JP2007031448A - Bacterial endotoxin adsorbent and method for screening the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an easily synthesizable new lipopolysaccharide adsorbent (bacterial endotoxin adsorbent), enabling easy adjustment of various relating structures, and usable for the adsorption of various lipopolysaccharides and provide a screening method for the adsorbent. <P>SOLUTION: The method for the screening of a bacterial endotoxin adsorbent comprises the binding assay (binding experiment) of a peptide library and a radioactive labeled lipid A produced by introducing a radioactive element to a phosphonoxyethyl derivative of a biosynthesis precursor-type lipid A or an E.coli-type lipid A. The invention further relates to a bacterial endotoxin adsorbent composed of a specific peptide library found by the screening method. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a screening / preparation method of endotoxin (bacterial endotoxin) adsorbent and an adsorbent having a specific structure found by the method.

  Higher animals have a system that activates their own immune system when bacteria enter the body, and this system is attracting attention as innate immunity. Lipopolysaccharide (LPS, endotoxin, endotoxin) is the main component of the cell surface layer of gram-negative bacteria such as Escherichia coli and Salmonella, and acts as a signal for bacterial invasion, lipids such as cytokines, prostaglandins and PAF, NO, etc. The immune system is activated by producing various mediators. When an appropriate amount of lipopolysaccharide acts, a beneficial immune response occurs, but when the lipopolysaccharide acts excessively due to infection, the immune system runs away, causing high fever and systemic blood clotting, causing fatal shock It is. On the other hand, mixing of lipopolysaccharide in the artificial dialysate may induce cytokines and cause various complications when lipopolysaccharide enters the body during dialysis. Another major problem is the incorporation of lipopolysaccharide into biological products. Therefore, development of a lipopolysaccharide adsorbent for removing lipopolysaccharide has been desired. However, up to now, what has been developed and put into practical use as an adsorbent for lipopolysaccharide is tremyxin (trade name of Toray Industries, Inc.) used for endotoxin adsorption therapy, in which the antibiotic polymyxin B is immobilized on a fiber. There is only about.

  The present invention has been made in view of the present situation as described above, and is a novel lipopolysaccharide adsorbent (bacterial endotoxin adsorption) that is easy to synthesize, can be easily adjusted to various similar structures, and can be used for adsorption of various lipopolysaccharides. Agent) and a screening method thereof.

  The present invention performs a binding assay (binding experiment) with a peptide library using a lipid A radiolabeled product in which a radioactive element is introduced into a phosphonooxyethyl derivative of biosynthetic precursor type lipid A or E. coli type lipid A. And a bacterial endotoxin adsorbent found by the screening method.

  In the present invention, a solid phase carrier having a functional group is bound to a compound having a functional group capable of binding to the functional group and having two or more other functional groups, and further, an oligopeptide is bound thereto. It is related with the peptide library.

  Furthermore, the present invention allows a compound having a functional group capable of binding to the functional group and having two or more other functional groups to be bound to a solid phase carrier having a functional group, and further to the other functional group. The present invention relates to a peptide library in which a compound having a functional group capable of binding to a group and having two or more other functional groups is bound, and then an oligopeptide is further bound thereto.

  Furthermore, the present invention relates to a lipid A radiolabeled product obtained by introducing a radioactive element into a phosphonooxyethyl derivative of synthetic precursor type lipid A or E. coli type lipid A.

That is, the 1-position glycosyl phosphate group of natural lipid A is chemically unstable, but the PE analog in which this is replaced with a phosphonooxyethyl (PE) group is chemically stable, It has been clarified that the activity is equivalent to that of the above. The present inventors have previously developed and reported an efficient method for synthesizing PE analogues (Bull. Chem. Soc. Jpn., 72 , 1377 (1999)). Was successfully synthesized using E. coli lipid A tritium-labeled PE analogues by using NaB ³ H ₄ for reduction of aldehyde intermediates, and these radiolabels were used to bind to specific peptide libraries (binding experiments). ), It was found that effective bacterial endotoxin adsorbent screening can be performed, and the present invention has been completed.

  The present invention relates to a screening / preparation method for endotoxin (bacterial endotoxin) adsorbent and an adsorbent having a specific structure found by the method, which is easy to synthesize and can easily adjust various similar structures, It has an effect in that it provides a new bacterial endotoxin adsorbent (endodoxin adsorbent) that can be used for adsorption of various bacterial endotoxins. In addition, the adsorbent of the present invention has a great advantage in using natural products (natural amino acids) as compared with existing adsorbents.

Examples of the radioactive element in the radiolabeled lipid A analog obtained by introducing a radioactive element into the phosphonooxyethyl derivative of biosynthetic precursor type lipid A or Escherichia coli type lipid A used in the present invention include tritium ( ³ H). It is done.
Examples of the radiolabeled lipid A analog obtained by introducing a radioactive element into the phosphonooxyethyl derivative of E. coli lipid A used in the present invention include those represented by the following structural formula [4].

Moreover, as a radiolabeled lipid A analog used by this invention which introduce | transduced the radioactive element into the phosphonooxyethyl derivative of biosynthetic precursor type lipid A, what is shown by following Structural formula [5] is mentioned, for example. .

  The method for producing the radiolabeled lipid A analog is as described in detail in Examples below.

As the peptide library used in the present invention, for example, a solid phase carrier having a functional group is bound to a compound having a functional group capable of binding to the functional group and having two or more other functional groups, A peptide library prepared by binding an oligopeptide to this can be mentioned.
Further, a compound having a functional group capable of binding to the functional group and having two or more other functional groups is bound to a solid phase carrier having a functional group, and further bonded to the other functional group. Peptide libraries prepared by binding a compound having a functional group to be obtained and having two or more functional groups other than this, and then further binding an oligopeptide thereto are also preferred.

Examples of the solid phase carrier having a functional group include a solid phase carrier having an amino group.
When the solid phase carrier having a functional group is a solid phase carrier having an amino group, examples of the compound having a functional group capable of binding to the amino group and having two or more other functional groups include the formula: HOOC (NH ₂ ) CH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 4), carboxylic acids having two or more hydroxyl groups such as deoxycholic acid and chenodeoxycholic acid Etc.
Specific examples of the amino acid include lysine, ornithine and the like.

Solid phase carriers having a functional group is a solid phase carrier having an amino group, a functional group capable of binding the amino group, and a compound having two or more other functional groups has the formula: HOOC (NH ₂₎ In the case of an amino acid represented by CH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 4), the amino acid has a functional group capable of binding to the other functional group, and other than this Examples of the compound having two or more functional groups include basic amino acids represented by the formula: HOOC (NH ₂ ) CH (CH ₂ ) _n NH ₂ (wherein n represents an integer of 1 to 4). It is done.
Specific examples of the amino acid include lysine, ornithine and the like.

In the peptide library used in the present invention, the solid phase carrier having a functional group has a functional group capable of binding to the functional group, and a compound having two or more other functional groups, or the functional group A compound having a functional group capable of binding to the functional group and having two or more other functional groups is bound to a solid phase carrier having a functional group capable of binding to the other functional group. As an oligopeptide to be bonded via a compound having a compound having two or more functional groups other than this, for example, the amino acid residue is 2 to 20, preferably 3 to 10, more preferably 4 to 8 oligopeptides.
The type of amino acid residue is not particularly limited. For example, Gly, D, L-Val, D, L-Phe, D, L-Pro, D, L-Ser, D, L-Gln, D, L- Glu, D, L-Lys and the like are preferable.

（式中、球状の部分は固相担体を表し、ＡＡ_１、ＡＡ_２、ＡＡ_３及びＡＡ_４はそれぞれ独立してアミノ酸残基を表す。）

（式中、球状の部分は固相担体を表し、ＡＡ_１、ＡＡ_２、ＡＡ_３及びＡＡ_４はそれぞれ独立してアミノ酸残基を表す。）

（式中、球状の部分は固相担体を表し、ＡＡ_１、ＡＡ_２、ＡＡ_３及びＡＡ_４はそれぞれ独立してアミノ酸残基を表す。） Specific examples of the peptide library according to the present invention include compounds represented by the following general formula [1], [2] or [3].

(In the formula, the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue.)

(In the formula, the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue.)

(In the formula, the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue.)

The general formula [1], in peptide libraries in [2] or _[3], AA _1, AA 2, AA ₃ and in particular restrictions on the type of amino acid residue represented by AA ₄ is not, either An amino acid residue may be used, but preferred examples include Gly, D, L-Val, D, L-Phe, D, L-Pro, D, L-Ser, D, L-Gln, D, L- Glu, D, L-Lys etc. are mentioned.

The bacterial endotoxin adsorbent of the present invention is searched and extracted by performing a binding assay with the above peptide library using the lipid A radioactive label, and the bacterium of the present invention thus obtained is extracted. As specific examples of the endotoxin adsorbent, for example, in the above general formula [1], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described in Table 1 below,

In the above general formula [2], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described in Table 2 below,

And in the above general formula [3], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described in Table 3 below,

Etc.
Correlation between the effectiveness of the oligopeptide structure with bacterial endotoxin adsorbent is not fully elucidated yet, it is more effective manner that has lysine AA _4.

Examples of the solid phase carrier having a functional group used in the present invention include a resin having a functional group such as an amino group, a carboxyl group, and a hydroxyl group, preferably a bead-like resin. Specific examples of such a resin include, for example, a polyethylene glycol-polystyrene resin having a functional group as described above [specific names include, for example, TentaGel S-NH ₂ (Rapp Polymere), Aminomethyl NovaGel HL ( Novabiochem). ] Or a hydrophilic vinyl polymer [as a specific trade name, for example, TSKgel AF-AminoTOYOPARRL 650S (Tosoh Corporation), etc.). ] Etc. are mentioned.

The outline of the binding experiment between the radiolabeled lipid A analog and the molecular tweezer library (peptide library) is described below.
Preparation of molecular tweezers library First, an encoded molecular tweezers library (peptide library) was prepared by split synthesis.
That is, using chenodeoxycholic acid as a template, a tetrapeptide library bound to its two hydroxyl groups via a Gly residue (diarm L1), and a tetrapeptide library bound to two amino groups of L-Lys. (Diarm L2) and L-Lys are linked to L-Lys, and a tetrapeptide library is bound to a total of four amino groups (tetraarm L3). In consideration of performing the assay in water, TentaGel, which is a swellable polyethylene glycol-polystyrene resin, was used as the solid phase carrier. When L1 was used as the core structure, deoxycholic acid was first introduced into the solid support, and then Fmoc-Gly was bound to the two hydroxyl groups. Lysine (L2) and trilysine (L3) cores were prepared by conventional peptide solid phase synthesis. 15 types of amino acids (Gly and D and L-Val, Phe, Ser, Gln, Glu, Lys and Pro) were used for library preparation. 4 by performing the steps of splitting coupling cycle was constructed libraries containing 3 × ¹⁵ 4 = 151,875 different peptides.

Binding experiment of radiolabeled lipid A analog and molecular tweezers library Radiolabeled lipid A analog represented by the above structural formula [4] (hereinafter abbreviated as [ ³ H] PE506), or the above structural formula [5] Using a radiolabeled lipid A analog (hereinafter abbreviated as [ ³ H] PE406), a binding experiment (binding assay) with a molecular tweezer library was performed. Nestler has already established a binding experiment with a molecular tweezer library using a radioactive compound, and the method was followed (Nestler HP; Wennemers H .; Sherlock R .; Dong DL-Y. Bioorg. Med. Chem Lett., 6 , 1327 (1996).).
For binding experiments with [ ³ H] PE506, the library (15.7 mg, containing about 19000 beaded resins) was shaken at 4 ° C. for 41 hours in the presence of [ ³ H] PE506. Resin recognizing molecules tweezers are bound to ^[3 H] PE506 This operation takes on radioactivity because adsorbs ^[3 H] PE506. When Kodak autoradiographic emulsion is applied to this, the area around the radioactive resin is exposed. After fixing, if the excess emulsion is removed, the radioactive resin will turn black. This was taken out and the peptide sequence on the resin was determined by the encoding method described later. As a result, 19 types of molecular tweezers that bind to [ ³ H] PE506 were found (Table 4).

As is clear from Table 4, up to 17 out of 19 molecular tweezers had the L3 structure. In addition, 9 types of L-lysine (K) residues (K-L3 structure) and 3 types of D-lysine (k) (k-L3 structure) were found at the C-end of each arm. It was done. Although the whole library contains 151,875 molecular tweezers, the number of bead-shaped resins used in the actual assay is about 19,000, and the number of molecular tweezers examined in the assay is 20 Less than%. Therefore, in addition to the peptide sequences found this time, it is predicted that there are many that bind to [ ³ H] PE506.

For binding experiments using [ ³ H] PE406, a similar binding experiment was performed using a library (24.2 mg, containing about 29000 beads of resin). As a result, 32 types of molecular tweezers were found (Table 5).

As is clear from Table 5, 20 out of 32 types had L3 structures, and only 5 of them had an L-lysine residue at the C-terminal end of the tetrapeptide. Although this portion is still a consensus sequence, [ ³ H] PE406 is thought to be able to bind various molecular tweezers because of increased conformational mobility.

Resynthesis of lipid A analog-recognizing molecular tweezers In order to confirm whether the molecular tweezers found as described above bind to natural lipid A or lipopolysaccharide, molecular tweezers are appropriately selected from the above molecular tweezers. Resins 6-17 were prepared.
^[3 H] molecular tweezers found in binding experiments with _{PE506 (k-skS) 4 L3} -TentaGel (6-TentaGel), (sSKk) 4 L3-TentaGel (7-TentaGel), (KKkk) 4 L3- TentaGel (8-TentaGel), (QEKk) ₄ L3-Tenta-gel (9-TentaGel), (KSKE) ₄ L3-TentaGel (10-TentaGel), (EqfK) ₄ L3-TentaGel (11-TentaGel)
Molecular tweezers found in binding experiments with [ ³ H] PE406 (S-sFv) ₄ L3-TentaGel (12-TentaGel), (FVEs) ₄ L3-TentaGel (13-TentaGel), (kKeE) ₄ L3- TentaGel (14-TentaGel), (KvEe) ₄ L3-TentaGel (15-TentaGel), (PESK) ₄ L3-TentaGel (16-TentaGel), (PPSk) ₄ L3-TentaGel (17-TentaGel)

まず分子ピンセット樹脂６〜１７-TentaGelのトリフルオロ酢酸塩を用いてＰＥ５０６及び５０６との結合実験を行った。樹脂への結合はアッセイ後の溶液ＴＬＣ分析を行って確認した。結果を表６に示す。 Binding experiment using re-synthesized molecular tweezers Next , using re-synthesized molecular tweezers, phosphonooxyethyl derivative of E. coli lipid A (hereinafter abbreviated as PE506), E. coli lipid A (hereinafter referred to as 506) Abbreviated)), phosphonooxyethyl derivative of biosynthetic precursor type lipid A (hereinafter abbreviated as PE406), biosynthetic precursor type lipid A (hereinafter abbreviated as 406), lipopolysaccharide of Escherichia coli Re mutant Binding experiments with (ReLPS) and E. coli lipopolysaccharide (E. coli O111: B4 LPS (Sigma)) were performed. ReLPS has a structure in which a sugar called Kdo is bonded to the lipid A part, and lacks most of the core polysaccharide part and the O-specific polysaccharide part.
For reference, the structural formula of ReLPS is shown below.

First, binding experiments with PE506 and 506 were performed using molecular tweezer resins 6 to 17-TentaGel trifluoroacetate. Binding to the resin was confirmed by solution TLC analysis after the assay. The results are shown in Table 6.

Next, the adsorption ability of the resins 6-TentaGel, 11-TentaGel, 12-TentaGel, 16-TentaGel, 17-TentaGel was confirmed by measuring the Limulus activity of the solution after the assay. The Limulus activity test is a method for colorimetric determination of endotoxin activity using a reaction in which lipopolysaccharide activates a blood cell extract component LAL (Limulus Amebocyte Lysate) coagulase of horseshoe crab (such as Limulus polyphemus).
Using PE506, 506, PE406, 406, ReLPS, and E. coli lipopolysaccharide (E. coli O111: B4 LPS (Sigma)), 6-TentaGel, 11-TentaGel, 12-TentaGel, 16- Binding experiments with TentaGel and 17-TentaGel were performed, and the Limulus activity was measured for the supernatant. The absorbance observed when these endotoxins are adsorbed to the resin decreases. Here, since each resin was washed with water before the assay, most amino groups on the resin are considered to be free. Therefore, the adsorption capacity of the resin for lipopolysaccharide and lipid A, which are acidic complex carbohydrates, is improved as compared with that before washing with water. In fact, when the above trifluoroacetate is used, 12-TentaGel does not adsorb PE 506 and 506, but both 12-TentaGel after washing with water adsorbs well. Resin 11-TentaGel has a weak adsorption force to 406. All the resins showed strong adsorption ability to natural ReLPS, but hardly showed adsorption ability to E. coli lipopolysaccharide (E. coli O111: B4 LPS (Sigma)) (see FIG. 1). ).

  TentaGel is a polyethylene glycol-polystyrene graft polymer, which is a highly hydrophobic resin that swells in water. E. coli lipopolysaccharide (LPS) has a long sugar chain, and it was thought that the hydrophilic sugar chain part hinders penetration into the resin. Therefore, 6-Toyopearl, 11-Toyopearl, 12-Toyopearl to which molecular tweezers 6, 11, 12, 16 and 17 are bonded using TSKgel AF-AminoTOYOPEARL 650S (Tosoh Corp.) as a carrier. , 16-Toyopearl, and 17-Toyopearl were synthesized, and binding experiments with 506, ReLPS, and E. coli lipopolysaccharide (E. coli O111: B4 LPS (Sigma)) were performed, and the binding ability was evaluated by Limulus activity. As a result, as expected, the resin based on Toyopearl adsorbed all of these efficiently with few exceptions (see FIG. 2).

From the above, 19 types of molecular tweezers (peptide library) described in Table 4 and 32 types of molecular tweezers (peptide library) described in Table 5 are both more effective as bacterial endotoxin adsorbents. Highly expected to be usable.
The adsorbent of the present invention has a great advantage in that natural products (natural amino acids) are used as compared with existing adsorbents.
In addition, in order to obtain the specific adsorbent of the present invention, as described above, 15 ⁴ × 3 = 1151875 were selected from 15 amino acids, and actually, a library consisting of 19000 and 29000 was obtained, From this, 51 effective adsorbents were obtained.
At this time, selection of 15 amino acids and narrowing down to 19000 and 29000 from 151875 were carried out in consideration of such points as hydrophobicity, hydrophilicity, and acid / basicity.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

なお、シリカゲルカラムクロマトグラフィーにはＫｉｅｓｅｌｇｅｌ ６０（Ｅ．Ｍｅｒｃｋ社 ０.０４０−０．０６３ｍｍ）を用いた。また、Ｓｅｐｈａｄｅｘ ＬＨ−２０はＰｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ，Ｓｗｅｄｅｎから購入した。無水ＣＨ_２Ｃｌ_２とＣＨＣｌ_３は水素化カルシウムを脱水剤に用いて蒸留して調製した。無水テトラヒドロフラン（ＴＨＦ）と無水ベンゼンは関東化学（株）より購入した。ＮａＢ^３Ｈ_４（ｓｐｅｃｉｆｉｃ ａｃｔｉｖｉｔｙ ２２２ ＧＢｑ ｍｍｏｌ^−１）はアマシャムライフサイエンスより購入した。オートラジオグラフィーにはイメージングプレートＢＡＳ−ＴＲ２０４０Ｓ（富士フィルム（株））を用い、検出にはバイオ・イメージングアナライザーＢＡＳ−１５００ ＭＡＣ（富士フィルム（株））を用いた。 Example 1 Synthesis of Radiolabeled Lipid A Analogues Two types of radiolabeled lipid A analogs were synthesized according to the following synthesis scheme.

In addition, Kieselgel 60 (E. Merck 0.040-0.063 mm) was used for silica gel column chromatography. Sephadex LH-20 was purchased from Pharmacia Biotech, Sweden. Anhydrous CH ₂ Cl ₂ and CHCl ₃ were prepared by distillation using calcium hydride as a dehydrating agent. Anhydrous tetrahydrofuran (THF) and anhydrous benzene were purchased from Kanto Chemical Co., Inc. NaB ³ H ₄ (specific activity 222 GBq mmol ⁻¹ ) was purchased from Amersham Life Sciences. An imaging plate BAS-TR2040S (Fuji Film Co., Ltd.) was used for autoradiography, and a bio-imaging analyzer BAS-1500 MAC (Fuji Film Co., Ltd.) was used for detection.

(1) 2- (phosphonooxy) [2- ³ H ₁ ] ethyl 2-deoxy-6-O- [2-deoxy-2-[(R) -3- (dodecanoyloxy) tetradecanoylamino] -4 -O-phosphono-3-O-[(R) -3- (tetradecanoyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3-hydroxytetradecanoyl] Synthesis of 2-[(R) -3-hydroxytetradecanoylamino] -α-D-glucopyranoside ([ ³ H] PE506: 4 *) (1-1) Formylmethyl 4-O-benzyl-6-O -[6-O-benzyl-2-deoxy-4-O- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphin-3-yl)- 2-[(R) -3- (dodecanoyloxy) tetradecanoylamino] -3-O-[(R) -3- (tetradecanoyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3 -O-[(R) -3- (Benzyloxy) tetradecanoyl] -2-[(R) -3- (benzyloxy) tetradecanoylamino] -2-deoxy of α-D-glucopyranoside (Compound 20) 4-Methylmorpholine N-oxide under vigorous stirring in a solution of Compound 19 (450 mg, 202 mmol) in THF / t-butanol / water (10: 10: 1, 12 mL) (NMO) (94 mg, 0.80 mmol) and an aqueous solution of osmium tetroxide (OsO ₄ ) (2.5%, 400 mL, 40 mmol) were added. After 6 hours, saturated aqueous Na ₂ S ₂ O ₃ (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL). The organic layer was washed with an aqueous Na ₂ S ₂ O ₃ solution (50 mL × 2) and saturated brine (20 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain a crude product diol (458 mg). . The diol was dissolved in anhydrous benzene (10 mL), lead tetraacetate (Pb (OAc) ₄ ) (purity 90%, 119 mg, 242 mmol) was added, and the mixture was stirred for 30 minutes. The reaction mixture was charged onto a silica gel column (3 g) and eluted with ethyl acetate. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography (20 g, toluene / ethyl acetate = 5: 1) to obtain Compound 20 as a colorless syrup (377 mg, yield: 84%).
FAB-MS (positive) m / z 2244 [(M + Na) ⁺ ]; ¹ H NMR (500 MHz, CDCl ₃ ) δ = 9.37 (s, 1H, CHO).
Compound 19 was synthesized according to the literature [Liu WC, Oikawa M, Fukase K, Suda Y, Kusumoto S. Bull Chem Soc Jpn 1999; 72: 1377-1385. Fukase K, Oikawa M, Suda Y, Liu WC, Fukase Y, Shintaku T, Sekljic H, Yoshizaki H, Kusumoto S. J Endotoxin Res 1999; 5: 46-51.]

(1-2) 2-hydroxy- [2- ³ H ₁ ] ethyl 4-O-benzyl-6-O- [6-O-benzyl-2-deoxy-4-O- (1,5-dihydro-3) -Oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphepin-3-yl) -2-[(R) -3- (dodecanoyloxy) tetradecanoylamino] -3-O- [(R) -3- (tetradecanoyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -2-[(R ) -3- (Benzyloxy) tetradecanoylamino] -2-deoxy-α-D-glucopyranoside (Compound 21 *) Compound 20 (150 mg, 62.4 mmol) in 2-propanol / methanol / CH ₂ Cl ₂ NaB ³ H ₄ (590 mL, 26.4 mmol / mL, 240 GBq mmol ⁻¹ ) was added to the (5: 1: 1, 3.5 mL) solution at 0 ° C. and stirred for 30 minutes. Saturated aqueous ammonium chloride solution was added to stop the reaction, and the mixture was extracted with ethyl acetate. The extract was dried over Na ₂ SO ₄ and concentrated under reduced pressure to give colorless syrup of compound 21 * (144 mg, quantitative). The product was used in the next reaction without purification.

(1-3) 2- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphin-3-yloxy)-[2- ³ H ₁ ] ethyl 4 -O-benzyl-6-O- [6-O-benzyl-2-deoxy-4-O- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphos Fepin-3-yl) -2-[(R) -3- (dodecanoyloxy) tetradecanoylamino] -3-O-[(R) -3- (tetradecanoyloxy) tetradecanoyl]- β-D-glucopyranosyl] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -2-[(R) -3- (benzyloxy) tetradecanoylamino] -2-deoxy- Synthesis of α-D-glucopyranoside (compound 22 *) N, N-diethyl-1,5-dihydro-3H-2 in a solution of compound 21 * (144 mg, 59.9 mmol) in CH ₂ Cl ₂ (14 mL) at 0 ° C. , 4,3-Benzodioxaphosphepin-3-amine (89 mg, 0.37 mmol) Tetrazole (25 mg, 0.32 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes and then cooled to -78 ° C. mCPBA (70%, 81 mg, 0.37 mmol) was added and stirred for 45 minutes. Saturated Na ₂ S ₂ O ₃ was added to terminate the reaction, followed by extraction with CHCl ₃ . The organic layer was washed with saturated aqueous NaHCO ₃ solution and saturated brine, and dried over Na ₂ SO ₄ . After concentration under reduced pressure, the residue was purified by silica gel column chromatography [3.0 g, toluene / ethyl acetate / hexafluoro-2-propanol (HFIP) = 100: 50: 1] to obtain colorless syrup of compound 22 * (134 mg) Yield: 87%). The obtained compound coincided with the standard unlabeled product 22 on TLC.
[α] _D ²⁵ = +17.8 (c 1.00, CHCl ₃ ); FAB-MS (positive) m / z 2428 [(M + Na) ⁺ ]. Found: C, 69.96; H, 9.21; N, 1.14%. Calcd for C ₁₄₀ H ₂₁₈ N ₂ O ₂₆ P ₂ : C, 69.85; H, 9.13; N, 1.16%.

(1-4) 2- (phosphonoxy) [2- ³ H ₁ ] ethyl 2-deoxy-6-O- [2-deoxy-2-[(R) -3- (dodecanoyloxy) tetradecanoylamino] -4-O-phosphono-3-O-[(R) -3- (tetradecanoyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3-hydroxytetradeca Synthesis of Noyl] -2-[(R) -3-hydroxytetradecanoylamino] -α-D-glucopyranoside ([ ³ H] PE506: 4 *) Compound 22 * (57 mg, 22 mmol) in THF (8 mL) Pd-black (130 mg) was added to the mixture, and catalytic reduction was performed at room temperature under pressure (7 kgcm ⁻² ) for 2 hours. After neutralizing by adding triethylamine, Pd-black was removed by filtration. The residue obtained by concentration under reduced pressure was subjected to liquid-liquid partition chromatography (Sephadex LH-20: 20 g, CHCl ₃ / methanol / water / 2-propanol = 9: 9: 9: 1) (the organic layer was used as the stationary phase and the aqueous layer was separated). Used as mobile phase) to give [ ³ H] PE506 (4 *) as a white powder (25 mg, 841 MBq, 62 GBq mmol ⁻¹ , yield: 63%). The obtained compound coincided with the standard unlabeled product 4 on chromatography.
FAB-MS (negative) m / z 1840 [(MH) ⁻ ]; ¹ H NMR (600 MHz, CD ₃ OD / CDCl ₃ = 1: 1) δ = 5.23 (m, 1H), 5.19 (t, J = 8.2 Hz, 1H), 5.17 (m, 1H), 5.14 (t, J = 8.2 Hz, 1H), 4.82 (d, J = 3.0 Hz, 1H), 4.56 (d, J = 7.4 Hz, 1H), 4.23 ( q, J = 8.0Hz, 1H), 4.18 (dd, J = 8.9, 3.0Hz, 1H), 4.08-3.93 (m, 5H), 3.92-3.82 (m, 4H), 3.80 (dd, J = 10.3, 4.5Hz, 1H), 3.74 (d, J = 10.6Hz, 1H), 3.63 (m, 1H), 3.56 (t, J = 8.1Hz, 1H), 3.37 (m, 1H), 2.72 (dd, J = 14.0, 6.6Hz, 1H), 2.64 (dd, J = 14.0, 4.5Hz, 1H), 2.52-2.46 (m, 2H), 2.44-2.36 (m, 2H), 2.36-2.27 (m, 6H), 1.66-1.39 (m, 12H), 1.38-1.20 (m, 108H), 0.89 (t, J = 5.6Hz, 18H).

(2) 2- (phosphonoxy) [2- ³ H ₁ ] ethyl 2-deoxy-6-O- [2-deoxy-2-[(R) -3-hydroxytetradecanoylamino] -4-O-phosphono -3-O-[(R) -3-hydroxytetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3-hydroxytetradecanoyl] -2-[(R) -3 Of 2-hydroxytetradecanoylamino] -α-D-glucopyranoside ([ ³ H] PE406: 5 *) (2-1) Formylmethyl 4-O-benzyl-6-O- [6-O-benzyl-2 -Deoxy-4-O- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphepin-3-yl) -2-[(R) -3- (Benzyloxy) tetradecanoylamino] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3- (benzyl Oxy) tetradecanoyl] -2-[(R) -3- (benzyloxy) tetradecanoylamino] -2-deoxy-α-D-glucopyranoside (compound 24) Compound 4-methylmorpholine N-oxide (NMO) (40 mg, 0.45 mmol) was stirred vigorously into a solution of compound 23 (230 mg, 0.15 mmol) in THF / t-butanol / water (10: 10: 1, 9 mL). ) And an aqueous solution of osmium tetroxide (OsO ₄ ) (2.5%, 230 mL, 20 mmol). After 6 hours, saturated aqueous Na ₂ S ₂ O ₃ (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL). The organic layer was washed with an aqueous Na ₂ S ₂ O ₃ solution (50 mL × 2) and saturated brine (20 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain a crude product diol (458 mg). . The diol was dissolved in anhydrous benzene (10 mL), lead tetraacetate (Pb (OAc) ₄ ) (purity 90%, 68 mg, 140 mmol) was added, and the mixture was stirred for 30 minutes. The reaction mixture was charged onto a silica gel column (3 g) and eluted with ethyl acetate. After concentration under reduced pressure, the residue was purified by medium pressure silica gel chromatography (6 g, toluene / ethyl acetate = 4: 1) to obtain Compound 24 as colorless syrup (190 mg, yield: 83%).
FAB-MS (positive) m / z 2032 [(M + Na) ⁺ ]; ¹ H NMR (500 MHz, CDCl ₃ ) δ = 9.30 (s, 1H, CHO).

(2-2) 2-hydroxy- [2- ³ H ₁ ] ethyl 4-O-benzyl-6-O- [6-O-benzyl-2-deoxy-4-O- (1,5-dihydro-3) -Oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphepin-3-yl) -2-[(R) -3- (benzyloxy) tetradecanoylamino] -3-O- [ (R) -3- (Benzyloxy) tetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -2-[(R) -3 Synthesis of-(benzyloxy) tetradecanoylamino] -2-deoxy-α-D-glucopyranoside (Compound 25 *) Compound 24 (100 mg, 49.8 mmol) in 2-propanol / methanol (5: 1, 1.5 mL) ) NaB ³ H ₄ (474 mL, 26.3 mmol / mL, 240 GBq mmol ⁻¹ ) was added to the solution at 0 ° C. and stirred for 30 minutes. The reaction was stopped by adding 2M aqueous HCl and saturated brine, and the mixture was extracted with ethyl acetate. The extract was dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain colorless syrup of compound 25 * (100 mg, quantitative). The product was used in the next reaction without purification.

(2-3) 2- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphosphin-3-yloxy)-[2- ³ H ₁ ] ethyl 4 -O-benzyl-6-O- [6-O-benzyl-2-deoxy-4-O- (1,5-dihydro-3-oxo-3λ ⁵ -3H-2,4,3-benzodioxaphos Fepin-3-yl) -2-[(R) -3- (benzyloxy) tetradecanoylamino] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -β-D -Glucopyranosyl] -3-O-[(R) -3- (benzyloxy) tetradecanoyl] -2-[(R) -3- (benzyloxy) tetradecanoylamino] -2-deoxy-α-D -Synthesis of glucopyranoside (compound 26 *) N, N-diethyl-1,5-dihydro-3H-2,4 was added to a solution of compound 25 * (100 mg, 49.8 mmol) in CH ₂ Cl ₂ (14 mL) at 0 ° C. 3-Benzodioxaphosphepin-3-amine (70 mg, 0.29 mmol) and tetrazo (20 mg, 0.25 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes and then cooled to -20 ° C. mCPBA (70%, 65 mg, 0.29 mmol) was added and stirred for 45 minutes. Saturated Na ₂ S ₂ O ₃ was added to terminate the reaction, followed by extraction with CHCl ₃ . The organic layer was washed with saturated aqueous NaHCO ₃ solution and saturated brine, and dried over Na ₂ SO ₄ . After concentration under reduced pressure, the residue was purified by silica gel column chromatography [2.3 g, toluene / ethyl acetate / hexafluoro-2-propanol (HFIP) = 100: 50: 1] to obtain a colorless syrup of compound 26 * (63 .9 mg, yield: 59%). The obtained compound was consistent with the unlabeled product 26 of the standard on chromatography.
[α] _D ²⁵ = +16.9 (c 1.00, CHCl ₃ ); FAB-MS (positive) m / z 2428 [(M + Na) ⁺ ].
Found: C, 69.98; H, 8.40; N, 1.33%. Calcd for C ₁₂₈ H ₁₈₂ N ₂ O ₂₄ P ₂ : C, 70.05; H, 8.36; N, 1.28%.

(2-4) 2- (phosphonoxy) [2- ³ H ₁ ] ethyl 2-deoxy-6-O- [2-deoxy-2 [(R) -3-hydroxytetradecanoylamino] -4-O- Phosphono-3-O-[(R) -3-hydroxytetradecanoyl] -β-D-glucopyranosyl] -3-O-[(R) -3-hydroxytetradecanoyl] -2-[(R)- Synthesis of 3-hydroxytetradecanoylamino] -α-D-glucopyranoside ([ ³ H] PE406: 5 *) Pd-black (120 mg) was added to a solution of compound 26 * (17 mg, 7.8 mmol) in THF (6 mL). In addition, catalytic reduction was performed at room temperature for 2 hours under pressure (7 kgcm ⁻² ). After neutralizing by adding triethylamine, Pd-black was removed by filtration. The residue obtained by concentration under reduced pressure liquid-liquid partition chromatography (Sephadex LH-20: 20g, CHCl 3 / methanol / water / 2-propanol = 8: 8: 9: 1) (the aqueous layer and the organic layer as a stationary phase (Used as mobile phase) to give a white powder of [ ³ H] PE406 (5 *) (7.5 mg, 331 MBq, 64 GBq mmol ⁻¹ , yield: 66%). The obtained compound coincided with the unlabeled sample 5 on the chromatography.
FAB-MS (negative) m / z 1448 [(MH) ⁻ ]; ¹ H NMR (500 MHz, SDS-d ₂₅ -D ₂ O) d = 5.18 (dd, J = 9.4, 7.5 Hz, 1H), 5.17 ( dd, J = 9.8, 9.6Hz, 1H), 4.87 (d, J = 3.8 Hz, 1H), 4.67 (d, J = 7.0Hz, 1H), 4.12 (dd, J = 9.8, 3.8 Hz, 1H), 4.11 (dd, J = 5.5,1.6Hz, 1H), 4.11 (q, J = 7.5,1H), 4.02 (dd, J = 9.4, 7.0Hz, 1H), 3.99 (m, 1H), 3.96 (m, 1H), 3.91 (m, 3H), 3.87 (ddd, J = 9.6, 1.6, 2.9Hz, 1H), 3.82 (dd, J = 9.8, 8.6Hz, 1H), 3.82 (dd, J = 15.5, 2.9Hz , 1H), 3.82 (m, 1H), 3.81 (m, 3H), 3.71 (m, 1H), 3.59 (m, 1H), 2.56-2.23 (m, 8H), 1.54-1.11 (m, 80H), 0.86 (m, 12H).

Example 2 Preparation of Molecular Tweezers Library (Peptide Library) The encoded molecular tweezers library was prepared by a split-mix synthesis method. As a solid support, 15 g of TentaGel S-NH ₂ (Rapp Polymer, particle size 90 μm, 0.29 mmol / g of NH ₂ ) was used. The peptide chain was elongated using a standard Fmoc / Boc (Fluorenylmethoxycarbonyl / Butyloxycarbonyl) peptide solid phase synthesis method. As a peptide condensation method, HOBt / DIC (Hydroxybenzotriazole / Diisopropylcarbodiimide) was used. The end point of the peptide condensation reaction was confirmed by the absence of blue color development by the bromophenol blue test. Catechol Tag was introduced into about 1% of the amino group by amide bond formation reaction by the HOBt / DIC method via the carboxyl group. When chenodeoxycholic acid (L1) is used as the core structure, deoxycholic acid is first introduced into the solid support, and then 6 equivalents of Fmoc-Gly-F and 6 equivalents of triethylamine in DMF with respect to the two hydroxyl groups. Fmoc-Gly was coupled by acting 0.06 equivalents of dimethylaminopyridine. Lysine (L2) and trilysine (L3) cores were prepared by conventional peptide solid phase synthesis. 15 types of amino acids (Gly and D and L-Val, Phe, Ser, Gln, Glu, Lys and Pro) were used for library preparation. 4 by performing the steps of splitting coupling cycle was constructed libraries containing 3 × ¹⁵ 4 = 151,875 different peptides. The protecting groups on the library were removed using 20% piperidine / DMF and 95% TFA / 2.5% water / 2.5% thioanisole and stored at 4 ° C.

EXAMPLE 3 Binding Experiment molecular tweezers library (the peptide library) and radiolabeled lipid A analogs ⁽¹⁾ [3 H] binding experiments with ^{PE506 [3 H] PE506 (62GBq} / mmol, 1.4mg, 47MBq ) Was dissolved in water (2 ml). The molecular tweezers library (15.7 mg, about 19000 beads) was suspended in water (200 μl), and [ ³ H] PE506 solution (100 μl, 2.4 MBq) was added. A suspension of this [ ³ H] PE506 (130 μM) and resin was gently shaken at 4 ° C. for 41 hours. The operation of centrifuging to precipitate the resin and removing the supernatant was repeated three times. After the glass plate was treated with the NaCIO solution, it was washed thoroughly with water, immersed in a 0.5 gelatin solution, taken out, and air-dried to prepare a glass plate coated with gelatin. The above resin was placed on this and allowed to air dry. This was covered with Kodak autoradiography emulsion which was heated and melted, and after it was confirmed that it had hardened sufficiently, it was allowed to stand at −78 ° C. for 12 days under light shielding. The glass plate was placed in the developing solution for 10 minutes and then washed with water. The fixative was soaked for 15 minutes and then washed with water. Around the radioactive resin, silver adheres to the gelatin and becomes black. Using this as an index, the radioactive resin is taken out, the tag bonded to the resin is separated from the resin, and the tag is detected and decoded by electronic supplementary gas chromatography (ECGC) to bind 19 types of [ ³ H] PE506. It found molecular tweezers to ⁽²⁾ [3 H] binding experiments with ^{PE406 [3 H] PE406 (64GBq} / mmol, 0.6mg, 26MBq) was dissolved in water (1 ml). A molecular tweezers library (24.2 mg, about 29000 beads) was suspended in water (200 μl), and a solution of [ ³ H] PE406 (100 μl, 2.6 MBq) was added. The [ ³ H] PE406 (130 μM) and resin suspension was gently shaken at 4 ° C. for 48 hours. The resin was precipitated by centrifugation and the supernatant was removed. The operation of adding 1 μl of water, shaking and centrifuging to precipitate the resin and removing the supernatant was repeated three times.
The resin was placed on a glass plate coated with gelatin and allowed to air dry. This was covered with Kodak autoradiography emulsion that had been heated and melted, and after confirming that it had hardened sufficiently, it was allowed to stand at -78 ° C. for 7 days under light shielding. The resin with radiation was taken out and subjected to ECGC analysis. Molecular tweezers that bind 32 types of [ ³ H] PE406 were found.

Example 4 Resynthesis of molecular tweezers TentaGel S-NH ₂ (Rapp Polymere, particle size 90 μm, 0.29 mmol / g of NH ₂ ) or TSKgel AF-AminoTOYOPARRL 650S (Tosoh Corp., Japan) 1 mmol / ml) was used. The peptide chain was elongated using a standard Fmoc / Boc (Fluorenylmethoxycarbonyl / Butyoxycarbonyl) peptide solid phase synthesis method. As a peptide condensation method, HOBt / DIC (Hydroxybenzotriazole / Diisopropylcarbodiimide) was used. The end point of the peptide condensation reaction was confirmed by the absence of blue color development by the bromophenol blue test.
As a typical example, a synthesis example of molecular tweezers 6-Toyopearl [(kskS) ₄ L3-Toyopearl] is shown.
(1) Introduction of Nα, Nε-di-t-butoxycarbonyl-L-lysine (Boc-L-Lys (Boc) -OH) TSKgel AF-AminoTOYOPEARL 650S (0.1 mmol / ml) (1 ml, 0.1 mmol) 10% diisopropylethylamine (DIEA) / DMF (4.0 ml) was added to the mixture, and the mixture was shaken for 30 seconds and then vortexed. After the resin was washed twice with DMF (4.0 ml), a BMF-L-Lys (Boc) -OH (104 mg, 0.300 mmol) solution in DMF (3 ml), diisopropylcarbodiimide (DIC) (47.0 μl, 0.0 300 mmol) of DMF (1 ml) and 1-hydroxybenzotriazole (HOBt) (40.5 mg, 0.300 mmol) in DMF (1 ml) were added, and the mixture was shaken at room temperature for 2 hours. After the filter vortex, the resin was washed 4 times with DMF (4.0 ml). The peptide condensation reaction with Boc-L-Lys (Boc) -OH was repeated once more. Acetic anhydride-DMF (1: 1) solution (2 ml) and triethylamine-DMF (1: 1) solution (2 ml) were added and shaken at room temperature for 15 minutes to completely block the remaining unreacted amino groups. After the filter vortex, the resin was washed 4 times with DMF (4 ml).
(2) Removal of Boc group 25% TFA / DMF (4.0 ml) was added and shaken at room temperature for 30 minutes. After the filter vortex, the resin was washed 4 times with DMF (4.0 ml).
(3) Condensation of Nα-fluorenylmethoxycarbonyl-Nε-t-butoxycarbonyl-L-lysine (Fmoc-L-Lys (Boc) -OH) Fmoc-L-Lys (Boc) -OH (281 mg, 0. (600 mmol) in DMF (3 ml), DIC (94.0 μl, 0.600 mmol) in DMF (1 ml) and HOBt (81.1 mg, 0.600 mmol) in DMF were added and shaken at room temperature for 2 hours. After the filter vortex, the resin was washed 4 times with DMF (4.0 ml). The peptide condensation reaction with Fmoc-L-Lys (Boc) -OH was repeated once more. Unreacted amino groups were similarly blocked using acetic anhydride.
(4) Removal of Fmoc group 20% piperidine / DMF (4 ml) was added and shaken at room temperature for 30 minutes to remove the Fmoc group. After filtration, the resin was washed 3 times with DMF (4.0 ml) and 2 times with methanol (4.0 ml). The operation of removing the Fmoc group was repeated twice.
(5) Removal of Boc group The Boc group was removed in the same manner as in (2) above.
(6) Condensation of Nα-fluorenylmethoxycarbonyl-ot-butyl-L-serine (Fmoc-L-Ser (tBu) -OH) Fmoc-L-Ser (tBu) -OH (460 mg, 1.20 mmol) ) In DMF (6 ml), DIC (188 μl, 1.20 mmol) in DMF (2 ml) and HOBt (162 mg, 1.20 mmol) in DMF (2 ml) were added, and the mixture was shaken at room temperature for 2 hours. After the filter vortex, the resin was washed 4 times with DMF (4.0 ml).
(7) Removal of Fmoc group The Fmoc group was removed in the same manner as in (4) above.
(8) Condensation of Fmoc-D-Lys (Boc) -OH Fmoc-D-Lys (Boc) -OH (562 mg, 1.20 mmol) in DMF (6 ml), DIC (188 μl, 1.20 mmol) in DMF (2 ml) and a DMF solution (2 ml) of HOBt (162 mg, 1.20 mmol) were added and shaken at room temperature for 2 hours. After filter vortex, the resin was washed with DMF (4.0 ml).
(9) Removal of Fmoc group The Fmoc group was removed in the same manner as in (4) above.
(10) Condensation of Fmoc-D-Lys (Boc) -OH Fmoc-D-Lys (Boc) -OH (562 mg, 1.20 mmol) in DMF (6 ml), DIC (188 μl, 1.20 mmol) in DMF (2 ml) and a DMF solution (2 ml) of HOBt (162 mg, 1.20 mmol) were added and shaken at room temperature for 2 hours. After filter vortex, the resin was washed with DMF (4.0 ml).
(11) Removal of Fmoc group The Fmoc group was removed in the same manner as in (4) above.
(12) Removal of Boc group and tBu group The side chain protecting group was removed in the same manner as in the removal operation of the Boc group in (2) above.
Above, (1) by sequentially performing an operation to (12), the molecular tweezers [(k-s-k- S) 4 L3-Toyopearl] was obtained.
Other molecular tweezer resins were synthesized in the same manner as described above.

Example 5 Molecular tweezers 6-TentaGel to 17-TentaGel, PE506, 506, PE406, 406, ReLPS and E. coli lipopolysaccharide (LPS)
Binding experiment with PE506 (0.06 mg) was dissolved in water (0.80 ml). Molecular tweezers 6-17 TentaGel (about 20 mg) was suspended in water (100 μl), and PE506 solution (25 μl) was added. This suspension was gently shaken overnight at room temperature. The resin was precipitated by centrifugation, and the supernatant was used for TLC analysis and measurement of Limulus activity.
506 (0.56 mg), PE406 (0.7 mg), 406 (0.7 mg), ReLPS (0.85 mg), LPS (1 mg) were similarly subjected to a binding experiment, and the resulting supernatant was measured for Limulus activity. (In the case of 506, it was also used for TLC analysis). The results of TLC analysis are as shown in Table 6.

Example 6 Binding Experiment of Molecular Tweezers 6-Toyopearl, 11-Toyopearl, 12-Toyopearl, 16-Toyopearl, and 17-Toyopearl with 506, ReLPS and E. coli Lipopolysaccharide (LPS) 506 (0.56 mg) in water (0.56 mg) The solution dissolved in 0.80 ml) was further diluted 3-fold. Each Toyopearl (about 20 mg) of molecular tweezers was suspended in water (100 μl), and 506 solution (25 μl) was added. This suspension was gently shaken overnight at room temperature. The resin was precipitated by centrifugation, and the supernatant was used for measuring Limulus activity.
Similarly, ReLPS (0.85 mg) and Escherichia coli lipopolysaccharide (LPS) (1 mg) were similarly diluted three-fold before binding experiments were performed, and the resulting supernatant was used for measuring Limulus activity.

Example 7 Measurement of Limulus Activity Limulus activity was measured using Endspace R (manufactured by Seikagaku Corporation).
1. Each sample (PE506, 506, PE406, 406, ReLPS and E. coli lipopolysaccharide (LPS)) was dissolved in water to prepare a 1 mg / ml solution.
2. The sample was diluted to an appropriate concentration on a 96-well microplate using distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.).
3. 20 μl of the sample solution was dispensed onto a microplate, and 30 μl of Endspacey ES-50M set LAL reagent was added while cooling in an ice-water bath, followed by heating at 37 ° C. for 20 minutes.
4). The following diazotizing reagents A, B, and C were sequentially added in an amount of 75 ml, and the absorbance at a wavelength of 540 nm was measured. Each sample concentration was used and the absorbance at a ¹⁰ -6 mg / ml in the standard.
(Diazotization reagent)
A: Solution in which 40 mg of sodium nitrite is dissolved in 4 ml of concentrated hydrochloric acid and 96 ml of water B: Solution in which 300 mg of ammonium sulfamate is dissolved in 100 ml of water C: Solution in which 70 mg of N-naphthylethylenediamine dihydrochloride is dissolved in 100 ml of water Each of the obtained samples was diluted in exactly the same manner, and a Limulus activity test was performed for each sample, and the absorbance was measured. The adsorption capacity of each resin was estimated by comparing with the standard absorbance of each sample. The results are as shown in FIG. 1 and FIG.

FIG. 1 shows the results of examining the binding ability of the peptide library of the present invention (using a polyethylene glycol-polystyrene resin TentaGel as a solid phase carrier) to lipid A, lipid A analog, and lipopolysaccharide. In FIG. 1, (a) is a phosphonooxyethyl derivative of E. coli lipid A (PE506), (b) is E. coli lipid A (506), and (c) is a phospho of biosynthetic precursor lipid A. Nooxyethyl derivative (PE406), (d) biosynthesis precursor lipid A (406), (e) E. coli Re mutant lipopolysaccharide (ReLPS), (f) E. coli lipopolysaccharide The results of binding experiments with (LPS) are shown respectively. FIG. 2 shows the results of examining the binding ability of the peptide library of the present invention (using the hydrophilic vinyl polymer TOYOPEARL as a solid phase carrier) to lipid A, lipid A analog and lipopolysaccharide. In FIG. 2, (a) shows the binding experiment with Escherichia coli lipid A (506), (b) with the lipopolysaccharide (ReLPS) of the Escherichia coli Re mutant, and (c) the binding experiment with Escherichia coli lipopolysaccharide (LPS). Each result is shown.

Claims (12)

A peptide library comprising a solid phase carrier having a functional group, a compound having a functional group capable of binding to the functional group, and a compound having two or more other functional groups, and an oligopeptide bonded thereto. The following general formula [1]

The peptide library according to claim 1, wherein the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue. The peptide library according to claim ₂ , wherein in the general formula [1], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described below.

The following general formula [2]

The peptide library according to claim 1, wherein the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue. The peptide library according to claim 4, wherein in the general formula [2], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described below.

A compound having a functional group capable of binding to the functional group and having two or more other functional groups is bound to a solid phase carrier having a functional group, and further, a function capable of binding to the other functional group. A peptide library obtained by binding a compound having a group and two or more functional groups other than this, and then further binding an oligopeptide thereto. The following general formula [3]

The peptide library according to claim 6, wherein the spherical portion represents a solid phase carrier, and AA ₁ , AA ₂ , AA ₃ and AA ₄ each independently represents an amino acid residue. The peptide library according to claim 7, wherein in the general formula [3], AA ₁ , AA ₂ , AA ₃ and AA ₄ are composed of any combination described below.

Peptide library according to any one of claims 2, 4 or 7 AA ₄ is lysine residues. A bacterial endotoxin adsorbent comprising the peptide library according to claim 3. A bacterial endotoxin adsorbent comprising the peptide library according to claim 5. A bacterial endotoxin adsorbent comprising the peptide library according to claim 8.

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