JPH07126186A - Chemically defined non-polymeric bonded hand platform molecule and its composite body - Google Patents

Abstract

PURPOSE: To obtain a composite material containing a non-polymeric bonded platform molecule and useful for the treatment of autoimmune diseases such as systemic lupus erythematosus. CONSTITUTION: This composite material contains a non-polymeric bonded platform molecule of formula I or formula II [G<(1)> and G<(2)> are each C, N, O, Si, P or S; the n<(1)> parts shown as T<(1)> and the P<(2)> ×n<(2)> parts shown as T<(2)> are each amine, hydrazide, carboxylic acid, etc.; the n<(2)> parts shown as L<(2)> are each O, S, etc.; the n<(2)> parts shown as J<(2)> are each C(=O) or C(=S); n<(1)> is 1-32; n<(2)> is 1-32; P<(2)> is 1-8; the n<(2)> parts shown as Z<(2)> are each groups containing 1-200 atoms]. The molecule forms the subject composite material by coupling with a biological or chemical molecule such as a polynucleotide. The biological or chemical molecule is carbohydrate, drug, lipid, lipopolysaccharide, peptide, protein, oligonucleotide, hepten, mimotope, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an organism such as a polynucleotide used for treating diseases such as systemic lupus erythematosus (SLE, which is simply referred to herein as "autoimmune disease"). A complex comprising a chemically defined non-polymeric valency platform molecule coupled to a biological or chemical molecule. The present invention also relates to the chemically defined non-polymeric valency platform molecule itself.

[0002]

BACKGROUND OF THE INVENTION Many compounds have been used as carriers of biologically useful molecules in the preparation of conjugates that are said to be immunotolerant. For example, Benacerraf, Katz and their co-workers were called " D- GL" in earlier work,
D is referred to herein as "D -EK" - glutamic acid / D - using a random copolymer of lysine, a complex of the hapten and various antigens was studied and presented a specific immune tolerance induced ing. See U.S. Pat. No. 4,191,668, and U.S. Pat. No. 4,220,565.

Others have found that nucleosides or DNA
, And studying complexes with other carriers. Borel et al ( Scie
nce (1973) 182 : 76) evaluated the ability of syngeneic mouse IgG-nucleoside complexes to reduce the antibody response to denatured DNA in young mice of the NZB strain. In another independent study, Parker et al. ( J. Immunol. (1974) 113 : 292).
Evaluated the effect of denatured DNA complexed with poly- D -lysine and / or cyclophosphamide on the progression of the above symptoms in NZB mice.

In a later report, Borel et al . ( Ann. NY Acad. Sci.
(1986) 475 : 296-306) describe an oligonucleotide-immunoglobulin complex. Borel et al. ( J. Cli
n. Invest. (1988) 82 : 1901-1907, and U.S. Pat.
650,675) describe an in vitro study with a complex of human immunoglobulins bound to DNA. Dint
US Pat. No. 5,126,131 to zis et al. also relates to complexes comprising a carrier and a molecule involved in the immune response.

Other references include non-immunogenic polymers,
A complex with an immunogen is described. (Saiki et al . , Scand.
J. Immun. (1982) 16 : 191-200; Sehon, Prog. Allerg
y (1982) 32 : 161-202; Wilkinson et al . , J. Immunol. (1
987) 139: 326-331; Borel et al . , J. Immunol. Methods.
(1990) 126 : 159-168. ) The inventors of the present invention, U.S. Patent Application No. 07 / 914,869, U.S. Patent No. 5,162,515, and U.S. Patent Application No. 07 / 652,658 include D- EK, polyethylene glycol, poly-D-lysine, polyvinyl. A complex consisting of a polymeric carrier such as alcohol, polyvinylpyrrolidone, etc. and an immunoglobulin is described.

[0006] To summarize the above-mentioned prior art, it should be pointed out that compounds of undefined definition or compounds having many non-specific binding sites are only used as a valency platform molecule of a complex. The inventors consider.
Since the bonds, position of binding sites, and number of binding sites of such compounds are unpredictable and vary widely, prior art conjugates of such compounds are reproducibly prepared. No, and its reported activity shows wide variation.

[0007]

SUMMARY OF THE INVENTION In contrast to the prior art described above, the inventors have developed a complex containing a chemically defined non-polymeric valency platform molecule. The valency of the platform molecule is predetermined and each binding site can be used for the binding of biological or chemical molecules. The bond platform molecules of the present invention have defined chemical groups, valences, and homogeneities, and have defined functional groups for efficient binding to appropriate biological and / or chemical molecules. There is.

Accordingly, the present invention in one aspect relates to a chemically defined non-polymeric valency platform molecule and a complex comprising a biological and / or chemical molecule. Examples of biological and / or chemical molecules suitable for attachment to chemically defined non-polymeric valency platform molecules to form the conjugates of the invention include carbohydrates, drugs, lipids, Examples include lipopolysaccharides, peptides, proteins, glycoproteins, single-stranded or double-stranded oligonucleotides and their chemical analogues, immunogenic analogues, haptens, mimotopes, aptamers and the like. Chemically defined non-polymeric valency platform molecules suitable for use in the present invention include, but are not limited to, the following biocompatible, non-antigenic carbon-based compounds of the formula: And derivatives thereof are included.

[0009]

[Chemical 8]

Alternatively,

[0011]

[Chemical 9]

However, when present, each of G ^[1] and G ^[2] is independently ¹ to 2000, more preferably selected from the group consisting of C, N, O, Si, P and S. It is a straight chain, branched chain or multi-branched chain containing 1-1000 chain atoms.

More preferably, if present, G
^[2] is a group derived from polyalcohol, polyamine or polyglycol, most preferably G ^[2]
Is-(CH ₂ ) _q- with q 0 to 20 and -CH ₂ (CH ₂ OCH with r 0 to 300
₂ ) _r CH _2- , and C (CH ₂ 0CH ₂ CH _2- ) _s (OH) ₄ -s where s is 1 to 4, more preferably s is 3 to 4.

The n ^[1] parts shown as T ^[1] and the p ^[2] × n ^[2] parts shown as T ^[2] are each independently -NHR ^SUB (Amine), -C (= O) NHNHR ^SUB (hydrazide), -NHNHR ^SUB (hydrazine), -C (= O) OH (carboxylic acid),
-C (= O) OR ^ESTER (active ester), -C (= O) OC (= O) R ^B (anhydride), -C (= O) X (acid halide), -S (= O) ₂ X (sulfonyl halide), -C (= NR ^SUB ) OR ^SUB (imidate ester), -NCO
(Isocyanate), -NCS (isothiocyanate), -OC (=
O) X (haloformate), -C (= O) OC (= NR ^SUB ) NHR ^SUB (carbodiimide adduct), -C (= O) H (aldehyde), -C (= O) R ^B (ketone), -SH (sulfhydryl or thiol), -OH
(Alcohol), -C (= O) CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂ OR ^ALK X (alkylsulfonate), R ¹ R ² is -C ( = O) CH = CHC (= O)-is -NR ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl), -R
^ALK -Hg-X (alkyl mercury), and -S (= O) CR ^B = CR ^B ₂ (α, β
-Unsaturated sulfone).

More preferably, n represented as T ^[1]
[1] number of partial and, p, shown as ^{T [2] [2] ×} n [2]
Independently, each of the moieties is -NHR ^SUB (amine), -C (= O) CH ₂
X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂
OR ^ALK X (alkyl sulfonate), R ¹ R ² is -C (= O) CH = CHC (=
O)-is -NR ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α, β-
Unsaturated carbonyl), -R ^ALK -Hg-X (alkylmercury), and -S (= O) CR ^B = CR ^B ₂ (α, β-unsaturated sulfone).

Even more preferably, the n ^[1] parts denoted as T ^[1] and p ^[2] xn denoted as T ^[2].
Each ^[2] part is independently -NHR ^SUB (amine), -C (= O)
CH ₂ X (haloacetyl), R ¹ R ² is -C (= O) CH = CHC (= O)-
NR ¹ R ² (maleimide) and —C (═O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl).

[0017] Most preferably, n, shown as T ^{[1] [1]}
All of these parts and p ^[2] × n denoted as T ^{[2]
All of the [2]} parts are the same.

In the formula, each X is independently halogen (Cl = 17, Br = 35, I = 53) with an atomic number greater than 16 and less than 54, or another good leaving group (ie Alkyl or alkyl-substituted sulfonates or sulphates, etc., aryl or aryl-substituted sulphonates or sulphates, etc., which behave similarly to halogens in some circumstances.

Each R ^ALK is independently a linear, branched, or cyclic (1-20C) alkyl group.

Each R ^SUB is independently H, straight chain, branched or cyclic (1-20C) alkyl, (6-20C) aryl, or (7-30C) alkylaryl.

Each R ^ESTER is independently N-succinimidyl, p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl, cyanomethyl, etc. Alternatively, it is another activating group such as 5-chloro-8-quinolone-1-yl, 1-piperidyl, N-benzotriazolyl and the like.

Each R ^B is independently a group containing 1-50 atoms selected from the group consisting of C, H, N, O, Si, P and S.

When present, each of the n ^[2] moieties designated L ^[2] is independently selected from the group consisting of O, NR ^SUB and S.

When present, each of the n ^[2] moieties designated J ^[2] is independently selected from the group consisting of C (= O) and C (= S).

N ^[1] is 1 to 32, more preferably n
^[1] is 2 to 16, more preferably n ^[1] is 2 to 8 and most preferably n ^[1] is 2 to 4.

N ^[2] is from 1 to 32, more preferably n ^[2] is from 1 to 16, provided that the product of n ^[2] × p ^[2] is greater than 1 and less than 33. , Even more preferably n ^[2] is 1 to 8, even more preferably n ^[2] is 1 to 4 and most preferably n ^[2] is 1 to 2; p ^[2] is , 1 to 8, more preferably p ^[2] is 1 to 4, and most preferably p ^[2] is 1 to 2.

Each of the n ^[2] moieties represented by Z ^[2] independently has a bonding site for at least p ^[2] functional groups as an alkyl carbon atom, an alkenyl carbon atom, or an aromatic carbon atom. A group containing 1 to 200 atoms selected from the group consisting of C, H, N, O, Si, P and S contained on the atom.

[0028] More preferably, n, shown as Z ^{[2] [2]}
All of the parts are the same.

More preferably, n represented as Z ^[2]
Each of the ^[2] parts is independently described by an expression selected from the following group.

[0030]

[Chemical 10]

[0031]

[Chemical 11]

Alternatively,

[0033]

[Chemical 12]

However, in the formula, the n ^[2] portions shown as W ^[3] , W ^[4] , or W ^[5] , if present, are each independently C, H, N, O. , Si, P and S are groups containing 1-100 atoms selected from the group consisting of:

N ^[2] parts shown as Y ^[3] , 2 × n ^[2] parts shown as Y ^[4] , and 2 × n ^[2] parts shown as Y ^{[5] . 2]} parts are each independently at least p ^[2]
Functional groups (for Y ^[3] ) or p ^[2] / 2 functional groups
(In the case of Y ^[4] and Y ^[5] , where p ^{[2 ]} / 2 is an integer) containing a binding site on an alkyl carbon atom, an alkenyl carbon atom, or an aromatic carbon atom, C, H, N, O, S
A group containing 1-100 atoms selected from the group consisting of i, P and S.

More preferably, when present, each of the n ^[2] moieties designated as W ^[3] is independently (CH ₂ ) _r , (CH ₂ CH ₂ O) _r , NR ^SUB (CH ₂ CH ₂ O) _r CH ₂ CH ₂ ,
And NR ^SUB (CH ₂ ) _r NR ^SUB C (= O).

More preferably, each of the n ^[2] moieties represented by Y ^[3] is independently a straight chain, branched chain or cyclic (1
-20C) alkyl, (6-20C) aryl, or (7-30
It is an alkylaryl of C).

Most preferably, each of the n ^[2] moieties represented by Y ^[3] is independently C ₆ H ₄ (1,4-disubstituted phenyl), C ₆ H ₃
(1,3,5, -trisubstituted phenyl), and r is 1 to 10 (CH
₂ ) selected from the group consisting of _r .

More preferably, when present, each of the n ^[2] moieties designated as W ^[4] is independently r is from 1 to 10, (CH ₂ ) _r C (= O) , And (CH ₂ ) _r NR ^SUB C (= O).

More preferably, Y^[Four]Shown as 2 × n
^[2]Each part independently, r is 1 to 10, more preferred
Where r is 2 to 6 and q is 1 to 10, more preferably q is 1
3 and so on, (CH₂)_r, (CH₂)_rNR^SUBC (= O) (CH₂)_q, (CH₂)_rC
(= O) NR^SUB(CH₂)_q, (CH₂)_rNR^{SU B}C (= O) (CH₂)_qNR^SUBC (= O)
(CH₂)_r, (CH₂)_rC (= O) NR^SUB(CH₂)_qNR^SUBC (= O) (CH₂)_r, (C
H₂)_rNR^SUBC (= O) (CH₂CH₂O)_qCH₂CH₂,, and (CH₂)_rC (=
O) NR^SUB(CH₂CH₂O)_qCH₂CH₂Is selected from the group consisting of

More preferably, when present, each of the n ^[2] moieties represented by W ^[5] is independently r is from 1 to 10, (CH ₂ ) _r C (= O) NR ^SUB and (CH ₂ ) _r NR ^SUB C (= O) NR ^SUB
Is selected from the group consisting of

More preferably, 2 × n ^[2] represented by Y ^{[5 ]}
Groups of (CH ₂ ) _r , and (CH ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q , where r is independently 1 to 10 and q is 1 to 10. Selected from.

In another preferred embodiment for treating lupus, the conjugate is a chemically defined non-polymeric valency platform molecule, and each is bound to said platform molecule, and human SLE Of a plurality of polynucleotide duplexes of at least about 20 base pairs having significant binding activity to anti-dsDNA autoantibodies. In these preferred embodiments, the polynucleotide duplex is substantially uniform in length, and one strand of the duplex is either directly or via a linker molecule. Bonded to the platform molecule.
Generally, the synthetic polynucleotide is attached to a linker molecule and then to a bond platform molecule.
Generally, the chain containing the linker in the duplex is either at its one end or at one end so that a pendant chain of at least about 20 base pairs is formed in each chain from the position where the linker molecule is attached in the chain. Bond in the vicinity (ie, within about 5 base pairs). The second strand is then annealed to the first strand to form a duplex. Thus, the conjugates of the invention may be generally described by the formula: [(PN) _n -linker] _m -bond platform molecule, where n is at least about 20 and m is 2-8 And PN is n
It is a double-stranded polynucleotide having 40 nucleotides.

An example of a linker molecule suitable for the present invention is thiol
6-carbon thiols, such as HAD, which is a -6 carbon chain phosphate, and HA, which is a thio-6 carbon chain phosphorothioate
It is D _p S. The chemically defined valency platform molecules of the present invention include, for example, amino-modified PEG and 3,5
-Bis- (iodoacetamido) benzoyl chloride (hereinafter referred to as "IA-DABA chloride"); 3-carboxypropionamide-N, N-bis-[(6'-N'-carbobenzyloxyaminohexyl) ) Acetamide] 4 "-nitrophenyl ester (hereinafter referred to as" BAHA "); 3-
Carboxypropionamide-N, N-bis-[(8'-N'-carbobenzyloxyamino-3 ', 6',-dioxaoctyl) acetamide] 4 "-nitrophenyl ester (hereinafter referred to as" BAHA _{O X} "); or PEG-bis-chloroformate and N, N-di [2- (6'-N'-carbobenzyloxyaminohexanoamido) ethyl] amine (main Hereinafter referred to as "AHAB").

The double-stranded nucleotide composition of the complex of the invention may be substantially uniform. The duplex length of the complex of the present invention may be 20 to 50 base pairs. The duplex of the complex of the present invention may bind to a valency platform molecule at or near its end. The polynucleotide duplex of the complex of the invention may be composed of 2 to 4 mer complementary repeat units of different bases. The polynucleotide duplex of the complex of the invention is: poly (CG): poly (C
G); poly (TA): poly (TA); poly (CI): poly (CI); poly (C
A): poly (TG); or poly (GA): poly (TC);

In the complex of the invention, the polynucleotide duplex may be (CA) ₁₀ : (TG) ₁₀ and the valency platform molecule may be derivatized triethylene glycol. In this complex, the molar ratio of the double strand and the bond platform molecule is preferably 2: 1 to 8: 1, and more preferably 4 :.
Is 1.

In the complex of the invention, the polynucleotide duplex may be (CA) ₁₀ : (TG) ₁₀ and the valency platform molecule may be a derivatized 1,2-bis- (2 '-
It may be aminoethoxy) ethane. In this complex, the molar ratio of the above double-stranded chain and 1,2-bis- (2′-aminoethoxy) ethane is preferably 2: 1 to 8: 1, and more preferably 4: Is 1.

The complex of the present invention may be a tolerogen of human SLE (systemic lupus erythematosus).

The polynucleotide duplex of the complex of the invention may have a B-DNA type helical structure, and the human SLE
It may have significant binding activity to anti-dsDNA autoantibodies (systemic lupus erythematosus).

According to the invention, a pharmaceutical composition for the treatment of lupus may comprise a complex of the invention formulated with a pharmaceutically acceptable injectable excipient. A method of treating an individual suffering from lupus may include the step of administering to the individual in need thereof a therapeutically effective amount of this composition.

Methods for making the composites of the present invention include:
(a) binding a plurality of single stranded polynucleotides, each of at least about 20 base pairs, onto the valency platform molecule; and (b) attaching a complementary single stranded polynucleotide to the valency platform molecule. Annealing the bound single stranded polynucleotide to form the double stranded. The method may further comprise first attaching the polynucleotide duplex to a linker molecule and then attaching the linker molecule to the bond platform molecule.

The biological or chemical molecule of the complex of the invention may be an analog of the immunogen, wherein (a) this analog is specific for the B cell to which the immunogen specifically binds. And (b) the complex may lack T cell epitopes. Where the immunogen is
Rh associated with biological agents, allergens, Rh hemolytic disease
/ D immunogen, α-sperm associated with male infertility, or
It may be a foreign immunogen, such as the carbohydrate complex associated with rheumatic fever. Alternatively, the immunogen can be an autoimmunogen such as those associated with thyroiditis, diabetes, stroke, or myasthenia gravis. The immunogen and the analog can be the same chemical species, eg, a polypeptide.
Alternatively, the immunogen and the analog can be different chemical species.

According to the invention, a pharmaceutical composition for the treatment of antibody-mediated diseases comprises a therapeutically effective amount of a conjugate of the invention in combination with a pharmaceutically acceptable carrier. obtain.

According to the invention, specific for the immunogen
The method of inducing B cell anergy in an individual may include the step of administering to the individual an effective amount of a complex of the invention, eg, in the form of a pharmaceutical composition described above.

According to the invention, a method of treating an individual having an antibody-mediated disease in which an unwanted antibody is produced in response to an immunogen comprises administering a therapeutically effective amount of a conjugate of the invention, eg It may include the step of administering to an individual in the form of a pharmaceutical composition as described above.

Methods for producing the conjugates of the invention include: (a) covalently conjugating an analog of the above immunogen lacking a T cell epitope to the chemically defined valency platform molecule and conjugating Forming a body; and (b)
Recovering the complex from the reaction mixture. Here, the immunogen may be a biological agent, an allergen, α-sperm associated with male infertility, or a foreign immunogen such as Rh / D immunogen associated with Rh hemolytic disease. Alternatively, the immunogen can be an autoimmunogen such as those associated with thyroiditis, diabetes, stroke, myasthenia gravis, or rheumatic fever. The immunogen and analog can be of the same chemical species.

Surprisingly, the chemically defined, non-polymeric valency platform molecule of the present invention,
The use of a complex consisting of a biological or chemical molecule (non-hapten) is expected to result in at least about 10 times to several hundred times more immunosuppression than the polymeric carriers described in the prior art. The unfulfilled results were achieved.
For example, using a complex of the invention that comprises a chemically defined, non-polymeric valency platform molecule as described herein, a carrier of undefined definition as described in the prior art. At least 100 times more anti-dsDN than
Immunosuppression of A autoantibodies was achieved.

Yet another aspect of the invention is (a) a chemically defined non-polymeric valency platform molecule, and (b) located at or near one end of one of the duplexes. It is a complex, consisting of multiple polynucleotide duplexes, each all bound to the valency platform molecule by a functional group, which is the immunotolerant of human SLE.

The pharmaceutical compositions of the above conjugates and pharmaceutically acceptable pharmaceutical vehicles are yet another aspect of the present invention.

Another aspect of the invention comprises the step of administering to an individual in need of treatment an effective amount of the above complex.
It is a method of treating SLE.

Yet another aspect of the invention is the step of administering to an individual an effective amount of the above complex in the individual,
This is a method of inducing specific B cell anergy to the immunogen.

Another aspect of the invention is treating an antibody-mediated disease in which an unwanted antibody is produced in response to an immunogen, comprising the step of administering to the individual an effective amount of the above complex. Is the way.

Another aspect of the present invention comprises the step of attaching a biological or chemical molecule to a chemically defined valency platform molecule to form a complex.
A method for producing the above composite.

Another aspect of the invention is that each is at least about
Reacting a plurality of single-stranded polynucleotides that are 20 nucleotides in length and that have a functional group that reacts with a chemically defined functional group on the bond platform molecule at or near one end The step of forming the complex and annealing the single-stranded polynucleotide bound to the chemically defined valency platform molecule and the complementary single-stranded polynucleotide to form a double-stranded DN
A method for producing a complex for treating SLE, which comprises the step of forming a pendant chain of A.

Yet another aspect of the present invention is a novel, chemically defined, non-polymeric valency platform molecule of the formula:

[0066]

[Chemical 13]

Alternatively,

[0068]

[Chemical 14]

However, when present, each of G ^[6] and G ^[7] is independently 1 to 2000, more preferably, selected from the group consisting of C, N, O, Si, P and S. 1-1000 is a straight chain, branched chain or multi-branched chain containing chain atoms.

More preferably, G ^[6] and G ^[7] are each a group derived from polyalcohol, polyamine or polyglycol.

Most preferably, G ^[6] and G ^[7] are each
q is from 0 to 20 _{- (CH 2) q -,} -CH 2 (CH 2 OCH 2) from r 0 300 _r C
H _2- , and s is 1 to 4, more preferably s is 3 to 4
Of C (CH ₂ 0CH ₂ CH _2- ) _s (OH) _4-s .

N ^[6] × p ^[6] parts, denoted as T ^[6] ,
And the p ^[7] × n ^[7] moieties shown as T ^[7] are each independently -NHR ^SUB (amine), -C (= O) NHNHR ^SUB (hydrazide), NHNHR ^SUB ( Hydrazine), -C (= O) OH (carboxylic acid),-
C (= O) OR ^ESTER (active ester), -C (= O) OC (= O) R ^B (anhydride), -C (= O) X (acid halide), -S (= O) ₂ X (Sulfonyl halide), -C (= NR ^SUB ) OR ^SUB (imidate ester), -NCO
(Isocyanate), -NCS (isothiocyanate), -OC (=
O) X (haloformate), -C (= O) OC (= NR ^SUB ) NHR ^SUB (carbodiimide adduct), -C (= O) H (aldehyde), -C (= O) R ^B (ketone), -SH (sulfhydryl or thiol), -OH
(Alcohol), -C (= O) CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂ OR ^ALK X (alkylsulfonate), R ¹ R ² is -C (= O) CH = CHC (= O)-is -NR ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl), -R ^{A LK}
-Hg-X (alkylmercury), and -S (= O) CR ^B = CR ^B ₂ (α, β-unsaturated sulfone).

[0073] More preferably, n, shown as T ^{[6] [6]}
× p ^[6] parts and n ^[7] × p shown as T ^[7]
Each ^[7] part is independently -NHR ^SUB (amine), -C (= O)
CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (=
O) ₂ OR ^ALK X (alkyl sulfonate), R ¹ R ² is -C (= O) CH = CH
C (= O)-is -NR ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α,
β-unsaturated carbonyl), -R ^ALK -Hg-X (alkylmercury), and -S (= O) CR ^B = CR ^B ₂ (α, β-unsaturated sulfone).

More preferably, n represented as T ^[6]
[6] × p ^[6] number of partial and, n, shown as T ^{[7] [7]}
× p ^[7] moieties are each independently -NHR ^SUB (amine), -C
(= O) CH ₂ X (haloacetyl), R ¹ R ² is -C (= O) CH = CHC (= O)-, -NR ¹ R ² (maleimide), -C (= O) CR ^B = It is selected from the group consisting of CR ^B ₂ (α, β-unsaturated carbonyl).

[0075] Most preferably, n, shown as T ^{[6] [6]}
All of the × p ^[6] parts and n, denoted as T ^{[ 7]
All of the [7]} × p ^[7] parts are the same.

Here, in the formula, each X is independently a halogen having an atomic number of 16 to 54, or a good leaving group.

Each R ^ALK is independently a linear, branched or cyclic (1-20C) alkyl group.

Each R ^SUB is independently H, straight chain, branched, or cyclic (1-20C) alkyl, (6-20C) aryl, or (7-30C) alkylaryl.

Each R ^ESTER is independently N-succinimidyl, p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl, cyanomethyl, etc. Alternatively, it is another activating group such as 5-chloro-8-quinolone-1-yl, 1-piperidyl, N-benzotriazolyl and the like.

Each R ^B is independently a group containing 1-50 atoms selected from the group consisting of C, H, N, O, Si, P and S.

N ^[6] is 1 to 32, and more preferably n ^[6] is 1 to 16, provided that the product of n ^[6] x p ^[6] is greater than 1 and less than 33. And more preferably n ^[6] is 1 to 8, more preferably n ^[6] is 1 to 4, most preferably n ^[6] is 1 to 2; p ^[6] is 1 to 8, more preferably p ^[6] is 1 to 4 and most preferably p ^[6] is 1 to 2.

N ^[7] is 1 to 32, and more preferably n ^[7] is 1 to 16, provided that the product of n ^[7] x p ^[7] is greater than 1 and less than 33. And more preferably n ^[7] is 1 to 8, more preferably n ^[7] is 1 to 4 and most preferably n ^[7] is 1 to 2; p ^[7] is 1 to 8, more preferably p ^[7] is 1 to 4 and most preferably p ^[7] is 1 to 2.

N ^[6] parts indicated by Q ^[6] , and Q
2 × n ^[7] shown in ^[7] more portions each, independently, at least p ^[6] number (for Q ^[6]) or p ^[7] / 2 pieces of (Q ^{[7 ]} , For example, d stock p ^[7] / 2 is an integer) containing a bonding site for a functional group on an alkyl carbon atom, an alkenyl carbon atom, or an aromatic carbon atom, C, H, N, O, S
A group containing 1-100 atoms selected from the group consisting of i, P and S.

[0084] More preferably, n, shown as Q ^{[6] [6]}
All of the parts are the same.

More preferably, 2 × n, denoted as Q ^{[7]
All of the [7]} parts are the same.

[0086] More preferably, n, shown as Q ^{[6] [6]}
Each of these moieties are independently CH [(CH ₂ ) _r (binding site)] ₂ , and CH [(CH ₂ ) _r C (= O) where r is 1 to 10 and q is 1 to 10. NR
^SUB (CH ₂ ) _q (binding site)] ₂ .

More preferably, 2 × n, denoted as Q ^[7]
Each of the ^[7] parts independently has r of 1 to 10, more preferably r of 2 to 6, and q of 1 to 10, more preferably q
1 to 3, (CH ₂ ) _r , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ ) _q , (C
H ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ ) _q NR ^SUB C
(= O) (CH ₂ ) _r , (CH ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q NR ^SUB C (= O) (C
H ₂ ) _r , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ CH ₂ O) _q CH ₂ CH ₂ , and (C
H ₂ ) _r C (= O) NR ^SUB (CH ₂ CH ₂ O) _q CH ₂ CH ₂ .

[0088]

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "bond platform molecule" is chemically defined, having multiple sites that allow the attachment of many distinct biological and / or chemical molecules. A non-polymeric, non-immunogenic molecule.

As used herein, the term “non-immunogenic”
Is used in the adjective of the valency platform molecule and means that the valency platform molecule does not substantially elicit an immune response when administered to an individual alone.

The term “individual” as used herein refers to many mammalian species, including humans, primates, mice, and farm animals such as cattle and sheep, sport animals such as horses, and , Including pets such as dogs and cats.

As used herein, the term “immunogen”
en) ”is a substance that induces a humoral immune response when injected into an animal. The immunogen has both B and T cell epitopes.

The term “analog” of an immunogen, as used herein, means a molecule that (a) specifically binds to an antibody to which the immunogen specifically binds, and (b) lacks a T cell epitope. Is. Usually, the analog is a fragment or derivative of the immunogen, so it belongs to the same chemical species as the immunogen (for example, the immunogen is a polypeptide and the analog is a polypeptide), but the chemical similarity is not important. . Therefore, above (a)
The analog may be a different chemical species than the immunogen (eg, the immunogen is a hydrocarbon and the analog is a polypeptide), provided that it has the functional properties of and (b). Analogs can be proteins, carbohydrates, lipids, lipoproteins, glycoproteins, lipopolysaccharides, nucleic acids, or other chemical or biological substances.

Immunogen analogs may also include "mimotopes." The term “mimotope” as used herein, competitively prevents an antibody from binding to an immunogen,
Means a synthetic molecule. Because they bind specifically to antibodies, mimotopes are believed to mimic the antigenic determinant sites of the immunogen. Like immunogen analogs, mimotopes (a) specifically bind to the antibody to which the immunogen specifically binds, and (b) lack a T cell epitope.

Immunogen analogs can also include “aptamers”. The term "aptamer" as used herein
Means a synthetic oligonucleotide that competitively prevents the antibody from binding to the immunogen. Aptamers, like analogs of immunogens, (a) specifically bind to the antibody to which the immunogen specifically binds, and (b) lack a T cell epitope.

As used herein, the term “B cell anergy” means, and is not limited to, a lack of B cell response that requires the help of T cells to produce and secrete antibodies. Clonally deficient in mature and / or immature B cells, and / or the B cells lacking the ability to produce antibodies. The term “lack of response” as used herein means a therapeutically effective reduction of the humoral response to an immunogen. In quantitative terms, the reduction (as measured by reduction in antibody production) is at least 50%, preferably at least 75%, and most preferably 100%.

As used herein, the term “antibody” means an antibody whose production is T cell dependent.

The valency of the chemically defined bond platform molecule of the present invention can be predetermined by the number of branched groups introduced into the platform molecule. Suitable branched groups are generally derived from diamino acids, triamines, and amino diacids. Examples of diamino acids and amino diacids include compounds of the formula:

[0098]

[Chemical 15]

The complexes of the invention are biologically stable; that is to say on the order of hours, days or months.
It shows the elimination half-life in vivo and contributes to the therapeutic effect.
The chemically defined valency platform molecules of the invention are also substantially non-immunogenic (i.e., do not show immunogenicity when administered to animals, or show only mild immunogenicity). ) Is nontoxic at the given dose (i.e.
Nontoxic enough to be useful as a therapeutic agent),
And preferably consists of a defined chemical structure. They provide non-immunogenic, non-toxic, multifunctional substrates to which multiple biological or chemical molecules, such as polynucleotide duplexes, can be covalently attached. These are usually
It has an average molecular weight in the range of about 200 to about 200,000, generally about 200 to about 20,000. These are more uniform than prior art polymers, which are mixtures of compounds with widely varying molecular weights. Examples of particularly suitable and uniform valency platform molecules of the present invention include derivatized 2,2′-ethylenedioxydiethylamine (EDDA), triethylene glycol (TEG), and polyethylene having a molecular weight of about 200 to 8,000. It is glycol (PEG).

The attachment of biological or chemical molecules to chemically defined platform molecules can be accomplished in a number of ways. Typically, this is done using biological or chemical molecules and functional groups on the bond platform molecule and one or more crosslinkers.

The synthetic polynucleotide duplex coupled to the valency platform molecule has at least about
It consists of 20 bp, and preferably 20-50 bp. The polynucleotides described herein are deoxyribonucleotides unless otherwise noted and are written in the 5'to 3'direction. The duplexes are preferably substantially uniform in length; that is, the variation in length in the population is about ± 20% of the average duplex length in number of base pairs, preferably Does not exceed ± 10%. The nucleotide composition is also preferably substantially uniform; that is, the base composition and base sequence are
Do not vary by more than about 10% between each duplex. Most preferably, the nucleotide composition is perfectly uniform for each duplex.

Based on the interpretation of circular dichroism (CD) spectra, the duplex of the present invention is presumed to be a B-DNA type helical structure. However, it is not intended that the present invention be limited by this speculation. Based on a more complete analysis, the duplex may be a Z-DNA and / or A-DNA type helical structure.

These polynucleotide duplexes can be synthesized from natural DNA, or chemically or by recombinant techniques. Longer-chain dsDNA, either natural or recombinantly produced, is digested (e.g., enzymatically digested, chemically digested, or mechanically sheared), and (e.g.,
Fractionation (on an agarose gel or Sephadex® column) can give the desired length of the polynucleotide.

Alternatively, pairs of complementary single-stranded polynucleotide chains of up to about 70 bases in length can be readily prepared by standard procedures using commercially available DNA synthesizers and then annealed. Form a double strand. Longer chain synthesis
dsDNA is an enzymatic stretch (5 '
-Phosphorylation followed by ligation).

Polynucleotides can also be produced by molecular cloning. For example, a polynucleotide of desired length and sequence is synthesized as described above. These polynucleotides may be digested and have suitable ends for ligation insertion into specific restriction enzyme sites.
The resulting construct is inserted into a standard cloning vector and the vector is transformed into a suitable microorganism / cell. Transformants are identified by standard markers and grown under conditions suitable for DNA replication. The polynucleotide is treated with a restriction enzyme, and
It can be isolated from the DNA of other microorganisms / cells by standard size fractionation methods (eg by agarose gel or Sephadex® columns).

Alternatively, the polynucleotide may be replicated by the polymerase chain reaction (PCR) technique. Saiki, RK et al., Science (1985) 230 : 1350; Sacki
Et al., Science (1988) 239 : 487, Sambrook et al., In Molecul.
ar Cloning Techniques: A Laboratory Manual , Vol 1
2, p 14.1-14.35 Cold Spring Harbor Press (1989).

Polynucleotides can be screened for binding activity to SLE antisera by the assay described in the Examples. A modified Farr assay, in which the binding activity can be expressed as an I ₅₀ (polynucleotide concentration resulting in half maximal inhibition, expressed in molar concentration of nucleotides) is a preferred assay. Polynucleotide duplexes with an I ₅₀ of less than or equal to about 500 nM, preferably less than or equal to 50 nM, are considered to have significant binding activity and are therefore useful in preparing the conjugates of the invention.

The polynucleotide is attached to a chemically defined valency platform molecule in such a way that its antibody binding activity is retained. This allows the polynucleotide to bind to a pendant platform molecule at a defined position on the polynucleotide chain such that it forms a pendant strand of at least about 20 base pairs from the binding site to the free (unbound) end of the strand. It is done by combining them.

In a particularly preferred embodiment, the polynucleotide of the complex of the invention is coupled with a linker molecule at or near one end thereof. This linker molecule is then coupled to a chemically defined valency platform molecule. For example,
The defined double-stranded PN can be attached to a valency platform molecule. First, a single strand consisting of nucleotides having a length of about 20 in which cytosine (C) and adenosine (A) are alternately present is prepared. Next, four CA chains can be covalently attached to four reactive sites on a derivatized platform molecule such as triethylene glycol via a linker such as HAD.
This bond platform molecule has been synthesized to include groups such as bromoacetyl. During this complexing, the leaving group is replaced by sulfur. Next, a second single-stranded nucleotide chain consisting of nucleotides having a length of about 20 in which thymidine (T) and guanosine (G) are alternately present is annealed to the CA chain, and the structural formula: [(PN ) ₂₀ -linker] can form a double-stranded PN complex of ₄ -bond platform molecules.

Alternatively, in another preferred embodiment, the polynucleotide may be coupled to the valency platform molecule at the 3'end of the polynucleotide via a morpholino bridge. The morpholino bond is formed by condensing the oxidized 3'-terminal ribose of one strand of the polynucleotide with the free amino group on the derivatized platform molecule, then exposing the adduct to reducing conditions. ,It is formed. This coupling requires that the derivatized valency platform molecule have at least as many amino groups as there are polynucleotide duplexes attached to the platform molecule.

The synthesis of such a complex takes place in two steps. The first step is the coupling of one strand of the polynucleotide duplex to the derivatized platform molecule via the condensation / reduction reaction described above. By treating the single-stranded polynucleotide with periodate to convert the ribose group at the 3 ′ end to oxidized ribose,
Form the oxidized 3'terminal ribose. This single-stranded polynucleotide is then slowly added to an aqueous solution of derivatized valency platform molecules at a pH of about 6.0 to 8.0 at 2-8 ° C. The molar ratio of polynucleotide to platform molecule is generally about 2: 1 to about 30: for all complex reactions.
It is in the range of 1, usually about 2: 1 to about 8: 1, and preferably about 4: 1 to 6: 1.

In this connection, the complex preferably does not have a very high molecular weight like macromolecules. In particular, macromolecules with repeating units of molecular weight greater than 200,000 can be T-cell dependent immunogens. Dint
zis et al., J. Immun. (1983) 131 : 2196; and J. Immun.
(1989) 143 : 1239. A strong reducing agent such as sodium cyanoborohydride is added during or after the condensation reaction (generally 24-48 hours reaction time) to form the morpholino group. The double stranded complementary strand is then added to the complex and the mixture is heated and annealed to anneal both strands. The complex can be purified by gel permeation chromatography.

One alternative to the ribose-based method described above is to form aldehyde groups on the polynucleotide, and then add these groups via reactive functional groups on the platform molecule to the polyamine. It is used to couple nucleotides and platform molecules. This reaction is 3'or
The gem -or vicinal -diol attached to the 5'end is
It utilizes that it can be oxidized with sodium periodate to an aldehyde, which can be condensed with the amino functional group of the platform molecule. When the diol is present in a cyclic moiety, such as a 5-membered ring, the resulting condensation product is a nitrogen-containing heterocyclic compound, such as a 6-membered morpholino or piperidino ring. The imino condensation product can be stabilized by reduction with a suitable reducing agent such as sodium borohydride or sodium cyanoborohydride. When the diol is an acyclic compound, the resulting oxidation product has only one aldehyde group and the condensation product is a secondary amine.

Another method is to react an alkylamino or alkylsulfhydryl group with the appropriate nucleotide.
(Eg, phosphoamidite reaction) is used to introduce the compound into either 3 ′ or 5 ′ end of the polynucleotide. This nucleophilic group is then used, for example, in the case of alkylamine derivatives, with a large excess of a homobifunctional cross-linking reagent such as dimethylsuberimidate, or in the case of alkylsulfhydryl derivatives, for example, m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
Alternatively, it may be reacted with an excess of a heterobifunctional cross-linking reagent such as succinimidyl (4-iodoacetyl) aminobenzoate (SIAB). After removing excess cross-linking reagent, the polynucleotide derivative is reacted with amino groups on the platform molecule. Alternatively, the sulfhydryl group can be reacted with an electrophilic center on the platform molecule such as a maleimide, or an α-haloacetyl group, or other suitable Michael addition acceptor.

Yet another method uses modified nucleotides. Standard DNA synthesis reactions can be used to incorporate the appropriate deoxynucleoside derivative at the desired position in the polynucleotide, preferably at the 5'or 3'end. These nucleoside derivatives can then be reacted specifically and directly with alkylamino groups on the platform molecule. Alternatively, side reactions that occur in the reaction of the above dialdehydes, such as amine-catalyzed β-elimination, can be prevented by introducing an appropriate nucleoside derivative at the 3'end of the attached chain. An example of this is a 5'methylene extension of ribose; that is, 5'instead of a 5'hydroxymethyl group.
The (2-hydroxyethyl) group is introduced. Alternatively, a phosphonate or phosphinate linkage is used for the dinucleotide at the 3'end of the polynucleotide that is attached to the platform molecule.

<Analog of immunogen> Immunogens involved in antibody-mediated diseases include allergens, α-sperm of male infertility, carbohydrate complex of rheumatic fever, erythrocyte Rh / D antigen of hemolytic disease of newborns, (Therapeutic proteins, peptides,
And foreign agents such as biological agents (including natural biological substances that are foreign to the individual) such as antibodies, thyroiditis (thyroglobulin), stroke (Cardiolipin), and myasthenia gravis (acetylcholine receptor), such as
(Self-antigen).

Such immunogen analogs screen candidate molecules to determine if they specifically bind to (a) serum antibodies to the immunogen, and (b) lack T cell epitopes. You can confirm by checking whether or not.
Specific binding to serum antibodies can be determined using conventional immunoassays, and the presence or absence of T cell epitopes can be determined using conventional T cell activation tests. In that sense, an analog that "specifically binds" to a serum antibody against the immunogen has sufficient affinity for that antibody. In that sense,
Testing for T cell epitopes can be performed by testing T
It is also understood that this may be done for individual purposes using cells, or T cells from various patients representing the target population of individuals. The presence or absence of T cell epitopes can be examined using the ³ H (tritylated) thymidine incorporation assay described in the Examples. The presence of T cell epitopes can also be determined by measuring secretion of T cell-derived lymphokines by methods well known in the art. Analogs that did not induce a statistically significant uptake of thymidine above background are considered to lack T cell epitopes. The quantitative amount of thymidine incorporation can vary from immunogen to immunogen. Generally, a stimulation index of less than about 2-3, and more typically less than about 1-2 indicates a lack of T cell epitopes.

The usual first step in identifying useful analogs is the preparation of a panel or library of candidates to be screened. For example, in the case of protein or peptide analogs, Geysen et al.
Synthetic Peptides as Antigens ; Ciba Symposium (19
86) 119 : 131-149; Devlin et al., Science (1990) 249 : 404.
-406; Scott et al., Science (1990) 249: 386-390; and Cwirla et al., PNAS USA (1990) 87 : 6378-6382, etc., may be prepared by synthetic techniques or recombinant methods.
In one synthetic method, peptides of about 5 to 30 amino acids are synthesized such that each peptide overlaps with the next peptide and all linear epitopes are expressed. This is done by overlapping both the carboxyl and amino termini by one residue less than the length expected for the B cell epitope. For example, if the minimum requirement for B-cell epitopes is assumed to be 6-amino acids, then each peptide must overlap with adjacent peptides by 5-amino acids. In this embodiment, each peptide is then screened for the presence of B cell epitopes with antisera raised against the natural immunogen, either by immunization of the animal or from the patient. Molecules with antibody binding activity are then screened for the presence of T cell epitopes, as described in the Examples. Molecules lacking T cell epitopes are useful as analogs of the invention.

If the T cell epitope (s) of the immunogen are known or can be identified, then random T cell screening of candidate analogs is not necessary. In that case, the T (s)
A cellular epitope may be altered such that it is non-functional (eg, by chemically derivatizing or removing one or more components of that epitope), or synthetic methods, such as in the case of peptides. Alternatively, it can be completely removed by a recombinant method or the like.

Mimotopes or aptamers can be synthesized by conventional methods and screened in the same manner as analogs of other immunogens.

These analogs are coupled with non-immunogenic valency platform molecules to prepare the conjugates of the invention. Conjugates composed of valency platform molecules and biologically active molecules such as carbohydrates, lipids, lipopolysaccharides, proteins, glycoproteins, drugs, and analogs of interest are exemplified herein. It is synthesized using a chemical reaction. A preferred synthetic method is to incorporate the linker molecule onto the biological molecule in a well-selected and well-known manner.

When a drug such as adriamycin (doxorubicin) is attached to a valency platform molecule,
The amino group on the sugar ring can react with the platform molecule containing the active ester. Adriamycin can also be modified to include a thiol group for attachment to haloacetylated platforms (Kanek).
o, T. Et al., Bioconjugate Chemi
stry , 2 : 133 (1991)).

Carbohydrates such as oligosaccharides can be modified to include a sulfhydryl-containing linker (Woo
d, SJ and Wetzel, R., Bioconjugate Chemistry , 3 :
391 (1992)). The sulfhydryl group is used to attach to the haloacetylated platform. Alternatively,
Oxidizes carbohydrates to form aldehydes, which are
It can be reacted with an amination platform in the presence of BH ₃ .

Lipids such as glycol lipids containing ethanolamine groups are reacted with active carboxylates on the platform molecule. Lipopolysaccharides containing sugar units oxidize to form aldehydes, which are
Reacted with an amination platform in the presence of BH ₃ to form a complex by reductive amination.

In the case of another protein, such as a Fab 'antibody fragment, the sulfhydryl group on the protein (Fab') is attached to the platform via the haloacetyl group. Glycoproteins are modified with thiol linkers using iminothiolate. This thiol reacts with platforms containing haloacetyl groups.

The ability of the complex to function as an immunotolerant,
And the ability to specifically suppress antibody production can be assessed in the mouse model described in the Examples.

The complex is usually formulated for administration by injection (eg, intraperitoneal, intramuscular, intravenous, etc.). Therefore, they are typically combined with pharmaceutically acceptable aqueous carriers such as saline, Ringer's solution, dextrose solution and the like. The complex is typically about 0.01% by weight of the formulation
% To 10% by weight. The complex is administered to the individual in an amount sufficient to at least partially reestablish immune tolerance to the SLE-causing self-antigen. Such an amount is sometimes referred to herein as a "therapeutically effective" amount.
Administration regimen in a particular case, i.e. dose, timing and frequency of administration, will vary with the particular individual and the individual's medical history. Normally, about 1 to 1000 μg complex / Kg body weight will be administered. Repeated administrations may be required to achieve and / or maintain a state of immune tolerance.

The following examples further illustrate the invention and its unexpected effects on the prior art. These examples do not limit the scope of the invention.

[0129]

【Example】

Example 1 The following reaction scheme illustrates a method of synthesizing a derivatized, chemically defined bond platform molecule of the present invention. The following abbreviations are used in this example: DMTr = 4,4'-dimethoxytriphenylmethyl;
Tr = trityl; Bz = benzoyl; Cp = deoxycytidine monophosphate; CE = cyanoethyl; CPG = controlled pore glass; DMF = dimethylformamide; DC
C = dicyclohexylcarbodiimide; TFA = trifluoroacetic acid; CDI = carbonyldiimidazole; Ts = tosyl (p-toluenesulfonyl); DIPAT = diisopropylammonium tetraazolide; TBDMSCl = tert-butyldimethylsilyl chloride; TBAF = tetrabutylammonium fluoride Ride; NMMO = N-methylmorpholine oxide.

[0130]

[Chemical 16]

[0131]

[Chemical 17]

[0132]

[Chemical 18]

[0133]

[Chemical 19]

[0134]

[Chemical 20]

[0135]

[Chemical 21]

[0136]

[Chemical formula 22]

[0137]

[Chemical formula 23]

[0138]

[Chemical formula 24]

[0139]

[Chemical 25]

(CA) ₈ , (CA) ₁₀ , (CA) ₁₂ and (CA) ₁₆ are
The synthesis of reagents used to modify with a disulfide linker is described in Scheme 11 below.

[0141]

[Chemical formula 26]

Synthesis of reagents used to modify (CA) ₂₅ with a vicinal-diol linker is described in Scheme 12 below.
Described in.

[0143]

[Chemical 27]

[0144]

[Chemical 28]

(Example 2: Chemically defined bond platform molecule) <Compound 1- [3,5-bis- (iodoacetamido) benzoic acid]> 2.93 g (8.28 mmol, 2.2 equivalents) of iodoacetic anhydride ) To N ₂
572 mg (3.76 mm) of 3,5-diaminobenzoic acid at room temperature in an atmosphere
ol) was added to a stirred suspension of dioxane (19 mL). The mixture was stirred, covered with foil for 20 hours, and EtOAc.
Partitioned (50 mL) and 1N HCl solution (50 mL). The EtOAc layer was washed with brine, dried over MgSO _4, filtered, and concentrated on a rotary evaporator to give a solid 3.3g of brown. Silica gel chromatography (94/5/1
Purification by _{CH 2 Cl 2 / MeOH / HOAc} ), to give the compound 1 in 992 mg (54%) as a white solid: NMR (DMSO) 3.84
(s, 4H), 7.91 (s, 2H), 8.14 (s, 1H), 10.56 (s, 2
H).

<Compound 2- [3,5-bis- (iodoacetamido) benzoyl chloride]> SOCl ₂ 117 μL (1.6 mmol,
190 mg) was added to a solution of 390 mg (0.8 mmol) of 1 in THF (34 mL). The mixture was refluxed under N ₂ atmosphere until all solids were dissolved (approximately 30 minutes) to give a clear reddish brown solution. The mixture was concentrated on a rotary evaporator and left under reduced pressure to give crude compound 2 as a foamy solid. This foamy solid was used directly in the next step.

<N-N'-bis of compound 3- [α, ω-bis- (N-2-aminoethylcarbamoyl) polyethylene glycol]
-(3,5-bis- (iodoacetamido) benzoyl) derivative]
> Α, ω-bis- (N-2-aminoethylcarbamoyl) polyethylene glycol (0.16mmol, 3350g / mol, Sigma) 570mg
Was placed in a tared flask. Toluene (20 mL) was added and water was removed by azeotropic distillation. The residue was dried under reduced pressure to give a solid 549 mg and was dissolved in 4 mL THF with 89 μL (0.64 mmol) diisopropylethylamine. Dissolve the crude acid chloride in 4 mL anhydrous THF,
Then added to the mixture over 30 seconds under N ₂ . The mixture was stirred at room temperature for 16 hours, and 0.1 N HCl (25 mL) and CH ₂ Cl ₂ (25 m
L) and divided. The aqueous layer was extracted again with CH ₂ Cl ₂ and the organic layers were combined and washed with 25 mL H ₂ O, then 50 mL NaHCO ₃ solution. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to give an orange oil 784 mg. 190 mg colorless oil by silica gel chromatography (9/1 CH ₂ Cl ₂ / MeOH)
Got The oil was crystallized from hot EtOH / Et ₂ O, collected on a sintered glass filter under N ₂ pressure and dried under reduced pressure to give 177 mg of compound 3 as a white solid: NMR (CDCl ₃ ) 3.40 (bd m, 8H), 3.59 (bd s, (CH ₂ CH
₂ O) _n , which is extremely larger than other integrals), 3.91 (s,
8H), 4.21 (m, 4H), 6.04 (bd m, 2H), 7.55 (bd m, 2
H), 7.78 (bds, 4H), 8.10 (bd s, 2H), 9.30 (bd m, 4
H): Iodoacetyl assay ( European Journal of Bioche
mistry (1984) 140 : 63-71): Calculated value: 0.92 mmol / g; Found value: 0.96 mmol / g.

<Compound 4- [Mono-N-carbobenzyloxy-3,6-dioxa-1,8-diaminooctane]> 14.3 mL (17.1 g, 100 mmol) of CH ₂ Cl ₂ (200 m) of benzyl chloroformate
L) solution, 1,2-bis- (2'-aminoethoxy) ethane (Fluk
a) Add 29.0 mL (29.6 g, 200 mmol) of CH ₂ Cl ₂ (100 mL) to 0 ° C.
It was added dropwise over 1 hour. The mixture was stirred at room temperature for 24 hours and 1N HCl was added until the aqueous layer remained acidic (pH <2). The aqueous layer was washed 3 times with CH ₂ Cl ₂ (50 mL each) and neutralized with 1 N NaOH until pH> 13. The basic aqueous layer was extracted 5 times with CH ₂ Cl ₂ (75 mL each). The CH ₂ Cl ₂ layers were combined, dried (MgSO ₄ ), filtered and concentrated to 1
2.7 g (45%) of compound 4 was obtained as a thick oil: ¹ HNMR
(CDCl ₃ ) d 2.82 (bd s, 2H), 3.30-3.60 (m, 12H), 5.
10 (s, 2H), 5.75 (bd s, 1H), 7.20-7.40 (m, 5H); ¹³
C NMR (CDCl ₃ ) d 41.1, 41.8, 66.5, 70.0, 70.2, 70.4,
73.5, 127.9, 128.0, 128.4, 136.9, 156.4.

<Compound 5- [N-tert-butyloxycarbonyliminodiacetic acid]> Garrigues, B. and Lazraq, EM
This compound was prepared by a procedure similar to that reported by Tetrahedron Letters (1986) 27 , 1685-1686. Et ₃ N 47mL (3
4.2 g, 338 mmol) and 22.0 g (169 mmol) of iminodiacetic acid and di-t
To a stirred 50/50 dioxane / H ₂ O (169 mL) solution with 36.8 g (169 mmol) of ert-butyl dicarbonate was added at room temperature. The mixture was stirred for 24 hours and most of the dioxane was removed on a rotary evaporator. The mixture was partitioned between 1N HCl (350 mL) and 5 EtOAc (100 mL each).
The combined The EtOAc layers were dried (MgSO _4), filtered, and concentrated to give a white solid. Recrystallization from hexane / EtOAc gave 35.3 g (90%) of compound 5 as crystals: m.
p. 131-132 ℃ Fused; ¹ H NMR (DMSO) d 1.35 (s,
9H), 3.87 (s, 2H), 3.91 (s, 2H), 12.6 (bd s, 2H);
¹³ C NMR (DMSO) d 27.9, 49.6, 79.6, 154.8, 171.2.

<Compound 6> 9.99 g (48.5 mmol) of dicyclohexylcarbodiimide was combined with 4.52 g (19.4 mmol) of Compound 5 .
With N-hydroxysuccinimide (NHS) 4.46 g (38.8 mmol)
It was added to a THF (100 mL) solution at 0 ° C. The mixture was stirred at 0 ° C. for 3 hours and 5.39 mL Et ₃ N (3.92 g, 38.8 mmol) and 10.9
A solution of g (38.7 mmol) of compound 4 in THF (83 mL) was added and the mixture was stirred at 5 ° C. for 17 hours. The mixture was filtered to remove solids and the filtrate was concentrated to give an oil. The oil was partitioned between EtOAc (400 mL) and 2 IN HCl (100 mL each). The EtOAc layer by 1N Na ₂ CO ₃ 100mL ₃
Wash once with 100 mL brine, dry (MgS
O ₄ ), filtered and concentrated to 14.2 g (96%) of compound 6
Was obtained as a thick oil: ¹ H NMR (CDCl ₃ ) d 1.41
(s, 9H), 3.30-3.70 (m, 24H), 3.70-3.90 (m, 4H), 5.1
0 (s, 4H), 5.50 (bd s, 2H), 7.12 (bd s, 1H), 7.30-
7.40 (m, 10H), 8.24 (bd s, 1H).

<Compound 7> 26.3 mL of trifluoroacetic acid (38.
9g, 156mmol) to 14.2g (18.6mmol) of compound 6 in CH ₂ Cl _2
2 (111 mL) solution and the mixture was stirred at room temperature for 3 hours. The mixture was concentrated on a rotary evaporator to give a viscous oil and this oil was dissolved in 93 mL THF. The solution is cooled to 0 ° C. and succinic anhydride 3.7
2 g (37.2 mmol) was added, then 5.18 mL Et ₃ N (3.76 g, 37.2 m
mol) was added. Remove the cooling bath and allow the mixture to stand at room temperature.
It was stirred for 18 hours. The solvent was removed under reduced pressure and the resulting oil was partitioned between CH ₂ Cl ₂ (300 mL) and 3 H ₂ O (100 mL each). The CH ₂ Cl ₂ layer was dried (MgSO ₄ ), filtered and concentrated to give an oil. The oil was purified by chromatography on silica gel (9/1 / 0.1 EtOAc / MeOH / acetic acid) to give 10.5 g (74%) of compound 7 as a viscous oil; ¹ H NMR (CDCl ₃ ). d 2.50-2.60 (m, 4H), 3.30-3.60
(m, 24H), 3.88 (s, 2H), 4.03 (s, 2H), 5.07 (s, 4
H), 5.77 (bd s, 2H), 7.20-7.30 (m, 10H), 7.91 (bd
s, 2H), 8.88 (bd s, 1H); ¹³ C (CDCl ₃ ) d 27.7, 29.0,
39.4, 41.0, 52.9, 53.8, 66.5, 69.3, 69.8, 70.0, 7
0.1, 127.8, 128.1, 128.3, 136.7, 156.6, 169.1, 16
9.6, 173.3, 174.5.

<Compound 8- [4-nitrophenyl ester of compound 7]> Dicyclohexylcarbodiimide 1.61 g
(7.83 mmol), 3.98 g (5.22 mmol) of 7 and 800 mg (5.75 mmol)
To a solution of 4-nitrophenol in CH ₂ Cl ₂ (26 mL) at 0 ° C. The mixture was stirred under N ₂ at room temperature for 64 hours. The mixture was cooled to 0 ° C., 1 mL HOAc was added, and the mixture was kept at 0 ° C. for 2 hours. The solid was removed by filtration and the filtrate was concentrated. The residue was chromatographed on silica gel (gradient, 91/8/1 to 84/15/1 CH ₂
Cl ₂ / IPA / HOAc) to give 2.58 g (56%) of compound 8 as a viscous oil: ¹ H NMR (CDCl ₃ ) d 2.66 (t, 2
H), 2.84 (t, 2H), 3.32-3.68 (m, 24H), 3.90 (bd s,
2H), 4.01 (bd s, 2H), 5.06 (s, 4H), 5.58 (bd m, 2
H), 6.91 (bdm, 1H), 7.27 (d, 2H), 7.33 (s, 10H),
8.23 (d, 2H), 9.01 (bdm, 1H). <Compound 10- [4-nitrophenyl bromoacetate]>
Dicyclohexylcarbodiimide 9.28 g (45 mmol), bromoacetic acid 5.0 g (35.9 mmol) and 4-nitrophenol 8.50 g (6
To a stirred solution of EtOAc (180 mL) with 1.1 mmol) was added at 0 ° C. The mixture was stirred at 5 ° C. for 16 hours and 1 mL acetic acid was added. The mixture was stirred at room temperature for 20 minutes then placed in the freezer for 20 minutes. The solid material was removed by filtration and the filtrate was concentrated to give a viscous oil and crystallized from Et ₂ O / hexane to give 7.73 g (83%) of compound 10 as flakes: mp 86-87 ℃; TLC Rf = 0.63
(50/50/1 hexane / EtOAc / HOAc); ¹ H NMR (CDCl ₃ ) d 4.
13 (s, 2H), 7.36 (d, J = 12Hz, 2H), 8.32 (d, J = 12Hz,
2H); ¹³ C NMR (CDCl ₃ ) d 24.9, 122.1, 125.3, 155.5,
164.9; ANALYSIS: Calculated for _{C 8 H 6 BrNO 4: C} , 36.95;
H, 2.33; N, 5.39. Found: C, 37.24; H, 2.33; N, 5.
42.

<Compound 9> NaHCO ₃ 300 mg (3.57 mmol), then 1,2-bis- (2′-aminoethoxy) ethane (Fluka) 162 mg
(1.09 mmol) was added to a solution of 2.37 g (2.68 mmol) of compound 8 in dioxane (15 mL) and H ₂ O (8 mL). The mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure to approximately 1/2 original volume. The concentrate was washed with CH ₂ Cl ₂ (40 mL) and saturated NaHCO 3.
Partitioned with ₃ solutions (40 mL). This CH ₂ Cl ₂ layer is then added to 0.5N HCl.
Each was washed twice. The CH ₂ Cl ₂ layer was washed with saturated NaCl solution, dried (MgSO ₄ ), filtered and concentrated to 2.8 g.
Got oil. The crude product was purified by silica gel chromatography (3/6/1 / CH ₂ Cl ₂ / THF / MeOH) to give 94
0 mg (59%) of compound 9 was obtained as an oil: TLC R _f = 0.21
(3/6/1 CH ₂ Cl ₂ / THF / MeOH); ¹ H NMR (CDCl ₃ ) d 2.45 (m,
4H), 2.59 (m, 4H), 3.25-3.55 (m, 60H), 3.87 (s, 4
H), 4.05 (s, 4H), 5.07 (s, 8H), 5.62 (bd s, 4H), 6.
78 (bd s, 2H), 7.34 (bd s, 20H), 8.56 (bd s, 2H);
¹³ C NMR (CDCl ₃ ) d 28.1, 30.3, 31.1, 39.4, 41.1, 52.
9, 53.9, 66.5, 69.4, 69.7, 69.9, 70.2, 125.3, 127.
8, 128.3, 136.8, 156.5, 168.8, 169.4, 172.1, 173.
Five.

<Compound 34> 110 mg of 10% Pd carbon,
To a solution of 281 mg (0.175 mmol) of compound 9 in EtOH (5 mL) and cyclohexene (2 mL) was added under nitrogen and the resulting mixture was refluxed under nitrogen for 2 hours. Upon cooling, the mixture was filtered through diatomaceous earth and concentrated under reduced pressure,
170 mg (92%) of compound 34 was obtained as an oil. This oil was used directly in the next step without purification; ¹ H NMR (CDCl
₃ ) d 2.45 (m, 4H), 2.53 (m, 4H), 2.62 (m, 4H), 2.8
6 (m, 8H), 3.42-3.60 (m, 52H), 4.00 (s, 4H), 4.14
^{(s, 4H); 1 3} C NMR (CDCl 3) d 28.2, 30.3, 31.1, 39.4,
41.1, 46.5, 48.6, 52.9, 53.8, 69.4, 69.7, 70.2, 72.
4, 168.9, 169.5, 172.3, 173.8.

<Compound 11> 165 mg (0.155 mmol) of Compound 10 of 128 mg (1.4 mmol) and 200 mg (0.85 mmol) of NaHCO ₃
Was added to a solution of compound 34 in dioxane (6 mL) and H ₂ O (3 mL). The resulting mixture was stirred at room temperature for 24 hours and concentrated under reduced pressure. This concentrate was added to Sephadex®.
Purified by chromatography on G-10 (MeOH) to give 114 mg (4
6%) of compound 11 was obtained as a viscous oil. Preparative HPLC (C ₁₈ ; gradient 15/85 / 0.1 to 30/70/0.
1 CH ₃ CN / H ₂ 0 / CF ₃ CO ₂ H, 50 min, 225 nm): ¹ H NM
R (CDCl ₃ ) d 2.58 (m, 4H), 2.65 (m, 4H), 3.43-3.62
(m, 60H), 3.92 (s, 8H), 4.03 (s, 4H), 4.16 (s, 4
H); MS (FAB) m / e (relative intensity) MNa + 1605 (100), MH + 1
579 (1), 1581 (5), 1583 (7), 1585 (6), 1587 (2).

<Compound 12- [Mono-N-carbobenzyloxy-1,6-diaminohexane]> A solution of 21 mL (25.7 g, 150 mmol) of benzyl chloroformate in dioxane (200 mL) was added.
17.49 g (150 mmol) of 1,6-hexanediamine and 19.58 g of KHCO ₃
To a stirred solution of dioxane (100 mL) with (196 mmol) and H ₂ O (300 mL) was added dropwise at 0 ° C. The mixture was stirred at room temperature for 18 hours then cooled to 0 ° C. Add this mixture to 12 NH
Acidified with Cl and extracted twice with 100 mL Et ₂ O each.
The aqueous layer was neutralized with 10 N NaOH and extracted 8 times with 100 mL Et ₂ O each. The basic extracts were combined, dried (Na ₂
SO ₄ ), and then concentrated to give 5.03 g (13%) of crude compound 12
Was obtained as a semi-solid residue: ¹ H NMR (DMSO) d 1.22-1.
51 (m, 8H), 2.54 (t, 2H), 3.02 (d of t, 2H), 5.05
(s, 2H), 7.30-7.48 (m, 5H).

<Compound 13> 918 mg (4.45 mmol) of dicyclohexylcarbodiimide and 417 mg (1.78 mmol) of compound 5
And a NHS solution (409 mg, 3.56 mmol) in THF (15 mL) were added at 0 ° C. The mixture was stirred at 0 ° C. for 4.5 hours and 1.02 g (4.
A solution of 08 mmol of compound 12 in THF (4 mL) was added. The mixture was stirred under N ₂ at 5 ° C. for 18 hours. This concentrate is EtO
Partitioned between Ac (30 mL) and two 1N HCl (30 mL each). The combined EtOAc layers were washed successively with H ₂ O (30 mL) and saturated NaHCO ₃ solution (30 mL), dried (MgSO ₄ ), filtered and concentrated to give 1.48 g of viscous residue. Obtained. Purification by silica gel chromatography (5/95 MeOH / CH ₂ Cl ₂ ) gave 1.04 g (84%) of compound 13 as a sticky solid: ¹ H NMR (CDCl ₃ ) d 1.33 ( m, 8H), 1.43 (s, 9H),
1.51 (m, 8H), 3.18 (m, 4H), 3.26 (m, 4H), 3.81 (s,
2H), 3.85 (s, 2H), 4.90 (bd s, 2H), 5.10 (s, 4H),
6.81 (bd s, 1H), 7.28-7.40 (m, 10H), 8.05 (bd s, 1
H).

<Compound 14> 14.9 mL of trifluoroacetic acid
Was added to 5.16 g (7.45 mmol) of compound 13 in CH ₂ Cl ₂ (14.9 mL) and the resulting mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure and redissolved in 57 mL THF. To this mixture was added Et ₃ N 2.07 mL (1.51 g, 14.9 mmo
l) was added. 1.5 g (14.9 mmol) of succinic anhydride was added to this mixture.
Was added and then the mixture was stirred for 18 hours. The mixture
Partitioned between 1N HCl (75 mL) and 4 CH ₂ Cl ₂ (75 mL each). C
The H ₂ Cl ₂ layers were combined, dried (MgSO ₄ ), filtered and concentrated to give a solid. Crystallization from CH ₂ Cl ₂ / EtOAc / hexanes gave 3.84 g (74%) of compound 14 as a white solid: mp 122 ° C .; ¹ H NMR (MeOH) d 1.32 (m,
8H), 1.48 (m, 8H), 2.56 (m, 4H), 3.10 (t, 4H), 3.
23 (m, 4H), 4.00 (s, 2H), 4.18 (s, 2H), 5.05 (s, 4
H), 7.33 (m, 10H).

<Compound 15- [4-nitrophenyl ester of Compound 14]> Dicyclohexylcarbodiimide 887
mg (4.30 mmol) in a THF (15 mL) solution of 2.0 g (2.87 mmol) of compound 14 and 4-nitrophenol 438 mg (3.15 mmol) at 0 ° C.
Added in. The mixture was brought to room temperature, stirred for 18 hours and then cooled to 0 ° C. Then 200 μL of acetic acid was added and the mixture was stirred at 0 ° C. for 1 hour. The solid was removed by filtration and the filtrate was concentrated to give an oil. Chromatography on silica gel (92/8 CH ₂ Cl ₂ / IP
Purified by A) and the solid obtained was recrystallized from CH ₂ Cl ₂ / hexane to give 1.52 g (64%) of compound 15 as a white solid: mp 65-68 ° C .; ¹ H NMR (CDCl ₃ ) d
1.30 (m, 8H), 1.47 (m, 8H), 2.71 (t, 2H), 2.90
(t, 2H), 3.17 (m, 4H), 3.25 (m, 4H), 3.92 (s, 2H),
4.08 (s, 2H), 4.86 (bd t, 1H), 4.95 (bd t, 1H),
5,09 (s, 4H), 6.28 (bd t, 1H), 7.23 (d, J = 9Hz, 2
H), 7.32 (m, 10H), 8.22 (d, J = 9Hz, 2H), 8.95 (bd
t, 1H).

<Compound 16> 830 mg (0.99 mmol) of compound
A solution of 15 in dioxane (7.5 mL) was added with 1,2-bis- (2'-aminoethoxy) ethane (Fluka) 58 μL (59 mg, 0.40 mmol) and NaHCO 3.
₃ 111 mg (1.31 mmol) in H ₂ O (7.5 mL) solution was added. The mixture was stirred at room temperature for 18 hours. Mix the mixture with 1N HCl (50 mL)
And CH ₂ Cl ₂ (50 mL). The CH ₂ Cl ₂ layer was dried (Na ₂ S
O ₄ ), filtered and concentrated to give 1.28 g of viscous oil. Silica gel chromatography (84/15/1 CH ₂ Cl ₂ / Me
OH / HOAc) to give 670 mg of compound
16 was obtained as a waxy solid: ¹ H NMR (CDCl ₃ ) d
1.32 (m, 16H), 1.49 (m, 16H), 2.46 (m, 4H), 2.58
(m, 4H), 3.10-3.23 (m, 16H), 3.34 (m, 4H), 3.48
(m, 4H), 3.53 (s, 4H), 3.85 (s, 4H), 4.02 (s, 4H),
5.05 (s, 8H), 5.07 (overlapping bd t, 2H), 5.15 (bd t,
2H), 7.30 (m, 20H), 7.40 (bd t, 2H), 8.60 (bd t, 2
H).

<Compound 35> 613 mg (0.41 mmol) of compound
A solution of 16 EtOH (20.3 mL) and cyclohexene (10.1 mL) was stirred and purged with nitrogen. 10% Pd Carbon (A
ldrich) 20 mg and add the mixture in an oil bath at 85 ° C.
Heated for 1.5 hours. On cooling, the mixture was filtered through diatomaceous earth with 50/50 H ₂ O / acetone to rinse the flask and filter. The filtrate was concentrated under reduced pressure to give 448 mg (114%) of compound 35 as a waxy solid: ¹ H NMR (D ₂ O) d 1.39 (m, 16H), 1.59 (m, 1
6H), 2.57 (t, 4H), 2.65 (t, 4H), 2.88 (t, 8H), 3.2
3 (t, 4H), 3.29 (t, 4H), 3.42 (t, 4H), 3.65 (t, 4
H), 3.71 (s, 4H), 4.06 (s, 4H), 4.30 (s, 4H).

<Compound 17> 546 mg (6.50 mmol) of NaHCO ₃
Was added to a solution of 445 mg (0.406 mmol) of compound 35 in H ₂ O (9.5 mL). 838 mg (3.25 mmol) of the compound was added to the resulting mixture.
A solution of 10 in dioxane (14.4 mL) was added. The mixture was stirred at room temperature for 7 hours, and 0.1 NH ₂ SO ₄ (50 mL) and CH ₂ Cl
Partitioned with ₂ (50 mL). Discard the CH ₂ Cl ₂ layer and add the aqueous layer to C
50 mL H ₂ Cl ₂ twice, 9/1 CH ₂ Cl ₂ / MeOH ₂ mL 50 mL each
Once with 50 mL of 4/1 CH ₂ Cl ₂ / MeOH and 3/2 CH ₂ Cl ₂ / MeO
Extracted once with 50 mL H 2. The extracts were combined, dried (Na ₂ S
O ₄ ), filtered and concentrated to give 282 mg of solid. Et
By crystallization from OH / EtOAc / Et ₂ O, 143 mg (24%)
Compound 17 was obtained as a white solid: ¹ H NMR (CDCl
₃ / MeOH) d 1.33 (m, 16H), 1.55 (m, 16H), 2.55 (m, 8
H), 3.21 (m, 16H), 3.39 (m, 4H), 3.55 (m, 4H), 3.81
(s, 8H), 3.95 (s, 4H), 4.12 (s, 4H). Analysis value: C ₅₄ H
Calculated for _{9 4} N ₁₂ 0 ₁₄ Br ₄ : C, 44.57; H, 6.51; N, 1
1.55; Br, 21.97. Found: C, 45.85; H, 6.49; N, 11.
37; Br, 19.90.

<Compound 18- [1,5-bis (N-carbobenzyloxy-6-aminohexanoamido) -3-azapentane]>
Carbonyldiimidazole 3.09 g (19.0 mmol), N-carbobenzyloxy-6-aminohexanoic acid 5.05 g (19.0 mmol)
In EtOAc (25 mL) at room temperature. The mixture was stirred for 15 hours and then 1.02 mL of diethylenetriamine (982 mg,
9.52mmol) was added, followed by Et ₃ N 2.65mL (1.93g, 19.0mmo
l) was added. The resulting mixture was stirred for 4 hours and the solid product was collected by filtration. Recrystallization (MeOH / EtOAc) gave 4.27 g (75%) of compound 18 as a finely divided solid: mp 132-133 ° C; ¹ H NMR (CDCl ₃ ) d 1.33 (m, 4H),
1.52 (m, 4H), 1.64 (m, 4H), 2.18 (t, 4H), 2.73
(t, 4H), 3.16 (m, 4H), 3.35 (m, 4H), 4.96 (bd s, 2
H), 5.09 (s, 4H), 6.13 (bd s, 2H), 7.33 (s, 10H);
ANALYSIS: Calculated for _{C 32 H 47 N 5 0 6} : C, 64.29; H, 7.5
0; N, 11.72. Found: C, 63.54; H, 7.75; N, 11.91.

<Compound 19> Triethylene glycol-
Bis-chloroformate (Aldrich) 657 μL (880 mg, 3.2 mmo
l), 4.86 g (8.1 mmol) of compound 18 pyridine (162 mL)
The solution was added in a 20 ° C. water bath. This mixture immediately formed a precipitate. The mixture was stirred for 16 hours, and the resulting cloudy yellow solution was concentrated under reduced pressure. Et the concentrate
Partitioned between OAc (150 mL) and two 1N HCl (150 mL each) to ensure the aqueous layer was acidic. The aqueous layers were combined and extracted with a second EtOAc (150 mL). The EtOAc layers were combined, dried (MgSO4), filtered and concentrated. The resulting residue was crystallized (EtOAc / hexane / CHCl ₃ ) to give 1.92 g (43%) of compound 19 as pale yellow fine crystals: mp 86-
91 ° C; ¹ H NMR (CDCl ₃ ) 1.31 (m, 8H), 1.52 (m, 8H),
1.62 (m, 8H), 2.20 (m, 8H), 3.20 (m, 8H), 3.39 (s,
16H), 3.62 (s, 4H), 3.68 (m, 4H), 4.26 (m, 4H),
5.08 (s, 8H), 5.32 (bd s, 4H), 7.31 (bd s, 4H), 7.
37 (s, 20H); ¹³ C NMR (CDCl ₃ ) d 25.1, 26.2, 26.4, 2
9.6, 36.0, 36.2, 38.5, 38.8, 40.8, 64.5, 66.4, 69.
1, 70.3, 128.0, 128.4, 136.7, 156.5, 156.9, 173.
6; ANALYSIS: Calculated for _{C 72 H 1 04 N 10 0} 18: C, 61.87;
H, 7.50; N, 10.02. Found: C, 61.68; H, 7.63; N,
9.95.

<Compound 36> 3.5 mL of cyclohexene was added to 8
00 mg (0.57 mmol) of compound 19 in anhydrous EtOH (5 mL) was added. The solution was allowed to stand under nitrogen and 10% Pd carbon 500m
g was added and the resulting mixture was refluxed with stirring for 2 hours. On cooling, the mixture is filtered through diatomaceous earth,
Then concentrated to give 500 mg (100%) of compound 36 as an oil: ¹ H NMR (50/50 CDCl ₃ / CD ₃ OD) d 1.21 (m, 8
H), 1.49 (m, 8H), 1.62 (m, 8H), 2.19 (t, J = 7.4Hz,
8H), 2.67 (t, J = 7.4Hz, 8H), 3.36 (bd s, 16H), 3.67
(s, 4H), 3.71 (m, 4H), 4.21 (m, 4H).

<Compound 20> 3.9 g (46.4 mmol) of NaHCO _{3 was} added.
5.0 g (5.8 mmol) of compound 36 dioxane (37.5 mL) and H ₂ O
(12.5 mL) and added to the solution. This mixture in an ice bath at 0 ° C
Cooled to and added 8.7 g (34.8 mmol) of 4-nitrophenyl bromoacetate (Compound 10 ). This mixture
Stir at 0 ° C. for 1 hour and add 50 mL of 1 N H ₂ SO ₄ gently. The mixture was extracted 3 times with EtOAc (50 mL each). EtOAc
The extracts were discarded and the aqueous layer was washed with 20/80 MeOH / CH ₂ Cl ₂ (50 mL
6 times). The combined MeOH / CH ₂ Cl ₂ layers were dried (Na ₂ SO _4), filtered, and concentrated. Silica gel chromatography (step gradient 9/1 CH
₂ Cl ₂ / MeOH then 85/15/5 CH ₂ Cl ₂ / MeOH / THF) to give 3.62 g (46%) of compound 20 as a white solid: mp 66.0-70.5 ° C. Preparative HPLC (C ₁₈ reverse phase column, gradient 25/75 / 0.1 to 35/65 / 0.1 CH ₃ CN / H ₂
O / CF ₃ CO ₂ H prepared at 225 nm for 50 minutes) to give a clear oil. The oil was left under reduced pressure to give a white solid: mp 87-89 ° C; ¹ H NMR (CDCl ₃ ) d 1.35.
(m, 8H), 1.55 (m, 8H), 1.64 (m, 8H), 2.26 (m, 8H),
3.28 (m, 8H), 3.42 (bd s, 16H), 3.66 (s, 4H), 3.7
0 (m, 4H), 3.89 (s, 8H), 4.19 (m, 4H); 1 3 C NMR (CD
Cl ₃ ) d 25.1, 26.2, 28.8, 29.0, 38.5, 39.1, 40.0, 4
7.8, 48.3, 64.7, 69.1, 70.3, 157.0, 166.3, 174.9;
MS (FAB) m / e (relative intensity) MH + [1341 (25), 1343 (60), 13
45 (70), 1347 (56), 1349 (21)], 705.6 (100); analytical value: C
Calculated for ₄₈ H ₈₄ N ₁₀ O _{1 4} Br ₄ : C, 42.86; H, 6.29;
N, 9.27; Br, 23.77. Found: C, 42.15; H, 6.28; N,
9.87; Br, 25.33.

<Compound 21- [Tetrakis- (2-cyanoethoxymethyl) methane]> The method reported (Bruson, HA,
(U.S. Pat.No. 2,401607; June 4, 1946),
This compound was prepared. Acrylonitrile 27.3mL (21.8mL
g, 411 mmol) to 8.0 g (58.8 mmol) of pentaerythritol
And 40% benzyltrimethylammonium hydroxide aqueous solution 1.7
Added to a stirred H ₂ O (50 mL) solution with 6 mL. A reflux condenser was provided and the mixture was heated under N ₂ atmosphere with stirring at 40 ° C. for 16 hours and then at 60 ° C. for 24 hours. On cooling, the mixture was acidified with 1 mL concentrated HCl and transferred to a separatory funnel. Collect the oil that has settled to the bottom, and CH the aqueous layer.
_It was extracted 3 times with 40 mL each of ₂ Cl ₂ . The oil and the combined extracts were dried (MgSO _4), filtered, and concentrated to an oil 23.5 g. Biscyanoethyl ether was subjected to Khugelrhor distillation at 110 ° C for 0.25 torr.
removed by r. The pot residue was crystallized from H ₂ O 1 L to give 8.4
3 g (41%) of compound 21 were obtained as white needles: m.
p. 42.5 ° C [( Macromolecules 1991, 24, 1443-1444.) reported 39-40 ° C]; ¹ H NMR (CDCl ₃ ) d 2.61
(t, J = 6Hz, 8H), 3.50 (s, 8H), 3.6 (t, J = 6Hz, 8
H).

<Compound 22- [Tetrakis- (2-carboxyethoxymethyl) methane]> 5.0 g (14.35 mmol) of compound
A solution of 21 in concentrated HCl (21.5 mL) was stirred at 75 ° C. for 3 hours. During this time, a white precipitate formed. The aqueous HCl solution was removed under reduced pressure and the mixture was concentrated twice from H ₂ O (25 mL each). The resulting solid material, 9.68 g, was loaded onto a column with an inner diameter of 45 mm containing a bed of DOW-1-X2 resin in the form of the hydroxide of 16.5 cm and the column was eluted with 200 mL of H ₂ O followed by 1N HCl. . TLC
The product-containing fractions (80/20/1 CH ₃ CN / H ₂ O / HOAc) were concentrated to give 1.21 g (21%) of 22 as an oil: ¹ HNMR (D ₂ O) d 2.46 (t, J = 6Hz, 8H), 3.22 (s,
8H), 3.55 (t, J = 6Hz, 8H).

<Compound 23> Thionyl chloride 3.71 mL (6.06
g, 50.8 mmol) to 1.12 g (2.85 mmol) of compound22THF
(7.0 mL) added to the solution. Stir this mixture at room temperature for 3 hours
And the solvent was removed under reduced pressure. Crude acid chloride
Was dissolved in THF (7 mL). The solution is then Et ₃N 2.1
2 mL (1.54 g, 15.24 mmol) was added. N this mixture₂Below
Stir and cool to 0 ° C. 3.60 g (12.74 mmol)
CompoundFourTHF solution (5 mL) was added over 1 minute. Cooling bath
Was removed, and the mixture was stirred at room temperature for 5.5 hours, then
, Partitioned between 1 N HCl (25 mL) and 4 EtOAc (25 mL each).
The EtOAc layers were combined, washed with brine, dried (MgSO 4_Four),
Filtered and concentrated to give 3.46 g of viscous oil.
Chromatography on silica gel (95/5 CH₂Cl₂/ MeOH)
Therefore, 1.26 g (30%) of the compound23To
Obtained as a viscous oil:¹H NMR (CDCl₃) d 2.40 (t,
8H), 3.29 (s, 8H), 3.35 (m, 16H), 3.48-3.77 (m, 48
H), 5.12 (s, 8H), 5.60 (bd, 4H), 6.85 (bd, 4H), 7.
34 (s, 20H).

<Compound 37> 4.0 mL of cyclohexene and 83 mg of 10% Pd carbon were added under N _{2 to} 142 mg (0.093 mmol).
Compound 23 in EtOH (8.4 mL) was added. The mixture was refluxed with stirring in a 90 ° C. oil bath for 3 hours, and when cool, filtered through diatomaceous earth to wash the filter and flask with CH ₂ Cl ₂ . The filtrate is concentrated to 70 mg
(78%) of compound 37 was obtained as an oil: ¹ H NMR (CDC
l ₃ ) d 2.90 (t, 8H), 3.33 (s, 8H), 3.45 (t, 8H), 3.
52-3.73 (m, 48H).

<Compound 24> 40 mg (0.48 mmol) of NaHCO ₃ and 104 mg (0.40 mmol) of Compound 10 were added to 70 mg (0.098 mmol)
Compound 37 from above was added to a solution of dioxane (2 mL) and H ₂ O (0.67 mL). The mixture was stirred at room temperature for 17 hours and 1N
Add 0.5 mL H ₂ SO _{4 to} bring the pH to 4. Concentrate the mixture,
The concentrate was then purified by chromatography on G-10 Sephadex® (MeOH). Fractions containing product were concentrated under reduced pressure to give 91 mg of oil. By purification of the crude product 36 mg by HPLC (C ₁₈ , gradient 20/80 / 0.1 to 35/65 / 0.1 CH ₃ CN / H ₂ O / CF ₃ CO ₂ H), 19 mg (4
4%) of compound 24 was obtained as an oil: ¹ H NMR (CDCl ₃ ).
d 2.50 (t, 8H), 3.31 (s, 8H), 3.36-3.72 (m, 56H),
3.91 (s, 8H); ¹³ C NMR (CDCl ₃ ) d 28.8, 36.5, 39.7, 4
0.0, 67.2, 69.3, 69.5, 70.3, 166.6, 173.0. MS (FAB)
m / e (relative intensity) MH ⁺ [1425 (15), 1427 (63), 1429 (75),
1431 (64), 1433 (12)], 577 (100).

<Compound 25a- [Bis-tosylate of PEG ₃₃₅₀ ]> 6.47 mL of pyridine was dried by azeotropic distillation (toluene). CH ₂ Cl ₂ 4
Added to the solution in 0 mL. The solution is placed under nitrogen and 0
Cooled to ° C. Tosyl chloride 7.63 g (40 mmol) CH ₂ Cl ₂ (40
(mL) solution was added over 25 minutes. The cooling bath was removed and the mixture was stirred at room temperature for 16 hours. Mix the mixture with 1N HCl 80m
Shake with L and CH ₂ Cl ₂ containing emulsion
The layers were washed with H ₂ O 100 mL. The CH ₂ Cl ₂ layer was dried (MgS
O ₄ ), filtered and concentrated. This residue was added to CH ₂ Cl ₂ / Et.
Crystallization from ₂ O gave 16.82 g (92%) of compound 25a as a white solid: ¹ H NMR (CDCl ₃ ) d 2.50 (s, 6H),
3.48 (t, J = 5Hz, 4H), 3.55-3.77 (m, greater than 600H,
Integral too large to be accurate), 3.83 (t, J = 5Hz, 4
H), 7.44 (d, J = 7Hz, 4H), 7.94 (d, J = 7Hz, 4H).

<Compound 26a- [diazide-PEG ₃₃₅₀ ]> 1
0.83 g (2.96 mmol) of compound 25a and NaN ₃ 1.92 g (29.6 mmo
A solution of l) in DMF (30 mL) was heated under N ₂ in a 120 ° C. oil bath for 3 hours. Upon cooling, the mixture was added to H ₂ O (100 mL) and CH ₂ Cl ₂
(100 mL) and divided. The CH ₂ Cl ₂ layer was diluted to 200 mL with CH ₂ Cl ₂ and washed with 100 mL 1N HCl, dried (Na ₂ SO 4
₄ ), filtered and concentrated. The waxy solid obtained was recrystallized (CH ₂ Cl ₂ / Et ₂ O) and the solid obtained was chromatographed on silica gel (gradient 98 / 2-95).
Further purification with / 5 CH ₂ Cl ₂ / MeOH) gave 4.75 g (47%) of compound 26a as a waxy solid: TLC Rf 0.41 (9
/ 1CH ₂ Cl ₂ ); ¹ H NMR (CDCl ₃ ) d 3.35 (t, J = 5Hz, 4H),
3.44 (t, J = 5Hz, 2H), 3.54-3.77 (m, about 300H, integral value too large to be accurate), 3.79 (t, J = 5Hz, 2H).

<Compound 27a- [diamino-PEG ₃₃₅₀ ]> 10
473 mg of% Pd carbon (Aldrich) was added to a solution of 4.75 g (1.39 mmol) of compound 26a in EtOH (140 mL). 6 this mixture
Shake for 30 hours under 0 psi H ₂ . The reaction was incomplete (T
LC, 9/1 CH ₂ Cl ₂ / MeOH) for an additional 10% Pd carbon 473m
g was added and the mixture was shaken under 60 psi H ₂ for a further 5 hours. The mixture was then filtered through diatomaceous earth, concentrated under reduced pressure and the concentrate crystallized (CH ₂ Cl _2
2 / Et ₂ O), 4.03 g (86%) of compound 27a was obtained as a white solid: ¹ H NMR (CDCl ₃ ) d 2.92 (t, 4H), 3.49 (t,
2H), 3.66 (t, 4H), 3.67 (m, about 300H, integral value too large to be accurate), 3.86 (t, 2H).

<Compound 28- [N-hydroxysuccinimidyl ester of compound 7]> 596 mg (2.89 mmol) of dicyclohexylcarbodiimide and 1.84 g (2.41 mmol) of compound
7 in NHS 278 mg (2.41 mmol) in THF (12 mL) under N _{2 at} 0 ° C.
Added in. The cooling bath was removed and the mixture was stirred at room temperature for 16 hours. 250 μL of acetic acid was added to this mixture. At room temperature
Stirring was continued for 1 hour. The mixture was then left in the freezer for 2 hours. Solids were removed by filtration and the filtrate was concentrated to give 2.27 g (110%) of crude compound 28 as a viscous oil. Since it is difficult to purify the compound 28 so as not to decompose, the compound 28 was used directly to acylate the diamino-PEG.

<Compound 29a> A solution of 900 mg (1.05 mmol) of Compound 28 in dioxane (4.68 mL) was added to 877 mg (0.26 mmol).
Of compound 27a and 176 mg (2.10 mmol) of NaHCO ₃ in H ₂ O (3.12
(mL) solution was added at 0 ° C. The mixture was stirred for 2 hours then partitioned between 1N HCl (25 mL) and two CH ₂ Cl ₂ (25 mL each). The CH ₂ Cl ₂ layers were combined, dried (Na ₂ SO ₄ ), filtered and concentrated to give a viscous oil. Silica gel chromatography (gradient, 95/5 to 87/13 CH ₂ Cl ₂ / MeO
H) to yield 695 mg (55%) of compound 2
9a was obtained as a waxy solid: ¹ H NMR (CDCl ₃ ) d
2.55 (bd, 8H), 3.39 (m, 16H), 3.44-3.72 (m, approx.
432H, integral too large to be accurate), 3.89 (s, 4H),
4.03 (s, 4H), 5.09 (s, 8H), 7.36 (s, 20H).

<Compound 38a> 7.1 mL of cyclohexene
Was added to 688 mg (0.142 mmol) of the compound 29a in EtOH (14.2 mL) under N ₂ . 284 mg of 10% Pd on carbon was added and the resulting mixture was refluxed for 2 hours. On cooling, the mixture was filtered through diatomaceous earth with EtOH and the filtrate concentrated under reduced pressure to give 550 mg (90%) of compound 38a as a waxy solid: ¹ H NMR (CDCl ₃ ) d 2.58 ( m, 8H), 2.93 (m,
8H), 3.38-3.76 (m, about 550H), 4.00 (s, 4H), 4.1
3 (s, 4H).

<Compound 30a> A solution of 268 mg (1.04 mmol) of Compound 10 in dioxane (4.65 mL) was added to 550 mg (0.13 mmol).
Compound 38a of Example 1 and 175 mg (2.08 mmol) of NaHCO ₃ and H ₂ O (3.11
(mL) solution was added at 0 ° C. The mixture was stirred for 20 hours and partitioned between 1N H ₂ SO ₄ (50 mL) and 2 CH ₂ Cl ₂ (50 mL each). The CH ₂ Cl ₂ layers were combined, dried (Na ₂ SO ₄ ), filtered and concentrated to give an oil. By purification with G-10 Sephadex® chromatography (MeOH),
An amorphous solid was obtained. Crystallize this solid (EtOH /
Et ₂ O), 378 mg (61%) of compound 30a was obtained as a white solid: ¹ H NMR (CDCl ₃ ) d 2.59 (bds, 8H), 3.38-3.82.
(m, about 500H, integral value too large to be accurate), 3.
88 (s, 8H), 3.98 (s, 4H), 4.10 (s, 4H); Bromoacetyl assay ( European Journal of Biochemistry , 1984,
140 , 63-71): Calculated value, 0.84mmol / g; Measured value, 0.50mmol / g
g.

<Compound 25b- [Bis-tosylate of PEG ₈₀₀₀ ]> 2.3 mL (16.5 mmol) of triethylamine, then TsCl
3.15 g (16.5 mmol) was dried by azeotropic distillation (toluene) PEG ₈₀₀₀ (Aldrich, average molecular weight 8000 g / mmol) 12.0 g
(1.5 mmol) was added to a solution of CH ₂ Cl _{2 in} 30 mL. The mixture was stirred at room temperature for 18 hours and 4N with 1N HCl (50 mL each).
Extracted once and then once with saturated NaCl solution (50 mL). CH ₂ Cl ₂
The layers were dried (Na ₂ SO ₄ ), filtered, and concentrated under reduced pressure to give a waxy solid. Recrystallization (CH ₂ Cl ₂ / Et ₂ O) gave 11.0 g (92%) of compound 25b as a white solid: ¹ H NMR (CDCl ₃ ) d 2.38 (s, 6H), 3.40-3.89. (m,
800H, integral value too large to be accurate), 4.14
(m, 4H), 7.34 (d, J = 8.2Hz, 4H), 7.79 (d, J = 8.2Hz, 4
H).

<Compound 26b- [PEG ₈₀₀₀ diazide]> N
aN ₃ 1.86 g (28.6 mmol) was added to 10.8 g (1.3 mmol) of the compound 25
b ) in dry DMF (30 mL) solution. Add this mixture under N ₂ to 1
Heated at 20 ° C. for 2.5 hours. Upon cooling, this mixture was added to CH ₂ Cl _2.
Partitioned between ₂ (240 mL) and three 0.5 N HCl (50 mL each). CH ₂ Cl
_{The two} layers were washed with saturated NaCl solution (50 mL), dried (Na ₂ SO ₄ ),
Filtered and concentrated to give a solid. Chromatography on silica gel (gradient 2/98 to 6/94 MeOH / CH
₂ Cl ₂ ) and recrystallize the purified product (MeOH / E
t ₂ O) gave 6.95 g (66%) of compound 26b as a white solid: TLC (Rf = 0.33, 12/88 MeOH / CH ₂ Cl _2
2 ); ¹ H NMR (CDCl ₃ ) 3.39-3.86 (m).

<Compound 27b- [diamino-PEG ₈₀₀₀ ]> 6.
9 g (0.86 mmol) of compound 26b Me saturated with ammonia
The OH (150 mL) solution was purged with nitrogen. 10% Pd carbon 1.
5 g was added and the mixture was shaken under 65 psi H ₂ . After 20 hours, TLC analysis showed the reaction was incomplete. As a result, 200 mg of 10% Pd carbon was added and shaking was continued for another 20 hours under 65 psi H ₂ .
The mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure. The resulting waxy solid was recrystallized (MeO
H / Et ₂ O), 6.0 g (89%) of compound 27b was obtained as a white solid: ¹ H NMR (CDCl ₃ ) d 2.96 (t, J = 5.1 Hz, 4H), 3.
40-3.89 (m, about 700H, integral too large to be accurate).

<Compound 29b> 221 mg (2.63 mmol) of NaHCO ₃
Was added to a solution of 3.0 g (0.375 mmol) of compound 27b in water (10 mL) and dioxane (3 mL). Then dioxane (10
1.3 g (1.51 mmol) of compound 28 dissolved in (mL) was added.
The mixture was stirred for 24 hours, then 0.5N HCl (40 mL) was added. The mixture was extracted 4 times with CH ₂ Cl ₂ (25 mL each). C
The H ₂ Cl ₂ layers were combined, dried (MgSO ₄ ), filtered and concentrated to give an oil. Crystallization from MeOH / Et ₂ O gave 2.0 g (58%) of compound 29b : ¹ H NMR (CDC
l ₃ ) d 2.52 (m, 8H), 3.40-3.64 (m, about 700H, integral value too large to be accurate), 3.89 (s, 4H), 4.02 (s, 4
H), 5.09 (s, 8H), 7.35 (s, 20H).

<Compound 38b> 10% Pd carbon 123 mg
600 mg (0.063 mmol) of compound 29b in absolute EtOH (5 mL)
And a solution of cyclohexene (2.5 mL) were added under nitrogen. The mixture was refluxed under nitrogen for 2 hours. The reaction mixture was filtered through diatomaceous earth and evaporated to 54%.
Obtained 9 mg (97%) of compound 38b as a white solid: ¹ H
NMR (CDCl ₃ ) d 2.58 (m, 8H), 2.90 (m, 8H), 3.39-3.
70 (m, about 700H, integral value too large to be accurate),
4.05 (s, 4H), 4.15 (s, 4H).

<Compound 30b> NaHCO ₃ 100 mg (1.2 mmo
l), followed by 84 mg (0.32 mmol) of compound 10 , 529 mg (0.05
9 mmol) of compound 38b was added to a solution of dioxane (2 mL) and water (5 mL). After stirring for 12 hours, the reaction was acidified with 1N H ₂ SO ₄ and extracted 4 times with CHCl ₃ (40 mL each). CHCl
The combined _three layers, dried (MgSO _4), filtered, and concentrated to give a residue of semisolid 503 mg. This residue is G-1
Purified by chromatography on Sephadex® (MeOH) and crystallized (MeOH / Et ₂ O / hexane), 215 m
g (39%) of compound 30b was obtained as a white solid: ¹ HN
MR (CDCl ₃ ) d 2.58 (m, 8H), 3.35-3.70 (m, about 700
H, integral too large to be accurate), 3.89 (s, 8H), 4.
01 (s, 4H), 4.16 (s, 4H); Bromoacetyl assay ( Europ
ean Journal of Biochemistry , 1984, 140 , 63-71): Calculated value, 0.42 mmol / g; Found value, 0.27 mmol / g.

<Compound 31- [PEG ₃₃₅₀ -bis-chloroformate]> 2 drops of dry pyridine and then 125 mg of triphosgene
(0.418 mmol) was added to a solution of 1.0 g (0.249 mmol) of polyethylene glycol (JT Baker, 3350 g / mol average molecular weight) dried in CH ₂ Cl ₂ by azeotropic distillation (toluene). The mixture was stirred at room temperature for 20 hours and the solvent was evaporated under reduced pressure to give 1.0 g (100%) of compound 3
1 was obtained as a white solid: ¹ H NMR (CDCl ₃ ) d 3.40-
3.65 (m, about 300H, integral value too large to be accurate),
3.77 (m, 4H), 4.46 (m, 4H).

<Compound 32> 1.0 g (0.25 mmol) of Compound 3
1 of 5: 1 CH ₂ Cl ₂ / dioxane (12 mL) solution, 600 mg (1.0 mm
ol) compound 18 dioxane (10 mL) and pyridine (1.5 mL)
And was added dropwise to the solution at 50 ° C. The resulting cloudy solution was stirred for 72 hours. CH ₂ Cl ₂ (25 mL) was added, then the mixture was filtered. The filtrate was evaporated and the semi-solid residue was purified by chromatography on G-10 Sephadex®. The solid obtained was crystallized (CH ₂ Cl ₂ / Et
₂ O), 829 mg (75%) of compound 32 was obtained as a slightly yellow solid: ¹ H NMR (CDCl ₃ ) d 1.30 (m, 8H), 1.40 (m, 8H),
1.61 (m, 8H), 2.18 (m, 8H), 3.17 (m, 8H), 3.40 (m,
16H), 3.62 (m, about 300H, integral value too large to be accurate), 4.15 (m, 4H), 5.07 (s, 8H), 7.33 (m, 20
H).

<Compound 39> 10 mg of 10% Pd carbon, 3
A solution of 00 mg (0.065 mmol) of compound 32 in absolute EtOH (5 mL) and cyclohexene (2 mL) was added under nitrogen. The mixture was refluxed under nitrogen for 2 hours. The mixture was filtered through diatomaceous earth and the solvent was evaporated to give 237 mg (90%) of compound 39 as a white solid: ¹ H NMR (CDCl ₃ ) d
1.37 (m, 8H), 1.48 (m, 8H), 1.65 (m, 8H), 2.21 (m,
8H), 2.50 (m, 8H), 3.39 (m, 16H), 3.64 (m, approx. 3
00H, integral too large to be accurate), 4.19 (m, 4H).

<Compound 33> 125 mg (0.67 mmol) of NaHCO ₃ and 115 mg (0.44 mmol) of compound 10 were added to 225 mg (0.055 mmo).
l) of 39 was added to a solution of dioxane (5 mL) and water (5 mL). The resulting yellow solution was stirred at room temperature for 12 hours. The solution was then extracted 3 times with CH ₂ Cl ₂ (30 mL each). The aqueous layer was acidified with 1 NH ₂ SO ₄ and extracted 3 times with CH ₂ Cl ₂ (30 mL each). The CH ₂ Cl ₂ layers were combined, dried (MgSO ₄ ), filtered and concentrated to give a yellow oil. G-10 Sepha
Purify by chromatography on dex® (MeOH) and recrystallize the resulting oil (EtOH / Et ₂ O) to give 1
82 mg (73%) of compound 33 was obtained as a white solid: ¹ H
NMR (CDCl ₃ ) d 1.35 (m, 8H), 1.55 (m, 8H), 1.65 (m,
8H), 2.22 (m, 8H), 3.28 (m, 8H), 3.42 (m, 16H), 3.
50-3.64 (m, about 300H, integral value too large to be accurate), 3.87 (s, 8H), 4.18 (m, 4H); Bromoacetyl assay ( European Journal of Biochemistry , 1984, 140 , 63
-71): Calculated value, 0.87 mmol / g; Actual value, 0.73 mmol / g. Analytical value: Calculated value for C ₁₉₁ H ₃₇₅ O ₈₇ N ₁₀ Br ₄ : C, 50.84; H,
8.33; N, 3.09; Br, 7.05. Found: C, 51.98; H, 8.3
4; N, 2.45; Br, 10.19.

<Compound 40- [4-Nitrophenyliodoacetate]> 5.15 g (2
EtOAc with 5 mmol) and 2.92 g (2.92 mmol) 4-nitrophenol
The (100 mL) solution was added to a solution of 3.72 g (20 mmol) of iodoacetic acid at 0 ° C. The mixture was stirred at 0 ° C. for 1 hour and room temperature for 2 hours. Solids were removed by filtration and the filtrate was concentrated under reduced pressure. The resulting yellow solid was recrystallized (EtOAc / hexane / trace HOAc) to give 4.82 g (78%) of compound 40 as a tan solid: ¹ H NMR (CDCl ₃ ).
d 4.00 (s, 2H), 7.39 (d, 2H), 8.40 (m, 2H).

<Compound 41> NaHCO ₃ 103 mg (1.22 mmol),
Then, 211 mg (0.692 mmol) of compound 40 was added to 110 mg (0.104 m
mol) of compound 34 in a solution of dioxane (5 mL) and H ₂ O (5 mL). The mixture was stirred for 18 hours then concentrated under reduced pressure. 140 mg (87%) of compound 4 by purification by Sephadex® chromatography (MeOH).
1 was obtained as an oil. Analytical samples were prepared by preparative HPLC (C ₁₈ , gradient 20/80 / 0.1 to 25/75 / 0.1 CH ₃ CN / H ₂ O / TFA at 225 nm over 60 min): ¹ H NMR (CDCl ₃ ) d
2.59 (m, 4H), 2.65 (m, 4H), 3.44-3.62 (m, 60H), 3.
77 (s, 4H), 3.78 (s, 4H), 4.02 (s, 4H), 4.21 (s, 4
H).

<Compound 42> 145 mg (0.935 mmol) of N-methoxycarbonylmaleimide and 171 mg (0.161 mmol) of compound
34 dioxane (8 mL) and saturated NaHCO ₃ solution (2 mL) and H ₂ O (2 m
L) and the solution with vigorous stirring at 0 ° C ( The
Practice of Peptide Synthesis , M. Bodansky and A.
Bodansky, Springer-Verlag, New York, 1984, 29
-Page 31. Keller, O., Rudinger, J. Helv. Chim, Acta 1
975, 58 , 531.). After 15 minutes, 25 mL of dioxane was added, the cooling bath was removed, and stirring was continued for 45 minutes at room temperature. The mixture was extracted twice with CHCl ₃ (30 mL each), and the CHCl ₃ layers were combined, dried (MgSO ₄ ), filtered and concentrated.
Got the oil. Purification by chromatography on G-10 Sephadex® (MeOH) gave 103 mg (45%) of compound 42 as an oil. HPLC for preparative analysis
(C ₁₈ , gradient 20/80 / 0.1 to 25/75 / 0.1 CH ₃ CN / H ₂ O /
Prepared at 225 nm) over 65 min TFA to give an oil: ¹ H NMR (CDCl ₃ ) d 2.57 (m, 4H), 2.67 (m, 4H), 3.
42-3.65 (m, 52H), 3.72 (m, 8H), 4.03 (s, 4H), 4.17
(s, 4H), 6.74 (s, 4H), 6.75 (s, 4H).

<Compound 43- [hydroxymethyl-tris
-(2-Cyanoethoxymethyl) methane]> KOH 0.30g (5.41mm
ol), and then 23 mL of acrylonitrile (18.6 g, 350 mmol)
Was added to a solution of 6.8 g (50 mmol) pentaerythritol in H ₂ O (50 mL). The mixture was stirred at room temperature for 16 hours and concentrated HCl
It was acidified with 1.5 mL of solution and extracted twice with CH ₂ Cl ₂ (50 mL each). The CH ₂ Cl ₂ layers were combined, dried (MgSO ₄ ),
Filtered and concentrated to give 16.97 g of liquid. Purification by chromatography on silica gel (EtOAc) gave 8.49 g (51%) of compound 43 as a viscous oil: TLC, Rf = 0.15 (EtOAc); ¹ H NMR (CDCl ₃ ) d 2.62 ( t,
6H), 3.54 (s, 6H), 3.68 (t, 6H), 3.70 (s, 2H).

<Compound 44- [hydroxymethyl-tris]
-(2-Carboxymethylethoxymethyl) methane]> 78 mL of a MeOH solution saturated with HCl was added to 5.45 g (15.6 mmol) of compound 4
Added to 3 . The mixture was heated at reflux for 1 hour and, on cooling, partitioned between H ₂ O (100 mL) and 4 Et ₂ O (100 mL each). The combined Et ₂ O layers were washed sequentially with saturated NaHCO ₃ solution (100 mL) and saturated NaCl solution (100 mL), dried (MgSO ₄ ),
Filtered and concentrated to give 4.74 g viscous liquid.
Purification by chromatography on silica gel gave 3.05 g (50%) of compound 44 as an oil: TLC,
Rf = 0.27 (80/20 EtOAc / hexane); ¹ H NMR (CDCl ₃ ) d
2.58 (t, 6H), 3.43 (s, 6H), 3.61 (s, 2H), 3.69 (t,
6H), 3.70 (s, 9H); ¹³ C NMR (CDCl ₃ ) d 34.8, 44.9, 5
1.6, 65.2, 66.9, 71.0, 172.1.

<Compound 45> 560 mg (1.4 mmol) of compound 4
4 and 1.69 g (6.0 mmol) of compound 4 at 150 ° C.
Heated under nitrogen for hours. This mixture was added to EtOAc (50 mL) and 1N.
Partitioned with HCl (25 mL) and the HCl layer was extracted with CH ₂ Cl ₂ (25 mL). The extracted layers of EtOAc and CH ₂ Cl ₂ were combined and saturated with N 2.
Washed with aHCO ₃ solution, dried (K ₂ CO ₃ ), filtered and concentrated to give a viscous residue. Purification by chromatography on silica gel (gradient 95/5 to 90/10 CH ₂ Cl ₂ / MeOH) gave 300 mg (19%) of compound 45 as a viscous oil: TLC, Rf = 0.24 (90 / 10 CH ₂ Cl ₂ / MeOH);
¹ H NMR (CDCl ₃ ) d 2.40 (t, 6H), 3.38 (s, 6H), 3.39-
3.48 (m, 12H), 3.52-3.67 (m, 32H), 5.13 (s, 6H), 5.
62 (bd s, 3H), 6.80 (bd s, 3H), 7.40 (s, 15H).

<Compound 46> 104 mg of 10% Pd carbon, 3
To a solution of 08 mg (0.269 mmol) of compound 45 in EtOH (10.4 mL) and cyclohexene (5.2 mL) was added under nitrogen. Attach a reflux condenser and add the mixture to an oil bath at 85 ° C. for 1.5 hours.
Heated for hours. On cooling, the mixture was filtered through diatomaceous earth and the filtrate concentrated to give 177 mg of residue. This residue was partially dissolved in 5.98 mL of dioxane.
The resulting mixture was added to 386 mg (1.49 mmol) of compound 10 , which was then added with 251 mg of NaHCO ₃ (2.99 mmol) of H ₂ O (3.99 m
L) added to the solution. The resulting mixture was stirred under nitrogen for 18 hours and partitioned between 1N HCl (25 mL) and 3 CH ₂ Cl ₂ (25 mL each). The aqueous layer was washed 3 times with 3/1 CH ₂ Cl ₂ / MeOH (25 mL each) and
It was extracted 3 times with 1/1 CH ₂ Cl ₂ / MeOH (25 mL each). Discard the first two CH ₂ Cl ₂ extracts and combine the remaining extracts, dry (Na ₂ SO ₄ ), filter and concentrate to 102 m
g viscous oil was obtained. By HPLC (C ₁₈ , 23/77 / 0.1 CH ₃ CN
/ H ₂ O / CF ₃ CO ₂ H, 234 nm detection)
Obtained g (14%) of compound 46 as a viscous oil: ¹ H NMR
(CDCl ₃ ) d 2.48 (t, 6H), 3.40 (s, 6H), 3.44-3.54
(m, 14H), 3.56-3.62 (m, 12H), 3.63 (s, 12H), 3.67
(t, 6H), 3.91 (s, 6H), 6.90 (t, 3H), 7.10 (t, 3H);
MS (FAB) m / e (relative intensity) MH ⁺ [1103 (17), 1105 (42),
1107 (41), 1109 (18)], MNa ⁺ [1125 (38), 1127 (100), 11
29 (99), 1131 (39)].

<Compound 47- [S- (6-hydroxyhexyl) isothiouronium chloride]> 11.1 g (14
6mmol) to 6-chlorohexanol 16.6mL (20.0g, 146mmol)
Solution in ethanol (49 mL) and add this mixture to
Reflux for hours. The mixture was cooled to 0 ° C. and the product crystallized. The crystals were collected by vacuum filtration and dried to give 28.4 g (92%) of compound 47 as a white solid: mp 122-124 ° C; ¹ H NMR (DMSO) d 1.40 (m, 4
H), 1.65 (m, 2H), 3.21 (t, 2H), 3.41 (t, 2H), 9.27
And 9.33 (overlapping wide singlets, 4H); analytic value: calculated value for C ₇ H ₁₇ ClN ₂ OS: C, 39.51; H, 8.06;
N, 13.17; S, 15.07. Found: C, 39.69; H, 8.00; N,
13.01; S, 15.16.

<Compound 48- [6-mercaptohexane-1-
All]> NaOH pellets 9.25 g were added to a solution of 17.8 mg (83.6 mmol) of compound 47 in H ₂ O (120 mL) and EtOH (120 mL). The mixture was refluxed for 4 hours. Approximately 75 mL of mixture
Was carefully concentrated to and the concentrate was purified by vacuum distillation to give 7.4 g (66%) of compound 48 : bp 95
-105 ° C (5 mmHg); ¹ H NMR (CDCl ₃ ) 1.41 (m, 9H), 2.59 (d
t, 2H), 3.69 (t, 3H overlapping with brd s); ¹³ C NMR (CDCl
₃ ) d 24.5, 25.2, 28.0, 32.5, 33.9, 62.7; analytical value: C
Calculated for ₆ H ₁₄ OS: C, 53.68; H, 10.51; S, 23.8
9. Found: C, 53.35; H, 10.72; S, 23.60.

<Compound 49- [bis- (6-hydroxyhexyl) disulfide]> I ₂ 4.02 g (15.8 mmol) of MeOH (90 mL)
The solution was treated with 4.26 g (31.7 mmol) of compound 48 in MeOH (10 mL) and E
A solution with t ₃ N (13.7 mL (9.97 g, 98.5 mmol)) was added dropwise under N ₂ atmosphere over 10 minutes and cooled in an ice bath. The cooling bath was removed and the mixture was stirred at ambient temperature for 4 hours. The mixture was concentrated on a rotary evaporator,
And silica gel chromatography (1: 1 hexane / Et
OAC) to give 3.12 g (73%) of compound 49 as a pale yellow solid: TLC R _f 0.18 (1: 1 hexane / EtOAc);
mp 38-48 ° C; ¹ H NMR (CDCl ₃ ) 1.15-2.20 (m, 16H), 2.73
(t, 4H), 3.70 ( t, 4H); _{analysis: C 12 H 26 S 2 0} 2 Calculated for:. C, 54.09; H, 9.84; S, 24.06 Found: C, 5
4.85, H, 9.86; S, 24.11.

<Compound 50- [mono-O- (4 ′, 4 ″ -dimethoxytriphenylmethyl) -bis- (6-hydroxyhexyl)
Disulfide]>4,4'-dimethoxytriphenylmethyl chloride 3.97 g (11.7 mmol), 3.12 g (11.7 mmol) of compound
49 was added to a solution of pyridine (45 mL). The mixture was stirred at ambient temperature for 16 hours. Most of the pyridine was removed on a rotary evaporator and the residue was saturated with NaHCO _3.
Partitioned between solution (100 mL) and EtOAc (100 mL). Saturate the EtOAc layer
Washed with 50 mL of NaCl solution, dried (Na ₂ SO ₄ ), filtered and concentrated to give an oil. 2.84 by purifying by silica gel chromatography (9: 1 CH ₂ Cl ₂ / EtOAc).
g (43%) of compound 50 was obtained as a viscous oil: TLC R _f
0.35 (9: 1 CH ₂ Cl ₂ / EtOAc); ¹ H NMR (CDCl ₃ ) 1.41 (m,
8H). 1.65 (m, 8H), 2.70 (two overlapping triplets, 4H), 3.08 (t, 2H), 3.65 (t, 2H), 3.81 (s, 6H),
6.85 (d, 4H), 7.32 (m, 7H), 7.47 (d, 2H); HRMS (F
AB, M +), C ₃₃ H ₄₄ 0 ₄ S ₂ Calculated: 568.2681. Found: 568.2665.

<Compound 5- [O- [14- (4 ', 4''-dimethoxytriphenylmethoxy) -7,8-dithiotetradecyl] -O-
(2-Cyanoethyl) -N, N-diisopropylphosphoramidite]> O-Cyanoethyl-N, N, N ', N'-tetra-isopropylphosphorodiamidite 458 mg (1.52 mmol) CH ₂ Cl ₂ (0.5 m
L) solution, 771 mg (1.36 mmol) of compound 50 and diisopropylammonium tetrazolide 116 mg (0.68 mmol) of CH ₂ Cl _2.
2 (6.8 mL) solution was added under N ₂ atmosphere. The mixture was stirred for 4 h, and was partitioned NaHCO ₃ and (25mL) CH ₂ Cl ₂ and (3 × 25mL). The CH ₂ Cl ₂ layers were combined, washed with saturated NaCl solution, dried (Na ₂ CO ₃ ), filtered and concentrated to an oil.
Purify by filtering through basic alumina (2 "plug) in a 25 mm column and eluting with 9: 1 CH ₂ Cl ₂ / Et ₃ N to give 831 mg (80%) of compound 51 as a viscous oil. Obtained as: ¹ H NMR (CDCl ₃ ) d 1.25 (m, 12H), 1.45 (m, 8H),
1.70 (m, 8H), 2.72 (m, 6H), 3.09 (t, 2H), 3.65 (m,
4H), 3.87 (s, 6H), 3.91 (m, 2H), 6.89 (d, 4H), 7.
35 (m, 7H), 7.49 (d, 2H); ³¹ P NMR (CDCl ₃ with 15% H ₃ PO ₄ as internal standard) 147,69; HRMS (FAB, MH +), C ₄₂
Calculated for H ₆₂ N ₂ O ₅ PS ₂ : 769.3839, Found: 769.38
53.

<Compound 52- [trityl-HAD alcohol]
> Trityl chloride 60 g (0.21 mol), 57 g (0.21 mol) of compound
49 was added to a solution of pyridine (60 mL). 100 this mixture
The mixture was stirred at ° C for 19 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was diluted with 300 mL methylene chloride and extracted with 200 mL saturated sodium bicarbonate.
The organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated to
Got the oil. 55 g (50%) of compound 5 by purification by silica gel chromatography (gradient 9: 1 hexane: ethyl acetate to 3: 1 hexane: ethyl acetate).
2 was obtained: ¹ H NMR (CDCl ₃ ) δ 1.38 (m, 8H), 1.63 (m,
8H), 2.66 (m, 4H), 3.04 (t, 2H), 3.62 (t, 2H), 7.
25 (m, 9H), 7.42 (m, 6H). HRMS (FAB, M +), C ₃₁ H ₄₀ O ₂ S
Calculated for: 508.2470. Found: 508.2482.

<Compound 53- [Trityl HAD phosphoramidite]> 10 g (19.7 mmol) of compound 52 and 6.3 mL (36.2 mmol) of diisopropylethylamine in methylene chloride (90 m
L) solution under argon at 0 ° C., 2-cyanoethyl-N, N-
4.5 mL of diisopropylchlorophosphoramidite (20.2 mm
ol) was added slowly. After stirring this mixture for 90 minutes, the reaction mixture was diluted with 100 mL of saturated sodium hydrogen carbonate 2 times each.
Extracted twice. The methylene chloride solution was dried over Na ₂ SO ₄ , filtered and concentrated to give an oil. Purified by basic alumina chromatography (75: 24: 1, hexane: ethyl acetate: triethylamine) to give 11.3 g.
(81%) of compound 53 was obtained as an oil: ¹ H NMR (CDC
l ₃ ) δ 1.18 (m, 12H), 1.37 (m, 8H), 1.62 (m, 8H),
2.6 (m, 6H), 3.04 (t, 2H), 3.60 (m, 4H), 3.82 (m,
2H), 7.26 (m, 6H), 7.44 (m, 9H). HRMS (FAB, MH +), C
Calculated for ₄₀ H ₅₈ N ₂ O ₃ PS ₂ : 709.3626. Found: 709.
3621.

<Compound 54- [O- (tert-butyldimethylsilyl) -5-hexenol]> 15.66 g (230 mmo) of imidazole
l) and tert-butyldimethylsilyl chloride 20.0 g (130 mm
ol), 5-hexen-1-ol 12.47 mL (10.4 g, 104 mmol)
Was added to the DMF (104 mL) solution. This mixture at ambient temperature
Stir for 4 h, then add EtOAc (200 mL) and saturated NaHCO ₃ solution (10
(0 mL). 100 mL of saturated NaHCO ₃ solution, saturated EtOAc layer
Wash with 100 mL of NaCl solution, dry (MgSO ₄ ), filter and concentrate to approximately 100 mL volume. 70.07 g (90%) of compound 54 was obtained by distillation under reduced pressure: bp 13
0-143 ℃ (100mmHg); ¹ H NMR (CDCl ₃ ) 0.11 (s, 6H), 0.9
5 (s, 9H), 1.48 (m, 2H), 1.57 (m, 2H), 2.11 (dt, 2
H), 3.66 (t, 2H), 5.03 (m, 2H), 5.86 (m, 1H); ¹³ CN
MR (CDCl ₃ ) -5.25, 18.40, 25.21, 26.01, 32.35, 33.6
0, 63.09, 114.40, 138.92; Analytical value: C ₁₂ H ₂₆ Calculated value for OSi: C, 67.22; H, 12.22. Actual value: C, 66.96; H,
12.16.

<Compound 55- [1-O- (tert-butyldimethylsilyl) -1,5,6-hexanetriol]> 9.86 g (46.0 mmo
To a solution of compound 54 in l) in acetone (92 mL) was added a solution of 6.46 g (55.2 mmol) of N-methylmorpholine oxide in H ₂ O (23 mL). To this mixture was added 443 μl of 2.5% OsO ₄ in tert-butyl alcohol (solution 360 mg, OsO ₄ 9.0 mg, 35 μmol) and 30% H ₂ O ₂ 50 μL. The mixture was stirred for 16 hours and a solution of 474 mg sodium dithionite in H ₂ O (14 mL) was added. After an additional 0.5 hours, the mixture was filtered through Celite. The filtrate was dried over MgSO ₄ and filtered through 1 ″ silica gel in 150 mL Buchner funnel, EtOAc 250 m
L was used for elution. Fractions containing product were concentrated to give 11.0 g (96%) of 55 as a viscous oil: TL
CR _f 0.2 (1: 1 hexane / EtOAc); ¹ H NMR (CDCl ₃ ) 0.05
(s, 6H), 0.89 (s, 9H), 1.25 (m, 4H), 1.55 (m, 2
H), 3.41 (dd, 2H), 3.62 (t, 2H), 3.71 (m, 1H); ¹³ C
_{NMR (CDCl 3) -5.23, 18.42} , 21.91, 26.02, 32.68, 3
2.81, 63.16, 66.74, 72.24; HRMS (FAB, MH +), C ₁₂ H ₂₉ O
Calculated for ₃ Si: 249.1886. Found: 249.1889.

<Compound 56- [5,6- (bis-O-benzoyl)]
-1-O- (tert-butyldimethylsilyl) -1,5,6-hexanetriol]> benzoyl chloride 6.18mL (7.48g, 53.2mmol)
Was added to a solution of 5.29 g (21.3 mmol) of 55 in pyridine (106 mL). The mixture was stirred for 18 hours and concentrated on a rotary evaporator. Mix the mixture with cold 1N HCl (100 mL) and E
Separated with tOAc (100 mL). The pH of the aqueous layer was checked to make sure it was acidic. The EtOAc layer was washed sequentially with 100 mL H ₂ O and 100 mL saturated NaCl, dried (MgSO ₄ ), filtered and concentrated to give 10.33 g (99%) of compound 56 as a viscous yellow oil. : TLC R _f 0.45 (1: 4 EtOAc / hexane); ¹ HNMR
(CDCl ₃ ) d 0.05 (s, 6H), 0.88 (s, 9H), 1.59 (m, 4
H), 1.85 (m, 2H), 3.14 (t, 2H), 4.49 (dd, 1H), 4.5
9 (dd, 1H), 5.54 (m, 1H), 7.45 (m, 4H), 7.58 (m, 2
H), 8.05 (m, 4H).

<Compound 57- [5,6- (bis-O-benzoyl)]
-1,5,6-hexanetriol]> 1N tetrabutylammonium fluoride 10.7mL (10.7mmol) in THF solution, 2.62g (5.3
6 mmol) of 56 in THF (10.9 mL) was added. The mixture was stirred for 16 hours. The mixture was saturated with NaHCO ₃ solution (25 mL) and Et.
Partitioned with OAc (3 x 25 mL). The combined EtOAc extracts were washed with saturated NaCl solution, dried (MgSO _4), filtered, and concentrated to give a viscous oil. This oil was purified by silica gel chromatography (1: 1 hexane / EtOAc) to give 823 mg (41%) of compound 57 as a viscous oil: R _f 0.14 (1: 1 hexane / EtOAc); ¹ H NMR (CDCl ₃ ) d
1.58 (m, 2H), 1.68 (m, 2H), 1.88 (m, 2H), 3.68 (t,
2H), 4.52 (dd, 1H), 4.62 (dd, 1H), 5.56 (m, 1H),
7.46 (m, 4H), 7.58 (m, 2H), 8.05 (m, 4H); ¹³ C NMR
(CDCl ₃ ) d 22.08, 31.20, 31.30, 32.88, 62.92, 66.17,
72.63, 128.93, 130.19, 130.57, 133.62, 166.72, 16
6.86; Calculated for HRMS (FAB, MH +), C ₂₀ H ₂₃ O ₅ : 34
3.1545. Found: 343.1553.

<Compound 58- [O- [5,6- (bis-O-benzoyloxy) -hexyl] -O- (2-cyanoethyl) -N, N-diisopropylphosphoramidite]> O-cyanoethyl-N , N,
N ', N'-Tetraisopropylphosphorodiamidite 989mg
CH ₂ Cl ₂ (2.0 mL) solution of (3.28 mmol), 1.02 g (2.98 mmol)
57 and diisopropylammonium tetrazolide 255mg
CH ₂ Cl ₂ (1.49 mmol) (prepared as a white solid by mixing diisopropylamine in acetonitrile with tetrazole in a 1: 1 molar ratio and concentrating).
(mL) solution. The mixture was stirred for 4 hours, then CH.
Partitioned between ₂ Cl ₂ (25 mL) and cold saturated NaHCO ₃ solution (25 mL). CH ₂ C
The ₁₂ layer was washed with saturated NaCl solution, dried (Na ₂ SO ₄ ), filtered and concentrated. Basic alumina in a 25 mm column
Filtered through (2 "plug) and purified by eluting with 9: 1 EtOAc / Et ₃ N to give 1.5 g (93%) of compound 58 as a viscous oil: ¹ H NMR (CDCl ₃ ) d 1.19 (m, 12H),
1.62 (m, 2H), 1.73 (m, 2H), 1.90 (m, 2H), 2.62 (d
d, 2H), 3.53-3.92 (m, 6H), 4.53 (dd, 1H), 4.62 (d
d, 1H), 5.58 (m, 1H), 7.48 (m, 4H), 7.60 (m, 2H),
8.09 (m, 4H); ³¹ P NMR (CDCl ₃ with 15% H ₃ PO ₄ as internal standard) d 148.2; HRMS (FAB, MH +), calculated for C ₂₉ H ₄₀ O ₆ N ₂ P: 543.2624. Found: 543.2626.

<Compound 59- [4 (iodoacetamido) benzoic acid]> Weltman, JK, 1983 Biotechniques 1: 148-1
This compound was prepared as described in 52. Briefly, 708 mg (2.0 mmol) iodoacetic anhydride was added to a solution of 137 mg (1.0 mmol) para-aminobenzoic acid in dioxane (10 mL). The mixture was stirred in the dark for 18 hours, and H ₂ O was added.
Partitioned between (25 mL) and EtOAc (25 mL). The EtOAc layer was washed with saturated NaCl solution, dried (MgSO _4), filtered, and concentrated to give a solid 797mg of pink. 4- (iodoacetamido) benzoic acid was obtained by recrystallization from hexane / EtOAc.
221 mg (72%) were obtained as a white solid: mp 220-230 ° C;
¹ H NMR (CDCl ₃ ) d 3.86 (s, 2H), 7.68 (d, 2H), 7.91
(d, 2H), 10.60 (s, 1H).

<Compound 60- [α, ω-bis- (N-2-aminoethylcarbamoyl) polyethylene glycol 4- (iodoacetamide) benzoyl derivative]> 188 mg (0.909 mmol) of dicyclohexylcarbodiimide was added to 4- (iodoacetamide). ) Benzoic acid 185 mg (0.606 mmol) and α, ω-bis- (N-2-
Aminoethylcarbamoyl) polyethylene glycol (Si
gma Chemical Co., St. Louis, MO., dried by azeotropic distillation with toluene) and added to a solution of 406 mg (0.121 mmol) in THF (2 mL). The mixture was stirred for 2 hours, then 6 drops of acetic acid were added. 10 mL CH ₂ Cl ₂ was added and the mixture was placed in the freezer for 30 minutes. The mixture was filtered to remove solids and the filtrate was concentrated to give a viscous residue. Silica gel chromatography (gradient 99/1 to 96/4 CH ₂ Cl ₂ / MeOH)
A solid was obtained by purification with. This solid is Me
Trituration with OH gave a pale yellowish white solid 292 mg: ¹ H
NMR (CDCl ₃ ) 3.48 (m, 8H), 3.63 (bd s, (CH ₂ CH ₂ O) _n ,
Too large to integrate), 3.98 (s, 4H), 4.18 (bd m, 4
H), 5.91 (bd m, 2H), 7.48 (bd m, 2H), 7.76 (d, 4
H), 7.88 (d, 4H), 9.38 (bdm, 2H): Iodoacetyl assay ( European Journal of Biochemistry 1984, 140 , 63
-71): Calculated, 0.46 mmol / g; Found, 0.37 mmol / g.

Example 3 Preparation of Active Bonding Platform Molecules and Complexes There are many ways to form a complex between a biological or chemical molecule and a bonding platform molecule. A particular method is to use a thiol attached to a biological or chemical molecule to nucleophilically react with a reactive "thiophyllic" group on a valency platform molecule to form a thioether bond. However, other combinations of reactive groups on the platform molecule and reactive groups on the biological or chemical molecule also share the biological or chemical molecule with the bond platform molecule. It can be used to bind binding. Table 1 lists some combinations of interactive groups. Which of the given methods is preferentially performed is determined by the nature of the biological or chemical molecule (solubility, the presence of other reactive groups, etc.).

[0211]

[Table 1]

The examples set forth below illustrate methods of synthesizing various valency platform molecules and methods of conjugating the molecules with biological or chemical molecules. These examples show how peptides and oligonucleotides are conjugated to valency platform molecules using the interactive groups in Table 1. In addition to peptides and oligonucleotides, other biological molecules (proteins, drugs, etc.) can also be conjugated to the valency platform molecule.

(Combination 1: Platform Thiol-Ligand Thiophyllic Group)

[0214]

[Chemical 29]

<Compound A> Compound 36 (861 mg, 1.0 mmo
l) and 252 mg (3.0 mmol) of NaHCO _{3 in} 1/1 dioxane
Dissolve in 20 mL of / H ₂ O. The mixture is cooled to 0 ° C and N-
Succinimidyl-S-acetylthioacetate (Prochem
Inc.) 1.16 g (5.0 mmol) in dioxane (40 mL) is added with stirring the mixture. After 1 hour, the mixture is extracted with CH ₂ Cl ₂ . The total extract is dried over MgSO ₄ , filtered and concentrated. The crude product is purified by silica gel chromatography to give A.

<Compound B-4 Platform Having Four Thiol Groups> A 732 mg (0.55 mmol) of DMSO (7.3 m
L) solution, buffered with helium at pH 10 (100 mM
Sodium carbonate, 10 mM NH ₂ OH) in 55 mL. The mixture is placed under N ₂ gas and stirred for 1 hour to give a solution of about 10 mM tetrathiol platform B.

<Compound X-Bromoacetylated peptide>
Peptides are synthesized by standard solid-phase method on Wang (p-alkoxybenzyl) resin using FMOC chemistry. FMOC (fluorenylmethoxycarbonyl) protected amino acids are sequentially added to the amino terminus. The final step involves conjugation of N-bromoacetylaminocaproic acid. The protecting group is removed and the peptide is removed from the resin with trifluoroacetic acid to give X , which is purified by preparative reverse phase HPLC.

<Peptide-Platform Complex, C
> About 10 mmol solution of tetrathiol platform B (in a buffer solution of pH 10) was added with bromoacetylated peptide X.
Add excess of DMSO solution. Peptide complex C is purified by preparative reverse phase HPLC.

(Combination 2: Platform amine-
Peptide active carboxylate)

[0220]

[Chemical 30]

<Compound Y-Peptide Having Active Carboxylate> Using a stable protecting group for TFA (benzyl ester for carboxyl group, CBZ for amino group), Wang
Peptides are synthesized on a (p-alkoxybenzyl) resin by the standard solid phase method. Amino acid residues are sequentially added to the amino terminus. Removal of the peptide from the resin by TFA yields a peptide with one free carboxyl group at the carboxy terminus and all other carboxyls and amines blocked. The protected peptide Y is purified by reverse phase HPLC.

<Peptide-Platform Complex D>
Compound Y (0.3 mmol) was dissolved in 1 mL of DMF, and in this solution,
0.3 mmol of diisopropylcarbodiimide and 0.3 mmol of HOBT (hydroxybenzotriazole) are added. Tetraamino platform 36 0.025 mmol
To DMF (1 mL) solution. When the reaction is complete, DMF is removed under reduced pressure to give the crude fully protected complex. The complex is dissolved in MeOH and the solution is placed in a Parr hydrogenator with 100 mg of 10% Pd carbon per gram of complex. The mixture is shaken under 60 psi H ₂ and the deprotected complex D is purified by preparative reverse phase HPLC.

(Combination 3: Platform amine-
Oligonucleotide aldehyde)

[0224]

[Chemical 31]

<Oligonucleotide-Platform Complex E> A 500 μL aliquot of NaIO ₄ (200 mM) solution (10
0 μmol) to ACT modified (CA) ₂₅ 1.0 g (total length 400 mg, ₂₅ μmo
l) in H ₂ O (19.5 mL) at 0 ° C. in the dark. The mixture was left at 0 ° C. for 40 minutes and 50 mL EtOH was added. The mixture is placed at -20 ° C for 30 minutes and centrifuged at 2000 RPM for 5 minutes. The supernatant is discarded and the remaining pellet is dried under reduced pressure. The pellet is dissolved in 3.3 mL H ₂ O and to the resulting solution is added a solution of 4.3 mg (0.005 mmol) 36 in 2.0 mL 100 mM sodium borate (pH 8.0). To the resulting solution, 250 μL (50 μmol) of a 200 mM solution of pyridine-borane complex (MeOH solution) is added, and the mixture is left at 37 ° C. for 4 days. This complex E can be purified by ion exchange chromatography.

(Combination 4: Platform Active Carboxylate-Ligand Amine)

[0227]

[Chemical 32]

<Compound F-4 Platform Having Four Carboxylic Acid Groups> Succinic anhydride (1.0 g, 10 mmol)
36 861 mg (1.0 mmol) and NaHCO ₃ 252 mg (3.0 mmo
l) to a solution of 1/1 dioxane / H ₂ O in 20 mL,
The mixture is stirred at room temperature for 16 hours. The mixture is acidified with 1N HCl and concentrated. The concentrate is purified by silica gel chromatography to give F.

<Compound G-4 Platform Having Four N-Succinimidyl Ester Groups> F 126 mg (0.1 mmo
l) and N-succinimide 46 mg (0.4 mmol) anhydrous THF
(5 mL) Prepare the solution. The mixture is cooled to 0 ° C. and 103 mg (0.5 mmol) dicyclohexylcarbodiimide are added.
Stir for a few hours until the mixture reaches room temperature. Solids are removed by filtration and the filtrate is concentrated to give G. This G
Can be purified by silica gel chromatography.

<Compound Z-amino group-containing peptide>
Standard solid-phase method on Wang (p-alkoxybenzyl) resin
Synthesize peptides. Lysine ε-amine as a CBZ group
Protect. Using the FMOC chemistry, at the amino terminus, the amino
Acid residues are added sequentially. The last residue added is N-FMOC-
Minocaproic acid. Cut from resin with trifluoroacetic acid
After the cleavage, the FMOC group is eliminated by piperidine,
A peptide having a linker is obtained. This peptideZ
Is purified by reverse phase HPLC.

<Peptide-Platform Complex H>
Prepare a solution of Z 0.05 mmol and Et ₃ N 0.1 mmol in DMF (1 mL). To this solution, 16.5 mg (0.01 mmol) G of DMF (1 m
L) Add the solution. The mixture is stirred until the reaction is complete. This complex was removed with MeOH to remove the protecting groups.
And 100 mg / gram of complex
Place in Parr hydrogenator with 10% Pd carbon. The mixture was shaken under 60 psi H ₂ and the deprotected complex
H is purified by preparative reverse phase HPLC.

(Combination 5: Platform Isothiocyanate-Ligand Amine)

[0233]

[Chemical 33]

<Compound I-4 Platform Having Four Isothiocyanate Groups> Thiophosgene (381 μL, 57
5 mg, 5.0 mmol) to 36 861 mg (1.0 mmol) of THF (10 m
L) Add to the solution and stir the mixture at room temperature until TLC shows the reaction is complete. The mixture is subjected to separating extraction between methylene chloride and 5% NaHCO ₃ solution. The extract was dried over MgSO _4, filtered and concentrated. The product I is purified by silica gel chromatography.

<Peptide-Platform Complex J>
Prepare a solution of Z 0.05 mmol and Et ₃ N 0.1 mmol in DMF (1 mL). To this solution, DMF for I 10.3mg (0.01mmol) (1m
L) Add the solution. The mixture is stirred until the reaction is complete. This complex was removed with Me to remove the protecting group.
Dissolve in OH, and add this solution to 100 mg / gram of complex
Place in a Parr hydrogenator with 10% Pd carbon. The mixture is shaken under 60 psi H ₂ and the deprotected complex J is purified by preparative reverse phase HPLC.

(Combination 6: Platform Chloroformate-Ligand Amine)

[0237]

[Chemical 34]

<Compound K-4 Platform Having Four Hydroxyl Groups> Triethylene glycol bis-chloroformate 205 μL (275 mg, 1 mmol) of CH ₂ Cl ₂ (5 m
L) solution, diethanolamine 497 μL (525 mg, 5 mmo
l) and Et ₃ N 696 μL (506 mg, 5 mmol) CH ₂ Cl ₂ (5 m
L) Add to solution at 0 ° C. The mixture was warmed to room temperature, until TLC showed the reaction was complete,
Stir this. The mixture is concentrated and the product K is separated by silica gel chromatography.

<Platform Having Compound L-Chloroformate Group> Pyridine (100 μL), then triphosgene 1.19 g (4 mmol), K 412 mg (1 mmol) CH ₂ Cl ₂
(20 mL) Add to solution. The mixture is stirred at room temperature for 20 hours and the solvent is evaporated under reduced pressure to give compound L.

<Peptide-Platform Complex M>
A solution of Z 1 mmol in pyridine (10 mL) was added to L 132 mg (0.2 mmo
l) in 1/1 THF / pyridine (5 mL) solution. The mixture is stirred until the reaction is complete. The solvent is removed under reduced pressure. To remove the protecting group, the complex was dissolved in MeOH and the solution was added to 100 mg of 10 mg / gram of complex.
Place in Parr hydrogenator with% Pd carbon. The mixture was shaken under 60 psi H ₂ and deprotected complex M
Is purified by preparative reverse phase HPLC.

Example 4 Synthesis of Conjugates Containing Two Different Biological Molecules It may be useful to conjugate more than one type of biologically active group to the platform molecule. This example describes the preparation of a platform containing two maleimide groups (reactive with thiol-containing peptides) and two activated ester groups (reactive with drugs containing free amines). The resulting conjugate contains two peptides and two drug molecules, as shown in Scheme 20.

<Preparation of Heteroactive Bond Platform Molecule Benzyl 6-aminocaproic acid tosylate K> 32 mmol of 6-aminocaproic acid, 51 mmol of p-toluenesulfonic acid,
A mixture of 40 mmol of benzyl alcohol and 40 mmol of benzyl alcohol in toluene (60 mL) was refluxed using a Dean-Stark trap,
Remove water. When the reaction is complete, the mixture is cooled and the product is precipitated. Collect the solids by filtration and use EtOH / Et ₂
Recrystallize from 0 to give compound K.

<Compound L> Dicyclohexylcarbodiimide (2 equivalents) was added to 1 equivalent of Compound 5 and 2 equivalents of N.
-Add to the solution of hydroxysuccinimide in THF. The mixture is stirred for 4 hours and compound K is added in 2.2 equivalents. TLC
The mixture is stirred until indicates that the reaction is complete. The mixture is filtered and concentrated. The product is purified by silica gel chromatography.

<Compound M> Compound L is treated with trifluoroacetic acid diluted with CH ₂ Cl ₂ . Once the reaction was complete, the mixture was concentrated under reduced pressure to give the compound as trifluoroacetate salt
Get M.

<Compound N> Compound (N-tert-butyloxycarbonyl-2,2'-bis- (N-carbobenzyloxy-6-
Aminohexanoamidoethyl) amine) is treated with trifluoroacetic acid diluted with CH ₂ Cl ₂ . When the reaction is complete,
The mixture is concentrated under reduced pressure to give compound N as the trifluoroacetate salt.

<Compound O> Triethylene glycol bis
3.2 mmol of -chlorochloroformate was added to a solution of compound M 4 mmol and compound N 4 mmol in pyridine (162 mL) at 20 ° C.
Add in water bath. The mixture is stirred until TLC shows the reaction is complete and concentrated under reduced pressure. This concentrate was dissolved in CH ₂ Cl ₂ , followed by 1N HCl solution, 5% NaH.
Wash with a CO ₃ solution and a saturated NaCl solution in this order. CH ₂ Cl ₂
Drying the layer over MgSO _4, filtered and concentrated. E for this concentrate
Dissolve in 10 mL of tOH and add 10 mL of 1M NaOH. The mixture is stirred for several hours until TLC shows no further reaction. The mixture is acidified to pH 1 with 1N HCl and extracted with CH ₂ Cl ₂ . The CH ₂ Cl ₂ layer was dried over MgSO ₄ ,
Filter and concentrate. The product O is separated by silica gel chromatography.

Compound P Compound O was dissolved in EtOH and placed in a Parr shaker at 100 mg of 10% Pd per gram of O 2.
Hydrogenate with carbon. Check the completion of the reaction by TLC. When the reaction is complete, the catalyst is removed by filtration,
Then, the mixture is concentrated to obtain the compound P.

<Compound Q> 3 mmol of N-methoxycarbonylmaleimide is added at 0 ° C. to a solution of 1 mmol of compound P in 20 mL dioxane and 5 mL saturated NaHCO ₃ . The mixture is stirred for 1 hour, acidified with 1N HCl and extracted with CH ₂ Cl ₂ . The CH ₂ Cl ₂ layer was dried over MgSO ₄ , filtered, concentrated,
Then, the product is purified by silica gel chromatography to obtain Q.

<Compound R> 2 mmol of DCC are added to a solution of Q 1 mmol and p-nitrophenol 2 mmol in CH ₂ Cl ₂ and the mixture is stirred for 16 hours. Solids are removed by filtration, the filtrate is concentrated and purified by silica gel chromatography to give R.

<Conjugate with two peptides and two drug molecules, compound S> Two equivalents of the thiol-containing peptide was added to the heteroactivation platform R at one equivalent pH.
7.5 Add to phosphate buffer solution. The mixture is stirred for 1 hour,
Add 2 equivalents of amine containing drug. This complex S
Isolate by reverse phase HPLC or ion exchange chromatography, or a combination thereof.

[0251]

[Chemical 35]

Example 5: Synthesis and Testing of Complex 3-II

[0253]

[Chemical 36]

<Preparation of DMTr-5'-modified (CA) ₁₀ > Polynucleotide d- [DMTr- (bzCp (CE) bzA) ₁₀ ] was prepared according to the manufacturer's protocol for DNA phosphoramidite synthesis according to Millig.
It was prepared on an en 8800 Prep Scale DNA synthesizer (see Figure 6). The synthesis was performed on 10 g DMTr-d-bzA-CPG support loaded with nucleosides at 30.0 μmol / g. The final DMTr blocking group was removed using a mechanical protocol. Milligen Activator Solution (Cat.No.MBS5040)
45 mL and compound 51 (see Reaction Scheme 11) 0.385
g was added to the reaction system, and this suspension was mixed by bubbling argon for 8 minutes. The mixture was oxidised by standard mechanical protocols, the support-bound polynucleotide was collected by filtration, air dried and concentrated with 100 mL concentrated ammonia.
Treated for 6 hours at 55 ° C. When the temperature drops, gel the mixture.
Filtered through a man 10μ polypropylene filter.
The filter was washed with 200 mL of 2 mM NaCl adjusted to pH 12 with NaOH. The filtrate is then filtered first with 3M NaCl and then 2m.
Q-Sepharose (Pharmacia) equilibrated to pH 12 with M NaCl
Amicon chromatography column (0.45 × 9.
4 cm, 150 mL). Column is a linear gradient (2
elution with 500 mL of mM NaCl, pH12 to 1.3M NaCl, pH12, then 1.3M NaCl until all UV absorbers are present.
It was washed with (pH 12). The 260 nm absorbance fractions are further analyzed by polyacrylamide electrophoresis and the fractions containing pure product are pooled. This pool (120 mL) was treated with 240 mL of cold isopropanol and kept at -20 ° C for 30 minutes. A Sorvall RC 3B centrifuge using a model H-6000A rotor was centrifuged for 15 minutes at 3000 rpm and 4 ° C., whereby the precipitate was collected and DMTr-5′-modified (CA) ₁₀ (14946 A ₂₆₀
Unit, 498 mg, 62.2 μM, 20% (based on 300 μM CPG nucleosides)).

<Synthesis of Tr-5′-modified (CA) ₁₀ >

[0256]

[Chemical 37]

[0257] The synthesis of Tr-5'modified (CA) _10, by replacing (prepared as described in Reaction Scheme 11) Compound 51 Compound 53, the synthesis of DMTr-5'modified (CA) ₁₀ As described above for.

<Complexation of DMTr-5′-Modified Polynucleotide with Compound 3 (IA-DABA-PEG, Reaction Scheme 1) —Preparation of Complex 3-I> In the complexation procedure shown below, All buffers and solutions used were sparged with sufficient helium and all reaction vessels were purged with argon before use. DMTr-5'-Modification (CA) ₁₀ of 11,568 A ₂₆₀ units (48.2
μmol, estimated molar absorbance at 260 nm = 240,000) water (7.7
The solution was treated with 1 mL 0.1 M NaHCO ₃ and 210 μL tributylphosphine (876 μmol, 18-fold molar excess) at room temperature for 0.5 h. This suspension was occasionally shaken. Add 0.8 mL of 3M NaCl and 1 ml of cold isopropanol to the suspension.
Treated with 6 mL. After 30 minutes at -20 ℃, this substance is 3000
Centrifuge at rpm for 20 minutes. 2 mL of the obtained pellet with water,
It was redissolved in 0.2 mL of 3M NaCl, treated with 4 mL of isopropanol and centrifuged again. The pellet was briefly dried under reduced pressure, helium-blown water 2.8 mL and 0.1 N NaHCO ₃
Dissolved in 1 mL. 6.7 mg of compound 3 (IA-DABA-PEG) was added and the mixture was left in the dark at room temperature for 16 hours. Final capacity
6 mL of the reaction mixture was passed through a 5 × 91 (1800 Ml) Pharmacia column packed with Sephacryl 200 (Pharmacia). The column was eluted with 0.5 M NaCl, 0.1 M sodium borate (pH 8.3). Using a peristaltic pump, the flow rate was set to about 2 mL / min and 15 ml fractions were collected. 260 nm of this fraction
The absorbance at was measured. Further, this fraction was analyzed by polyacrylamide gel electrophoresis, and the fractions containing pure complex were pooled.

<Hybridization of Complex 3-I-
Preparation of Complex 3-II> The pooled fraction was a fraction containing 726 A ₂₆₀ units. An equivalent amount of (TG) ₁₀ was added and the tube was heated at 90 ° C. for 10 minutes then cooled to room temperature over 1.5 hours. An equal amount of isopropanol was added and the mixture was placed at -20 ° C for 3 hours. After centrifugation at 3000 rpm for 20 minutes, the pellet was dissolved in 0.15 M NaCl, 0.01 M sodium citrate (pH 6.8). Got 53 mg of hybrid. A portion of this material was diluted with the above buffer and the melting temperature (Tm) of the double helix was measured with a Carey 3E spectrophotometer. This material had a Tm of 73.4 ° C and a dark color of 24.3%. A 10 A ₂₆₀ unit portion of the product was annealed with excess (TG) ₁₀ as described above. Similar to this material, non-annealed complex and (TG) ₁₀ standard were treated with Rainin H
It was analyzed by gel permeation HPLC on a Shodex Protein KW 8025 column using a PLC device. Column, 0.05M
It was eluted isocratically with NaH ₂ PO ₄ (pH 6.5) and 0.5 M NaCl. The elution time was 12 minutes. Product retention time
6.9 minutes and (TG) ₁₀ was 9.2 minutes. Comparison of peak areas indicated that 98.09% of this product was double-stranded DNA. This complex is represented by the structure shown as "Complex 3-II" in FIG.

Example 6 Preparation of PN-KLH Complex PN-KLH
The complex was prepared according to the scheme shown below:

[0261]

[Chemical 38]

<Synthesis of ACT-Modified (CA) ₂₅ > As the final step of the automated synthesis, Compound 58 was bound to (CA) ₂₅ . 30.0μ
10 g of DMT-d- loaded with nucleoside at mol / g
Starting from the bzA-CPG support, 49 sequential steps were performed using alternating dC and dA phosphoramidites. The DMTr blocking group was removed from the resulting d- [DMTr- (bzCp (CE) bzA) ₂₅ ] and the activator solution (Milligen, Cat. No. MBS50
40) 40 mL and compound 58 800 mg were added to the reaction mixture. The suspension was mixed for 8 minutes by sparging with argon and the usual oxidation steps were performed. The support-bound polynucleotide was removed from the reaction vessel, air dried, and treated with 100 mL concentrated ammonia for 40 hours at 55 ° C. When the temperature had dropped, the mixture was filtered through a Gelman 10 μm polypropylene filter, then the filtrate was purified by conventional ion exchange chromatography. Fractions showing absorbance at 260 nm were further analyzed by polyacrylamide electrophoresis and the fractions containing the pure product were combined and precipitated with isopropanol, ACT-modified (CA) ₂₅ 510 mg (31.9%).
μmol, 10%) was obtained.

<Synthesis of Single-Chain PN-KLH Complex> 100 mg (2.5 μmol) of ACT-modified (CA) ₂₅ treated with NaIO ₄ was dissolved in 1.33 mL of 50 mM sodium borate (pH 8.0) to obtain KLH. 3
1.3 mg (0.208 μmol) and pyridine-borane 2.0 mg (3
1.8 μmol) was added. This mixture is kept at 37 ° C for 72 hours,
The product was purified by chromatography on S-200.

<Hybridization of single-stranded PN-KLH complex with (TG) ₂₅ > An equivalent amount of (TG) ₂₅ was added to the single-stranded PN-KLH complex, and the tube was heated at 90 ° C. for 10 minutes. , Then 1.5
Cooled to room temperature over time. Precipitation with isopropyl alcohol gave 53 mg of PN-KLH; Tm (0.15 M NaC
l, 0.01M sodium citrate, pH 6.8) 73.4 ℃, 31.1%
Dark color; HPLC comparison to a standard consisting of excess (TG) ₁₀ annealed sample, non-annealed complex, and non-annealed (TG) ₁₀ confirmed 98% double-stranded (Shodex Protein KW 8025 column, 0.05MN
aH ₂ PO ₄ , pH 6.5, 0.5 M NaCl). This complex can be represented by the following formula: KLH- [NH (CH ₂ ) ₅ OPO ₂ · O- (CA) ₂₅ : (TG) ₂₅ ] _-5 (KLH molecular weight is estimated to be 10 ⁵ ) , "PN-KLH".

Testing Complex 3-II as an Immunotolerogen Complex 3-II was tested for its ability to elicit immunotolerance in mice immunized with the polynucleotide in the form of the immunogen.

<Materials and Methods> Mice: C57BL / 6 female mice (6 weeks old) were tested by Jackson Laboratories (Bar Harbo).
r, ME). Mice are National Institutes of Health (NIH)
It was bred by the improved method of. Immunization: Iverson's method (Handbook of Experimental Immunology, Volume 2, Cel
lular Immunology , (DM Weir, LA Herzenberg, C.
Blackwell and A. Herzenberg, 4th edition, Blackwe
ll Scientific Publications, Oxford) Assay in for
According to the in vivo Adoptive Immune Response ), 100 μg of alum-precipitated PN-KLH and 2 × 10 ⁹ of formalin-fixed B. pertussis as an adjuvant were intraperitoneally injected into the mouse to immunize the mouse for the first time. The mice were boosted with 50 μg PN-KLH in saline (intraperitoneally).

Binding of PN to SRBC: Sheep red blood cells (SRB
Olseever solution of (C) was added to Colorado Serum Co. (Denver,
CO) and used within 2 weeks. This SRBC is Ki
pp and Miller's method ( Selected Methods in Cellular
Immunology , (1980), BBMishell and SM Shiigi.
Ed., WH Freeman and Co. (San Francisco), “Preparation of protein-complexed red blood cells using ECDI (modification),” page 103, (CA) ₂₅ : (TG) ₂₅ (CA: GT
0 mer). Briefly, SRBC was washed 4 times with cold saline and 0.01M NaC containing 10 mg carbodiimide.
l, 0.35 M D- EK bond in mannitol (CA) ₂₅ : (TG) ₂₅ 2 mg
And mixed for 30 minutes at 4 ° C. Coated SRBC
Was washed twice with cold balanced salt solution and resuspended in 10% (v / v).

Plaque assay: Anti-PN plaque forming cells
The number of (pfc) is determined by the Cunningham method ( Selected Methods in Cel
lular Immunology , (1980), BBMishell and SM Sh
iigi, WH Freeman and Co. (San Francisco), 86
Page, Marbrook, J., "Liquid Matrix (Slide Method)"). The number of IgG pfc is described by Henry ( Selected Methods in Cellular Immunolog
y , (1980), edited by BB Mishell and SM Shiigi, WH F
Reeman and Co., San Francisco, “Evaluation of IgG response by removal of IgM plaques” on page 91)), by removing IgM plaques with rabbit anti-mouse IgG. Briefly, spleens were harvested and single cell suspensions were prepared in balanced salt solution (BSS). Guinea pig serum was added to the polynucleotide-coated SRBC and the final dilution was
It was 1: 9 guinea pig serum and sufficient rabbit anti-mouse IgG was added to make the final dilution 1: 100 rabbit anti-mouse IgG. The SRBC mixture and diluted spleen cells were mixed in equal amounts in microtiter wells,
Transferred to Cunningham chamber. Each spleen was tested individually three times. The edges of the chamber were sealed with paraffin and the chamber was incubated at 37 ° C for 1 hour. The number of plaques was counted by observing the chamber under an inverted microscope.

<Results> Pertussis as an adjuvant (A &
Mice were primed with alum-precipitated PN-KLH together with P) and 7 weeks later, divided into 3 groups each. This mouse was treated with PN-DABA-PEG, that is, a diluted solution in which the concentration of the complex 3-II was changed by 2 times (intraperitoneal), and after 5 days, all mice including the control were treated with physiological saline. Boosted with 50 μg of injected PN-KLH (intraperitoneal). 4 days later
The spleen was collected and the number of IgG pfc was measured. As shown in Table 2, the test body to which the complex 3-II was administered showed a significant decrease in the number of pfc as compared with the control group.

Mice were treated with complex 3-II and D- EK binding (PN) ₅₀ under the same conditions as above, and anti-dsPN antibody was measured by the Farr assay. The results are shown in Fig. 14.
Compared to LJP-105 ( D- EK coupling (PN) ₅₀ ), LJP-249A and LJP
-249B (complex 3-II) was significantly more effective in reducing anti-dsPN antibody.

[0271]

[Table 2]

(Example 7: Preparation and test of complex 20-II) <Complexation of 5′-modified (CA) ₁₀ to bond platform molecule 20—Preparation of single-stranded complex 20-I> Tri-n-butylphosphine 969 μL (789 mg, 3.89 mol) was treated with 5'-modified (C
A) To a solution of ₁₀ 918 mg (0.14 mmol) of H ₂ O (30 mL) was added under argon gas. The mixture was stirred for 1 hour, then 3M
2.4 mL of NaCl solution was added, followed by 42 mL of isopropanol which was purged with oxygen by blowing helium. The mixture was placed in the freezer at −20 ° C. for 1 hour and then centrifuged at 3000 rpm for 30 minutes. The supernatant was removed and the oily residue was dissolved in 15.5 mL H ₂ O sparged with helium. 3M NaCl 1.24
mL and isopropanol 21.7 mL bubbling helium
Was added to the mixture. The resulting mixture was then placed in a freezer at -20 ° C for 1 hour and centrifuged at 3000 rpm for 20 minutes. The obtained oily pellet was dried under reduced pressure for 18 hours to obtain a solid. This solid was dissolved in 6 mL of helium-blown H ₂ O to give a total volume of 6.4 mL. The amount of DNA was 863 mg as measured by UV absorbance at 260 nm (pH 7.5).
0.333 mg per absorption unit in phosphate buffered saline. This solution was transferred to a 50 mL three neck flask under argon gas. One port of the flask was used as an inlet for argon gas,
The other two were plugged. Helium-blown 1M sodium phosphate buffer (pH 7.8) 0.87mL, MeOH 0.97mL and H ₂ O
And were added to adjust the total volume to 7.7 mL. Compound 1 of 20
1.9 mL of 7.7 mg / mL MeOH solution (33.63 mg, 0.025 mmol) was added to the mixture. The resulting mixture was stirred under argon gas for 20
Stir for hours and then dilute to 100 mL with a solution containing 0.1 M NaCl, 0.05 M sodium phosphate (pH 7.5), and 10% MeOH. Purification was performed by chromatography on Fractogel® (equilibrium: 0.1M NaCl, 0.05M
Sodium phosphate, pH 7.5, 10% MeOH: Elution gradient 0.5M NaCl, 0.05M Sodium phosphate, pH 7.5, 10% M
eOH to 0.8M NaCl, 0.05M sodium phosphate, pH 7.5, 1
Up to 0% MeOH). Fractions containing pure complex 20-I , as shown by HPLC and polyacrylamide gel electrophoresis, were collected in 232 mL of eluent. The product and salts were precipitated by adding an equal amount of isopropanol and placing it in the freezer at -20 ° C for 1 hour. Dialyzed against H ₂ O (2 × 100 vol), 335 mg of complex 20-I (concentration 10.47 mg
32 mL, 0.033 mg / absorption unit (260 nm)) was obtained.

<Annealing of Complex 20-I and (TG) ₁₀ to Form Double-Stranded Complex 20-II> Complex 2
0-I 150 mg (0.033 mg / absorption unit (260 nm) based on concentration 1
(14.33 mL at 0.47 mg / mL) and (TG) ₁₀ 157.5 mg (0.033 mg / mL)
Based on the absorption unit (260 nm), 1.50 mL at 104.6 mg / mL)
Place in a 0 mL polypropylene centrifuge tube. pH 7.2 10 x PBS 2.
By adding 0 mL and 2.17 mL H ₂ O, the concentration was increased to 15 mg / m 2.
Adjusted to L. The mixture is placed in a 90 ° C water bath and 1.5
Cooled to room temperature over time. The concentration was confirmed to be 17.7 mg / mL by absorbance at 260 nm (0.050 mg / absorption unit); transition melting temperature 67.5 ° C .; dark color 27%; osmolality 346; pH 7.2. For the final formulation of Complex 20-II , add 7.23 mL of pH 7.2 1/2 x PBS and add 0.22
The solution was diluted to a final concentration of 12.7 mg / mL and an osmolality of 299 by filtering through a μ filter.

<5'-Modification (CA) Another complexation of _10-20 , preparation of single-chain complex 20-I> 10 equivalents of tri-n-butylphosphine was added to 5'-modification (CA) ₁₀ is sparged with He and added to a 10 mg / mL solution in 100 mM sodium acetate (pH 5). The mixture is stirred for 1 hour and then precipitated with 1.4 volumes of isopropyl alcohol (IPA). The mixture is placed in the freezer at −20 ° C. for 1 hour and then centrifuged at 3000 rpm for 20 minutes. The supernatant was removed, and the pellet was placed in a helium-blown IPA for 10 m.
Dissolve at g / mL. The mixture is placed in the freezer at -20 ° C for 1 hour and then centrifuged at 3000 rpm for 20 minutes. The pellet is dried under reduced pressure for 18 hours to give a solid. 50 of this solid
A mg / mL solution is prepared in 100 mM sodium borate buffer (pH 10) bubbled with helium. 9/1 40 in MeOH / H ₂ O
As a mg / mL solution, 0.25 equivalents of compound 20 are added to this mixture. The mixture is stirred at room temperature for 3-20 hours and diluted (0.1M NaCl, 0.05M sodium phosphate (pH 7.
5), 10% MeOH). Purify by Factogel chromatography. (Equilibrium: 0.1M NaCl, 0.05M sodium phosphate, pH 7.5, 10% MeOH: elution gradient 0.5M NaC
l, 0.05M sodium phosphate, pH 7.5, 10% MeOH to 0.8M
NaCl, 0.05M sodium phosphate, pH 7.5, up to 10% MeOH). Fractions containing pure complex 20-I as indicated by HPLC and polyacrylamide gel electrophoresis were collected.
The product and salt are precipitated by adding an equal amount of IPA and placing it in the freezer at -20 ° C for 1 hour. It is dialyzed against H ₂ O (2 × 10 vol) to obtain the complex 20-I .

<Another Annealing of 20-I with (TG) _10-20 to Form Double-Stranded Complex 20-II> Except that the annealing was done at 70 ° C instead of 90 ° C. It is substantially the same as the above method.

<Addition of another complex of 5'-modified (CA) _10-20 , preparation of single-chain complex 20-I> 4.8 mL of tri-n-butylphosphine was Ar-blown with 100 mM sodium acetate (pH 5). )
To a solution of 7.75 g of 5'-modified (CA) ₁₀ in 104 mL was added under N ₂ gas. The mixture was stirred for 1 hour, then IPA 23
Precipitated at 2.5 mL. Mix the mixture in a freezer at -20 ° C for 1.5
Every hour, then centrifuge at 3000 rpm for 20 minutes,
Then it was frozen at -20 ° C for 24 hours. The supernatant was removed, and the pellet was dissolved in 170 mL of 0.3 M NaCl solution blown with helium. The mixture was reprecipitated with 232 mL of IPA blown with Ar. The mixture is then placed in a freezer at -20 ° C for 2 hours, 3
Centrifuge at 000 rpm for 20 minutes and at −20 ° C. for 11 hours. The supernatant was removed, and the pellet was dried under reduced pressure for 12 hours to obtain a solid. This solid solution is blown with Ar 10
Prepared in 110 mL of 0 mM sodium borate buffer (pH 10). 9/1 MeOH / H ₂ O (4.4 mL) as a solution, 406 mg of compound
20 was added to this mixture. The mixture was stirred at room temperature for 2 hours. The resulting mixture was shown by high pressure ion chromatography to contain 62% of 20-I . This chromatography is based on the Waters Gen Pak Fax
Column (100 x 4 mm), 60 ° C, linear gradient 65%
A / 35% B to 18% A / 82% B; A = 0.05M NaH ₂ PO ₄ , p
H7.5 1 mM EDTA, 10% MeOH (v / v); B = 0.05 M NaH ₂ PO ₄ , p
H7.5, 1M NaCl, 1mM EDTA, 10% MeOH (v / v), 20
-I eluted at 19.5 minutes.

<Complex 20-II and non-complex control studies> C57BL / 6 mice were immunized with PN-KLH and A & P. After 3 weeks, groups of 5 mice / group were given various doses of Complex 20-II or 4.5, respectively.
nM HAD-AHAB-TEG (derivatized valency platform molecule, AHAB-TEG with linker HAD, see Figure 7),
Or 18nM (4 × 4.5) (CA) ₁₀ : (TG) ₁₀ or 4.5nM HAD
A mixture of AHAB-TEG and 18 nM (CA) ₁₀ : (TG) ₁₀ was administered intraperitoneally; and one group received no treatment. Each group was injected and boosted, serum was collected and the assay described in Example 6 was performed. The reduction in anti-PN response (expressed as a percentage) is shown in FIG. These mouse anti
The KLH response was normal and was not significantly different from the anti-KLH response shown in Figure 2. From the results of this example,
PN response is (i) bond platform molecule only, (ii) P
It is clear that N alone or (iii) was not affected by the mixture of the two. PN must be bound to a non-immunogenic valency platform molecule in order to induce immune tolerance.

<The fact that the complex 20-II causes a decrease in the number of PN-specific antibody-producing cells> C57BL / 6 mice were treated with PN-KLH.
And immunized with A & P. After 3 weeks, groups of 3 mice / group were each intraperitoneally administered with different doses of complex 20-II; and one group was untreated. After 5 days, all mice were boosted by intraperitoneal injection of PN-KLH in saline, then 4 days later their spleens were harvested and PN specific IgG production using hemolytic plaque assay. The number of cells was assayed. The results are shown in Table 3. From this result, it is clear that this complex reduced the number of PN-specific IgG-producing cells.

[0279]

[Table 3]

(Example 8: Test of complex as immunotolerance) <Test of complex 17-II as tolerogen> C57BL / 6 mice were immunized with PN-KLH and A & P. After 3 weeks, groups of 5 mice / group were each intraperitoneally dosed with different doses of complex 17-II; and one group was untreated. After 5 days, all mice were boosted by intraperitoneal injection of PN-KLH in physiological saline, and after 7 days, blood was collected from these mice. Serum was analyzed for anti-PN antibody by the Farr assay at a PN concentration of 10 ⁻⁸ M. The reduction in anti-PN response (expressed as a percentage) is shown in FIG. The sera were also analyzed for anti-KLH antibody using an ELISA assay. The results are expressed as a percentage of anti-KLH compared to a standard pool of anti-KLH sera and are shown in Figure 2. The data in Figure 1 show that this complex reduced the anti-PN response. All of these mice are anti-KL
The H (platform molecule) response was normal (Fig. 2
reference).

<Test of various 11 series of complex as immunotolerance> A group of C57BL / 6 mice / group was
Immunized with PN-KLH and A & P. After 3 weeks, 3 different doses of conjugate 11 each for 2 groups
-IV, complex 11-II, complex 11-VI, or complex 1
1-VIII was given intraperitoneally and one group received no treatment. These conjugates are described in Figure 6 and were prepared according to the method of Example 7 above. 5 days later,
All mice were boosted by intraperitoneal injection of PN-KLH in saline, and after 7 days, blood was collected from these mice. Serum was analyzed for anti-PN antibody by the Farr assay at a PN concentration of 10 ⁻⁸ M. The reduction in anti-PN response (expressed as a percentage) is shown in FIG. The anti-KLH response of these mice was not significantly different from the anti-KLH response shown in FIG. Four
All three conjugates significantly reduced the anti-PN response at all doses tested.

<The fact that complex 11-II causes a decrease in the number of PN-specific antibody-producing cells> C57BL / 6 mice were treated with PN-KLH.
And immunized with A & P. After 3 weeks, various doses of complex 11-II were intraperitoneally administered to groups of 3 mice / group and one group was untreated. 5 days later, all mice received PN-K in saline
LH was boosted by intraperitoneal injection, then 4 days later, their spleens were harvested and assayed for the number of PN-specific IgG-producing cells using a hemolytic plaque assay. The results of the experiments with various doses of Complex 11-II are shown in Table 4. The results demonstrate that this complex reduces the number of PN-specific IgG producing cells and that the reduction in antibody titer is not due to clearance of serum antibody bound to the complex.

[0283]

[Table 4]

Example 9: HADpS- (CA) ₁₀ -Complex 20-IV
Preparation of) A modified polynucleotide having a phosphorothioate having a linker attached to the 5'end was prepared. 20-mer
Synthesis of (CA) ₁₀ and addition of the HAD linker to the polynucleotide was performed according to the method of Example 5, except as noted below. In the final oxidation step, the iodine solution was replaced with a 0.05M acetonitrile solution of 3H-1,2-benzodithiol-3-one 1,1 dioxide (Glen Reseach, Sterling, VA). The sulfurization treatment was performed according to the manufacturer's instructions. Ammonia treatment and purification were performed in the same manner as in Example 5. The conjugation of the polynucleotide to the AHAB-TEG valency platform was performed according to the method of Example 5.

Testing Complex 20-IV as an Immunotolerogen Since the 5'phosphate of PN is susceptible to enzymatic degradation, one of the terminal phosphate oxygen molecules was replaced with sulfur (hence, HAD _p S Name)). C57BL / 6 mice were immunized with PN-KLH and A & P. Three weeks later, groups of 5 mice / group were treated with different doses of complex 2
0-IV was given intraperitoneally and one group received no treatment. Groups were given booster injections, serum was collected and assayed as above. The results showing the reduction of anti-PN response (expressed in percentage) are shown in FIG. The anti-KLH response of these mice (data not shown) was normal and not significantly different from the anti-KLH response shown in FIG. These results indicate that this complex significantly reduced the anti-PN response.

<The fact that complex 20-IV causes a decrease in the number of PN-specific antibody-producing cells> C57BL / 6 mice were treated with PN-KLH.
And immunized with A & P. After 3 weeks, groups of 3 mice / group were intraperitoneally injected with various doses of complex 20-IV and one group was untreated. 5 days later, all mice received PN-K in saline
LH was boosted by intraperitoneal injection, then 4 days later, their spleens were harvested and assayed for the number of PN-specific IgG-producing cells using a hemolytic plaque assay. The results are shown in Table 5. From this result, the PN
It is clear that the number of specific IgG producing cells was reduced.

[0287]

[Table 5]

Example 10: LJP394, in complex 20-II
Treatment of BXSB mouse) <Mouse treatment protocol> Male BXSB mice (Jackson Laboratory, Bar Harbor, Me) aged 6 to 9 weeks were treated with La Jolla P.
Raised in a harmaceutical facility. Food and water were provided ad libitum. Animals were allowed to rest for 1 week before being used in the experiment. Initial blood samples were obtained and initial body weights were measured prior to the first conjugate treatment. Complex processing is 7 ~
Starting at 9 weeks of age, intraperitoneal administration was performed twice a week from 59th to 150th. Blood is collected from animals on a regular basis and its anti-DN
The A antibody titer was determined.

<Assay for IgG Anti-DNA Antibody Production> Serum samples obtained from each mouse were tested for the presence of anti-DNA antibody by ELI.
It was evaluated by SA. Falcon Probind 96 Well Microtitration Assay Plate (Becton Dickerson,
Oxnard, CA) was coated with 100 μL / well of (PN) ₅₀ -D -EK (copolymer of D-glutamic acid and D-lysine) at a concentration of 50 μg / mL at 4 ° C. overnight. The plate was washed with PBS containing no calcium and magnesium and 0.05% Twe.
With en20 (wash buffer), M96V plate washer (ICN Biome
(dical, Inc., Irvine, CA). Plates with 1% gelatin (Norland Products, Inc, New
Blocked with PBS containing Brunswick, NJ) and 0.05% Tween 20 for 1 hour at room temperature. Plates were washed twice with wash buffer before adding serum samples or standards. Serum samples and standards were prepared as a diluent containing PBS with 1% gelatin, 0.05% Tween 20, and 10% goat serum. The plates were incubated with serum samples for 60-90 minutes at 37 ° C, then the wells were washed 4 times with wash buffer. Biotinylated goat anti-mouse IgG
(Sigma Chemical Co., St. Louis, MO) was diluted 1/1000 in blocking solution containing 10% goat serum. Plate at 37 ° C
Incubate for 1 hour and wash 4 times. Serve as a substrate
OPD (Sigma Chemical Co. (St. Louis, MO)) was added.
ELISA plate reader at OD 450nm until the highest reading of the highest standard was about 1 OD unit,
The plates were incubated in the dark (Bio-Tek Inst
ruments, Winooski, VT). The reaction was stopped with 50 μL of 3M HCl and the plate read at 490 nm. Each microtitration plate contained a reference positive serum and the positive wells from each assay were sensitive within the 95% range of this reference time curve. In the blood collected later, some of the positive samples exceeded the reference curve. However, most diluted mouse serum samples were within the reference curve range. No significant binding was observed with normal control negative sera. The results are shown in Fig. 15.

(Example 11: Preparation of melittin peptide and complex) The melittin molecule consists of 26 amino acids and is one of the main components of bee venom. One third of honey bee venom susceptible individuals have melittin-specific antibodies. Melittin is highly immunogenic in several mouse strains (Balb / c, CAF1). Many (> 80%) of the responding mouse strains of melittin-specific antibodies bind to the C-terminal seven peptides of melittin, the B cell epitope.

<Melittin>

[0292]

[Table 6]

<Melittin peptides that stimulate T cells>

[0294]

[Table 7]

<Peptide Synthesis> Melittin peptide is a glycine resin (Advanced ChemTech # SG5130) or its equivalent (Advanced ChemTech, 2500 Seventh Street R).
Oad, Louisville, KY) by standard Fmoc chemistry. A 2.3 M excess of amino acid derivative was used in each coupling step. Binding completion was monitored with bromophenol blue and confirmed with ninhydrin.

<Melittin peptide used for complex>

[0297]

[Table 8]

Since it is necessary to attach the peptide to the valency platform molecule by a thioether bond,
Cysteine was added. The peptide was purified by reverse phase HPLC after synthesis and dried by lyophilization. The appropriate amount of peptide was then weighed for each conjugation.

<Reduction of preformed disulfide bonds
(Tributylphosphine method)> Helium was blown into all the buffer solutions. Minimum volume of peptide (about 10-20 mg / mL)
Of 0.05M NaHCO ₃ (pH8.25). 0.7M tributylphosphine (TBP; MW = 202.32g / mol; d-0.812g / m
1) solution of TBP 174.4 μL in isopropanol (iPr
OH) 825.6 μL. Then, to the peptide solution prepared as above, TBP was added at an equivalent ratio of (1: 1), mixed well, and then stirred occasionally to dissolve and / or disperse TBP in the solution for 30 minutes to 1 minute. Reacted for hours. HPLC confirmed complete reduction.

<Conjugation of Peptide to Bond Platform Molecule # 3 or # 60> Helium was blown into all buffer solutions. Polyethylene glycol (PEG) derivative # 3 or # 60 with a minimum volume of about 20 mg / mL 0.05M
It was dissolved in NaHCO ₃ (pH8.25). About 3 equivalents of peptide were used per iodoacetyl group of the PEG derivative. p-Aminobenzoic acid (PABA) -PEG (2 iodoacetyl groups; MW = about 410)
At 0 g / mol), 6 equivalents of peptide were used for each equivalent of PABA-PEG. Diaminobenzoic acid (DABA) -PE
For G (4 iodoacetyl groups; MW = about 4300 g / mol), 12 equivalents of peptide were used for each equivalent of DABA-PEG. The PEG solution was added to the reduced peptide solution, and the mixture was allowed to react in the dark for at least 1 hour. HPL for preparing peptide complexes
Purified by C. 15% before pooling and lyophilization
Fractions were confirmed by electrophoresis using Tricine gel.

[0301]

[Table 9]

<Mouse Lymph Node Proliferation Assay> Female Balb
/ c mouse (6-8 weeks old; Jackson Laboratory, Bar Harb
or, Maine) to obtain La Jolla Pharmaceutical animal
The animals were bred at the facility according to the guidelines of the National Institutes of Health. Food and water were provided ad libitum. Balb / c mice were treated with Complete Freund's Adjuvant (CFA) (Sigma Chemica
The back of each hind foot was immunized with 50 μg of melittin in Co., St. Louis, MO). Seven days later, popliteal lymph nodes were collected aseptically. Lymph nodes were gently separated by mincing the cells through a 50 mesh sieving screen. Single cell suspensions were loaded with RPMI-1640 (Irvine Scient) containing glutamine, penicillin, and streptomycin.
ific, Irvine, CA). Round bottom 96 well Cornin
g in microtiter plate quadruplicate wells in RPMI medium supplemented with 10% fetal bovine serum,
5 × 10 ⁵ cells were cultured with 10, 1.0, or 0.1 μg / mL melittin or melittin peptide. Cells in positive control wells were cultured with 100 or 50 U / mL mouse interleukin 2 (IL-2), 1 μg / mL PHA (phytohemagglutinin). Negative control wells
Had lymph node cells in RPM-1640 and 10% FCS. The cells were cultured for 4 days in a 37 ° C. incubator with 5% CO ₂ . Add 1 μCi of [ ³ H] thymidine (ICN Biochem
icals, Costa Mesa, CA) for another 18 hours. The cells were collected on a glass fiber filter mat by a semi-automatic cell collector (Scatron, Sterling, VA). [ ³ H] thymidine incorporation was measured by liquid scintillation. Results are expressed as average counts per minute.

<In Vivo Protocol> Balb / c mice were
By intraperitoneally administering 4 μg of melittin in CFA,
I was immunized for the first time. One month later, a latent tolerogen or formulation buffer was administered intraperitoneally. After 3 days, incomplete Freund's adjuvant (ICF) (Sigma Chemi
Cal Co., St. Louis, MO) was injected intraperitoneally with 4 μg of melittin. After 10 days, 100-200 μL of blood was collected from the retro-orbital venous plexus. Serum samples were assayed for anti-peptide or anti-melittin IgG antibody.

Assay for IgG Anti-Melittin or Total Anti-Melittin Antibodies Individual mouse serum samples were evaluated serially for the presence of anti-melittin antibodies by ELISA. Place the Falcon Probind 96-well microtitration plate in phosphate-buffered saline (PBS) (p
H7.2) melittin or melittin peptide 10μg /
Precoated with mL overnight at 4 ° C. The plate was placed in PBS, 0.02% Tween-20, and 1% gelatin (Norla
nd Products Inc., New Brunswick, NJ). The plates were blocked with 200 μL of PBS containing 5% gelatin for 1 hour at 37 ° C. Serum samples were prepared as a dilution of PBS containing 5% gelatin. Samples were tested by diluting 1: 100 to 1: 1000. After incubation at 37 ° C for 1 hour, the plate was washed 4 times. Extraavidin peroxidase (Sigma Chemical Co., St. Louis, MO) was diluted 1: 1000 in PBS containing 5% gelatin. The plate was incubated at 37 ° C for 1 hour and then washed 5 times. Wells were filled with ο-phenylenediamine (OPD) (Sigma Chem
ical Co., St Louis, MO) developed in the dark for 15-30 minutes and the reaction was interrupted with 3M HCl. Optical density (OD) was measured using a microplate reader (Bio-tek Instrument, Winoos
ki, VT) at 450 nm.

<Antibody-Producing Cell Assay> Cellulose Microtitration Plate (Millipore Co., Bedfo
rd, MA) was prepared as described above for the IgG antibody (ELISA) assay. However, when serum samples were added to the wells in the IgG antibody assay above, splenocytes (5 x 1
0 ⁵ / well) was added instead of serum and incubated overnight. The remaining steps of the ELISA assay were performed as above.

<T cell epitope> T cells obtained from mice sensitized with melittin showed T cell proliferation in response to whole melittin molecule and C-terminal melittin peptides 3, 4, and 5 (FIG. 8). . However, C-terminal peptides 1 and 2 did not induce significant T cell proliferation. Melittin peptides 2 and 5 were conjugated to PEG. Like melittin peptide 2, the PEG conjugate of melittin peptide 2 also did not induce significant T cell proliferation.

<Experiment with melittin-complexed peptide to tolerize mice primed and boosted with melittin> Mice treated with the above conjugates (10 mg / kg, 200 μg / mouse) were treated with formulation buffer. Control Balb / c
They had significantly lower levels of anti-melittin peptide 2 antibody (FIG. 9) and lower levels of anti-melittin antibody compared to mice (FIG. 10). Spleen cells from mice treated with buffer control or the above complex,
The ability of antibody-producing cells to produce anti-melitin antibody or anti-melitin peptide 2 antibody was assayed by measurement in a soluble ELISA assay. As shown in FIG.
The level of anti-melitin peptide 2 antibody-producing cells in the group treated with the complex was significantly lower than that in the control group administered with the formulation buffer. Mice treated with complex 4 (a complex of peptide 5 containing a T cell epitope) did not reduce the titer of antibodies against peptide 5 in treated mice. Therefore, the complex containing the T cell epitope is not a tolerogen (Figure 12). In fact, rather than diminishing the response, the level of anti-peptide antibody may be slightly elevated.

Example 12 Further Experiments with Melittin Peptide Conjugates Tolerize Tolerance in Mice Preprimed and Boosted with Melittin Female C57BL / 6 mice (5-8 weeks old) were tested by The Jackson Laboratory, Bar Harbor, Purchased from ME. Animals were maintained and handled in accordance with National Institutes of Health guidelines.

<Immunization Protocol> Alum-precipitated melittin (5 μg) and B. pert.
ussis ) (Michigan Department of Public Health, Lans
ing, MI) 2 × 10 ⁹ was intraperitoneally injected into mice. To this mouse, 5 μg of melittin in PBS was intraperitoneally administered for boosting.

<Pfc assay> Sheep red blood cells (SRBC)
(Colorado Serum Co., Denver, Colorado) was complexed with melittin peptide 2 using carbodiimide.
Fresh SRBC (<2 weeks old) were washed 4 times with cold saline and once with mannitol (0.35M mannitol, 0.01M NaCl). This SRBC was suspended in mannitol to a concentration of 10% (v / v). Melittin peptide # 3 30 μg
Was added to a 1 mL aliquot of 10% SRBC, which was then incubated in ice for 10 minutes. Then, 100 μL of a 100 mg / mL solution of 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide HCl (EDCI).
Was added and incubated in ice for 30 minutes. SRBC is balanced salt solution (BSS) (Irvine Scientific Co., Irvine, C
It was washed twice with A) and resuspended to 10% (v / v).
Freeze-dried Guinea pig complement (GIBCO, New York, NY) B
Reconstituted with SS, then diluted 1: 3 with BSS. 1 mL of diluted guinea pig complement was added to 3 mL of complexed SRBC. Rabbit anti-mouse IgG was added to give a final 1: 100 dilution of rabbit antiserum. This concentration was predetermined to increase the maximum number of IgG pfc while inhibiting all IgG pfc. This complement / anti-mouse IgG / SRBC solution and a cell suspension of mouse spleen cells taken from one mouse were mixed in equal amounts. 50 μL of each mixture was transferred to Cunningham slide chambers (3 chambers per slide). The ends were then sealed with paraffin and incubated at 37 ° C for 1 hour. The number of plaques per chamber was counted using a dissecting microscope. Each spleen suspension was assayed using uncomplexed SRBC as a control. The number of viable cells was measured in each spleen cell suspension. Spleen cells 10 ⁶
The number of pfc per piece was measured for each chamber and the average of triplicates was calculated. The number of peptide-specific pfc was determined by subtracting the pfc number in the uncomplexed SRBC from the pfc number in the complexed SRBC.

<Determination of Optimal Time for pfc Measurement> Mice were primed with melittin. Groups of sensitized mice (3 mice per group) were 2, 4, 6, and 8
Boosted with melittin on the day. On day 10, mice were killed and their spleens were harvested. Cell suspensions were prepared and assayed for determination of the number of peptide specific pfc. pfc
The optimal number of was obtained with a melittin boost after 6 days.

<Orientation of Peptide in PEG Conjugate Does Not Affect the Complex's Ability to Induce Tolerance> Whether the peptide orientation of the PEG complex affects the ability of the complex to induce immune tolerance. Two different tolerogens were constructed to determine. The peptide was covalently attached to the valency platform molecule 3 through its C-terminus to generate melittin complex 3. Groups of mice (3 / group) primed with melittin were treated intraperitoneally with different conjugates or saline. After 5 days, all mice, including untreated control mice, were boosted with 5 μg of melittin. After 6 days, the mice were killed, their spleens were harvested and the number of peptide-specific pfc was determined. As shown in Table 10, peptide-specific pf was observed in mice primed and boosted with the parent protein melittin in both directions.
It had the effect of reducing the number of c / 10 ⁶ spleen cells.

[0313]

[Table 10]

<Number of peptides per PEG conjugate affects the ability of the conjugate to induce tolerance> Three different conjugates were determined to determine if the ratio of peptide to PEG molecule was important. The bodies (each having different number of peptides per PEG complex) were constructed. Complex 1 is PEG
Only had 2 peptides per complex. Another conjugate had 4 peptides per PEG conjugate (conjugate 2). The third conjugate had 8 peptides per PEG conjugate (conjugate 5). Groups of mice (3 / group) primed with melittin were treated intraperitoneally with conjugate or saline. After 5 days, all mice, including untreated control mice, were boosted with 5 μg of melittin. After 6 days, the mice were killed and their spleens were harvested,
The number of peptide specific pfc was measured. As shown in Table 11, complex 1 (containing 2 peptides per PEG molecule)
Was not effective in reducing the number of peptide-specific pfc / 10 ⁶ spleen cells in mice primed and boosted with the parent protein melittin. This result indicates that both melittin complexes 2 and 5 were effective as tolerogens. However, complex 5 (containing 8 peptides) was effective at a lower dose than complex 2 (containing 4 peptides per valency platform molecule).

[0315]

[Table 11]

As described above, the present invention provides chemically defined non-polymeric valency platform molecules, as well as chemically defined non-polymeric valency platform molecules and biological Alternatively, a complex containing a chemical molecule is provided. As a biological molecule,
Included is a polynucleotide duplex of at least 20 base pairs that has significant binding activity to human lupus anti-dsDNA autoantibodies.

Modifications of the above embodiments for practicing the invention which are obvious to those skilled in polynucleotide chemistry, complex chemistry, immunology, and related fields are within the scope of the appended claims. Is intended.

[0318]

[Sequence list]

[0319]

[SEQ ID NO: 1] Sequence length: 26 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25

[0320]

[SEQ ID NO: 2] Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Lys Arg Lys Arg Gln Gln Gly 1 5

[0321]

[SEQ ID NO: 3] Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Trp Ile Lys Arg Lys Arg Gln Gln Gly Gly 1 5

[0322]

[SEQ ID NO: 4] Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 10

[0323]

[SEQ ID NO: 5] Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 10

[0324]

[SEQ ID NO: 6] Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Cys Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 10

[0325]

[SEQ ID NO: 7] Sequence length: 10 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Cys Trp Ile Lys Arg Lys Arg Gln Gln Gly 1 5 10

[0326]

[SEQ ID NO: 8] Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Trp Ile Lys Arg Lys Arg Gln Gln Lys Cys Gly 1 5 10

[Brief description of drawings]

FIG. 1: PN-KLH-primed immunization, treated with [(PN) ₂₀ -BAHA] -EDDA (complex 17-II) at the dose shown in the figure, and PN-KL
Anti-PN response of mice that received a booster injection of H and bled 5 days later is shown. Three dilution series of sera were tested in the Farr assay with PN radiolabeled at 10 ^-8 M and the data expressed as percent reduction of anti-PN antibody. Five mice were used for one group.

FIG. 2. PN-KLH primed, treated with the dose of [(PN) ₂₀ -BAHA] -EDDA (complex 17-II) shown in the figure, and PN-KL.
Anti-KLH response is shown in mice that received a booster injection of H and were bled 5 days later. Anti-KLH antibodies were tested by enzyme linked immunosorbent assay (ELISA). Results were expressed as a percentage of the standard pool of antisera. Five mice were used for one group.

[Fig. 3] Fig. 3 shows that [(PN) ₁₆ -BAHA _OX ] -EDDA (complex 11-IV) and [(PN) ₂₀ -BAH were immunized with PN-KLH at the doses shown in the figure.
A _OX ] -EDDA (complex 11-II), [(PN) ₂₄ -BAHA _OX ] -EDDA (complex 11-VI), or [(PN) ₃₂ -BAHA _OX ] -EDDA (complex 11-VI)
Anti-PN response of mice treated with either II), boosted with PN-KLH and bled 5 days later is shown. Farr with serum-labeled PN radiolabeled at 10 ^-8 M
Tested in the assay. Five mice were used for one group.

FIG. 4 is a figure showing the dose of (PN) ₂₀ -HAD _p -AHAB-TEG (complex 20-II), or H, which was given by the first immunization with PN-KLH.
Shown are anti-PN responses of mice treated with AD-AHAB alone or with PN alone or a mixture of each and then boosted with PN-KLH and bled 5 days later. Serum was tested in the Farr assay with 10 ⁻⁸ M radiolabeled PN. Percentage reduction was calculated and this data is shown.
Five mice were used for one group.

FIG. 5: Primary immunization with PN-KLH, treatment with the indicated dose of (PN) ₂₀ -HAD _p S-AHAB-TEG (complex 20-IV), followed by PN-KLH. Figure 7 shows the anti-PN response of mice boosted with and bled 5 days later. Serum was tested in the Farr assay with 10 ⁻⁸ M radiolabeled PN. Five mice were used for one group.

FIG. 6 shows complexes 3-I, 3-II, 11-I, 11-II, 11-I.
V, 11-VI, 11-VIII, 17-I, 17-II, 20-I, 20-II, 20-II
1 shows structures of derivatized valency platform molecules for I and 20-IV, and linkers that link polynucleotides to the valency platform molecules.

FIG. 7 shows the structure of the derivatized valency platform molecule “HAD-AHAB-TEG”.

FIG. 8 compares the levels of T cell proliferation induced by melittin peptides.

FIG. 9 compares the levels of anti- (melitin peptide 2) antibody produced in mice treated with melittin peptide conjugate and in control mice treated with formulation buffer.

FIG. 10 compares the levels of anti- (melitin) antibody produced in mice treated with melittin peptide conjugates and in control mice treated with formulation buffer.

FIG. 11 shows mice treated with melittin peptide conjugates in control mice treated with formulation buffer.
The levels of anti- (melitin peptide 2) antibody-producing cells are being compared.

Figure 12: Peptide # containing T cell epitopes
Illustrating that melittin peptide complex 4, a complex of 5, is not a tolerogen.

FIG. 13 illustrates a melittin complex included in the present invention.

FIG. 14 shows the complex of the present invention (LJP-249A and LJP
JP-249B (complex 3-II)) achieved an increased percentage decrease in anti-dsPN antibody and D- EK and polynucleotide (PN) _50.
Figure 9 illustrates a comparison with that of a prior art complex containing (LJP-105).

FIG. 15 is a complex LJP-394, complex 2 of the present invention.
Anti-DNA I in circulating serum of male BXSB mice treated with 0-II
It shows suppression of gG antibody. An IgG antibody against (PN) ₅₀ bound to D- EK was measured in an ELISA assay. Serum from each 8 mice in each group was assayed individually.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor David S. Jones United States California 92127, Florin Road, San Diego 11265 (72) Inventor Douglas Alan Livingstone United States California 92122, San Diego, number 115, Fiore Terrace 5260 (72) Inventor Lin Yu United States California 92122, San Diego, APT. 228, Lombard Place 8933

Claims (91)

[Claims] 1. A complex comprising: (a) reacted with a biological or chemical molecule, (b) a chemically defined non-polymeric valency platform molecule of the formula: ] Alternatively, However, if present, G ^[1] and G ^[2] are each independently C, N,
A linear, branched or multiply branched chain containing 1-2000 chain atoms selected from the group consisting of O, Si, P and S; n ^{[1 ]} designated as T ^{[1] ]} Parts and T ^[2]
The p ^[2] × n ^[2] parts shown as are independently -N
HR ^SUB (amine), -C (= O) NHNHR ^SUB (hydrazide), -NHNHR
^SUB (hydrazine), -C (= O) OH (carboxylic acid), -C (= O) OR
^ESTER (active ester), -C (= O) OC (= O) R ^B (anhydride), -C (=
O) X (acid halide), - S (= O) 2 X ( sulfonyl halide), - C
(= NR ^SUB ) OR ^SUB (imidate ester), -NCO (isocyanate), -NCS (isothiocyanate), -OC (= O) X (haloformate), -C (= O) OC (= NR ^SUB ) NHR ^SUB (carbodiimide adduct), -C (= O) H (aldehyde), -C (= O) R ^B (ketone), -SH (sulfhydryl or thiol), -OH (alcohol),
-C (= O) CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂ OR ^ALK X (alkylsulfonate), R ¹ R ² is -C
(= O) CH = CHC (= O)-, -NR ¹ R ² (maleimide), -C (= O) C
R ^B = CR ^B ₂ (α, β-unsaturated carbonyl), -R ^ALK -Hg-X (alkylmercury), and -S (= O) CR ^B = CR ^B ₂ (α, β-unsaturated sulfone) Selected from the group consisting of: wherein each X is independently a halogen having an atomic number of 16 to 54, or another good leaving group; each R ^ALK is independently a straight chain, a moiety A branched or cyclic (1-20C) alkyl group; each R ^SUB independently represents H, straight chain, branched or cyclic (1-20C) alkyl, (6-20C) aryl , Or (7
-30C) alkylaryl; each R ^ESTER is independently N-succinimidyl, p-nitrophenyl, pentafluorophenyl, or other activating group;
R ^B is independently a group containing 1-50 atoms selected from the group consisting of C, H, N, O, Si, P and S; and, if present, n represented by L ^{[2] [2]} The parts are O, N
Selected from the group consisting of R ^SUB and S; when present, each of the n ^[2] moieties denoted by J ^[2] is independently C (=
O) and C (= S); n ^[1] is 1 to 32; provided that the product of n ^[2] × p ^[2] is greater than 1 and less than 33. , N ^[2] is 1 to 32; p ^[2] is 1 to 8; Z ^[2]
The n ^[2] parts represented by are independently at least p
An attachment site for the ^[2] functional group is an alkyl carbon atom,
A group containing 1 to 200 atoms selected from the group consisting of C, H, N, O, Si, P and S on an alkenyl carbon atom or an aromatic carbon atom. 2. The G ^[1] and G ^[2] are each independently C,
The complex according to claim 1, which is a straight chain, a branched chain, or a multi-branched chain containing 1-1000 chain atoms selected from the group consisting of N, O, Si, P and S. 3. The G ^[2] is a group derived from polyalcohol, polyamine, or polyglycol,
The composite according to claim 1. 4. The G ^[2] has a value of-(CH ₂ ) _{q-in which} q is 0 to 20, and r is 0.
To 300 of -CH ₂ (CH ₂ OCH ₂ ) _r CH _2- , and s of 1 to 4 of C (CH
_{2 0CH 2 CH 2 -) s} (OH) is selected from the group consisting of _4-s, composite according to claim 1. 5. The complex according to claim 4, wherein the s is 3 to 4. 6. The n ^[1] parts shown as T ^[1] and the p ^[2] × n ^[2] parts shown as T ^[2] are each independently, -NHR ^SUB (amine), -C (= O) CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂ OR ^ALK X (alkylsulfonate), R ¹ R ² is- C (= O) CH = CHC (= O) -is-N
R ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl), -R ^ALK -Hg-X (alkylmercury), and -S (= O) CR ^B
The complex according to claim 1, which is selected from the group consisting of: = CR ^B ₂ (α, β-unsaturated sulfone). 7. The n ^[1] part shown as T ^[1] and the p ^[2] × n ^[2] part shown as T ^[2] are each independently, -NHR ^SUB (amine), -C (= O) CH ₂ X (haloacetyl), R ¹ R ² is -C (= O) CH = CHC (= O)-, -NR ¹ R ² (maleimide), And a complex according to claim 6, which is selected from the group consisting of -C (= O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl). 8. All of the n ^[1] parts shown as T ^[1] and all of the p ^[2] × n ^[2] parts shown as T ^[2] are: The composite of claim 1, which is the same. 9. The complex according to claim 1, wherein the n ^[1] is 2 to 16. 10. The complex according to claim 9, wherein the n ^[1] is 2 to 8. 11. The method of claim 10, wherein n ^[1] is 2 to 4.
The complex according to. 12. The method of claim 1, wherein n ^[2] is 1 to 16.
The complex according to. 13. The method of claim 12, wherein n ^[2] is 1 to 8.
The complex according to. 14. The method of claim 13, wherein n ^[2] is 1 to 4.
The complex according to. 15. The method of claim 14, wherein the n ^[2] is 1 to 2.
The complex according to. 16. The complex according to claim 1, wherein the p ^[2] is 1 to 4. 17. The method of claim 16, wherein p ^[2] is 1 to 2.
The complex according to. 18. The complex of claim 1, wherein all of the n ^[2] moieties designated as Z ^[2] are the same. 19. The complex of claim 1, wherein each of the n ^[2] moieties designated as Z ^[2] are independently described by a formula selected from the following group: Chemical 3] [Chemical 4] Alternatively, However, if present, the n ^[2] moieties denoted as W ^[3] , W ^[4] , or W ^[5] are each independently C, H, N, O, Si, P and A group containing 1-100 atoms selected from the group consisting of S; and n ^[2] moieties shown as Y ^[3] , 2 × n ^[2] shown as Y ^{[4 ]} , And 2 × n ^[2] parts, denoted Y ^[5] , are each independently at least p ^[2]
Functional groups (for Y ^[3] ) or p ^[2] / 2 functional groups
(Where Y ^[4] and Y ^[5] , where p ^[2] / 2 is an integer) containing a binding site on an alkyl carbon atom, an alkenyl carbon atom, or an aromatic carbon atom, C, H, N, O, S
A group containing 1-100 atoms selected from the group consisting of i, P and S. 20. The complex of claim 19, wherein each of the n ^[2] moieties designated as W ^[3] , when present, independently has r of 1-10. CH ₂ ) _r , (CH ₂ CH ₂ O) _r , NR
^SUB (CH ₂ CH ₂ O) _r CH ₂ CH ₂ and NR ^SUB (CH ₂ ) _r NR ^SUB C (= O); and each of the n ^[2] parts indicated by Y ^[3] is Independently, straight chain, branched or cyclic (1-20C) alkyl,
Aryl of (6-20C), or alkylaryl of (7-30C); when present, each of the n ^[2] moieties designated as W ^[4] are independently r 1 to 10 , (CH ₂ ) _r C (=
O), and (CH ₂ ) _r NR ^SUB C (= O); the 2 × n ^[2] moieties denoted as Y ^[4] are each independently r = 1 To 10 and q is 1 to 10, (CH ₂ ) _r , (CH ₂ ) _r NR
^SUB C (= O) (CH ₂ ) _q , (CH ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q , (CH ₂ ) _r NR
^SUB C (= O) (CH ₂ ) _q NR ^SUB C (= O) (CH ₂ ) _r , (CH ₂ ) _r C (= O) NR ^SUB (C
H ₂ ) _q NR ^SUB C (= O) (CH ₂ ) _r , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ CH ₂ O) _q CH
₂ CH ₂ , and (CH ₂ ) _r C (= O) NR ^SUB (CH ₂ CH ₂ O) _q CH ₂ CH ₂ ; if present, indicated by W ^[5]
Each of the n ^[2] parts independently has r of 1 to 10, (C
H ₂ ) _r C (= O) NR ^SUB and (CH ₂ ) _r NR ^SUB C (= O) NR ^SUB ; 2 × n ^[2] indicated by Y ^[5] Independently, each part of r is 1 to 10 and q is 1 to 10, (C
H ₂ ) _r and (CH ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q . 21. The n ^[2] moieties represented by Y ^[3] are each independently C ₆ H ₄ (1,4-disubstituted phenyl), C ₆ H ₃ (1,3, The complex of claim 20, wherein the complex is selected from the group consisting of (CH ₂ ) _r , wherein r is 1-10. 22. For the Y ^[4] , r is 2 to 6,
The composite according to claim 20. 23. For said Y ^[4] , q is 1 to 3,
The composite according to claim 20. 24. The biological molecule comprises a double-stranded polynucleotide of at least about 20 base pairs, each bound to the valency platform molecule, each of the duplexes comprising a human SLE (systemic erythema). Lupus) anti-dsD
2. It has a remarkable binding activity to NA autoantibodies.
The complex according to. 25. The biological or chemical molecule is a carbohydrate, lipid, lipopolysaccharide, peptide, protein, glycoprotein, single-stranded or double-stranded oligonucleotide, hapten or mimotope,
The complex of claim 1, selected from the group consisting of these chemical analogs, such as aptamers. 26. The complex according to claim 1, wherein said biological or chemical molecule is an analog of an immunogen, provided that (a) said analog is B to which said immunogen specifically binds. Binds specifically to cells, and (b) the complex lacks T cell epitopes. 27. The bond platform molecule is DA
The complex of claim 1, wherein the complex is derived from a reagent selected from the group consisting of BA, BAHA, BAHA _OX , and AHAB. 28. The complex of claim 24, wherein a linker molecule attaches the duplex to the valency platform molecule. 29. The complex of claim 28, wherein the linker molecule is selected from the group consisting of HAD and HAD _p S. 30. The complex of claim 24, wherein the length of the double strands is substantially uniform. 31. The complex of claim 24, wherein the double-stranded nucleotide composition is substantially uniform. 32. The complex of claim 24, wherein the duplex length is 20 to 50 base pairs. 33. The complex according to claim 24, wherein the double strand is bound to the bond platform molecule at or near the end thereof. 34. The complex according to claim 24, wherein the polynucleotide duplex is composed of 2 to 4 mer complementary repeating units of different bases. 35. The polynucleotide duplex is: poly (C
G): poly (CG); poly (TA): poly (TA); poly (CI): poly (C
I); poly (CA): poly (TG); or poly (GA): poly (T
C); The complex according to claim 24. 36. The polynucleotide duplex is (C
25. The complex of claim 24, wherein A) ₁₀ : (TG) ₁₀ and the bond platform molecule is derivatized triethylene glycol. 37. The molar ratio of the duplex to the bond platform molecule is from 2: 1 to 8: 1.
7. The complex according to item 6. 38. The complex according to claim 36, wherein the molar ratio of the double chain and triethylene glycol is 4: 1. 39. The polynucleotide duplex is (C
25. The complex of claim 24, wherein A) ₁₀ : (TG) ₁₀ and the bond platform molecule is a derivatized 1,2-bis- (2′-aminoethoxy) ethane. 40. The complex according to claim 39, wherein the molar ratio of the double strand and 1,2-bis- (2′-aminoethoxy) ethane is 2: 1 to 8: 1. . 41. The molar ratio of the duplex to 1,2-bis- (2′-aminoethoxy) ethane is 4: 1.
The complex according to. 42. The complex of claim 24, wherein the complex is a human SLE (systemic lupus erythematosus) immunotolerogen. 43. The polynucleotide double strand is B-DNA.
25. The complex of claim 24 having a helical structure of the type and having significant binding activity to human SLE (systemic lupus erythematosus) anti-dsDNA autoantibodies. 44. A pharmaceutical composition for the treatment of lupus comprising the conjugate of claim 24 formulated with a pharmaceutically acceptable injectable excipient. 45. Administering a therapeutically effective amount of the composition of claim 44 to an individual in need thereof.
A method of treating an individual suffering from lupus. 46. A method for producing a complex according to claim 24, comprising the steps of: (a) providing a plurality of single stranded polynucleotides, each of at least about 20 base pairs, to said bond platform. Conjugating onto the molecule; and (b) annealing a complementary single-stranded polynucleotide with the single-stranded polynucleotide bound to a valency platform molecule to form the duplex. 47. The method of claim 46, further comprising first attaching the polynucleotide duplex to a linker molecule and then attaching the linker molecule to the bond platform molecule. 48. The complex of claim 26, wherein the immunogen is a foreign immunogen. 49. The foreign immunogen is a biological drug, allergen, Rh / D immunogen associated with Rh hemolytic disease, α-sperm associated with male infertility, or a carbohydrate complex associated with rheumatic fever. 49. The complex of claim 48 which is a body. 50. The complex of claim 26, wherein the immunogen is an autoimmunogen. 51. The autoimmunogen is thyroiditis, diabetes,
6. Associated with stroke or myasthenia gravis.
The complex according to 0. 52. The complex of claim 26, wherein the immunogen and the analog are the same chemical species. 53. The complex of claim 52, wherein the immunogen and the analog are polypeptides. 54. The complex of claim 26, wherein the immunogen and the analog are different chemical species. 55. A pharmaceutical composition for the treatment of antibody mediated diseases comprising a therapeutically effective amount of the complex of claim 26 in combination with a pharmaceutically acceptable carrier. 56. A method of inducing B cell anergy specific for an immunogen in an individual, comprising the step of administering to the individual an effective amount of the complex of claim 26. 57. A method of inducing anergy in a B cell specific for an immunogen in an individual, comprising the step of administering to said individual an effective amount of the composition of claim 55. 58. An individual having an antibody-mediated disease in which a unwanted antibody is produced in response to an immunogen comprising the step of administering to the individual a therapeutically effective amount of the complex of claim 26. How to treat. 59. An individual having an antibody-mediated disease in which an undesired antibody is produced in response to an immunogen, comprising the step of administering to the individual a therapeutically effective amount of the composition of claim 55. How to treat. 60. A method of producing the complex of claim 26, comprising the steps of: (a) binding the chemically defined analog of the immunogen lacking a T cell epitope. Covalently binding to a hand platform molecule to form a complex; and
(b) A step of recovering the complex from the reaction mixture. 61. The method of claim 60, wherein the immunogen is a foreign immunogen. 62. The foreign immunogen is a biological agent, an allergen, α-sperm associated with male infertility, or Rh.
62. The method of claim 61, which is a Rh / D immunogen associated with hemolytic disease. 63. The method of claim 60, wherein the immunogen is an autoimmunogen. 64. The autoimmunogen is thyroiditis, diabetes,
64. The method of claim 63, which is associated with stroke, myasthenia gravis, or rheumatic fever. 65. The method of claim 60, wherein the immunogen and the analog are the same chemical species. 66. A chemically defined non-polymeric valency platform molecule of the formula: Alternatively, However, when present, G ^[6] and G ^[7] are each independently C, N,
A linear, branched or multiply branched chain containing 1-2000 chain atoms selected from the group consisting of O, Si, P and S; n ^{[6 ]} designated as T ^{[6] ]} × p ^[6] moieties and p ^[7] × n ^[7] moieties denoted as T ^[7] are each independently -NHR ^SUB (amine), -C (= O ) NHNHR ^SUB (hydrazide), -NHNHR ^SUB (hydrazine), -C (= O) OH (carboxylic acid),
-C (= O) OR ^ESTER (active ester), -C (= O) OC (= O) R ^B (anhydride), -C (= O) X (acid halide), -S (= O) ₂ X (sulfonyl halide), -C (= NR ^SUB ) OR ^SUB (imidate ester), -NCO
(Isocyanate), -NCS (isothiocyanate), -OC (=
O) X (haloformate), -C (= O) OC (= NR ^SUB ) NHR ^SUB (carbodiimide adduct), -C (= O) H (aldehyde), -C (= O) R ^B (ketone), -SH (sulfhydryl or thiol), -OH
(Alcohol), -C (= O) CH ₂ X (haloacetyl), -R ^ALK X (alkyl halide), -S (= O) ₂ OR ^ALK X (alkylsulfonate), R ¹ R ² is -C (= O) CH = CHC (= O)-is -NR ¹ R ² (maleimide), -C (= O) CR ^B = CR ^B ₂ (α, β-unsaturated carbonyl), -R ^ALK
-Hg-X (alkylmercury), and -S (= O) CR ^B = CR ^B ₂ (α, β-unsaturated sulfone); wherein each X is independently an atom. Halogen numbered from 16 to 54, or other good leaving group; each R ^ALK is independently a linear, branched, or cyclic (1-20C) alkyl group; each R ^SUB Are independently H, linear, branched, or cyclic (1-20C) alkyl, (6-20C) aryl, or (7
-30C) alkylaryl; each R ^ESTER is independently N-succinimidyl, p-nitrophenyl, pentafluorophenyl, or other activating group;
R ^B is independently a group containing 1-50 atoms selected from the group consisting of C, H, N, O, Si, P and S; the product of n ^[6] × p ^[6] , Greater than 1 and less than 33, n ^[6] is 1 to 32; p ^[6] is 1 to 8; n ^[7]
X ^[7] is greater than 1 and less than 33, n ^[7] is 1 to 32; p ^[7] is 2, 4, 6 or 8; Q ^{[6 ] [} N ^{] [6]} parts and Q ^[7] 2 x n ^[7] parts are each independently at least p
^[6] (for Q ^[6] ) or p ^[7] / 2 (for Q ^[7] ,
Where p ^[7] / 2 is an integer) containing a bonding site for a functional group on an alkyl carbon atom, an alkenyl carbon atom, or an aromatic carbon atom, such as C, H, N, O, Si, P and It is a group containing 1-100 atoms selected from the group consisting of S. 67. The G ^[6] and G ^[7] , if present,
A straight chain, branched or multiply branched chain, each independently containing 1-1000 chained atoms selected from the group consisting of C, N, O, Si, P and S, A bond platform molecule according to 66. 68. The bond platform molecule of claim 66, wherein each of G ^[6] and G ^[7] is a group derived from a polyalcohol, polyamine, or polyglycol. 69. The G ^[6] and G ^[7] each have a q from 0 to 20.
-(CH ₂ ) _q- , r from 0 to 300 -CH ₂ (CH ₂ OCH ₂ ) _r CH _2- , and s from 1 to 4 C (CH ₂ 0CH ₂ CH _2- ) _s (OH ) A bond platform molecule according to claim 66 selected from the group consisting of _4-s . 70. The bond platform molecule of claim 69, wherein said s is 3 to 4. 71. The T^[6]Indicated as n^[6]× p^[6]Of
Part and T^[7]Indicated as n^[7]× p^[7]Pieces
Each independently -NHR^SUB(Amine), -C (= O) CH₂X (Haloa
Cetyl), -R^ALKX (alkyl halide), -S (= O)₂OR^ALKX
(Alkyl sulfonate), R¹R²Is -C (= O) CH = CHC (= O)-
Yes-NR¹R²(Maleimide), -C (= O) CR^B= CR ^B ₂(α, β-Fatigue
Carbonyl), -R^ALK-Hg-X (alkyl mercury), and -S
(= O) CR^B= CR^B ₂(α, β-unsaturated sulfone)
67. The bond platform of claim 66, which is selected.
molecule. 72. The n ^[6] × p ^[6] parts shown as T ^[6] and the n ^[7] × p ^[7] parts shown as T ^[7] Each independently, -NHR ^SUB (amine), -C (= O) CH ₂ X (haloacetyl), R ¹ R ² is -C (= O) CH = CHC (= O)-, -NR ¹ R ² (maleimide), - C (= O) CR B = CR B 2 (α, β- unsaturated carbonyl) selected from the group consisting of a bond platform molecule of claim 66. 73. All of the n ^[6] × p ^[6] parts shown as T ^[6] and the n ^[7] × p ^[7] parts shown as T ^[7] 67. The bond platform molecule of claim 66, wherein all of the moieties are the same. 74. The n ^[6] is 1-16.
A bond platform molecule according to item 6. 75. The n ^[6] is 1-8.
A bond platform molecule described in. 76. The method of claim 75, wherein n ^[6] is 1 to 4.
A bond platform molecule described in. 77. The n ^[6] is 1 to 2 above.
A bond platform molecule described in. 78. The method of claim 66, wherein p ^[6] is 1 to 4.
A bond platform molecule described in. 79. The p ^[6] is 1 to 2;
A bond platform molecule described in. 80. The method of claim 6, wherein n ^[7] is 1 to 16.
A bond platform molecule according to item 6. 81. The n 80 wherein n ^[7] is 1-8.
A bond platform molecule described in. 82. The method of claim 81, wherein n ^[7] is 1 to 4.
A bond platform molecule described in. 83. The method of claim 82, wherein n ^[7] is 1 to 2.
A bond platform molecule described in. 84. The method of claim 66, wherein p ^[7] is 1 to 4.
A bond platform molecule described in. 85. The p ^[7] is 1 to 2;
A bond platform molecule described in. 86. A valency platform molecule according to claim 66, wherein all of the n ^[6] moieties designated as Q ^[6] are the same. 87. A valency platform molecule according to claim 66, wherein all 2 × n ^[7] moieties designated as Q ^[7] are the same. 88. CH [(CH wherein CH is [1] and q is 1 to 10, wherein each of the n ^[6] moieties shown as Q ^[6] is independent.
₂ ) _r (binding site)] ₂ , and CH [(CH ₂ ) _r C (= O) NR ^SUB (CH ₂ ).
_q (binding site)] ₂ is selected from the group consisting of
A bond platform molecule described in. 89. The 2 × n ^[7] portions shown as Q ^[7] are each independently, r is 1 to 10 and q is 1 to 10.
H ₂ ) _r , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ ) _q , (CH ₂ ) _r C (= O) NR ^SUB (C
H ₂ ) _q , (CH ₂ ) _r NR ^SUB C (= O) (CH ₂ ) _q NR ^SUB C (= O) (CH ₂ ) _r , (C
H ₂ ) _r C (= O) NR ^SUB (CH ₂ ) _q NR ^SUB C (= O) (CH ₂ ) _r 、 (CH ₂ ) _r NR ^SUB C
(= O) (CH ₂ CH ₂ O) _q CH ₂ CH ₂ and (CH ₂ ) _r C (= O) NR ^SUB (CH ₂
CH ₂ O) is selected from the group consisting of _q CH ₂ CH _2, bond platform molecule of claim 66. 90. For the Q ^[7] , r is 2 to 6,
90. The bond platform molecule of claim 89. 91. For the Q ^[7] , q is 1 to 3,
90. The bond platform molecule of claim 89.