CN104725484A - Glycosylated polypeptide, preparation method and application thereof - Google Patents
Glycosylated polypeptide, preparation method and application thereof Download PDFInfo
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- C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
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- C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
- C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
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Abstract
The invention discloses a glycosylated polypeptide, a preparation method and application thereof. The glycosylated polypeptide has a structure shown as formula I in the specification, wherein the X group is preferably anti-HIV polypeptide T-20, Y is selected from arbitrary sugar chain compound, and Z is preferably mercapto containing cysteine. Compared with T-20, the partially glycosylated polypeptide has enhanced anti-HIV activity, prolonged half-life in vivo, long efficacy and high stability. The polypeptide site-directed galactosylated modification method disclosed by the invention can be carried out under mild reaction conditions, and the method is simple and efficient.
Description
Technical field
The invention belongs to field of biological pharmacy, have and relate to a kind of AntiHIV1 RT activity polypeptide and preparation method thereof and its purposes.
Background technology
Report acquired immune deficiency syndrome (AIDS) so far from U.S.'s Late Cambrian in 1981, the control of acquired immune deficiency syndrome (AIDS) is never effectively solved.Acquired immune deficiency syndrome (AIDS) is infected by human immunodeficiency virus (HIV) and causes, the curative drug of the HIV of current listing mostly is some small molecule enzyme inhibitor for viral reverse transcriptase, intergrase, proteolytic enzyme etc., due to the superelevation mutagenicity of HIV, these small molecules specific medicaments are made to deteriorate to normal effect, even ineffective agents gradually.In recent years, along with HIV invades progressively illustrating of host cellular molecules mechanism, the polypeptide drugs of this process are suppressed to become the study hotspot in AIDS preventing and controlling field gradually, this polypeptide drugs can fusion rotein on specific binding HIV cyst membrane thus suppress cell entry host cell, the T-20(trade(brand)name that Switzerland Roche and Trimeris company of the U.S. develop jointly: Fuzeon) be the AntiHIV1 RT activity polypeptide drugs uniquely gone on the market at present, compared with traditional small molecules inverase, T-20 has the ability of very strong antagonism mutant strain, and have no side effect, so far in treating AIDS, irreplaceable position is occupied from listing in 2003.
In recent years, along with the fast development of biotechnology, increasing polypeptide drugs are applied in prevention and treatment of diseases.Although the action site of polypeptide drug is single-minded, determined curative effect, solubleness is low, immunogenicity is high, be easily easily degraded by proteases and seriously constrained its clinical application by defects such as kidney removings.Also there is same problem in AntiHIV1 RT activity polypeptide, very soon can by plasma proteins enzyme liberating in patient body after T-20 injection, patient needs 1 day usually more than the injection for curing of 2 times, in the U.S., injection T-20 will spend 20,000 dollars in 1 year, within 1 year, will spend 25,000 dollars in Europe, expensive expenses for medicine makes the application of T-20 be extremely restricted.Therefore, study and set up that to extend T-20 and HR212 Half-life in vivo, the chemical modification method improving its bioavailability and technology most important for the clinical application of AntiHIV1 RT activity polypeptide drugs.
Summary of the invention
The object of this invention is to provide a kind of glycosylated polypeptides and preparation method thereof and its application.
A kind of glycosylated polypeptides provided by the invention, has structure shown in formula I:
In formula I, X is 1) or 2) described in polypeptide: the polypeptide 1) with the aminoacid sequence shown in SEQ ID № .1 in sequence table; 2) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have HIV (human immunodeficiency virus)-resistant activity by 1) derivative polypeptide; Y substituting group is selected from substituting group shown in formula II, and in formula II, R substituent is selected from any sugared substituting group, a and b be greater than zero integer;
In formula I, Z is selected from the amino acid containing sulfydryl; M is 0 or 1; N is 0 or 1; P is 0 or 1; Q is 0 or 1; Be 0 when p with q is different; Be 0 when m with n is different; When p is 0, m is also 0; When q is 0, n is also 0; Preferably, in formula I, m is 0 or 1; N is 0 or 1; P is 0 or 1; Q is 0 or 1; Be 1 or 0 when p with q is different; Be 0 or 1 when m with n is different; When p is 0, m is also 0; When q is 0, n is also 0; Preferred again, in formula I, m is 1; N is 0; P is 1; Q is 0.
In formula I, described Zp forms peptide bond by the carboxyl in its structure with the amino of the nitrogen terminal amino acid of described X polypeptide and realizes being connected, and described Zq forms peptide bond by the amino in its structure with the amino acid whose carboxyl base of the carbon teminal of described X polypeptide and realizes being connected;
In formula I, described Z is connected with the structure in formula I except X by the sulfydryl in its structure; Described connection particular by the hydrogen atom of described sulfydryl replace by structure in formula I except X and realize.
In described formula I, Z is selected from halfcystine; In Y substituting group, in formula II, a is 1, b is 3.
In described formula II, R substituent is selected from lactose Lac substituting group or sialyl lactose SiaLac substituting group, and the substituent structural formula of described SiaLac is that the Ac in formula represents ethanoyl:
Described lactose Lac substituting group is the substituting group formed after losing a hydrogen atom in the hemiacetal hydroxyl of lactose Lac.
Another object of the present invention is to provide a peptide species and fixes a point glycosylation modified method, and described method comprises: reacted by compound shown in compound cotype IV shown in formula III:
In formula III, Y substituting group is selected from substituting group shown in formula II, and in formula II, R substituent is selected from any sugared substituting group, a and b be greater than zero integer;
In formula IV, X is 1) or 2) described in polypeptide: the polypeptide 1) with the aminoacid sequence shown in SEQ ID № .1 in sequence table; 2) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have HIV (human immunodeficiency virus)-resistant activity by 1) derivative polypeptide; Z is selected from the amino acid containing sulfydryl; P is 0 or 1; Q is 0 or 1; P and q is not all 0.
In formula IV, described Zp forms peptide bond by the carboxyl in its structure with the amino of the nitrogen terminal amino acid of described X polypeptide and realizes being connected, and described Zq forms peptide bond by the amino in its structure with the amino acid whose carboxyl base of the carbon teminal of described X polypeptide and realizes being connected;
In described method, the preparation method of compound shown in described formula III for: compound shown in compound and formula VI shown in formula V is reacted compound shown in production VII, and compound shown in formula VII reacts with compound shown in formula VIII and obtains compound shown in formula III; Wherein, in compound shown in formula VIII, R substituent is selected from any sugared substituting group, and a is arbitrary integer;
In compound shown in formula V and formula VII, b is arbitrary integer:
The reaction process that shown in described formula V, shown in compound and formula VI, compound reacts comprises: compound shown in formula V is dissolved in saturated sodium bicarbonate, namely obtains formula VII shown in compound after adding the reaction of compound shown in formula VI under ice-water bath condition;
In described reaction, shown in described formula V, shown in compound and formula VI, the mol ratio of compound is 1:1-1:3, preferred 1:2;
Whether described reaction adopts TLC to detect and reacts completely; Neutralization, filtration, dry and purification step is also comprised after described reaction is complete; Described neutralization specifically uses acidic ion exchange resin to be neutralized to pH6.5-7.0, preferred pH6.8; Described drying is specially lyophilize; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 50% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, flow velocity 3mL/min, 10min sample introduction, and sample size is 50 microlitres, reverse phase semi-prep column.
The reaction process that shown in described formula VII, compound reacts with compound shown in formula VIII again comprises: under nitrogen protection, shown in formula VII, the mixture of compound shown in compound and formula VIII is dissolved in the mixing solutions of tetrahydrofuran (THF) and water, under stirring at room temperature, add cupric sulfate pentahydrate, add sodium ascorbate again, after transferring 35 DEG C of-50 DEG C of oil baths reaction to and get final product, preferably 35 DEG C;
In described reaction, shown in described formula VII, shown in compound and formula VIII, the mol ratio of compound is 1:1-3:1, preferred 1:1; In the mixing solutions of described tetrahydrofuran (THF) and water, the volume ratio of tetrahydrofuran (THF) and water is 2:1; The amount that described cupric sulfate pentahydrate adds is the 2%-5% of compound molar weight shown in formula VIII, preferably 5%, and the add-on of described sodium ascorbate is the 10%-25% for compound molar weight shown in formula VIII, preferably 15%;
Whether described reaction adopts TLC to detect and reacts completely; Concentrating under reduced pressure, purifying and drying step is also comprised after described reaction is complete; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 15% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, 10min sample introduction, and sample size is 100 microlitres, reverse phase semi-prep column, flow velocity 2.5-3mL/min; Preferred 2.5mL/min and 3mL/min of flow velocity; Described drying adopts lyophilize.
In described method, in described formula IV, Z is selected from halfcystine;
In described method, shown in described formula III, the preparation method of compound is: by compound 3 and compound 11 reacting generating compound 4, compound 4 reacts with compound shown in formula Ⅸ and obtains compound shown in formula III; Wherein, in compound shown in formula Ⅸ, R substituent is selected from any sugared substituting group;
Shown in the formula III that described method prepares, the concrete structure formula of compound is as follows, and wherein, R substituent is selected from any sugared substituting group:
The reaction process that described compound 3 and compound 11 react comprises: formula compound 3 is dissolved in saturated sodium bicarbonate, adds compound 11 and react rear i.e. compound 4 under ice-water bath condition;
In described reaction, described compound 3 is 1:1-1:3 with the mol ratio of compound 11, preferred 1:2;
Whether described reaction adopts TLC to detect and reacts completely; Neutralization, filtration, dry and purification step is also comprised after described reaction is complete; Described neutralization specifically uses acidic ion exchange resin to be neutralized to pH6.5-7.0, preferred pH6.8; Described drying is specially lyophilize; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 50% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, flow velocity 3mL/min, 10min sample introduction, and sample size is 50 microlitres, reverse phase semi-prep column.
The reaction process that described compound 4 and compound shown in formula Ⅸ react comprises: under nitrogen protection, compound 4 is dissolved in the mixing solutions of tetrahydrofuran (THF) and water with the mixture of compound shown in formula Ⅸ, under stirring at room temperature, add cupric sulfate pentahydrate, add sodium ascorbate again, after transferring 35 DEG C of-50 DEG C of oil baths reaction to and get final product, preferably 35 DEG C;
In described reaction, described compound 4 is 1:1-3:1 with the mol ratio of compound shown in formula Ⅸ, preferred 1:1; In the mixing solutions of described tetrahydrofuran (THF) and water, the volume ratio of tetrahydrofuran (THF) and water is 2:1; The amount that described cupric sulfate pentahydrate adds is the 2%-5% of compound molar weight shown in formula Ⅸ, preferably 5%, the 10%-25% that the add-on of described sodium ascorbate is compound molar weight shown in formula Ⅸ, preferably 15%;
Whether described reaction adopts TLC to detect and reacts completely; Concentrating under reduced pressure, purifying and drying step is also comprised after described reaction is complete; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 15% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, 10min sample introduction, and sample size is 100 microlitres, reverse phase semi-prep column, flow velocity 2.5-3mL/min; Preferred 2.5mL/min and 3mL/min of flow velocity; Described drying adopts lyophilize.
In described method, in described formula II, R substituent is selected from lactose Lac substituting group or sialyl lactose SiaLac substituting group, and the substituent structural formula of described SiaLac is that the Ac in formula represents ethanoyl:
Described lactose Lac substituting group is the substituting group formed after losing a hydrogen atom in the hemiacetal hydroxyl of lactose Lac.
In described method, the pH scope of the reaction of compound shown in compound cotype IV shown in described formula III is 7.2-9.0; Preferably 7.2,8.0 or 9.0; Most preferably 7.2.
In described method, the temperature of the reaction of compound shown in compound cotype IV shown in described formula III is room temperature; Be specially 20-30 DEG C.
In described method, the reaction solvent of the reaction of compound shown in compound cotype IV shown in described formula III is damping fluid.
Described damping fluid is weak acid and/or weak base salt damping fluid; Be specially phosphate buffered saline buffer; Described phosphate buffered saline buffer is specifically made up of solvent and solute, and described solvent is water, and described solute is SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate dibasic, and the concentration of described SODIUM PHOSPHATE, MONOBASIC is 5mM, and the concentration of described Sodium phosphate dibasic is 5mM.
An also object of the present invention is to provide a kind of glycosylated polypeptides prepared by above-mentioned arbitrary described method.
The application of glycosylated polypeptides in the following at least one product of preparation that another object of the present invention is to provide above-mentioned arbitrary described glycosylated polypeptides or is prepared by above-mentioned arbitrary described method:
1) AntiHIV1 RT activity product;
2) product of acquired immune deficiency syndrome (AIDS) is treated and/or prevented;
Described product is specially medicine or vaccine.
Another object of the present invention is to provide compound shown in following formula VII, compound 4, compound 8 or compound 10, and wherein, in compound shown in formula VII, b is arbitrary integer:
Another object of the present invention is to provide the preparation method of compound shown in formula VII, and wherein, in compound shown in formula VII, b is arbitrary integer, and described preparation method comprises: compound shown in compound and formula VI shown in formula V is reacted compound shown in production VII;
The reaction process that shown in described formula V, shown in compound and formula VI, compound reacts comprises: compound shown in formula V is dissolved in saturated sodium bicarbonate, namely obtains formula VII shown in compound after adding the reaction of compound shown in formula VI under ice-water bath condition;
In described reaction, shown in described formula V, shown in compound and formula VI, the mol ratio of compound is 1:1-1:3, preferred 1:2;
Whether described reaction adopts TLC to detect and reacts completely; Neutralization, filtration, dry and purification step is also comprised after described reaction is complete; Described neutralization specifically uses acidic ion exchange resin to be neutralized to pH6.5-7.0, preferred pH6.8; Described drying is specially lyophilize; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 50% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, flow velocity 3mL/min, 10min sample introduction, and sample size is 50 microlitres, reverse phase semi-prep column.
Another object of the present invention is to provide the preparation method of compound 4, and described preparation method comprises: by compound 3 and compound 11 reacting generating compound 4;
The reaction process that described compound 3 and compound 11 react comprises: formula compound 3 is dissolved in saturated sodium bicarbonate, adds compound 11 and react rear i.e. compound 4 under ice-water bath condition;
In described reaction, described compound 3 is 1:3 with the mol ratio of compound 11, preferred 1:2;
Whether described reaction adopts TLC to detect and reacts completely; Neutralization, filtration, dry and purification step is also comprised after described reaction is complete; Described neutralization specifically uses acidic ion exchange resin to be neutralized to pH6.5-7.0, preferred pH6.8; Described drying is specially lyophilize; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 50% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, flow velocity 3mL/min, 10min sample introduction, and sample size is 50 microlitres, reverse phase semi-prep column.
Another object of the present invention is to provide the preparation method of compound 8 or 10, and described preparation method comprises: compound 4 and compound 7 or compound 9 are reacted and be get final product;
The reaction process that described compound 4 and compound 7 or compound 9 react comprises: under nitrogen protection, compound 4 is dissolved in the mixing solutions of tetrahydrofuran (THF) and water with the mixture of compound 7 or compound 9, under stirring at room temperature, add cupric sulfate pentahydrate, add sodium ascorbate again, after transferring 35 DEG C of-50 DEG C of oil baths reaction to and get final product, preferably 35 DEG C;
In described reaction, described compound 4 is 1:1-3:1 with the mol ratio of compound 7 or compound 9, preferred 1:1; In the mixing solutions of described tetrahydrofuran (THF) and water, the volume ratio of tetrahydrofuran (THF) and water is 2:1; The amount that described cupric sulfate pentahydrate adds is the 2%-5% of compound 9 molar weight, preferably 5%, and the add-on of described sodium ascorbate is the 10%-25% of compound 9 molar weight, preferably 15%;
Whether described reaction adopts TLC to detect and reacts completely; Concentrating under reduced pressure, purifying and drying step is also comprised after described reaction is complete; Described purifying specifically adopts HPLC purifying; The condition of described HPLC purifying is: moving phase to be volumn concentration be 15% CH
3the CN aqueous solution, moving phase contains the trifluoroacetic acid that volumn concentration is 0.1%, 10min sample introduction, and sample size is 100 microlitres, reverse phase semi-prep column, flow velocity 2.5-3mL/min; Preferred 2.5mL/min and 3mL/min of flow velocity; Described drying adopts lyophilize.
Another object of the present invention is to provide the application in the arbitrary described glycosylated polypeptides of preparation claim 1-3 or glycosylated polypeptides according to claim 10 of compound shown in formula VII, compound 4, compound 8 or compound 10, wherein, in compound shown in formula VII, b is arbitrary integer.
The present invention's AntiHIV1 RT activity polypeptide that drug effect is long to obtain, stability is high is for target, according to mechanism of drug action and structure sequence feature, fixed point carried out to T-20 glycosylation modified, we introduce the amino acid sites selectively modified by synthesis in the non-active structure territory of T-20, under the reaction conditions of gentleness, (neutral system, lesser temps, gentle reaction reagent) coupling imports glycosyl, obtain long-acting AntiHIV1 RT activity polypeptide drugs, establish simple, efficiently AntiHIV1 RT activity polypeptide to fix a point glycosylation modified method, and it is active to have studied the polypeptide drugs after modification.The present invention can be the long-acting AntiHIV1 RT activity polypeptide drugs that development has a broad prospect of application and provides solid theoretical basis and technical support, and also contributing to (1) multipoint random solved in current polypeptide drugs chemical modification technology, to modify its lytic activity reduction, complicated component, purification difficult, the effective component that cause few; (2) the polypeptide conformation change that acutely causes of modification reaction condition and even a series of bottleneck problem such as inactivation.
Accompanying drawing explanation
Fig. 1 is the Mass Spectrometric Identification result figure of glycosylated polypeptides LacCF.
Fig. 2 is the Mass Spectrometric Identification result figure of glycosylated polypeptides SiaLacCF.
Fig. 3 is the Mass Spectrometric Identification result figure of glycosylated polypeptides YCLac.
Fig. 4 is the Mass Spectrometric Identification result figure of glycosylated polypeptides YCLacSia.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain or can refer to material prepared by document from commercial channels.
1h NMR by Bruker ARX400 at CDCl
3or CD
3recording in OD, is interior mark with tetramethylsilane.Mass spectrum adopts VG PLATFORM mass spectrograph, with ESI or MALDI-TOF technology sample introduction.Thin-layer chromatography (TLC) by HF254 silica-gel plate with 30%(V/V) methanolic solution or ultraviolet (UV) detector detect.Column chromatography adopts 200-300 object silica gel, with ethyl acetate-light petrol (60-90 DEG C) or methanol-ethyl acetate as leacheate, and solution underpressure distillation when being less than 60 DEG C.
In following embodiment if no special instructions, the ratio of described liquid and liquid is volume and volume ratio; The ratio of described solid and liquid is amount of substance and volume ratio, and described amount of substance is in mmol, and described volume is in ml; The ratio of described solid and solid is quality and mass ratio.
In following embodiment if no special instructions, described room temperature reaction is specially and controls within the scope of 20-30 DEG C by temperature of reaction, comprises 20 DEG C and 30 DEG C.
In following embodiment if no special instructions, the method for calculation of described yield are: (amount of the amount/raw material of product materials) if * 100%(has two or more raw materials, then the raw material selecting amount of substance less is as standard).
The preparation of embodiment 1, glycosylated polypeptides LacCF
(1) synthesis of sugar chain
1) compound 2 is prepared by compound 1
Reaction formula is as follows:
Under nitrogen protection, by compound 1 (3.2mmol), (preparation method of compound 1 can reference: Natara jan, A., Du, W., Xiong, C.-Y., DeNardo, G.L., DeNardo, S.J., Gervay-Hague, J., Construction of di-scFv through a trivalent alkyne – azide1, 3-dipolarcycloaddition.Chem.Commun.2007, (7), 695-697.) be dissolved in N, dinethylformamide (3mL), under ice-water bath condition, add sodium hydride (3.84mmol), stir 10min, add propargyl bromide (8mmol) again, find that reaction solution becomes Vandyke brown, stir 10min, transfer room temperature reaction to, after 8h, TLC (developping agent is the petrol ether/ethyl acetate of 2/1) finds that raw material reaction is complete, add two methyl alcohol cancellation reactions, concentrating under reduced pressure, silica gel chromatography is separated (eluent is the petrol ether/ethyl acetate of 8/1), obtain the faint yellow oily compound 2 of 380mg, yield 55%.Compound 2 nuclear-magnetism appraising datum is:
1h-NMR (400MHz, CDCl
3): δ 2.41 (d, 1H, J=1Hz, H-1), 3.36 (t, 2H, J=4.68Hz, H-9), 3.65 (m, 10H, H-4-8), 4.18 (s, 2H, H-3).
2) preparation of compound 3
Reaction formula is as follows:
Compound 2 (1.78mmol) is dissolved in the tetrahydrofuran (THF)/water (5.5mL) of 10/1, stirred at ambient temperature 10min, add triphenylphosphine (3.56mmol), after stirring 3.5h, TLC (developping agent is the ethyl acetate/methanol of 4/1) finds that raw material reaction is complete, concentrating under reduced pressure, and silica gel chromatography is separated (eluent is the ethyl acetate/methanol of 4/1), obtain 290mg tawny oily compound 3, yield 87%.
3) preparation of compound 4
Reaction formula is as follows:
Compound 3 (0.27mmol) is dissolved in saturated sodium bicarbonate (2mL), compound 11(is added purchased from lark prestige chemical reagents corporation by criticizing under ice-water bath condition, production code member: M261300, CAS:55750-48-6) (0.54mmol), after reaction 3h, TLC (developping agent is the petrol ether/ethyl acetate of 1/1) finds that raw material reaction is complete, acidic ion exchange resin (Amberlite IR120, H form) be neutralized to pH about 6.8, filter, lyophilize, HPLC purifies and separates: differential detects, moving phase to be volumn concentration be 50% CH
3the CN aqueous solution aqueous solution, 10min sample introduction, sample size is 50 microlitres, reverse phase semi-prep column (Agilent Zorbax Eclipse CSD-C18,5 μ, 250 × 9.4mm), flow velocity 3mL/min, moving phase contains the trifluoroacetic acid of 0.1%), obtain yellow oil 4 (60mg, 84%).
1H-NMR(400MHz,CDCl
3):δ2.39(s,1H,CH,H-1),3.58-3.71(m,12H,CH
2,H-3-8),4.16(d,2H,J=2Hz,CH
2,H-2),6.67(s,2H,CH,H-10).ESI-MS:Calcd forC
13H
17NO
5:267.11[M]
+;Found290.1[M+Na]
+,306.0[M+K]
+.
4) preparation of compound 6
Reaction formula is as follows:
Compound 5 purchased from Shanghai Mai Ruier chemical technology company limited, article No.: L115000, CAS:6291-42-5.Under nitrogen protection; compound 5 (1.47mmol) is dissolved in dry methylene dichloride (10mL); under ice-water bath condition; add ethylene bromohyrin (2.94mmol); slowly add boron trifluoride diethyl etherate (2.2mmol) again; stir 10min; transfer room temperature reaction to; after 3h, TLC (developping agent is the petrol ether/ethyl acetate of 1/1) finds that raw material reaction is complete, saturated sodium bicarbonate/dichloromethane extraction; collect organic phase; drying, concentrating under reduced pressure, obtains yellow oil 6.
5) preparation of compound 7
Reaction formula is as follows:
Above-mentioned oily compound 6 is dissolved in N, dinethylformamide (10mL), be placed in 65 DEG C of oil baths, add sodiumazide (4.41mmol), after 2h, TLC (developping agent is the petrol ether/ethyl acetate of 1/1) finds that raw material reaction is complete, drain solvent, dissolve with methylene dichloride, filter, concentrating under reduced pressure, obtaining oily matter is dissolved in the methylene chloride/methanol solution (9mL) of 1/2, dropwise add the methanol solution of 1.0mol/L sodium methylate, make the pH value of reaction system between 9 to 11, room temperature reaction 3h, TLC (developping agent is the ethyl acetate/methanol of 4/1) finds that raw material reaction is complete, add acidic ion exchange resin, being neutralized to pH is 7, filter resin, lyophilize, HPLC purifies and separates, obtain white powdery solids 7 (213mg, the overall yield 35% of three-step reaction).
1H-NMR(400MHz,MeOH-d
4):δ3.44-3.58(m,8H),3.69-3.88(m,6H),3.98(dd,1H,J=10.9Hz),4.32(t,2H,J=6.8Hz).MALDITOF-MS:Calcd for C
14H
25N
3O
11:411.15[M]
+;Found434.2[M+Na]
+,450.1[M+K]
+.
5) preparation of sugar chain 8
Reaction formula is as follows:
Under nitrogen protection, the mixture of compound 7 (0.224mmol) and compound 4 (0.224mmol) is dissolved in the tetrahydrofuran (THF)/water (altogether 4.5mL) of 2/1, under stirring at room temperature, add cupric sulfate pentahydrate (amount that cupric sulfate pentahydrate adds accounts for 5% of compound 7 molar weight), add sodium ascorbate (amount that sodium ascorbate adds accounts for 5% of compound 7 molar weight) again, transfer 35 DEG C of oil bath reactions to, after 18h, TLC (developping agent is the ethyl acetate/methanol/water of 5/2/1.5) finds that raw material reaction is complete, concentrating under reduced pressure, HPLC purifies and separates: differential detects, moving phase to be volumn concentration be 15% CH
3the CN aqueous solution, 10min sample introduction, sample size is 100 microlitres, reverse phase semi-prep column (Agilent ZorbaxEclipse CSD-C18,5 μ, 250 × 9.4mm), flow velocity 2.5mL/min, and moving phase contains the trifluoroacetic acid of 0.1%, lyophilize, obtains white powder compound sugar chain 8 (82mg, 54%).
1H-NMR(400MHz,MeOH-d
4):δ3.24(t,1H,J=8Hz),3.42(d,1H,J=8.6Hz),3.49-3.57(m,3H),3.59(s,5H),3.61-3.64(m,6H),3.68-3.73(m,3H),3.77-3.85(m,3H),3.89-3.92(d,1H,J=11.8Hz),4.01-4.06(m,1H),4.22-4.27(m,1H),4.36(d,2H,J=7.5Hz),3.64(s,2H),4.66-4.68(t,2H,J=5Hz),6.82(s,2H,CH in maleimide),8.14(s,1H,CH in triazole).MALDITOF-MS:Calcd for C
27H
42N
4O
16:678.26[M]
+;Found679.4[M+H]
+;701.4[M+Na]
+;717.4[M+K]
+.
(2) peptide C F's is glycosylation modified
The aminoacid sequence of T-20 polypeptide is as shown in SEQ ID № .1 in sequence table.Peptide C F is buied by Peptide systhesis company, and its aminoacid sequence is as shown in SEQ ID № .2 in sequence table.Peptide C F is that a newly-increased halfcystine obtains before the nitrogen terminal amino acid tyrosine of T-20 polypeptide, and newly-increased cysteine residues is as the site of glycosylation.
Be connected on the nitrogen terminal amino acid halfcystine of peptide C F by the Michael addition reaction that chemo-selective is high by the above-mentioned sugar chain 8 prepared, detailed process is as described below.
1, the synthesis of glycosylated polypeptides LacCF
0.006mmol sugar chain 8 and the mixture of 0.002mmol peptide C F be dissolved in 5 milliliters, concentration to be 5mM, pH be 7.2 SODIUM PHOSPHATE, MONOBASIC/disodium hydrogen phosphate buffer solution (solvent of described SODIUM PHOSPHATE, MONOBASIC/disodium hydrogen phosphate buffer solution is water, the concentration of described SODIUM PHOSPHATE, MONOBASIC is that 5mM(mmole often rises), the concentration of described Sodium phosphate dibasic is 5mM) in, with the Na of 5mM
2hPO
4solution regulates reacting solution pH value to 7.2, and HPLC monitors reaction, until polypeptide reacts completely.
2, the purifying of glycosylated polypeptides LacCF and sign
Adopt Agilent 1200 RP-HPLC look to boil instrument and successful purification is carried out to the glycosylated polypeptides according to above-mentioned steps gained.Chromatographic column model: Angilent Eclipse XDB-C18Semi-Prep, 5 μm, 9.4 × 250mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 40%A to 70%A, and 11 minutes time, elution flow rate is per minute 3 milliliters, ultraviolet detection wavelength 220 nanometer.The glycosylated polypeptides LacCF sterling of the fluffy state obtained after freezing solvent.
LacCF chemical structure is characterized by ESI mass spectrum, and the ESI mass spectral characteristi of LacCF the results are shown in Figure 1.
LacCF purity is provided by analysis mode high performance liquid chromatograph (flow velocity: per minute 1 milliliter).Wherein, the model of analytical high performance liquid chromatograph: Agilent 1200, adopt the model of chromatographic column: Angilent EclipseXDB-C18Analytical, 5 μm, 4.6 × 150mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 10%A to 100%A, and 25 minutes time, elution flow rate is per minute 1 milliliter, ultraviolet detection wavelength 220 nanometer.Analysis mode high performance liquid chromatograph detected result shows, and the purity of gained LacCF is 98.8%.
The glycosylation modified polypeptide LacCF obtained by above-mentioned preparation process, its structure is as follows:
The preparation of embodiment 2, glycosylated polypeptides SiaLacCF
(1) synthesis of sugar chain
1) preparation of compound 9
Reaction formula is as follows:
The preparation process of compound 7 is with described in embodiment 1.By compound 7 (42mg) and Cytidine-5-phosplate-sialic acid (CMP-NANA, mixture 5mM) is dissolved in the tris-HCI buffer (Tris-HCl that pH is 8.0,20mL), be placed in 37 DEG C of shaking tables, after 10min, add α-2,6-sialytransferase (100 μ L), after 24h, TLC (developping agent is the methanol/ethyl acetate/water of 6/3/1) finds that raw material reaction is complete, (note: ultraviolet detection wavelength 220nm, moving phase is 10%CH to HPLC purifies and separates
3cN to 25%CH
3cN, 20min sample introduction, sample size is 20 microlitres, reverse phase semi-prep column, 2mL quantitative loop, flow velocity 1mL/min, and moving phase contains the trifluoroacetic acid of 0.1%), lyophilize, obtains compound 9 (61mg, 85%).MALDITOF-MS:Calcd for C
25H
42N
4O
19:702.24[M]
+;Found725.4[M+Na]
+.
2) preparation of sugar chain 10
Reaction formula is as follows:
The preparation process of compound 4 is with described in embodiment 1.Under nitrogen protection, the mixture of compound 9 (0.092mmol) and compound 4 (0.092mmol) is dissolved in the tetrahydrofuran (THF)/water (altogether 1.5mL) of 2/1, under stirring at room temperature, add cupric sulfate pentahydrate (amount adding cupric sulfate pentahydrate accounts for 5% of compound 9 molar weight), add sodium ascorbate (amount adding sodium ascorbate accounts for 15% of compound 9 molar weight) again, transfer 35 DEG C of oil bath reactions to, after 18h, TLC (developping agent is the ethyl acetate/methanol/water of 5/2/1.5) finds that raw material reaction is complete, concentrating under reduced pressure, HPLC purifies and separates: differential detects, moving phase to be volumn concentration be 15% CH
3the CN aqueous solution, 10min sample introduction, sample size is 100 μ L, reverse phase semi-prep column (Agilent Zorbax Eclipse CSD-C18,5 μ, 250 × 9.4mm), flow velocity 3mL/min, moving phase contains the trifluoroacetic acid of 0.1%, lyophilize, obtain white powder compound 10 (13.8mg, 50%).ESI-MS:Calcd for C
38H
59N
5O
24:969.35[M]
+;Found970.4[M+H]
+;992.3[M+Na]
+;1008.3[M+K]
+.
(2) peptide C F's is glycosylation modified
The design preparation process of peptide C F is with described in embodiment 1.The above-mentioned sugar chain 10 prepared is connected on the nitrogen terminal amino acid halfcystine of peptide C F by the Michael addition reaction that chemo-selective is high, obtain glycosylated polypeptides SiaLacCF, detailed process is with described in embodiment 1, sugar chain 10 is changed into unlike by the sugar chain 8 in embodiment 1, in addition, with the Na of 5mM
2hPO
4solution regulates reacting solution pH value to 8.0.
The purifying of glycosylated polypeptides SiaLacCF and sign:
Adopt Agilent 1200 RP-HPLC look to boil instrument and successful purification is carried out to the glycosylated polypeptides according to above-mentioned steps gained.Chromatographic column model: Angilent Eclipse XDB-C18Semi-Prep, 5 μm, 9.4 × 250mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 40%A to 70%A, and 11 minutes time, elution flow rate is per minute 3 milliliters, ultraviolet detection wavelength 220 nanometer.The glycosylated polypeptides SiaLacCF sterling of the fluffy state obtained after freezing solvent.
SiaLacCF chemical structure is characterized by ESI mass spectrum, and the ESI mass spectral characteristi of SiaLacCF the results are shown in Figure 2.
SiaLacCF purity is provided by analysis mode high performance liquid chromatograph (flow velocity: per minute 1 milliliter).Wherein, the model of analytical high performance liquid chromatograph: Agilent 1200, adopt the model of chromatographic column: Angilent EclipseXDB-C18Analytical, 5 μm, 4.6 × 150mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 10%A to 100%A, and 25 minutes time, elution flow rate is per minute 1 milliliter, ultraviolet detection wavelength 220 nanometer.Analysis mode high performance liquid chromatograph detected result shows, and the purity of gained SiaLacCF is 98.2%.
The glycosylation modified polypeptide SiaLacCF obtained by above-mentioned preparation process, its structure is as follows:
The preparation of embodiment 3, glycosylated polypeptides YCLac
Polypeptide YC is buied by Peptide systhesis company, and its aminoacid sequence is as shown in SEQ ID № .3 in sequence table.Polypeptide YC is that a newly-increased halfcystine obtains after the carbon teminal amino acid phenylalanine of T-20 polypeptide, and newly-increased cysteine residues is as the site of glycosylation.
The sugar chain 8 embodiment 1 prepared is connected in the carbon teminal amino acid cysteine of polypeptide YC by the Michael addition reaction that chemo-selective is high, obtain glycosylated polypeptides YCLac, detailed process is with described in embodiment 1, polypeptide YC is changed into unlike by the peptide C F in embodiment 1, in addition, with the Na of 5mM
2hPO
4solution regulates reacting solution pH value to 9.0.
The purifying of glycosylated polypeptides YCLac and sign:
Adopt Agilent 1200 RP-HPLC look to boil instrument and successful purification is carried out to the glycosylated polypeptides according to above-mentioned steps gained.Chromatographic column model: Angilent Eclipse XDB-C18Semi-Prep, 5 μm, 9.4 × 250mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 40%A to 70%A, and 11 minutes time, elution flow rate is per minute 3 milliliters, ultraviolet detection wavelength 220 nanometer.The glycosylated polypeptides YCLac sterling of the fluffy state obtained after freezing solvent.
YCLac chemical structure is characterized by ESI mass spectrum, and the ESI mass spectral characteristi of YCLac the results are shown in Figure 3.
YCLac purity is provided by analysis mode high performance liquid chromatograph (flow velocity: per minute 1 milliliter).Wherein, the model of analytical high performance liquid chromatograph: Agilent 1200, adopt the model of chromatographic column: Angilent EclipseXDB-C18Analytical, 5 μm, 4.6 × 150mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 10%A to 100%A, and 25 minutes time, elution flow rate is per minute 1 milliliter, ultraviolet detection wavelength 220 nanometer.Analysis mode high performance liquid chromatograph detected result shows, and the purity of gained YCLac is 98.5%.
The glycosylation modified polypeptide YCLac obtained by above-mentioned preparation process, its structure is as follows:
The preparation of embodiment 4, glycosylated polypeptides YCLacSia
The design preparation process of polypeptide YC is with described in embodiment 3.The sugar chain 10 embodiment 2 prepared is connected in the carbon teminal amino acid cysteine of polypeptide YC by the Michael addition reaction that chemo-selective is high, obtain glycosylated polypeptides YCLacSia, detailed process is with described in embodiment 1, sugar chain 10 is changed into unlike by the sugar chain 8 in embodiment 1, peptide C F changes polypeptide YC into, in addition, with the Na of 5mM
2hPO
4solution regulates reacting solution pH value to 7.2.
The purifying of glycosylated polypeptides YCLacSia and sign:
Adopt Agilent 1200 RP-HPLC look to boil instrument and successful purification is carried out to the glycosylated polypeptides according to above-mentioned steps gained.Chromatographic column model: Angilent Eclipse XDB-C18Semi-Prep, 5 μm, 9.4 × 250mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 40%A to 70%A, and 11 minutes time, elution flow rate is per minute 3 milliliters, ultraviolet detection wavelength 220 nanometer.The glycosylated polypeptides YCLacSia sterling of the fluffy state obtained after freezing solvent.
YCLacSia chemical structure is characterized by ESI mass spectrum, and the ESI mass spectral characteristi of YCLacSia the results are shown in Figure 4.
YCLacSia purity is provided by analysis mode high performance liquid chromatograph (flow velocity: per minute 1 milliliter).Wherein, the model of analytical high performance liquid chromatograph: Agilent 1200, adopt the model of chromatographic column: Angilent EclipseXDB-C18Analytical, 5 μm, 4.6 × 150mm.Operation condition of chromatogram: linear gradient elution, elutriant is made up of mobile phase A and Mobile phase B.Mobile phase A is contain the acetonitrile solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Mobile phase B is contain the aqueous solution that concentration expressed in percentage by volume is 0.1% trifluoroacetic acid.Linear gradient elution is by 10%A to 100%A, and 25 minutes time, elution flow rate is per minute 1 milliliter, ultraviolet detection wavelength 220 nanometer.Analysis mode high performance liquid chromatograph detected result shows, and the purity of gained YCLacSia is 98.3%.
The glycosylation modified polypeptide YCLacSia obtained by above-mentioned preparation process, its structure is as follows:
The preparation of embodiment 5, peptide C C
Peptide C C is buied by Peptide systhesis company, and its aminoacid sequence is as shown in SEQ ID № .4 in sequence table.Peptide C C is before the nitrogen terminal amino acid of T-20 polypeptide and after carbon teminal amino acid, each newly-increased halfcystine obtains.
The glycosylated polypeptides that embodiment 6. embodiment 1-4 prepares and T20, CF, YC and CC polypeptide are to HIV-1
sF33the restraining effect of C-type virus C
TZM-bl cell is inoculated, 10 in 96 orifice plates
4/ hole, is placed in 37 DEG C, 5%CO
2spend the night in incubator.
Dilution medicine (i.e. the glycosylated polypeptides for preparing of embodiment 1-4 and T20, CF, YC and CC polypeptide) and viral SF33.Medicine is diluted to 8 extent of dilution with DMEM substratum by a certain percentage; The DEAE nutrient solution of virus to be measured containing DEAE-dextran is diluted to 2000TICD50/ml, and the concentration of DEAE-dextran is 30ug/ml.Liquid in 96 orifice plates is inhaled and abandons, add the medicine diluted successively, 100ul/ hole, and establish 3 multiple holes.The virus of having diluted is added in 96 orifice plates, 100ul/ hole (i.e. 200TICD50/ hole).Establish virus control group (VC) and cell controls group (CC), virus control group adds the DMEM substratum of 100ul and the virus dilution of 100ul, adds the DMEM nutrient solution of 200ul in cell control well simultaneously.
96 orifice plate surroundings are posted sealed membrane, is placed in 37 degree and cultivates 48 hours, then take out 96 orifice plates, the careful sucking-off 100ul in every hole, adds 100ul luminous detection liquid, and room temperature lucifuge places 2min, draw 150ul supernatant to be transferred in black 96 orifice plate, detect in luminometer.The inhibiting rate of medicine is calculated according to Reed and Muench method:
The IC of every peptide species medicine is calculated by the non-linear regression of GraphPad Prism Software5.0 software
50value and IC
90value.The IC of the glycosylated polypeptides that embodiment 1-4 prepares, CF, YC, CC and T-20 polypeptide
50value and IC
90be worth as shown in table 1.
Table 1
| Polypeptide | T-20 | CF | YC | CC |
| IC 50(μM) | 0.003±0.000 | 0.006±0.002 | 0.006±0.000 | 0.051±0.009 |
| IC 90(μM) | 0.023±0.002 | 0.035±0.004 | 0.044±0.002 | 0.350±0.028 |
| Polypeptide | LacCF | SiaLacCF | YCLac | YCLacSia |
| IC 50(μM) | 0.002±0.000 | 0.002±0.000 | 0.011±0.000 | 0.017±0.000 |
| IC 90(μM) | 0.016±0.001 | 0.018±0.001 | 0.095±0.004 | 0.111±0.006 |
Table 1 result shows: 1) antiviral activity of CF and YC is suitable with T20, and the peptide C C activity decrease that two ends add halfcystine is simultaneously obvious; 2) antiviral activity of nitrogen end glycosylated polypeptides is better than the glycosyl modified polypeptide of carbon teminal; 3) antiviral activity of LacCF and SiaLacCF glycosylated polypeptides is better than T20; .
In the SD rat body of embodiment 7. polypeptide, plasma half-life is evaluated
1. experimentation
For reagent thing: T20, LacCF, SiaLacCF
The making of typical curve: preparing each confession reagent thing (T20, LacCF, SiaLacCF) concentration with borate buffer solution (pH9.5) is the storing solution of 1mg/mL.Get this storing solution is mixed with 25,37.5,50,75,100,150,250 μ g/mL in right amount typical curve working fluid with 50% acetonitrile solution.Get ready typical curve working fluid 20 μ l, add blank mouse blood plasma 100 μ l, the typical curve sample of preparation 5,7.5,10,15,20,30,50 μ g/mL, add 20 μ l20% phosphoric acid solutions and 300 μ l methanol-acetonitrile (1:1) in typical curve sample, vortex mixes about 2min; 4000 revs/min centrifugal 10 minutes, gets the analysis of supernatant liquor loading, obtains each typical curve for reagent thing (T20, LacCF, SiaLacCF), and configuration quality-control sample detects its precision in accordance with the law.
Medicine ordinance: configuration before administration, 0.9% sodium chloride injection with 50% and 50% 5 mmole Na
2hPO
4be dissolved into transparent and homogeneous solution, T20, LacCF, SiaLacCF final concentration is respectively 3.9mg/ml, 4.6mg/ml, 4.8mg/ml, for subcutaneous administration.
Experimental animal: male and female SD rat, body weight 160 ~ 180 grams, source: Beijing HFK Bio-Technology Co., Ltd..
Experimentation on animals:
Administration: each medicine four SD rats, each two of male and female.Weighed body weight before administration, administration metering is 2mg/kg.
Sample collecting: be designated as zero moment before administration, after zero moment and administration, 30min, 1h, 2h, 4h, 6h, 10h, 12h, 24h are by tail venous blood sampling 0.3ml in the centrifuge tube that 6uL Trypsin inhibitor,Trasylol and 5uL heparin sodium are housed respectively, and 4500 revs/min of centrifugal 5min are separated upper plasma and are placed in-80 ° of Refrigerator stores.
Sample preparation: the blood plasma getting 100ul sample to be tested, add 20ul20% phosphoric acid solution, 20ul50% acetonitrile solution and 300ul methanol-acetonitrile (1:1) solution, vortex mixes about 2min, and 4000 revs/min centrifugal 10 minutes, gets the analysis of supernatant liquor loading.
Chromatographic condition: chromatographic column: XSELECT CSH C18,4.6 × 150mm, 5 μm, moving phase: A phase: the 0.1%TFA aqueous solution, B phase: 0.1%TFA acetonitrile, determined wavelength: 220nm, sampling volume: 20 μ L.
2. experimental result
1) by the drug level of the typical curve gained of medicine T20, LacCF, SiaLacCF and the relational expression of peak area in table 2.
Table 2
2) each time point each drug level establishing criteria curve obtains, and pharmacokinetic parameters is calculated by the non-compartment model in Winnonlin pharmacokinetics software.The results are shown in Table 3.
Table 3
Table 3 result shows:
Glycosylation medicine terminal elimination half-life in animal body (t1/2) obviously extends, and the polypeptide Increased Plasma Half-life effect of sialylated modification (SiaLacCF) is more obvious.
The clearance rate (Cl) of glycosylation medicine is lower than parent drugs T20, and the polypeptide of sialylated modification (SiaLacCF) clearance rate is starkly lower than parent drugs T20; The average retention time (MRT) of glycosylation medicine is longer than parent drugs T20, and the polypeptide of sialylated modification (SiaLacCF) to extend effect more obvious.
Above result can illustrate that the medicine elimination in animal body of glycosylation especially after sialic acid glycosylation is slower than parent drugs.This structural modification not only obviously can strengthen that it is water-soluble, can also reach the object of prolong drug action time.
Claims (10)
1. a glycosylated polypeptides, has structure shown in formula I:
In formula I, X is 1) or 2) described in polypeptide: the polypeptide 1) with the aminoacid sequence shown in SEQ ID № .1 in sequence table; 2) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have HIV (human immunodeficiency virus)-resistant activity by 1) derivative polypeptide; Y substituting group is selected from substituting group shown in formula II, and in formula II, R substituent is selected from any sugared substituting group, a and b be greater than zero integer;
In formula I, Z is selected from the amino acid containing sulfydryl; M is 0 or 1; N is 0 or 1; P is 0 or 1; Q is 0 or 1; Be 0 when p with q is different; Be 0 when m with n is different; When p is 0, m is also 0; When q is 0, n is also 0; Preferably, in formula I, m is 0 or 1; N is 0 or 1; P is 0 or 1; Q is 0 or 1; Be 1 or 0 when p with q is different; Be 0 or 1 when m with n is different; When p is 0, m is also 0; When q is 0, n is also 0; Preferred again, in formula I, m is 1; N is 0; P is 1; Q is 0.
2. glycosylated polypeptides according to claim 1, is characterized in that: in described formula I, Z is selected from halfcystine; In Y substituting group, in formula II, a is 1, b is 3.
3. glycosylated polypeptides according to claim 2, is characterized in that: in described formula II, and R substituent is selected from lactose Lac substituting group or sialyl lactose SiaLac substituting group, and the substituent structural formula of described SiaLac is that the Ac in formula represents ethanoyl:
4. a peptide species is fixed a point glycosylation modified method, and described method comprises: reacted by compound shown in compound cotype IV shown in formula III:
In formula III, Y substituting group is selected from substituting group shown in formula II, and in formula II, R substituent is selected from any sugared substituting group, a and b be greater than zero integer;
In formula IV, X is 1) or 2) described in polypeptide: the polypeptide 1) with the aminoacid sequence shown in SEQ ID № .1 in sequence table; 2) by the amino acid residue sequence of the SEQ ID № .1 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have HIV (human immunodeficiency virus)-resistant activity by 1) derivative polypeptide; Z is selected from the amino acid containing sulfydryl; P is 0 or 1; Q is 0 or 1; P and q is not all 0.
5. method according to claim 4, it is characterized in that: the preparation method of compound shown in described formula III for: compound shown in compound and formula VI shown in formula V is reacted compound shown in production VII, and compound shown in formula VII reacts with compound shown in formula VIII and obtains compound shown in formula III; Wherein, in compound shown in formula VIII, R substituent is selected from any sugared substituting group, and a is arbitrary integer; In compound shown in formula V and formula VII, b is arbitrary integer:
6. the method according to claim 4 or 5, is characterized in that:
In described formula IV, Z is selected from halfcystine;
Shown in described formula III, the preparation method of compound is: by compound 3 and compound 11 reacting generating compound 4, compound 4 reacts with compound shown in formula Ⅸ and obtains compound shown in formula III; Wherein, in compound shown in formula Ⅸ, R substituent is selected from any sugared substituting group;
Shown in the formula III that described method prepares, the concrete structure formula of compound is as follows, and wherein, R substituent is selected from any sugared substituting group:
7. the method according to claim 4-6, is characterized in that: in described formula II, and R substituent is selected from lactose Lac substituting group or sialyl lactose SiaLac substituting group, and the substituent structural formula of described SiaLac is that the Ac in formula represents ethanoyl:
8. according to the arbitrary described method of claim 4-7, it is characterized in that: the pH scope of the reaction of compound shown in compound cotype IV shown in described formula III is 7.2-9.0; Preferably 7.2,8.0 or 9.0; Most preferably 7.2.
9. the arbitrary described glycosylated polypeptides of claim 1-3 or the application of glycosylated polypeptides in the following at least one product of preparation that prepared by the arbitrary described method of claim 4-9:
1) AntiHIV1 RT activity product;
2) product of acquired immune deficiency syndrome (AIDS) is treated and/or prevented.
10. compound shown in following formula VII, compound 4, compound 8 or compound 10, wherein, in compound shown in formula VII, b is arbitrary integer:
Or, the preparation method of compound shown in formula VII, wherein, in compound shown in formula VII, b is arbitrary integer, and described preparation method comprises: compound shown in compound and formula VI shown in formula V is reacted compound shown in production VII;
Or, the preparation method of compound 4, described preparation method comprises: by compound 3 and compound 11 reacting generating compound 4;
Or, the preparation method of compound 8 or 10, described preparation method comprises: compound 4 and compound 7 or compound 9 are reacted and be get final product;
Or the application in the arbitrary described glycosylated polypeptides of preparation claim 1-3 or glycosylated polypeptides according to claim 10 of compound shown in formula VII, compound 4, compound 8 or compound 10, wherein, in compound shown in formula VII, b is arbitrary integer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108264542A (en) * | 2016-12-30 | 2018-07-10 | 四川本原作物科技有限公司 | Covalent trimeric polypeptides of Hrps of sodium alginate modification and preparation method thereof |
| CN108264563A (en) * | 2016-12-30 | 2018-07-10 | 四川本原作物科技有限公司 | Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof |
| CN110551179A (en) * | 2018-05-31 | 2019-12-10 | 中国科学院微生物研究所 | Modified anti-HIV polypeptide and preparation method and application thereof |
| CN112266410A (en) * | 2020-09-30 | 2021-01-26 | 河南师范大学 | Adenosine diphosphate ribose polypeptide and synthetic method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108264542A (en) * | 2016-12-30 | 2018-07-10 | 四川本原作物科技有限公司 | Covalent trimeric polypeptides of Hrps of sodium alginate modification and preparation method thereof |
| CN108264563A (en) * | 2016-12-30 | 2018-07-10 | 四川本原作物科技有限公司 | Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof |
| CN110551179A (en) * | 2018-05-31 | 2019-12-10 | 中国科学院微生物研究所 | Modified anti-HIV polypeptide and preparation method and application thereof |
| CN112266410A (en) * | 2020-09-30 | 2021-01-26 | 河南师范大学 | Adenosine diphosphate ribose polypeptide and synthetic method and application thereof |
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| CN104725484B (en) | 2018-03-13 |
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