CN1061990C - Schistosome vaccine peptide No.1 - Google Patents

Abstract

The present invention belongs to organic compounds.

Polypeptides having the following basic sequence:

X-Trp-Y-Ala-Phe-Z-Lys-OH

In the formula, X and Y are both amino acid residues, and the peptide has a protective immune effect on schistosomiasis, and can be used to prepare vaccine drugs against schistosomiasis.

Description

New Schistosoma Vaccine Peptides (1)

The present invention belongs to organic compounds.

The current research on schistosomiasis vaccines mainly focuses on two aspects: live attenuated irradiated vaccines and soluble antigen vaccines. The research history of irradiated attenuated live vaccines is long, and mature experience has been accumulated in preparation and application, and good results have been obtained in laboratory and animal experiments. But this kind of vaccine also has the following problems to be solved, (1) local inflammation caused by a large amount of injection, (2) safety of the vaccine, such as anaphylaxis, (3) pathogenicity of the vaccine, such as incomplete inactivation caused Infection, (4) Insufficient sources of worms when preparing vaccines on a large scale. In order to avoid the above-mentioned shortcomings, people are actively engaged in the research of synthetic soluble antigen vaccines. The significance of this kind of vaccine is that it can be synthesized artificially in large quantities, and the amount of inoculation of artificially synthesized vaccines is small, which can reduce allergic reactions to foreign proteins and can It is prepared in large quantities, and there is no risk of pathogenicity caused by incomplete inactivation, and has practical application prospects. The prior art, such as Chinese Patent Application No. 94105038.6, provides a peptide that can be used to prepare a schistosomiasis vaccine.

The object of the present invention is to provide a kind of polypeptide that can be used for preparing multivalent vaccine of schistosomiasis, and its advantage is that the multivalent vaccine made with it has small amount of inoculation, and has immune effect to various schistosomiasis, can reduce the allergic reaction of heterologous protein , without the risk of pathogenicity caused by incomplete inactivation, and can be artificially synthesized in large quantities.

The organic compound-polypeptide involved in the present invention is a schistosome soluble antigen vaccine polypeptide, which is characterized in that the chemical structure is as follows:

In the above formula:

A is hydrogen, C1-12 alkyl, C2-12 alkenyl and alkynyl, phenyl or C7-10 phenylalkyl or a group in the form of RCO. it's here:

(i) R is hydrogen, C1-11 alkyl, C2-11 alkenyl and alkynyl, phenyl or C7-10 phenylalkyl.

(ii) RCO can also be

(a) Natural amino acid residues may also be corresponding D-configuration amino acid residues.

(b) a dipeptide residue, the two amino acid residues of which may be the same or different, and the amino acid residues used are as defined in (a).

(iii) RCO can also be

(a) Natural four-, five-, and six-carbon sugar residues.

(b) a two, three, four oligosaccharide residues, the monosaccharides can be the same or different, and the monosaccharide residues used are natural monosaccharide residues, but the monosaccharide residues linked to the peptide for sugar residues.

A' is hydrogen, or when A is C1-12 alkyl, C2-12 alkenyl and alkynyl, phenyl or C7-10 phenylalkyl, also C1-12 alkyl, C2-12 alkenyl and alkynyl Base, phenyl or C7-10 phenylalkyl.

B is -Glu-, -Asp-, -Gln-, -Asn-, -Tbr-, -Ser-,

C is -Gly-, -Pro-,

D is -Glu-, -Asp-, -Gln-, -Asn-,

E is -Ser-, -Thr-, -His-, -Ala-,

F is -Pro-, -Gly-,

X can be of the form: -COOR ₁ , -CH ₂ OR ₂ or

it's here:

R ₁ is hydrogen, C1-3 hydrocarbon group, natural four, five, six carbon monosaccharides or two, three, four oligosaccharides, the monosaccharides can be the same or different, and the monosaccharide residues used For natural four, five, six carbon monosaccharide residues.

_R2 is hydrogen, or a physiologically acceptable and hydrolyzable ester group under physiological conditions.

R ₃ and R ₄ are hydrogen, C1-3 hydrocarbyl, phenyl, benzyl, C9-10 benzene hydrocarbyl. Or R ₃ is hydrogen, R ₄ is a natural amino acid residue, or a corresponding D-configuration amino acid residue. R ₄ can also be a dipeptide residue, and its two amino acid residues can be the same or different, and the amino acid residues used are natural amino acid residues or corresponding D-configuration amino acid residues. R ₄ can also be a natural four-, five-, six-carbon sugar residue, or a two, three, or four-oligosaccharide residue, and its monosaccharides can be the same or different, and the monosaccharides used The residues are natural four, five and six carbon monosaccharide residues.

The polypeptide involved in the present invention may exist in its free form, or in the form of a salt or a complex. For example, it can form salts with organic acids, polymeric acids and inorganic acids, such as hydrochloride and acetate. As another example, it can form Ca ²⁺ or Zn ²⁺ salts with inorganic substances, such as inorganic salts or hydroxides. It can also form complexes with high-polymeric organic substances, as well as complexes formed with pharmaceutically acceptable diluents and carriers (such as pharmaceutically acceptable protein carriers).

The polypeptides involved in the present invention can be synthesized and produced by known polypeptide chemical methods, such as the following examples, and chemical synthesis methods that are obviously equivalent to this example, solution method or solid phase synthesis method (including tert-butoxy Carbonyl protection method and fluorenyl moxycarbonyl protection method) to synthesize and produce.

The polypeptide involved in the present invention and its pharmaceutically acceptable salts and complexes have been shown to have valuable pharmaceutical activity in biological activity testing and animal experiments. Especially in the aspect of anti-schistosomiasis, the experiment of immunizing mice shows that there is a high rate of worm reduction. The dosage range of the polypeptide involved in the present invention in animal experiments is 0.001 to 1000ug/kg body weight.

Example 1

1. Preparation of Boc-Lys(Cbz)-OCH ₂ -Resin

1.1. Preparation of Boc-Lys(Cbz)-OCs

Dissolve 2.374g (6.24mmole) Boc-Lys(Cbz)-OH in 40mL ethanol and 8mL water, stir to dissolve, add 1.017g (3.12mmole) Cs ₂ CO ₃ , stir until the reaction is complete, vacuum dry, add 4× 20mL of dry benzene was dried under reduced pressure to remove water. Put it in a vacuum desiccator containing phosphorus pentoxide and dry overnight to obtain white cesium salt.

1.2. Preparation of Boc-Lys(Cbz)-OCH ₂ -Resin

Dissolve the above-prepared cesium salt in 10 mL of refined DMF, add 3.0 g (chlorine content: 3.12 mmole) of chloromethyl resin, keep it dry, and react with moderate stirring at 80°C for 24 hours, filter and drain , washed with 3×10mL DMF, 3×10mL DMF/H ₂ O (9:1), 3×10mL DMF, 3×10mL anhydrous EtOH, drained, and dried in a vacuum desiccator containing phosphorus pentoxide Overnight, white Boc-Lys(Cbz)-OCH ₂ -Resin was obtained.

1.3. Chlorine blocking of Boc-Lys(bz)-OCH ₂ -Resin

Add 2.558g (31.2mmole) of anhydrous sodium acetate and 30mL of refined DMF to the above-mentioned Boc-Lys(Cbz)-OCH ₂ -Resin, stir and react at 70°C for 24 hours, filter and drain, and then use 2×10mL DMF, 2×10mL DMF/H ₂ O (9:1), 7×10mL DMF, 2×10mL anhydrous EtOH, wash, drain, put in a vacuum desiccator containing phosphorus pentoxide and dry overnight to obtain a white seal Chlorine Boc-Lys(Cbz)-OCH ₂ -Resin.

1.4. Hydroxyl blocking of Boc-Lys(Cbz)-OCH ₂ -Resin

Add 10 mL (0.105 mole) redistilled acetic anhydride, 20 mL (0.25 mole) treated anhydrous anhydrous anhydrous pyridine, 20 mL dry benzene to the above chlorine-sealed Boc-Lys(Cbz)-OCH ₂ -Resin, room temperature Moderately stirred and reacted for 24 hours, filtered and drained, washed with 4×50mL DCM, 3×10mL anhydrous MeOH successively, drained, put in a vacuum desiccator containing phosphorus pentoxide and dried overnight to obtain white sealed chlorine and hydroxyl Boc-Lys(Cbz)-OCH ₂ -Resin.

The amino group content was detected by ninhydrin to be 0.89 mmole/g resin.

2. Preparation of Boc-Pro-Lys(Cbz)-OCH ₂ -Resin

1.5 g (amino group content: 1.335 mmole) of Boc-Lys(Cbz)-OCH ₂ -Resin with blocked chlorine and hydroxyl groups was added to 15 mL of DCM to swell for 5 minutes, and drained.

2.1. Deprotection

Add 15mL TFA/DCM (3:7) to the above resin, shake for 30 minutes, drain, wash with 10mL DCM, 10mL MeOH three times each, and drain.

2.2. Neutralization

Add 10mL TEA/DCM (1:9), shake for 5 minutes, drain, then add 10mL TEA/DCM (1:9), shake for 10 minutes, drain, wash three times with 10mL DCM, and drain.

2.3. Coupling

Add 0.645g (3mmlole) Boc-Pro, 0.608g (4.5mmoLe) HOBt, 0.727g (4.5mmole) DCC, 15mL DMF, shake for 6 hours, drain, wash three times with 10mL DMF, wash three times with 10mL MeOH, wash with 10mL DCM Wash three times and drain.

2.4. Detection

Take a little resin and put it into a test tube, add 3 drops of ninhydrin reagent 1, 2 and 3, and heat at 105°C for 5 minutes. If it is light yellow, it means that the coupling reaction is complete, and the next reaction can be carried out; if it is blue or brownish red, it means that the coupling reaction is incomplete. The situation reduces the feeding.

According to the above method, the following amino acids Boc-Pro, Boc-His (Tos), Boc-Asp (OBzl), Boc-Gly, Boc-Gly, Boc-Gly, Boc-Phe, Boc-Thr (Bzl), Boc- Ala, Boc-Gln, Boc-Trp.

When the previous amino acid is Pro, Gln and Asn, it is not necessary to detect because it is yellow when detected by ninhydrin reagent, but in order to ensure the complete coupling reaction, it should be re-coupled 1-2 times. When the amino acid used has Tos as a side-linked protecting group, change HOBt to HOSu, so as to prevent the basicity of HOBt from removing the Tos protecting group. After connecting Trp, a few drops of sulfide anisole and ethanedithiol should be added to TFA/DCM when removing Boc protection.

Finally, the side-linked protective peptide Trp-Gln-Ala-Thr(Bzl)-Phe-Gly-Gly-Gly-Asp(OBzl)-His(Tos)-Pro-Pro-Lys(Cbz)-Resin linked on the resin was obtained . Attachment: ninhydrin detection reagent

1. Ninhydrin reagent 1,1mL 0.001N KCN+40mL pyridine;

2. Ninhydrin reagent 2, 40gB phenol + 10mL absolute ethanol;

3. Ninhydrin reagent 3, 2g ninhydrin + 40mL absolute ethanol.

3. H-Trp-Gln-Ala-Thr-Phe-Gly-Gly-Gly-Asp-His-Pro-Pro-Lys-OH

Deprotection was carried out in a PTFE peptide deprotection unit, and Trp-Gln-Ala-Thr(Bzl)-Phe-Gly-Gly-Gly-Asp(OBzl)-His(Tos)-Pro- Pro-Lys(Cbz)-Resin, 0.5mL anisole, 0.5mL sulfide anisole and 0.5mL ethanedithiol, put 10-15mL hydrogen fluoride dried with anhydrous cobalt trifluoride under ice-water bath cooling , stirred and reacted for 45 minutes, dried under reduced pressure, washed with cold anhydrous ether to obtain resin and sticky matter, dissolved in 10% acetic acid solution, filtered to remove residue, freeze-dried to obtain dry powder, and used 5% acetic acid as eluent on SephadexG15 column Purified above, purified twice, and freeze-dried to obtain 1.30 g of solid, with a yield of 70%, [α] _D ¹⁸ =-40.6° (C=1,0, water). Product peptide purity was determined by HPLC and amino acid composition analysis.

The following polypeptides can also be synthesized and produced by the similar method given in Example 1, HA-Trp-B-Ala-Thr-Phe-(C) ₃ -DE-(F) ₂ -Lys-OH Example No. A B C D E F [α] _D ¹⁸ 2 Gly Ser Gly Asp Ala Pro -64.9(c=0.69,H ₂ O)3 Gln-Gly Asp Gly Asp Thr Pro -59.5(c=0.64,H ₂ O)4 - Ser Gly Asp Ala Pro -63.0 (c=0.66, H ₂ O) Abbreviations Ala Alanine Asn Asn Asp Asp Aspartate Boc tert-Butoxycarbonyl Bzl Benzyl Cbz Benzyloxycarbonyl DCC N,N′-Dicyclohexylcarbodi Imine DCM Dichloromethane DMF N,N-Dimethylformamide EtOH Ethanol Gln Glutamine Glu Glutamic acid Gly Glycine His Histidine HOBt 1-Hydroxybenzotriazole HOSu N-Hydroxysuccinimide HPLC HPLC Lys Lysine MeOH Methanol Phe Phenylalanine Pro Proline Resin Ser Serine Thr Threonine Tos p-Toluenesulfonyl Trp Tryptophan

Claims (8)

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that its chemical structure is shown in the following formula: In the formula: A is hydrogen or acetyl, A' is hydrogen, B is -Glu-, -Asp-, -Gln-, -Asn-, -Thr-, -Ser-, C is -Gly-, -Pro-, D is -Glu-, -Asp-, -Gln-, -Asn-, E is -Ser-, -Thr-, -His-, -Ala-, F is -Pro-, -Gly-, X can be of the form: -COOR ₁ , or it's here: R ₁ is hydrogen, C _1-3 hydrocarbyl, R ₂ and R ₃ are hydrogen, C _1-3 hydrocarbyl. 2. The polypeptide or pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that B is -Gln-, C is -Gly-, D is -Asp-, E is -His-, F is for -Pro-. 3. As claimed in claim 1, it is a peptide or a pharmaceutically acceptable salt thereof, characterized in that B is -Ser-, C is -Gly-, D is -Asp-, E is -Ala-, F is for -Pro-. 4. The polypeptide or pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that B is -Asp-, C is -Gly-, D is -Asp-, E is -Thr-, F is for -Pro-. 5. The polypeptide or pharmaceutically acceptable salt thereof as claimed in claim 1 or 3, characterized in that B is -Gln-, C is -Gly-, D is -Asp-, E is -His-, F is for -Pro-. 6. The polypeptide or pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that it has the following formula: H-Trp-Gln-Ala-Thr-Phe-Gly-Gly-Asp-His-Pro-Pro-Lys-OH. 7. The polypeptide or pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that the chemical structure is as follows: H-Gly-Trp-Ser-Ala-Thr-Phe-Gly-Gly-Gly-Asp-Ala-Pro-Pro-Lys-OH, H-Gln-Gly-Trp-Asp-Ala-Thr-Phe-Gly-Gly-Gly-Asp-Thr-Pro-Pro-Lys-OH, H-Trp-Ser-Ala-Thr-Phe-Gly-Gly-Gly-Asp-Ala-Pro-Pro-Lys-OH, H-Trp-Thr-Ala-Thr-Phe-Pro-Pro-Pro-Asp-Ala-Gly-Gly-Lys-OH, H-Trp-Ser-Ala-Thr-Phe-Gly-Gly-Gly-Gln-Ala-Pro-Pro-Lys-OH. 8. A pharmaceutical composition for treating and preventing schistosomiasis, which contains any one of the polypeptides or salts thereof according to claims 1-7 as an active ingredient and pharmaceutically acceptable diluents and carriers thereof.