CN1061990C

CN1061990C - Schistosome vaccine peptide No.1

Info

Publication number: CN1061990C
Application number: CN94105973A
Authority: CN
Inventors: 许家喜; 蔡孟深
Original assignee: Beijing Medical University
Current assignee: Beijing Medical University
Priority date: 1994-06-08
Filing date: 1994-06-08
Publication date: 2001-02-14
Anticipated expiration: 2014-06-08
Also published as: CN1112929A

Abstract

The present invention belongs to an organic compound with polypeptide with the following basic sequence: X-Trp-Y-Ala-Phe-Z-Lys-OH, wherein X, Y, Z are amino acid radicals. The polypeptide has a protective immunity function to schistosomiasis, and can be used for preparing vaccine medicaments against the schistosomiasis.

Description

Peptide for vaccine of schistosomiasis (one)

The invention belongs to organic compound.

Research to blood fluke vaccine at present mainly concentrates on irradiation attenuated live vaccine and soluble antigen vaccine two aspects.The research history of irradiation attenuated live vaccine is longer, has accumulated more sophisticated experience in preparation and application facet, has obtained better result in laboratory and experimentation on animals.But problem to be solved below also existing, this kind vaccine is arranged, the local inflammation that (1) a large amount of injections cause, the security of (2) vaccine, as anaphylaxis, (3) vaccine is pathogenic, as the infection that deactivation not exclusively causes, and the source deficiency of insect during (4) mass preparation vaccine.For fear of above-mentioned shortcoming, the throwing oneself into research of synthetic soluble antigen vaccine of people, the significance of this kind vaccine is, can adopt artificial a large amount of synthetic, vaccine inoculation amount through synthetic is little, can reduce the anaphylaxis of foreign protein, and can prepare in a large number, and do not have the pathogenic risk that deactivation not exclusively causes, actual application prospect is arranged.Prior art, as Chinese patent, application number 94105038.6 has provided a kind of peptide that can be used for preparing blood fluke vaccine.

The object of the present invention is to provide a kind of polypeptide that can be used for preparing the schistosomicide polyvalent vaccine, its advantage is that the polyvalent vaccine inoculum size made from it is little, and multiple schistosomicide there is immunization, can reduce the anaphylaxis of foreign protein, the pathogenic risk that no deactivation not exclusively causes, and can adopt artificial a large amount of synthetic.

Organic compound-polypeptide involved in the present invention promptly is a schistosomicide soluble antigen vaccine polypeptide, it is characterized in that chemical structure is as follows:

In following formula:

A is a hydrogen, C1-12 alkyl, C2-12 thiazolinyl and alkynyl, the group of phenyl or C7-10 benzene alkyl or RCO-form.Here:

(ⅰ) R is a hydrogen, C1-11 alkyl, C2-11 thiazolinyl and alkynyl, phenyl or C7-10 benzene alkyl.

(ⅱ) RCO can also be

(a) natural amino-acid residue also can be corresponding D-configuration amino-acid residue.

(b) dipeptide residue, two amino-acid residue can be identical also can be different, its used amino-acid residue is as definition in (a).

(ⅲ) RCO can also be

(a) natural four, five, hexose acid residue.

(b) one two, three, four oligosaccharides residues, its monose can be identical, also can be different, and its used monosaccharide residue is natural monosaccharide residue, but the monosaccharide residue that is connected with peptide is the saccharic acid residue.

A ' is a hydrogen, or when A be the C1-12 alkyl, C2-12 thiazolinyl and alkynyl when phenyl or C7-10 benzene alkyl, also are the C1-12 alkyl, C2-12 thiazolinyl and alkynyl, phenyl or C7-10 benzene alkyl.

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

C is-Gly-,-Pro-,

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

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

F is-Pro-,-Gly-,

X can be following form :-COOR ₁,-CH ₂OR ₂Or

Here:

R ₁Be hydrogen, the alkyl of C1-3, four, five, six natural carbon monose or two, three, four oligosaccharides, its monose can be identical, also can be different, its used monosaccharide residue is natural four, five, six carbon monosaccharide residues.

R ₂Be hydrogen, or physiology is acceptable, and under physiological condition hydrolyzable ester group.

R ₃And R ₄Be hydrogen, C1-3 alkyl, phenyl, benzyl, C9-10 benzene hydrocarbon base.Or R ₃Be hydrogen, R ₄Being natural amino-acid residue, also can be corresponding D-configuration amino-acid residue.R ₄Also can be a dipeptide residue, two amino-acid residue can be identical, also can be different, and its used amino-acid residue is natural amino-acid residue or corresponding D-configuration amino-acid residue.R ₄Can also be natural four, five, the hexose residue also can also be one two, three, and four oligosaccharides residues, its monose can be identical, also can be different, and its used monosaccharide residue is natural four, five, six carbon monosaccharide residues.

The polypeptide that the present invention relates to can exist with its free form, also can exist with the form of salt or the form of mixture.For example can and organic acid, superpolymer acid and mineral acid formation salt, the salt example hydrochloric acid salt of this form, acetate.For another example, can and inorganics, as inorganic salt or oxyhydroxide, form and to resemble Ca ²⁺Or Zn ²⁺Salt.Also can form mixture with the poly-organism of height, and and the mixture that forms of medicine acceptable thinner and carrier (as medicine acceptable protein carrier).

The polypeptide that the present invention relates to can synthesize and production with known chemiluminescent polypeptide method; as following embodiment; and the also available chemical synthesis process obviously of equal value with present embodiment, solution method or solid-phase synthesis (comprising tertbutyloxycarbonyl protection method and fluorenylmethyloxycarbonyl protection method) come synthetic and produce.

Polypeptide that the present invention relates to and medicine acceptable salt thereof and mixture show in biological activity assay and experimentation on animals all the valuable drug activity.Particularly aspect anti-schistosomiasis, show that by the immune mouse experiment very high worm reduction rate is arranged.The amount ranges of the polypeptide that the present invention relates in experimentation on animals is 0.001 to the 1000ug/kg body weight.

Embodiment 1

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

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

2.374g (6.24mmole) Boc-Lys (Cbz)-OH is dissolved in 40mL ethanol and the 8mL water, stirring and dissolving adds 1.017g (3.12mmole) Cs ₂CO ₃, being stirred to and having reacted, decompressing and extracting adds 4 * 20mL dry benzene, and decompressing and extracting is anhydrated to remove.Put into the vacuum drier dried overnight that contains Vanadium Pentoxide in FLAKES, get white cesium salt.

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

The above-mentioned cesium salt that makes is dissolved among the refined DMF of 10mL, adds 3.0g (chlorinity is 3.12mmole) chloromethyl resin, keeping dry state down, in 80 ℃ of appropriate stirring reactions 24 hours, filtration was drained, and uses 3 * 10mLDMF successively, 3 * 10mL DMF/H ₂O (9: 1), 3 * 10mL DMF, the anhydrous EtOH washing of 3 * 10mL is drained, and puts into the vacuum drier dried overnight that contains Vanadium Pentoxide in FLAKES, gets white Boc-Lys (Cbz)-OCH ₂-Resin.

1.3.Boc-Lys (bz)-OCH ₂The envelope chlorine of-Resin

To above-mentioned Boc-Lys (Cbz)-OCH ₂Add 2.558g (31.2mmole) anhydrous sodium acetate among the-Resin, the refined DMF of 30mL, 70 ℃ of appropriate stirring reactions 24 hours, filtration was drained, and uses 2 * 10mL DMF successively, 2 * 10mLDMF/H ₂O (9: 1), 7 * 10mL DMF, the anhydrous EtOH washing of 2 * 10mL is drained, and puts into the vacuum drier dried overnight that contains Vanadium Pentoxide in FLAKES, gets white Boc-Lys (the Cbz)-OCH that seals chlorine ₂-Resin.

1.4.Boc-Lys (Cbz)-OCH ₂The envelope hydroxyl of-Resin

To above-mentioned Boc-Lys (the Cbz)-OCH that seals chlorine ₂Add the diacetyl oxide that 10mL (0.105mole) heavily steamed among-the Resin, the anhydrous no Fampridine that 20mL (0.25mole) handled, the 20mL dry benzene, room temperature appropriateness stirring reaction 24 hours, filtration is drained, and uses 4 * 50mL DCM successively, the anhydrous MeOH washing of 3 * 10mL, drain, put into the vacuum drier dried overnight that contains Vanadium Pentoxide in FLAKES, get white Boc-Lys (the Cbz)-OCH that seals chlorine and hydroxyl ₂-Resin.

Detecting amino content through triketohydrindene hydrate is the 0.89mmole/g resin.

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

1.5g (amino content is 1.335mmole) seals Boc-Lys (the Cbz)-OCH of chlorine and hydroxyl ₂-Resin added 15mL DCM swelling 5 minutes, drained.

2.1. deprotection

Add 15mL TFA/DCM (3: 7) in above-mentioned resin, shake and stir 30 minutes, drain, use 10mL DCM successively, 10mL MeOH respectively gives a baby a bath on the third day after its birth inferior, drains.

2.2. neutralization

Add 10mL TEA/DCM (1: 9), shake and stir 5 minutes, drain, add 10mL TEA/DCM (1: 9) again, shake and stir 10 minutes, drain, it is inferior to give a baby a bath on the third day after its birth with 10mL DCM, drains.

2.3. coupling

Add 0.645g (3mmlole) Boc-Pro, 0.608g (4.5mmoLe) HOBt, 0.727g (4.5mmole) DCC, 15mL DMF shakes and stirs reaction 6 hours, drains, and 10mL DMF gives a baby a bath on the third day after its birth inferior, and 10mL MeOH gives a baby a bath on the third day after its birth inferior, and 10mL DCM gives a baby a bath on the third day after its birth inferior, drains.

2.4. detect

Get a little resin and add in the test tube, add respectively 3 of ninhydrin reagents 1,2 and 3,105 ℃ were heated 5 minutes.If faint yellow, illustrate that linked reaction is complete, can carry out next step reaction; If blueness or red-brown illustrate that linked reaction is incomplete, by in 2.2 and after, carry out 2.3 linked reactions again, feed intake but can reduce according to the reaction particular case.

Meet following amino acid Boc-Pro as stated above successively, Boc-His (Tos), Boc-Asp (OBzl), Boc-Gly, Boc-Gly, Boc-Gly, Boc-Phe, Boc-Thr (Bzl), Boc-Ala, Boc-Gln, Boc-Trp.

When previous amino acid is Pro, when Gln and Asn, owing to itself be yellow when ninhydrin reagent detects, so can not detect, but complete in order to guarantee linked reaction, coupling is 1-2 time again.When used amino acid has Tos is side when connecting protecting group, HOBt is changed into HOSu, in order to avoid the alkalescence of HOBt is taken off the Tos protecting group.After connecting Trp, should in TFA/DCM, add several thioanisoles and dithioglycol when deviating from the Boc protection.

The side that obtains at last being associated on the resin is even protected peptide Trp-Gln-Ala-Thr (Bzl)-Phe-Gly-Gly-Gly-Asp (OBzl)-His (Tos)-Pro-Pro-Lys (Cbz)-Resin.Attached: the triketohydrindene hydrate detection reagent

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

2. ninhydrin reagent 2,40gB phenol+10mL dehydrated alcohol;

3. ninhydrin reagent 3,2g ninidrine+40mL dehydrated alcohol.

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

In tetrafluoroethylene peptide deprotection device, carry out deprotection; in reactor, add Trp-Gln-Ala-Thr (Bzl)-Phe-Gly-Gly-Gly-Asp (OBzl)-His (Tos)-Pro-Pro-Lys (Cbz)-Resin; 0.5mL methyl-phenoxide; 0.5mL thioanisole and 0.5mL dithioglycol; under the ice-water bath cooling, put into 10-15mL through the dry hydrogen fluoride of crossing of anhydrous cobaltic fluoride; stirring reaction 45 minutes; decompressing and extracting; with cold anhydrous diethyl ether washing; get resin and sticky thing, the dissolving of 10% acetic acid solution is crossed and is filtered to remove residue; lyophilize gets dry powder; with 5% acetate is eluent purifying on the SephadexG15 post, purifying twice, and lyophilize gets solid 1.30g; yield 70%, [α] _D ¹⁸=-40.6 ° (C=1,0, water).Product peptide purity is determined by HPLC and amino acid composition analysis.

Following polypeptide also can synthesize and produces H-A-Trp-B-Ala-Thr-Phe-(C) with the similarity method that provides among the embodiment 1 ₃-D-E-(F) ₂-Lys-OH embodiment numbers A B C D E F [α] _D ¹⁸2 Gly Ser Gly Asp Ala Pro-64.9 (c=0.69, H ₂O) 3 Gln-Gly Asp Gly Asp Thr Pro-59.5 (c=0.64, H ₂O) 4-Ser Gly Asp Ala Pro-63.0 (c=0.66, H ₂O) abbreviation word Table A la alanine Asn asparagine Asp aspartic acid Boc tertbutyloxycarbonyl Bzl benzyl Cbz benzyloxycarbonyl group DCC N; N '-dicyclohexylcarbodiimide DCM carrene DMF DMF EtOH ethanol Gln glutamine Glu glutamic acid Gly glycine His histidine HOBt 1-hydroxy benzo triazole HOSu N-hydroxy-succinamide HPLC high pressure liquid chromatography Lys lysine MeOH methyl alcohol Phe phenylalanine Pro proline Resin resin Ser serine Thr threonine Tos p-toluenesulfonyl Trp tryptophan

Claims

1. acceptable salt on a peptide species or its pharmacology is characterized in that chemical structure is shown below:

In formula:

A is hydrogen or ethanoyl,

A ' is a hydrogen,

B is-Glu-,-Asp-, and-Gln-,-Asn-,-Thr-,-Ser-,

C is-Gly-,-Pro-,

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

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

F is-Pro-,-Gly-,

X can be following form:

-COOR ₁, or

Here:

R ₁Be hydrogen, C _1-3Alkyl, R ₂And R ₃Be hydrogen, C _1-3Alkyl.

2. acceptable salt on the polypeptide described in claim 1 or its pharmacology is characterized in that

B is-Gln-,

C is-Gly-,

D is-Asp-,

E is-His-,

F is-Pro-.

3. described in claim 1, be acceptable salt on peptide or its pharmacology, it is characterized in that

B is-Ser-,

C is-Gly-,

D is-Asp-,

E is-Ala-,

F is-Pro-.

4. acceptable salt on the polypeptide described in claim 1 or its pharmacology is characterized in that

B is-Asp-,

C is-Gly-,

D is-Asp-,

E is-Thr-,

F is-Pro-.

5. acceptable salt on the polypeptide described in claim 1 or 3 or its pharmacology is characterized in that

B is-Gln-,

C is-Gly-,

D is-Asp-,

E is-His-,

F is-Pro-.

6. acceptable salt on the polypeptide described in claim 1 or its pharmacology is characterized in that following formula:

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

7. acceptable salt on the polypeptide described in claim 1 or its pharmacology is characterized in that chemical structure is shown below:

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. the pharmaceutical composition of treatment and prevention schistosomicide, wherein contain any polypeptide among the with good grounds claim 1-7 or its salt as effective ingredient with and pharmacology acceptable thinner and carrier.