiDr cells using mAb In C3 analysis, no surface MHC class ■ expression could be detected, but anal^- Abundant expression of DR, -DP and -DQ in Raji cells Expression of DP was detected in interferon-treated Co1o205 cells. Co1o 205 cells were incubated at 1000 units/ml for 48 hours before use in the CTL assay. Treated with recombinant interferon-γ.

Figure 2: SE^-C215IIAb conjugate and SEA-C242mAb conjugate. The targeting of CTLs to colon cancer cells thus induced is dependent on the antigen selectivity of the mAb. Dependent. SEA-C215-^b conjugate and 5EA-C242mAb conjugate of Co1o205 cells by SEA-responsive CTL in the presence of (3 pg/111) Lysis was performed using unconjugated C215 and C242 mAb conjugates (30 pg/m J) is blocked by the addition of J). Unconjugated mAb or control medium (-) represents conjugate 10 min before addition to target cells.

Figure 3: Lysis of colon cancer cells coated with SE^-C215 mAb conjugate Mediated by A response CTL, but not SEB response CTL . Autologous SE^ and SEB selective T cell lines are 5EA-C215 mAb conjugates, A mixture of unconjugated C215 mAb and SEA (C215+SE^) and unconjugated In the absence of a mixture of C215 mAb and SEB (C215+5EB) (control), or or at a 10:1 effector to target cell ratio in the presence of 1xg/IIl concentration. , 5W620 and Raji target cells.

Figure 4: SE^-C242 mAb conjugate and 5EA-anti-Thy-1,2 mAb The zygotes target cells (Co1o205 tumor cells and EL, respectively) -4 tumor cells) induced cytotoxicity.

Table 1 SEA-C215mAb Co1o205 Po5RajiPos C215mAb Co1o205 Po5Raji Neg SEA-C242mAb Co1o205 Po5RajiPos C242mAb Co1o205 Po5Raji Neg SEA-anti-Thy-1,2a+Ab EL~4 Pos anti-Thy-1,2I IAb　EL-4Po5SEA　Co1o205　Neg Raji Pos Control Co1o205 Neg Raji Neg EL-4Neg Cells were incubated on ice for 30 min with various additives of control (PBS-BSA). washed, processed as described below. Binds to Co1o205 cells C215 and C242 mAbs and anti-Thy-1 bound to EL-4 , 2 staining was detected using FITC-labeled rabbit anti-mouse 1g. I? aji Cells were stained with SEA using rabbit anti-3EA serum, followed by FITC-bloat serum. It was detected by anti-rabbit 1g. For Co1o205 and Raji cells Staining of the SEA-C215 mAb conjugate was performed for C215 mAb and SEA. and detected using the procedure described above. EACS analysis by Becton & Dicki Performed on a FACS Star Plus from Nson. 2nd and 3rd step dyeing was used only to clarify the background.

”Honour! ! 4. Soviet Union---- CD4” 5W620 2 5 50 CD4” Raji O4143 CD8” 5W620 0 1 23 CD8+I? aji 2 72 68 A) CTL (SEA-3) c. With an effector to target ratio of 30:1, SEA and used in the absence of C215-SEA (control) or in the presence of 1 g/mA' did.

Effector/target ratio SEA/C215-5; EA (ng/m1) 10' 10' 10' 10' 1o27(7310' 105SEA/CC215-5EA(n/m/)SE A/C: 215-SEA (ng/m1) SEA/CC215-5EA (rr/c =l/specific cytotoxicity (%) international search report m+*+vn++ee*Illemk*l+aelle, PCT/SE 91 100497l11. Yumma+ & Eng, -+I-w+-PCT/SE 911 004972. ClaLm119-17: polyeeehers ftnt erme+natss Ln the production north @ btr uncttonai coupHng reagents+.

international search report PCT/SE　91100497 Continuation of front page (51) Int, C1, S Identification symbol Internal office reference number A61K 39/39 5 C9284-4CL 9284~4C Y9284~4C 47/48 Z 7433-4C 49100A 9164-4C C07C205/14 7188-4H2171087457-4H 2351067106~4H 271/16 7188-4H C07D 209/48 243/32 7167-4C CO7K 17106 8517-4HGOIN 331532 A 8310 -2J331547 9015-2J (81) Designation diagram EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, NL, SE), AU , CA, JP, US I (72) Inventor Åkerblom, Eva Sweden Varnish-75252U Psara.

Targuxveien 15

Claims (1)

[Claims] 1. A and A' of the conjugate material (A-B-A') are organic compounds F and A residue from F', at least one of which is a polymer (carrier) and these properties of the compound are maintained in the conjugate, -B- covalently bonds A and A' In the joined body that is a bridge, the bridge -B- has a structure -SrRCONHCH2CH2(OCH2CH2)nO(CH2)mCOY-( I) [In the formula, (i) n is an integer, for example 1 to 20, preferably >2, and the bridge n is uniform in order to connect the same positions in the individual molecules of the coalescent substance), (ii) m is 1 or 2; (iii) R has 1 to 4 carbon atoms, preferably <2 carbon atoms and no more than 1 or substituted with two or more (1 to 3, preferably <2) hydroxy (OH) groups. is an alkylene group which may be (iv) Sr is bonded to the saturated carbon atom in both directions, r is 1 or 2; (v) Y is -NH-, -NHNH- or -NHN=CH-, and its left end is bonded to CO, and at its right end is a saturated carbon atom or carbonyl group (-NHNH-). A zygote characterized by the following: 2. Claim 1, wherein the polymer has a polypeptide or polysaccharide structure. The zygote described. 3. Claims characterized in that the polymer is an antibody or an antibody active fragment thereof. The zygote according to any one of claims 1 to 2. 4. 4. The method according to claim 1, wherein the polymer is soluble in an aqueous medium. The zygote described in any of the above. 5. Entities of A and A' that are not antibodies or antibody-active fragments thereof are analytically 4. The conjugate according to claim 3, wherein the conjugate is a group that can be detected. 6. The carrier polymer is an antibody or an antibody active fragment thereof; The entities A and A' that are not, for example, are characterized as immunostimulants of bacterial origin. The zygote according to any one of claims 1 to 5. Type 7 -Z1RCONHCH2CH2(OCH2CH2)nO(CH2)mZ1'(I II) [In the formula, (i) n is an integer, for example 1 to 20, preferably >2; (ii) m is 1 or 2, preferably 1, (iii) Z1 is SH-reactive electrophilic groups or thiols (SH-) or protected thiols, e.g. acylation a thiol such as AcS-; (iv) R is one or more (1 to 3, preferably <2) hydroxy ( an alkylene group (1 to 4 carbon atoms, preferably has <2 carbon atoms), and (v) a bifunctional coupling reagent represented by Z1' is activated carboxy . 8. Z1' is a reactive disulfide in which the - side sulfur atom is bonded to R: 2,5-dioxo-1-aza-cyclopent-3-en-1-yl; and and halo, preferably bromo or iodo. Item 7. The bifunctional coupling reagent according to item 7. 9. formula XCH2CH2(OCH2CH2-)nOCH2Y(IV) [wherein, n is an integer from 2 to 20, preferably from 3 to 20, and X is H2N- or preferably is a substituted H2N- which can be converted to H2N- by hydrolysis or reduction, for example ( a) Nitro; amide (=carboxamide) e.g. lower saturated acylamide; phthalate imidoyl; (b) carbamate; the substituted carbon atom is α to the aromatic system; lower alkylamino; and (d) 4-oxo-1,3,5-triazine-1 -Il, Y is preferably carboxy (-COOH or -COO-) or carboxy is a group convertible by hydrolysis or oxidation, for example (a) carbonyl carbon an atom or a corresponding atom is bonded to the methylene at the right end of formula (I) Ter group, or (b) -CHO, (c) -CN, -CONH2, -CONR1' R2' (wherein R1' and R2' are lower alkyl, especially secondary and tertiary alkyl) Alkyl group, methyl substituted with 1 to 3 phenyl groups, the ring of which may be substituted A polyether characterized by the following: 10. Polyether according to claim 9, characterized in that X is NH2-. 11. X is an acylamid, e.g. CH3CONH-, and an electron at the α carbon atom. CH3CONH- with an attractive substituent (e.g. CF3CONH- or CH3CONH- COCH9CONH-) and formylamide (HCONH-) The polyether according to claim 9, characterized in that: 12. X is phthalimidoyl or carbamate, e.g. R1'OCONH- and (R1'OCO)(R2'OCO)N- (wherein R1' and R2' are Lower alkyl groups, especially secondary and tertiary alkyl groups, or rings are substituted methyl substituted with 1 to 3 phenyl groups) The polyether according to claim 9. 13. 13. The method according to claim 9 or 12, wherein X is Boc, Z or DiZ. The polyether described in any of the above. 14. X is an alkyl amine in which the carbon atom replacing the nitrogen atom is α with respect to the aromatic system. The polymer according to claim 9, characterized in that it is, for example, N-monobenzylamino. Riether. 15. Claims 9 to 14, wherein Y is a carboxy group. polyether. 16. Y is a carbonyl carbon atom or its corresponding atom of formula (IV) The point according to claim 9, characterized in that it is an ester group bonded to the right-most methylene. Riether. 17. Y is an alkyl ester group (-COOR1'): an orthoester group [-C (OR3')3] or a reactive ester group, such as N-succinimide-1- yloxycarbonyl, 4-nitrophenyloxycarbonyl and 2,4-di Nitrophenyloxycarbonyl (wherein R1' and R2' are lower alkyl groups, especially secondary and tertiary alkyl groups, and rings optionally substituted 1- Claim 9 characterized in that it is methyl substituted with three phenyl groups). or the polyether described in 16.