CN104114575A

CN104114575A - Compound and composition having hypoglycemic effect and use thereof

Info

Publication number: CN104114575A
Application number: CN201280061395.5A
Authority: CN
Inventors: 秦树林
Original assignee: Adedian Beijing Biotechnology Co ltd
Current assignee: Adedian Beijing Biotechnology Co ltd
Priority date: 2011-12-15
Filing date: 2012-12-14
Publication date: 2014-10-22
Anticipated expiration: 2032-12-14
Also published as: CN109180802A; CN104011070A; WO2013086786A1; WO2013086785A1; CN104114575B

Abstract

Provided in the present invention are a compound and composition having hypoglycemic effect, and an application of the compound or composition in treating diabetes and hyperglycemia. Also provided in the present invention is a method for treating diabetes and hyperglycemia, comprising administration to patients in need of the compound or composition of the present invention. Compared to existing insulin and other analogues, the compound of the present invention has great aqueous solubility, long in vivo cycle, high activity for combining with an insulin receptor, significantly reduced toxic effect on individuals, and is easy to prepare.

Description

COMPOUND AND COMPOSITION HAVING HYPOGLYCEMIC EFFECT AND USE THEREOF

Compound, composition with hypoglycemic effect and application thereof technical field

The invention belongs to field of biological pharmacy, and in particular to compound, composition and their purposes with hypoglycemic effect.Background technology

Up to the present insulin have been subjected to the historical development of three generations as the special efficacy and choice drug for the treatment of diabetes：First generation product is extracted from the animal pancreas such as pig or ox.Due to heterologous allergic reaction, such product curative effect is poor.Second generation product is rh-insulin, is that insulin gene is obtained from human cell, is then inserted into saccharomycete or Escherichia coli and is cultivated, and is produced by complicated modernization biological gene technology.The third generation is insulin analog, is to carry out structural modification acquisition, including Semilente Insulin and protamine zine insulin by the insulin to people.

The large-scale clinical researches such as the beginning of this century NHANS in the U.S. is studied, the CODE researchs in Europe show, to the increasingly stricter control of diabetes and its complication, so that the hypertension of diabetic, high fat of blood, T-CHOL compliance rate more and more higher, and most basic blood glucose fluctuation rate does not rise anti-drop, subject matter is that existing insulin medicament is still not able to fine simulation physiological insulin secretion pattern.Therefore, curative effect, anti-immunity source, treatment be up to standard, in terms of simulation physiologic secretion, the novel insulin analogs that are all improved largely will improve one of main solution for the treatment of diabetes effect for exploitation.

American diabetes association（ADA) with European diabetology meeting（EASD) guide is advised, after lifestyle modification and oral hypoglycemic drug therapy, if the glycemic control of diabetic is still undesirable, should start insulin therapy as early as possible, and preferred basal insulin is shared with OHA.If this therapy can not still control blood glucose, it is proposed that add when having dinner use Semilente Insulin on this basis.

Basal insulin is used to maintain euglycemia secretion on an empty stomach.Many metabolism researchs are found between dinner and night hours, keep basic insulin level to reduce the decomposition of the phosphide of glycerine three, are suppressed liver output glucose, fasting blood-glucose is kept stable, so as to reduce overall blood sugar level.Preferable basal insulin, such as Recent Development of Long-acting Insulin Analogs, it should physiological insulin secretion pattern can be simulated, it is to avoid occur hypoglycemia especially Nocturnal hypoglycemia, do not put on weight.

The most middle protamine zine insulins used at present can be divided into three big species.The first kind is actrapid monotard and zinc ion or the suspension of the crystal of basic nucleoprotamine formation, such as NPH insulin, lente insulin etc..The curative effect of these insulin preparations is unstable, is just gradually replaced by Recent Development of Long-acting Insulin Analogs.

Second is insulin detemir（detemir ).It is the insulin analog that tetradecanoic acid links to B29 lysines.It is slow that insulin detemir absorbs Slow after injection.Extinction time T at injection_50%About 10 hours.It is combined by Β 29 aliphatic acid with the albumin in blood, and then Slow is dissociated slowly from complex.Double six is multimerizing（), Dihexamerization six aggressiveness and dimer and albumin combination all extend remaining time of the insulin detemir at injection.Insulin detemir enters after blood circulation, with albumin combination, further extends the internal residence time.

The third is that insulin glargine medicine dissolves in the formulation in pH 3.0, and is crystallized after injecting when pH rises to about 7.4.The Slow of injection site decomposes the effect for bringing delay slowly.But absorbent properties and pharmacokinetics are in crowd and individual variation in vivo is all very big.

Insulin glargine and insulin detemir are only two kinds of Recent Development of Long-acting Insulin Analogs in the market, and most long action time is no more than 24 hours.Insulin glargine activity in insulin-like growth factor-1 receptor (IGF-1R) is far above natural human insulin.Because the generation of insulin-like growth factor-1 receptor and kinds cancer is closely related, whether disputable long-term use insulin glargine can increase the cancered risk of patient always.Bioactivity of the insulin detemir in human body is about the confirmation sheet of natural human insulin 20%, therefore its dosage is 5 times of insulin regular dosage, this dramatically increases production and use cost.

Insulin requirements when rh-insulin is difficult to meet meal.Human insulin molecule is usually formed six dimeric structures, and dimer is gradually depolymerized to after hypodermic injection, and circulation could be entered through capillary by being further dissociated into monomer, play blood sugar reducing function.Due to there is depolymerization, absorption process, rh-insulin just works for about 30 minutes after hypodermic injection, and peak time is long, effect lasts about 6 ~ 7 hours.And because of individual difference, the amount that circulation is eventually entered into after injection same dose actrapid monotard also has notable difference.

The limitation of actrapid monotard brings two negative consequences.On the one hand, to ensure reduction postprandial blood sugar, actrapid monotard must be subcutaneously injected at 30 minutes before the meal in diabetes patient, and meal time is easily caused poor blood glucose control, delayed in advance, easily causes hypoglycemia.On the other hand, there is depolymerization and individual absorption difference after being subcutaneously injected due to actrapid monotard, eventually entering into the amount of insulin of circulation can not accurately estimate, easily cause insulin excessive or not enough.

Insulin Asp（Such as insulin aspart, insulin lispro）Research and development precisely in order to making up the deficiency of actrapid monotard.Insulin Asp absorbs fast, and peak time is short, and peak value is higher, and Cmax continues 1-3 hour, and acting duration is 3 ~ 5 hours, hence it is evident that better than actrapid monotard.But the onset time of insulin aspart and insulin lispro is about 20 minutes, still not enough facilitate for diabetes patient, be still improved leeway.

Therefore, novel insulin analogs are developed, bioactivity and bioavilability, extension action time is improved（Protamine zine insulin）Or shorten onset time（Semilente Insulin）, individual difference when improving water solubility, reducing medication, more efficiently prevent from the important directions that risk of hypoglycemia, increase stability are trypsin class medicine exploitations.The content of the invention

It is an object of the invention to provide with insulin active, can highly be combined with insulin receptor, the compound with hypoglycemic effect, pharmaceutically acceptable composition and its application in hypoglycemic.

The first aspect of the invention is to provide a kind of compound with hypoglycemic effect, and the amino acid sequence of the compound is：

Xi₀7HLC[i]GSX₁o₈LVEALYLVC[₂]GEXi₀9GFX₁₁₀X₁₁iX₁₁2X₁i₃Xn4Xn₅-CL-GIVEQC[₃]C[4]Xii6 SIC_[5]SLYQLENYC_[6]X₁₁₇X₁₁₈, wherein,

Xl07 is phenylamino acid-valine-asparagine-glutamin tetrapeptide, valine-asparagine-glutamin tripeptides, asparagine-glutamin dipeptides or glutamine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； X₁₍₎₈It is histidine, phenylalanine, essence Gas acid or glutamine； X_1Q9It is arginine, alanine, glutamic acid or aspartic acid；Xuo is phenylalanine, tyrosine or histidine；X, is tyrosine, phenylalanine or missing； X₁₁₂It is threonine, asparagine, glutamic acid, aspartic acid or missing； X₁₁₃It is proline, lysine, glutamic acid, aspartic acid or missing； X₁₁₄It is lysine, proline, arginine, glutamic acid, aspartic acid or missing； X₁₁₅It is threonine or missing； X₁₁₆It is threonine, histidine or arginine； X₁₁₇It is alanine, glycine or asparagine； X₁₁₈It is lysine, arginine-lysine dipeptides or missing； C_LIt is junction fragment as defined herein.

In second aspect, the present invention further provides a kind of compound with hypoglycemic effect, being modified on the basis of polypeptide, solubility, stability, body-internal-circulation action time further to improve the compound etc..The modification is will to modify the alpha-amido that side chain is connected to the -terminal amino acid residue of compound of the invention, or is connected to the epsilon-amino of lysine present in the compound of the present invention.The structure of the compound is：

X3₀oVNQHLC[i]GSHLVEALYLVC[2]GERGFX₃oiX₃₀₂X₃₀₃X304X₃₀₅X₃₀6GX₃o7X308X309X3ioX3ii

X312X313X314 315X31₆ 317Gr EQC[₃]C[₄]X₃₁₈X₃19 32₀C[5]X₃₂lLX₃₂₂X₃₂₃LX₃₂₄X₃₂₅YC[ ]X₃₂6X₃27' wherein,

X₃₀O is phenylalanine or U_L- phenylalanine； X₃₍₎₁It is phenylalanine, histidine or tyrosine； X₃O2 is tyrosine, benzene 2 001700

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Alanine or missing； X₃03 is threonine, asparagine, glutamic acid, aspartic acid or missing； X₃04 is proline, lysine, glutamic acid, aspartic acid or missing； X₃。₅It is aspartic acid, glutamic acid, proline, arginine, lysine or missing, or formula（I) structure； X₃o₆It is threonine, formula（I) structure or missing； X₃₍₎₇It is lysine, serine, alanine, glycine, formula（I) structure or missing； X_3Q8It is glycine, formula（I) structure or missing； X_3G9It is lysine, glycine, serine, formula（I) structure or missing； X_31QIt is lysine, glycine, serine, formula（I) structure or missing； x₃₁₁It is lysine, glycine, serine, alanine, formula（I) structure or missing； x₃₁₂It is lysine, arginine, alanine, proline, glycine, formula（I) structure or missing； x₃₁₃It is glycine, alanine, arginine, lysine, glutamine, proline, formula（I) structure or missing； x₃₁₄It is smart Number acid, alanine, proline, threonine, glutamine, glycine, formula（I) structure or missing； x₃₁₅It is proline, glutamine, arginine, glycine or missing or formula（I) structure； x₃₁₆It is glutamine, threonine, arginine, glycine or missing or formula（I) structure； x₃₁₇It is threonine, arginine, lysine or missing； X₃₁₈It is threonine, histidine, arginine or formula（I) structure； x₃₁₉It is serine or formula（I) structure； X_32QIt is isoleucine or formula（I) structure； X_32IIt is serine or formula（I) structure； x₃₂₂It is tyrosine or formula（I) structure； X₃₂₃It is glutamine or formula（I) structure； x₃₂₄It is glutamic acid or logical formula (I) structure； X₃₂₅It is asparagine or formula（I) structure； x₃₂₆It is alanine, glycine or asparagine； x₃₂₇It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；Work as X₃₂₇During for dipeptides, one of amino acid is formula（I) structure； U_LAnd formula（I) structure is as defined herein.

The third aspect of the invention is to provide a kind of pharmaceutical composition, mixed by the compound with blood sugar reducing function and pharmaceutically acceptable carrier of the present invention, mixed proportion can be about 90/10%, about 80/20%, about 70/30%, about 60/40%, about 50/50%, about 40/60%, about 30/70%, about 20/80% or about 10/90%;Preferably, the composition further includes Insulin Asp；The Insulin Asp can be Asp^B28Actrapid monotard, Lys^B28Pro^B29Actrapid monotard or Lys^B3Glu^B29Actrapid monotard.

The fourth aspect of the invention is to provide application of the compound of the present invention in the treatment medicine such as diabetes or hyperglycemia is prepared.

The fifth aspect of the invention is to provide a kind of method for treating diabetes or hyperglycemia etc., including the compound of the invention of the sufferer administration to needs or composition.

Compared with existing insulin and the like, compound water soluble of the invention is good, and the high activity with bound insulin acceptor, toxic side effect is low, and it is easy to prepare.The circulation time of compound in vivo after modification is obviously prolonged.Brief description of the drawings

Fig. 1 be mouse subcutaneous injection physiological saline, actrapid monotard and the present invention II -2 compounds after blood glucose change over time value；Fig. 2 is that mouse subcutaneous injection physiological saline changes over time value with blood glucose after II -17 compounds of the present invention；

Fig. 3 be mouse subcutaneous injection physiological saline, actrapid monotard and the present invention II -11 compounds after blood glucose change over time value.

Embodiment

Definition and term

Unless otherwise indicated, following definitions are applied to the present invention in full.Undefined term can understand according to definition sanctified by usage in industry.

" amino acid " refers to any while the molecule comprising amino and carboxyl functional group, the amino and carboxyl of a-amino acid are connected on same carbon atom（α carbon).α carbon can have 1-2 organic substituent.Amino acid is mixed comprising L and dexiroisomer and racemization 0

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Compound.Unless otherwise instructed, the amino acid residue in the present invention in peptide sequence is all L isomers i.e. l-amino acid, and D- amino acid is before amino acid name or abbreviation plus lowercase " d " is represented, such as dK.

Expression way " codified atmosphere base acid " or " codified amino acid residue " are used for the amino acid or amino acid residue for representing to be encoded by nucleotide triplet.

HGlu is high glutamic acid；

- hGlu is-HNCH (CO-) CH₂CH₂CH₂COOH L isomers；

δ-hGlu are-H CH (COOH) CH₂CH₂CH₂CO- L isomers；

A-Asp is-H CH (CO-) CH₂COOH L isomers；

β-Asp are-HNCH (COOH) C CO- L isomers；

A-Glu is-HNCH (CO-) CH₂CH₂COOH L isomers；

γ-Glu are-HNCH (COOH) CH₂CH₂CO- L isomers；

β-Ala are-HN-CH₂-CH₂-COOH;

Sar is methyl amimoacetic acid.

Amino acid residue can be represented with three letter amino acid code or one letter amino code；Amino acid table is as follows：Table one：Amino acid name and cylinder are write

" natural insulin " refers to the mammalian islet element (such as actrapid monotard, bovine insulin, pork insulin etc.) from natural, chemical synthesis, genetic engineering production.The B chains that A chain and 30 amino acid of the actrapid monotard comprising 21 amino acid compositions are constituted.Two chains are connected by 3 disulfide bond：A7 and B7, A20 and B19, A6 and AI L B7, A7 refer to natural insulin B chains position 7 (from N-terminal number) amino acid residue and INSULIN A chain position 7 (from N-terminal number）Amino acid residue.

" insulin analog " is the common name for the insulin polypeptides changed, including with the duplex molecule that A chain and B chain is made up of of the natural insulin by homologous sequence, and single-chain insulin analogues." insulin analog;; part, whole with natural insulin or enhancing activity; or can be converted into the polypeptide of the part with natural insulin, whole or enhancing activity in vivo or in vitro, such as than natural insulin increase, reduce or replace the polypeptides of one or more amino acid residues.People, the proinsulin of animal or even nonmammalian, preproinsulin, insulin precurosor, single-chain insulin precursor and analog are referred to as " insulin analog ".Many insulin analogs are seen in document.Except non-specifically illustrates in addition, " insulin analog " broad sense includes natural insulin and insulin analog.

Unless otherwise specified, the insulin being related in the application refers to actrapid monotard.Actrapid monotard's A chain-orderings are SEQ ID NO:Sequence shown in 124, actrapid monotard's B chain-orderings are SEQ ID NO:Sequence shown in 125. 1700

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The amino acid number rule of compound:

Single chain compound is referred to general structure B chains-C_LThe peptide sequence of-A chains or the peptide sequence of modification, wherein B chains are B chains of insulin or the like, and A chains are A chains of insulin or the like, C_LIt is the peptide chain for connecting B chain C-terminal amino acid residues and A chain N-terminals.

Unless otherwise specified, in the application with A chains or the amino acid of B chain position descriptions, the A chains or the amino acid of B chain opposite positions of such as A14, B28 expression and insulin or its change, the wherein A chains of insulin or the numbering of B chains is since 1.INSULIN A chain and B chain-orderings are shown in SEQ ID NO respectively:124 and 125.

The numbering of cysteine in compound:

For convenience of describing, the cysteine in each compound in the present invention is numbered, respectively C_n] ~C_[6], it is corresponding in turn to 6 cysteines of the single chain compound from N-terminal to C-terminal.

The compound of the present invention is the structure based on insulin, therefore all includes disulfide bond in the tertiary structure of any one compound of the invention, and to form disulfide bond with insulin identical mode, i.e. C_n]With C [_4]Form disulfide bond,._[2] and._[6] form disulfide bond, C_[3]And C_[5]Form disulfide bond.Those skilled in the art are appreciated that and know the disulfide bond position of the compound of any one in the present invention completely based on above-mentioned explanation and common knowledge.

" modification group "

Insulin analog can include one or more modification groups.Modification group can provide the feature of insulin analog needs.For example, modification group can reduce insulin analog under circumstances（Such as alimentary canal, blood）Degradation rate.It is preferred that modification group be that those allow insulin analogs to retain the groups of suitable insulin receptor binding activity.It is preferred that modification group include amphiprotic group, water soluble group, or make insulin analog than the non-modified lower lipophilicity of analog, more highly lipophilic, more highly-water-soluble group.Modification group can include degradable linker.Such as PAG;Linker susceptible to hydrolysis, such as lactide, glycolide, carbonic acid, ester, amino Yue acid esters can be included.This method can make depolymerization into small-molecular-weight fragment.

Modification group can include the combination of one or more hydrophilic radicals, lipophilic group, amphiprotic group, salt forming group, spacer group, linking group, end-capping group or these groups.Various groups can be with covalent bond, or is linked together with the key of hydrolyzable or non-hydrolysable.Representative hydrophilic radical and lipophilic group are described below.

Hydrophilic radical

The example of hydrophilic radical includes the composition of PAG groups, polysaccharide, polysorbate and these groups.

Polyalkylene glycol（Polyalkylene Glycol, PAG) it is made up of multiple aklylene glycol monomers.In one embodiment, all monomers are identicals（Such as polyethylene glycol（) or polypropylene glycol PEG（PPG ) ).In another embodiment, aklylene glycol is different.Condensate can be random copolymer（The copolymer of such as oxirane and expoxy propane）, or branch or graft copolymer.

" PEG " or polyethylene glycol used herein refer to any water-soluble polyethylene glycol or polyethylene glycol oxide.The chemical structural formula of polyethylene glycol is-(CH₂CH₂0)_n-, wherein n can be the integer from 2 to 2000.PEG one end is typically the functional group for being relatively free of activity, such as alkyl or alkoxy.Alkyl includes the straight or branched alkyl of saturation.The example of alkoxy stands is Yue epoxides, ethyoxyl, propoxyl group（Such as 1- propoxyl group and 2- propoxyl group）, butoxy（Such as 1- butoxy, 2- butoxy and 2- methyl -2- propoxyl group）, amoxy, hexyloxy etc..The PEG blocked using Yue epoxides is named as mPEG, structural formula CH₃0(CH₂CH₂0)_n-, but general still referred to as PEG.PEG20K refers to molecular weight for 20,000 peg molecules.

The PEG other ends are typically activating functional group or the functional group for being easily formed Gong Jia Key, such as amino, carboxyl, hydroxyl, sulfydryl, ^.PEG- maleimides, PEG- vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan)s and PEG- iodoacteyls（CO-CH₂- I) etc. can be with half Guang 0

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Sulfydryl-the SH of propylhomoserin side chain reacts to form stable covalent bond；PEG-NHS (succinimides）Nucleophilic substitution can be passed through with polypeptide N-terminal α amino or lysine side chain amino groups（It is acylated）Engagement；The amino of PEG- aldehyde and polypeptide is in reducing agent（Such as sodium cyanoborohydride）It can be reacted and engaged by standard reductive alkylation under effect.

PEG molecules in the present invention can be straight chain, the PEG of side chain, bifurcated or dumbbell shaped.In one embodiment, side chain PEG can use general formula R (- PEG-_nOH)_mRepresent, wherein R (is typically polyhydroxy）It is core group, such as pentaerythrite, sugar, lysine or glycerine.M represents side chain number, can play core group attachment site maximum number from 2, the quantity that n represents the PEG fragments on the quantity of PEG fragments, each side chain can not be waited.Generally, n is 2-1800 integer.In another embodiment, side chain PEG can use formula (CH₃0-PEG-_n)_pR-Z represents that p is equal to 2 or 3, R are lysine or glycerine, and Z represents the activating functional group that can be reacted.In one embodiment, bifurcated PEG formula PEG (- L-X)_nRepresent, L is linker, and X is terminal activating functional group.

PEG-as be polydispersion, many ^ I purports numbers be less than 1.05.PEG group can also be single ^:'s.Single dispersing, which refers to PEG, has single length（Molecular weight）, rather than various length（Molecular weight）Mixture.

Glycosyl group

Representative glycosyl group includes, but are not limited to, glycerine, monose, disaccharides, trisaccharide, oligosaccharides and polysaccharide such as starch, glycogen, cellulose and/or polysaccharide gum.Special monose include C6 and more than（Particularly C6 and C8) sugar such as glucose, fructose, mannose, galactolipin, ribose or sedoheptose；Disaccharides and trisaccharide are included containing two or three monosaccharide units（Particularly C5 to C8) group, such as sucrose, cellobiose, maltose, lactose and/or melitriose.

Other hydrophilic radicals

Biocompatible polycation group includes the polyamine groups on skeleton or side chain with multiple amino, the amino acid polymer with multiple positive charges of such as polylysine and other natural or synthetic Amino acid profiles, including poly ornithine, poly arginine, polyhistidyl, for example poly- aminostyryl of non-polypeptide polyamine, poly- amino acrylates, poly- N Yue bases amino acrylates, quaternary polyamines etc..Biocompatible polyanion group includes the group on skeleton or side chain with multiple carboxyls, such as poly-aspartate, polyglutamic acid.Other hydrophilic radicals include natural or synthetic polysaccharide, such as chitosan, glucan.

Polyanion bioadhesive polymer

Some hydrophilic radicals have potential bio-adhesive properties.Such example is found in United States Patent (USP) US 6,197,346.There is the polymer of multiple carboxyls to show bio-adhesive properties for these.The fast degraded biologically polymer of multiple carboxyls is manifested during degraded, such as lactic-co-glycolic acid, polyanhydride, poe are also all bioadhesive polymers.These polymer can deliver insulin analog and arrive intestines and stomach.The carboxyl being exposed during depolymerization can be firmly attached to intestines and stomach, assist to deliver insulin analog.

Lipophilic group

In one embodiment, modification group includes one or more lipophilic groups.Lipophilic group can be those skilled in the art it is well known that including but is not limited to：Alkyl, alkenyl, alkynyl, aryl, aryl alkyl, alkylaryl, aliphatic acid, cholesterine and lipophilic polymer and oligomer.

Alkyl can be saturation, unsaturation, straight chain, side chain or cyclic hydrocarbon, with one or more carbon atoms.In one embodiment, alkyl has 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more carbon atom.Alkyl can be unsubstituted or have one or more substituent, and these substituents will not preferably make conjugate lose bioactivity.

Lipophilic group can also be aliphatic acid, such as natural, synthesis, saturation, undersaturated, straight chain or side chain aliphatic acid.In one embodiment, aliphatic acid has 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or more carbon atoms. With reference to (Conjugation) strategy

The selection of the combination degree, binding site of modification group and polypeptide, the selection of modification group will be varied as desired, for example, make conjugate not degradable in vivo, so as to extend plasma half-life.For example after insulin analog modification there are one, two, three, four or more modification groups.Binding site potentially includes an amino acid residue, such as lysine residue.In one embodiment, insulin bonder is unijunction compound.In another embodiment, insulin bonder is many conjugates.In another embodiment, insulin bonder is the mixture of unijunction compound, binode compound, three conjugates, four conjugates etc..Modification group can be with identical, can also be different.When insulin bonder has multiple modification groups, one or more modification groups are connected preferably by hydrolyzable bond with insulin bonder and other one or more modification groups are connected preferably by non-hydrolysable key with insulin bonder.Or, all modification groups are all connected by hydrolyzable bond with insulin bonder, but the hydrolysis rate faster or slower of each modification group in vivo.

It is conjugate is had the part or all of bioactivity of Proinsulin analog preferably to combine strategy.It is preferred that binding site include N-terminal α amino and lysine side chain amino groups and other binding sites can be created by the junction fragment in single chain compound or Α chains, the sour residue of natural or non-natural Number base of the Β chains insertion with amino Huo Pistol bases in addition.

Modification group can pass through hydrolyzable bond with insulin analog（Such as ester, carbonic acid, hydrolyzable amino Yue acid esters）With reference to.Hydrolyzable bond makes insulin bonder have the effect of prodrug.If it is desired to modification group and the no activity of insulin bonder, such as the binding site of modification group is in insulin analog to insulin receptor land, and prodrug strategies are exactly method for optimizing.When one or more modification groups depart within a period of time from insulin bonder, so as to discharge biologically active insulin analog, the effect that delay release or Slow are released can be provided using hydrolyzable bond.

In one embodiment, insulin analog passes through non-hydrolytic key（Such as amido link, ehter bond）It is connected with modification group.If necessary, non-Shui Xie Key help to extend circulation time of the insulin bonder in blood plasma.

Insulin homologue can be connected by various nucleophilic functional groups with modification group, including but not limited to, nucleophilic hydroxy or amino.Such as serine, threonine, tyrosine have nucleophilic hydroxy, and histidine, lysine or insulin analog A chains, B chain N- ends all have nucleophilic amino.Insulin homologue can also be connected by free sulfhydryl group-SH with modification group, for example, formed and dredge ester, thioether, sulfanilamide (SN) key.

Circulation time of the less polypeptide compound of molecular weight in a blood plasma short key factor is exactly that kidney is removed.Increase polypeptide compound molecular weight until removing critical point more than kidney, can significantly reduce renal clearance, extension polypeptide action time in vivo.Conventional method is to make polypeptide and natural or synthetic macromolecular formation hydrolyzable or non-hydrolysable key.Large biological molecule includes albumin, polysaccharide, antibody（Such as IgG).70% albumin is to dredge base albumin in blood vessel（Mercaptalbumin), the side chain coloured glaze base of its cysteine -34 is activity most strong coloured glaze base in blood plasma.Insulin analog can carry Ma by an one end and react generation insulin-albumin conjugates Lai the linker of the activating functional groups such as zhen imines.Linker can be long chain fatty acids or PEG molecules in itself.Instantiation is referred to Bioconjugate Chem. 2005,16,1000-1008.Synthetic macromolecule includes polyethylene glycol and glucan.Another way is fatty-acylation, will be discussed in acylated insulin analog part.

Occur in that in recent years and the method for making two molecules be combined by amido link is catalyzed with protease sortase.Sortase is a kind of mediation gram-positive bacteria cell membrane and the covalently bound transpeptidase of surface protein, is primarily present in gram-positive bacteria.The sequential analysis of protein of GeneBank CDS and NCBI data bank is shown, sortase families include 150 multiple protein sequences, wherein Staphylococcus aureus sortase A (SrtA or SrtA_StapH) be current most study isotype（ isoform ).There are many documents to disclose the molecular mechanism of the transpeptidation reaction of SrtA catalysis.SrtA recognizes the substrate for including LPXTG (Leu-Pro-X-Thr-Gly) motif, and thus the peptide bond Thr-Gly that its 184 cysteine is attacked in LPXTG motifs as nucleophilic group produces an acyl-enzyme intermediate.The thioesters intermediate of threonine carboxyl passes through the oligomeric glycine with substrate（It is pentaglycine (the Gly of branched lipids II precursor commissure bridge in S. aureus₅)) amino group occur necleophilic reaction, produce new connection product. Another related ■ Str^ptococciw/^ogew y sortase can receive the nucleophilic group being made up of two alanine, but aureus enzymes can not.This sortase (SrtA_strep) cut-out LPXTA motifs in peptide bond Thr-Ala, it is allowed to the nucleophilic group based on alanine. SrtA_strepLPXTG motifs can also be recognized, but activity is relatively low.LPXTA motifs will not be by SrtA_StaphCut-out.For the sake of simplicity, following methods are discussed with SrtA_StaphExemplified by, but SrtA_strepSame or analogous method can also be used Deng isotype.

SrtA is repeated to the glycine of LPXTG motifs and N-terminal with free amino group（ a-Gly_n) highly single-minded.X can be in addition to cysteine and tryptophan（Not yet test）All natural amino acids.Show that polypeptide of the N-terminal with a glycine can participate in the transpeptidation reaction of sortase catalysis in spite of experiment, substrate of the N-terminal with two or more glycine can reach maximum reaction efficiency.Sortase auxiliary connections（Sortase mediated ligation) a main application be exactly by non-natural functional group introducing albumen or polypeptide.Non-natural functional group can be small molecule, synthesis polypeptide or albumen, polymer etc..These functional groups and LPXTG or a-Gly_nThe molecule of fusion can turn into SrtA substrate.Specific method and reaction condition be referred to, close document (：δ mouthfuls of Tsukiji etc., " Sortase-Mediated Ligation: A Gift from Gram-Positive Bacteria to Protein Engineering", ChemBioChem, 2009,10,787-798;Popp etc., " Sortase-catalyzed transformations that improve the properties of cytokines ", PNAS, 2011,108,3169-3174).

SrtA can introduce non-natural functional group on insulin analog, and specific strategy will be depending on the structure according to insulin analog.For two-chain insulin analog, a ^:The binding site of little effect bioactivity is the N-terminal or A chains of B chains, the C-terminal of B chains.If binding site is the N-terminal of B chains, the N-terminal of so insulin B chain is preferably introduced multiple glycine, such as GGGGG- insulin B chains, and the modification group to be introduced such as PEG, long chain fatty acids or albumin etc. C-terminal will have LPXTG motifs, such as PEG-LPATGGGG, albumin-LPETGGG or aliphatic acid LPGTGGGGG etc..If binding site is the C-terminal of A or B chains, so the c terminal amino acid sequence of A or B chains will include LPXTG motifs, INSULIN A-LPATGGGGG or insulin B-LPGTGGGG etc. can be such as changed into, and the modification group to be introduced such as PEG, long chain fatty acids or albumin etc. N-terminal will have one or more glycine, such as GGG-PEG, GGGG- long chain fatty acids, GGGGG- albumin.For single-chain insulin analogues, the binding site being easiest to is the N-terminal of B chains or the C-terminal of A chains, and method and two chain analogs are essentially identical.Single chain compound（Single chain compound based on pancreas ^ elements）

In mammal, synthesized in the β cells of pancreas islet of the insulin in pancreas.Proinsulin is the single chain precursor containing 86 amino acid, is configured to：Β chain-ArgArg-C peptide-LysArg-A chains." connection peptide " that C peptides are made up of 31 amino acid.Arg-Arg and Lys-Arg are the split points that followed by action of proteolytic enzymes makes C peptides divide from A and B chains, it is known that proteolytic enzyme is prohormone convertase（PC1 and PC2), and external form protease carboxypeptidase 5.These changes of proinsulin remove C peptides, and remaining B chains and A chains are combined together by Er Liu Key.

The duplex structure of insulin causes insulin to have a variety of conformations.Insulin has the potential of sizable conformation change, and the limitation to these changes significantly reduces affinity of the insulin receptor to part.Closing GlyAl aminoterminal equally slackens receptor binding capacity.Proinsulin only has the 1-2% of insulin with insulin receptor affinity.

Effect of the current not clear C peptides in proinsulin folding.The length of C peptides changes between 26-38 amino acid in different animals species.In B chain-C peptides（) and C peptide-A chains B-C（C-A) the binary amino acid residue of junction is conservative, and it is minimum to think the need for for insulin conservative.The three-dimensional structure of insulin shows that A chains and B chains can be combined by the connection peptide more much smaller than the C peptides of 31 amino acid.

In insulin molecule physics and chemical stability be diabetes insulinization prerequisite, be also the basis of insulin conformation, the pot-life of applicable insulin administration methods and pharmaceutical preparation and preservation condition.In insulin administration 1700 use solution to cause insulin molecule to be exposed to many factors, and such as elevated temperature, gas-liquid-solid alternate change and shearing force may cause the expendable conformation change of insulin molecule, and such as fibrillation is acted on.This is in close relations with insulin solutions in syringe pump, because either external application is still implanted into, all by insulin molecule exposed to these factors and in the generation shearing force in the long-term moving process of pump.Therefore, when using syringe pump as insulin delivery system, the problem of fibrillation effect is one very big.In addition, the solubility of insulin is affected by many factors, and substantially reduced in the range of pH4.2-6.6.Generally limitation is brought to formula in pH decanting zones.

Therefore, the stability of insulin and solubility are the key factors of current insulin therapy.This invention address that these problems, provide stable single chain compound, reduction molecular flexibility simultaneously reduces fibrillation tendency, limitation or modification pH decanting zones, so as to be that preparation and formula provide broader selection by introducing C peptides between B and A chains simultaneously.In addition, genetic engineering life at present and then the plain precursor of pancreas generate double-strand pancreas islet ^ after being digested：.If directly producing single-chain insulin analogues, production process is enormously simplify, cost is reduced.

As the member of insulin family, insulin-like growth factor-i (IGF-1) is the single-stranded peptide with 70 amino acid residues, includes A, B, C and D domain.IGF-1 A domains and the basic structure in B domains are highly similar to the A chains and B chains of insulin, there is 52% and 45% homology respectively.Their three-dimensional structure is also closely similar.

IGF-1 C domains act on very little in insulin receptor combination.Remove whole IGF-1 C domains, the bridge constituted with 4 glycine replaces, combination rate of insulin receptor is caused to increase by twice compared with wild type, and the C-terminal that IGF-1 C domains are added into insulin B chain causes insulin receptor affinity to reduce 3.5 times compared with wild type.Insulin adhesion and the natural human insulin for the single-chain insulin/IGF-1 mixtures being made up of insulin and IGF-1 C domains are not significantly different.Ironically, IGF-1 CII mixtures all have increased affinity to IR-A and IR-B, and IGF-2 CI have weaker affinity, and display C domains determine IR binding specificities.

Tyr31 is for keeping IGF-1's most important by high affinity in IGF-1, but it seems to hinder to be combined with insulin receptor, because when tyrosine is replaced by alanine, result in the double increase that very little but obvious Human plactnta insulin receptor are combined.

Junction fragment

Inventor is had found under study for action, and the double-strand of insulin molecule is connected into the single chain compound to be formed by junction fragment, equally with insulin active, and with advantages such as easily preparation, the more peptide modified sites of offer.

Junction fragment CL is the peptide sequence of 6-60 amino acid composition, and each of which amino acid is all independently selected from glycine, alanine, serine, threonine, proline.Applicable junction fragment C_LWith three point features：First, junction fragment needs appropriate length.When B chains are 30 amino acid total lengths, junction fragment length is preferably equivalent at least 6 amino acid；When B chains are 25 amino acid, junction fragment length is preferably equivalent at least 10 amino acid.Junction fragment is shorter in length than above-mentioned amino acid number, or when being longer than 60 amino acid, the insulin receptor binding ability of single chain analogs has reduction trend；Second, junction fragment is preferably no secondary structure, and space conformation can flexibly change；3rd, junction fragment is in itself without bioactivity, but peptide modified site can be provided, such as acylated, glycosylation.

The junction fragment C designed with above method_LIt can be replaced by amino acid residue and inserted comprising 1 or more than 1 aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine. C_LCan include 1,2,3,4 aspartic acid, glutamic acid, arginine or lysines to adjust the charge balance of peptide sequence, improve solubility.The sequence can include 1,2,3,4, the serine or threonine of 5 asparagines and identical quantity, so as to constitute the N-X-S/T consensus sequences constituted needed for N glycosylations（X is the natural amino acid of codified）.Further, the peptide can also comprising 1,2,3 or 4 lysines or cysteines, its side-chain amino group or sulfydryl can be connected by hydrolyzing key or non-hydrolytic key with the natural or synthetic modification group such as aliphatic acid, polyethylene glycol, albumin, so that the insulin molecule after modification has different physics, chemistry and biological nature. According to a kind of embodiment, C_LC-terminal amino acid can be selected from the group that is made up of glycine-lysine, glycine-arginine, Arg-Arg, lysine-lysine, arginine-lysine, Lys-Arg, proline-glutamine-threonine, proline-glutamine-lysine or proline-glutamine-arginine.According to a kind of embodiment, 0_'s.End amino acid is selected from lysine or arginine.

In a particular embodiment, C_LIt can be all or part of sequence of following polypeptide fragment, or have 1 with following polypeptide fragment, the difference of 2 or 3 amino acid residues, or have 70%, 80%, 90% similar with following polypeptide fragment, or following polypeptide fragment all or part of sequence 1,2,3,4 or 5 repetitive sequences：

(GASPGGSSGS) GR, wherein n are 1,2,3,4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT;

GSGGAPSRSGSSR; GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR; GSGSSSRAPPSAPSPQR； GSSSESPSGAPQT； GAGTPASGSAPGR； GSSPSGGSSAPQT；

GSTSSTARSPGR； GAGPSGTASPSR； GSSTPSGAPQT； SSSSAPPPSAPSPSRAPQR；

GSGSSSAAAPQT ; GSGSSSAAPQT ; GASPGTSSTSGR ; GSGSSSAPQT ; GSGSSSRRA ;

GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;

GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR; GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT;

GASTSSRGAPSR; GSGSSSAGR; GPSGTSTSAPGR; GAGSSSAPQT; SSSSAPSAPSPSRPQR;

GSGASSPTSPQR; GAGGSGSGR; GSSPATSATPQT; GAGSSSAPPPSAPSPSRAPGPSAPQR;

GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;

GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSG SSSPGR; GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR；

RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK;

SSSAPPPSAPSPSRAPGPSPQR； SAASSSASSSSASSASAGR； GAGGPSSGAPPPSPQT；

GSGSSGGR; GAGSPAAPASPAPAPS AGR； SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR;

GSGSSSRRAPQT； SSSSAASAASASSSASGR； SSSRAPPSAPSPQR； GGPSSGAPPPSR； SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT;

GAGPSSGAPPPSPQT; GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPSRPGPSPQR;

SSASSASSSSAGR; GAGSSR; SSASSSAASSSASSSASGR; SSSGAPPPSPSRAPGPSPQR;

GSGSASRGR ； SSSSAASSASGR ； SASASASASSASSGR ； SASSPSPSAPSSPSPAS ；

GPSSPSPSAPSSPSPASPSSGR ； SSSAPPPASPSPSRAPGPQR ； SASASASASASSAGR ； GSGASSRGR ; GSGAAPASPAAPAPS AGR； GGPSSGAPPPSGR ; SSPSASPSSPASPSSGR ;

GAPASPAPSAPAPAAPSGR； GPSSPSPSAPSSPSPASPSSAPQT； SSASSASSSSSASAGR；

SAPSSPSPSAPSSPSASPSGR ； SSSAPPPSAPSPSAPQR ； GASSPSPSAPSSPSPASGR ；

SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR;

GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT ； GGGGAPQR ； GSGSSSAAR ； GSGSSAAPQR ； SSSSRRAPQR ；

SSSGSGSSAPQR； SSGSGSSSAPQR； GSGSSSRS； SSSSRAPQR； GASPGGSSGSGR；

GSGSSSAAAPQR; GAGSSSAAAPQR; GAGSSSAAAPQT; GSSGGSGR; GAGGGSSGR;

GSGSSGSR ； GSGSSSSR ； GSGSGGGR ； GAGSSGR ； GSGSSGR ；

SSSSRAPPPSAPSPSRAPGPSAPQR； GGGSSR； GSGSSSAAPQR； GASPGGSSGSSR； GSGSSSRSGR ； GTGPSSATPASR ； GAGPSGTASPSS ； SSSSAPSAPSPSRAPQR ； GSPSSPTRGSAT ； GPETPSGPSSAT ； GSSPATSGTPQT ；

GSGSSSRAPPPSAPSPSRAPGPSPAPQR; GSSTPSGAGPQT; GSGSSSRAPPPSAPSPSRAPQR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPAR; GAGSSSRAPPPSAPSPSRAPGPSPQR; GSGSSSRAPPSAPSAPQR; GSTAGSRTSTAR; GSSPSGRSSSPAR; SSASSASSSSSAASAGR; GSSSGSSGSPSAR ； SSSAPSPSRAPGPSPQR ； GAGSSSRAPPPSAPSPSRAPQR ； GSPAAPAPASPAAPAPSAGR; SSSAPSAPSPSAPQR; GGPSSGAPPPSPSPSRPGPSDTPPQR; SASASASASASASSASSGR ； SASSPSPSAPSSPSPASGR ； SASASASASASASSAGR ； SSPSASPSSPASPSPSSGR; GAPASPAPAAPSAPAPAAPSGR; GAGSPAAPAPASPAPAPSAGR; SSSRAPPPSAPSPSAPQT； GASPAAPSAPPAASPAAPSAGR； SSSAPPPSPSRAPGPSPQR； SSPSAPSPSSPASPSPSSGR; SSSSGPSSGAPPPSGR; GSSSRSPSGSPR; GGGPGAGSSPQR.

Based on the above, the present invention provides a kind of single-stranded compound with blood sugar decreasing effect, and the compound is transformed according to the structure of insulin, and the structure of the compound is：

X₁₀₇HLC [！] GSXjQsLVEALYLVC^GEXiogGFXnQXniXinXinXiHXns-CL-GIVEQC jC^jXneS IC_[5]SLYQLENYC_[6]X₁₁₇X„₈, wherein,

X_1Q7It is phenylalanine-valine-asparagine-glutamin tetrapeptide, valine-asparagine-glutamin tripeptides, asparagine-glutamin dipeptides or glutamine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； X₁₍₎₈It is histidine, phenylalanine, arginine or glutamine； Χπ)₉It is arginine, alanine, glutamic acid or aspartic acid； X₁₁₀It is phenylalanine, tyrosine or histidine； X_mIt is tyrosine, phenylalanine or missing； X₁₁₂It is threonine, asparagine, glutamic acid, aspartic acid or missing； X₁₁₃It is proline, lysine, glutamic acid, aspartic acid or missing； X₁₁₄It is lysine, proline, arginine, glutamic acid, aspartic acid or missing； X₁₁₅It is threonine or missing； X₁₁₆It is threonine, histidine or arginine； X₁₁₇It is alanine, glycine or asparagine；X is lysine, arginine-lysine dipeptides or missing； C_LStructure is as defined herein.

Similarly, in the tertiary structure of the compound, key is dredged in the cysteine formation two during this is single-stranded, is specially： C_n]And C_[4] form two keys, C_mWith C [_6]Form disulfide bond, C_[3] and.[_5]Form disulfide bond.

It is important to note that X₁₀₈、 X₁₀9th, X, X, X amino acid residue are related to whether compound is produced from connection as actrapid monotard（ self association ).Actrapid monotard is general to be stored in beta Cell of islet by forming six aggressiveness from connection.Rh-insulin's molecule is gradually depolymerized to dimer after being subcutaneously injected by six aggressiveness, and circulation could be entered through capillary by being further dissociated into monomer, play blood sugar reducing function.Due to there is depolymerization, absorption process, rh-insulin onset time length (Brange etc. after hypodermic injection, " Monomelic insulins and their experimental and clinical implications " Diabetes Care, the No.9 of Vol 13,923-54,1990).If X₁₀₈It is histidine, then be conducive to compound to form six dimeric structures under the assistance of zinc ion.If X₁₍₎₈It is the amino acid residues such as aspartic acid, glutamic acid, phenylalanine, glutamine, arginine, then can not forms six stable dimeric structures.If Χ κ)₉、 X₁₁₂、 X₁₁₃、 X_U4Amino acid residue Deng site is aspartic acid or glutamic acid, is not easy to form stable connection certainly.Therefore, if X₁₀₈It is non-histidine amino acid residue, or Xl09,

One or more sites of the amino acid residue in the sites such as X, Xii3, X are aspartic acid or glutamic acid, then respective compound is easier to dimer or monomeric form presence, blood is rapidly entered after hypodermic injection, can reach reduces the effect of blood glucose in a short time.

In a kind of specific embodiment, the structure of the single chain compound is：

FVNQHLCGSHLVEALYLVCGERGFFXi！ d₁₂Χ! ₁₃Xi "Xi i₅-C_L- GIVEQCCTSICSLYQLEN YCN, wherein,

X is tyrosine or missing； x₁₁₂It is threonine or missing； x₁₁₃It is proline or missing； x₁₁₄It is lysine or missing； X_U5It is threonine or missing； C_LIt is junction fragment as defined herein.

In further embodiment, the structure of the single chain compound is：

FVNQHLCGSHLVEALYLVCGERGFFYTPKT-C_L-GIVE(5CCTSICSLYQLENYCN.According to the embodiment of this aspect, the single chain compound with hypoglycemic effect of the invention is selected from following compound: I -1:

FVN

YCN (SEQ ID NO:l );

I -2:

YCN ( SEQ ID NO:2 )；

I -3:

FVN'

CN ( SEQ ID NO:3 );

1-4:

YCN ( SEQ ID NO:4 );

1-5:

F\

YCN ( SEQ ID NO:5 );

I -6:

FVNC YCN ( SEQ ID NO:6 );

1-7:

FVNi

( SEQ ID NO:7 )；

1-8:

( SEQ ID NO:8 );

1-9:

( SEQ ID NO:9 );

I -10:

FVNQ

(SEQ ID NO: 10);

I -11:

FVNC

N (SEQ ID NO: 11 );

1-12:

FVNQHLCGSHLVEALYLVCGERGFFYTPKTGAGGGSSGRGIVEQCCTSICSLYQ LENYCN ( SEQ ID NO: 12)； ■91- 1

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:8ε- 1

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■LZ- 1

；( 9ϋΌΝ ai 0HS ) a00/Zl0Z 3/X3d 98,980/εΐΟΖ OAV I -40:

CN ( SEQ ID NO:40 );

1-41:

FVNQ

YCN ( SEQ ID NO:41 );

1-42:

FVNQ YCN ( SEQ ID NO:42 );

I -43:

FVNQ YCN ( SEQ ID NO:43 );

I -44:

FVNQ]

NYCN ( SEQ ID NO:44 );

1-45:

FVNQ

NYCN ( SEQ ID NO:45 );

I -46:

YCN ( SEQ ID NO:46 );

1-47:

FVNQ YCN ( SEQ ID NO:47 );

I -48:

FVNQ]

SLYQLENYCN ( SEQ ID NO:48 )； I -49:

FVNQ YCN ( SEQ ID NO:49 );

I -50:

YCN ( SEQ ID NO: 50 );

I -51:

FVNC CN (SEQIDNO:51 );

1-52:

CN ( SEQ ID NO:52 );

I -53:

Q； ( ) ID N53CN SEO: Y

I -80:

( SEQ ID NO:80 );

1 -81 :

FVNQ

YCN ( SEQ ID NO:81 );

I -82:

FVNQ

QLENYCN ( SEQ ID NO: 82 );

I -83:

FVNQ

YQLENYCN ( SEQ ID NO:83 );

I -84:

FVNQ]

NYCN ( SEQ ID NO:84 );

I -85:

FVNQ CN ( SEQ ID NO:85 );

I -86:

CN ( SEQ ID NO: 86 );

I -87:

FVNQ]

QLENYCN ( SEQ ID NO: 87 );

I -88:

FVNQ]

SLYQLENYCN ( SEQ ID NO:88 );

I -89:

FVNQ

( SEQ ID NO:89 );

1 -90:

YCN ( SEQ ID NO:90 );

1 -91 :

FVNQ ID NO:91 );

I -92:

I -93: o

Q (O3 SE ID N:9 LYQLENYCN ( SEQ ID NO: 106 );

I -107:

FV Ql-

YQLENYCN ( SEQ ID NO: 107 );

I -108:

FVNQf

ENYCN (SEQ ID NO: 108 );

I -109:

FV Qi

YCN (SEQ ID NO: 109);

I -110:

SICSLYQLENYCN ( SEQ ID NO: 110 )；

I -in:

TSICSLYQLENYCN ( SEQ ID NO:lll );

I -112:

FVNQH YCN (SEQ ID NO: 112);

I -113：

FV QI

SLYQLENYCN ( SEQ ID NO: 113 );

I -114:

FVNQI CSLYQLENYCN ( SEQ ID NO: 114 )； I -115:

FVNQP NYCN (SEQ ID NO:115);

I -116:

NYCN (SEQIDNO:116);

I -117:

FVNQI

SICSLYQLENYCN ( SEQ ID NO: 117 );

I -118：

YQLENYCN ( SEQ ID NO:118 );

I -119: ENYCN ( SEQ ID NO: 119 )； I -120:

( SEQ ID NO: 120 );

I -121 :

FVNQH

( SEQ ID NO: 121 );

I -122:

FVNQP

N ( SEQ ID NO: 122 )；

I -123 :

FVNQH CN ( SEQ ID NO: 123 ).The compound of modification（The modification of compound based on insulin）

Chemiluminescent polypeptide man is used for several method to solve the problem that drug molecule of the blood plasma middle-molecular-weihydroxyethyl less than 67kDa is quickly removed by kidney.1st, injection site is built " storehouse,（ depot );2nd, combined with the carrier protein in blood plasma with non-covalent bond with Zu Zhi Kidney glomerular filtrations；3rd, it is connected with carrier protein with covalent bond；4th, combined with macromolecule modification group, for example macromolecule PEG, polysaccharide etc.（As disclosed in the chapters and sections before present specification）." hydrophobic to store sth. in a cellar " (hydrophobic depoting) increases considerably the hydrophobicity of peptide to reduce solubility, and makes it in injection portion formation " storehouse ".Peptide storehouse Slow is slowly after dissociation, and polypeptide is bound to the carrier protein of cell membrane and/or whole body（Such as albumin）.Carrying agent molecular weight of albumen is more than glomerular filtration maximum molecular weight, therefore is difficult to be removed by kidney, can be circulated in blood plasma many days.Therefore, the polypeptide Bu Yi Bei Kidney glomerular filtrations that are combined with carrier protein or by the proteasome degradation on inner membrance.

Aliphatic acid is general to extend polypeptide action time in vivo by three kinds of modes.First, aliphatic acid can be combined in drug injection site with albumin with non-covalent bond, and polypeptide-aliphatic acid of formation-albumin macromolecule conjugate Dry puts Slow is slow；Second, the macromolecule Shi Kidney clearance rates reduction of polypeptide-aliphatic acid-albumin conjugates；3rd, albumin provides protection for polypeptide, is difficult to be easily degraded by proteases.4th, aliphatic acid reduces the immunogenicity of polypeptide.Three features are similar with the effect that long-chain PEG is modified afterwards.More mechanism and experiment support are referred to Biochem. J. (1995) 312,725-731；Pharmaceutical Research, (2004), 21,8,1498-1504; Current Medicinal Chemistry ( 2009 ), 16 , 4399-4418; WO95/07931 ; Diabetes, Obesity and Metabolism, 2007, 9 , 290-299; Diabetes, 1997, 46, 637-642.

Exemplary is treating diabetes polypeptide drugs insulin detemir (detemir) and Liraglutide（ liraglutide ).They make use of based on fatty acid modifying it is hydrophobic store sth. in a cellar, make internal extended durations of action（Insulin detemir t_1/2=14 hours）.And the action time of unmodified insulin only has several hours.

In addition, using polyethylene glycol（PEG, molecular weight is not less than 20K) and the macromolecular such as human albumin modify insulin, the effect of action time in the extension body similar with above-mentioned fatty acid modifying can also be reached.Therefore, the site of fatty-acylation can be used all, can be modified with macromoleculars such as polyethylene glycol or human albumins.

The present invention is based on such a understanding：The bulk hydrophobicity of the compound with hypoglycemic effect of the present invention plays an important role in terms of the in vivo efficacy of the compound.The present invention further provides a kind of compound with hypoglycemic effect, being modified on the basis of polypeptide, further to improve the compound body-internal-circulation action time.The modification is will to modify alpha-amido or the epsilon-amino for the lysine being connected in single chain compound that side chain is connected to the -terminal amino acid residue of single chain compound of the invention.

The compound of the modification of the present invention can also be modified and formed based on insulin or its analog.In this regard, originally Invention provides the single chain compound of the modification based on insulin, and the structure of the compound is：

X₃₀₀VNQHLC[i]GSHLVEALYLVC[₂]GERGFX3₀₁X302 3₀3X3₀4 3₀5X3₀6GX307X3₀8X3₀9 31₀X311

X312X313X314X315X310 31₇GIVEQC[₃]C[4] X₃1 gX₃₁9X₃₂₀C[₅]X₃₂iLX₃₂₂X₃₂3LX3₂4X32₅Y [6]X₃₂₆X₃₂7, wherein,

Χ₃₀ο is phenylalanine or phenylalanine； X₃Oi is phenylalanine, histidine or tyrosine； X₃₀₂It is tyrosine, phenylalanine or missing； X₃03 is threonine, asparagine, glutamic acid, aspartic acid or missing； X30₄It is proline, lysine, paddy atmosphere acid, aspartic acid or missing； X_3Q5It is aspartic acid, glutamic acid, proline, arginine, lysine or missing, or formula（I) structure； X₃o₆It is threonine, formula（I) structure or missing； X₃₍₎₇It is lysine, serine, alanine, glycine, formula（I) structure or missing； X₃。_sIt is glycine, formula（I) structure or missing； X₃o₉It is lysine, glycine, serine, formula（I) structure or missing； X_31QIt is lysine, glycine, serine, formula（I) structure or missing； X₃₁₁It is lysine, glycine, serine, alanine, formula（I) structure or missing； X₃₁₂It is lysine, arginine, alanine, proline, glycine, formula（I) structure or missing； X₃13 be glycine, alanine, arginine, lysine, glutamine, proline, formula（I) structure or missing； X₃₁₄It is arginine, alanine, proline, threonine, glutamine, glycine, formula（I) structure or missing； X₃₁₅It is proline, glutamine, arginine, glycine or missing or formula（I) structure； X₃₁₆It is glutamine, threonine, arginine, glycine or missing or formula（I) structure； x₃₁₇It is threonine, arginine, lysine or missing； X₃₁₈It is threonine, histidine, arginine or formula（I) structure； x₃₁₉It is serine or formula（I) structure； X_32QIt is isoleucine or formula（I) structure； X₃₂₁It is serine or formula（I) structure；X322 is tyrosine or formula（I) structure； X₃₂₃It is glutamine or formula（I) structure； X₃₂₄It is glutamic acid or formula

(I) structure； X₃₂₅It is asparagine or formula（I) structure； x₃₂₆It is alanine, glycine or asparagine； x₃₂₇It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；Work as x₃₂₇During for dipeptides, one of amino acid is formula（I) structure；

Wherein, the logical structure is：

U_LIt is-W-X-Y-Z structures, aliphatic acid, polyethylene glycol, albumin, L-M_LStructure, hydrogen atom or N^a-(N^a-(HOOC(CH₂)_nCO)-Y-Glu)- , N^a-(N^a-(CH₃(CH₂)_nCO)-Y-Glu)-, wherein η is 8-20 integer, such as 8,10,12,14,16,18 or 20, Ν^αThe a- amino of amino acid or amino acid residue is represented, or is formula（II) structure.The formula（Π) structure is：

J is-W-X-Y-Z structures, L_n-M_tStructure or hydrogen atom.

M_LIt is modification group, including but not limited to-W-X-Y-Z, aliphatic acid, polyethylene glycol, albumin, IgGFc, glycosyl group etc..In the present invention, it is optional linker, covalent bond or is not present.Optional linker includes but is not limited to： Polyethylene glycol, long chain fatty acids or one or more peg molecules and long-chain fat acid molecule connect the long-chain to be formed by covalent bond.L can be-NH- (CH₂)_n-£0-、 -NH-(CH₂CH₂0)_n-CH₂-CO- , -NH-(CH₂CH₂0)„-(CH₂)_r- CO-, n are 1-20 integers, and r is 1-10 integer；In one embodiment, it is-NH- (C CH₂0)₂-CH₂-CONH -(CH₂CH₂0)₂-CH₂-CO-;In one embodiment, be-NH-iCHz^-C CHzCHzO CH nj- O-, nl, n2, η 3 be respectively 1-16 integer；In one embodiment, L_nIt is-NH- (CH₂)_nl-(OCH₂CH₂) 2- ￡ 0-, nl, n2 be respectively 1-16 integer.In embodiment of above, L forms amido link, the other end and M by the amino of the key from the carbonyl carbon underlined and polypeptide compound_LForm covalent bond.In one embodiment, L with the amino of polypeptide compound by the key from the carbonyl carbon underlined with forming amido link, and the other end forms amido link with-W-X-Y-Z.

Herein ,-W-X-Y-Z structures are：

W is the a-amino acid residue that side chain has carboxyl, and the residue forms amide groups with a carboxyl with the alpha-amido of Ν-terminal amino acid residue of polypeptide compound in the present invention or together with the epsilon-amino of the lysine residue of polypeptide compound；

Or W be by 2, the chain that 3 or 4 a-amino acid residues are connected by amido link, the chain is connected to the epsilon-amino of the alpha-amido of Ν-terminal amino acid residue of polypeptide compound or the lysine residue of polypeptide compound by amido link, W amino acid residue is selected from the group that amino acid residue and side chain with neutral side chain have the amino acid residue composition of carboxyl so that W contains the amino acid residue that at least one has carboxyl in side chain；

Or W be Ν-terminal amino acid residue from X to polypeptide compound alpha-amido or to polypeptide compound lysine residue epsilon-amino covalent bond；

X is-￡ 0- ,-CH (COOH) CO- ,-N (CH₂COOH)CH₂CO- 、 -N(CH₂COOH)CH₂CON (CH₂COOH)CH₂CO- 、 -N(CH₂CH₂COOH)CH₂CH₂CO- 、 -N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂CO-、 -NHCH(COOH)(CH₂)₄NHCO-、 -N(CH₂CH₂COOH)CH₂￡ 0- or-N (CH₂COOH)CH₂CH₂CO-, wherein

A) when W is amino acid residue or amino acid residue chain, above-mentioned X by the amino in the key and W of the carbonyl carbon underlined by forming amido link；Or

B) when W is covalent bond, the epsilon-amino formation amido link of the lysine residue of alpha-amido or polypeptide compound that above-mentioned X passes through the key from the carbonyl carbon underlined and the -terminal amino acid residue of polypeptide compound；

Υ is-(CH₂)_m, wherein m is 6-32 integer；

Or comprising 1,2 or 3-CH=CH- groups and multiple-CH₂The bivalent hydrocarbon chain of-group, the multiple-CH₂The total number of carbon atoms scope that the number of-group is met in hydrocarbon chain is 10-32;

Or formula-(CH₂)_VC₆H₄ (CH₂) _W- bivalent hydrocarbon chain, wherein V and w are integers, or they one of be zero so that the scope of V and w summations is 6-30;And

Z is-COOH ,-CO- Asp ,-CO-Glu ,-CO-Gly ,-CO-Sar ,-CH (COOH)₂、 -N(CH₂COOH)₂、 -S0₃H、 -P0₃H is not present；Condition is that, when W is covalent bond and X is-CO-, Z is not -COOH.

Side chain-W-X-Y-Z middle W can be covalent bond.On the other hand, W can be that side chain has the a-amino acid residue of carboxyl, including have 4-10 carbon atom altogether.W can be the a-amino acid residue encoded by genetic codon.For example, the group that W can be constituted selected from a-Asp, β-Asp, α-Glu and γ-Glu.W other selections are, for example, α-hGlu or 5-hGlu.

In another embodiment, the chain that W is made up of two a-amino acid residues, one of a-amino acid residue has 4-10 carbon atom and side chain has carboxyl, and another has 2-11 carbon atom but no free carboxyl group.The described a-amino acid residue without free carboxyl group can be the a-amino acid residue of neutral codified.It is according to the W of this embodiment example： a-Asp-Gly、 Gly-a-Asp, β-Asp-Gly, Gly-P.Asp、 a-Glu-Gly, Gly-a-Glu、 y-Glu-Gly Gly-y-Glu>A-hGlu-Gly. Gly-a-hGlu δ-hGlu-Gly and Gly-S-hGlu. In another embodiment, the chain that W is made up of two a-amino acid residues, two a-amino acid residues have on 4-10 carbon atom, side chain respectively is respectively provided with carboxyl.One or two of these a-amino acid residues can be the a-amino acid residue of codified.It is according to the W of this embodiment example： a-Asp-a-Asp、 a-Asp-a-Glu , a-Asp-a-hGlu、 a-Asp-P-Asp、 a-Asp-y-Glu、 a-Asp-6-hGlu、 β-Asp-a-Asp、 β-Asp-a-Glu、 β-Asp-a-hGlu、 β-Αβρ-β-Αβρ、 β-Asp-y-Glu、 -Asp-6-hGlu、 a-Glu-a-Asp、 a-Glu-a-Glu、 a-Glu-a-hGlu、 a-Glu-P-Asp a-Glu-y-Glu, a-Glu-5-hGlu, γ-Glu-a-Asp, γ-Glu-a-Glu, γ-Glu-a-hGlu, y-Glu-P_Asp、 γ-Glu-y-Glu ^ γ-Glu-S-hGlu、 a-hGlu-a- Asp、 a-hGlu-a-Glu、 a-hGlu-a-hGlu> a- hGlu-P-Asp、 a-hGlu-y-Glu, a-hGlu-S-hGlu、 δ-hGlu-a-Asp, 6-hGlu-a-Glu>5-hGlu-a-hGlu, 6- hGlu-P-Asp, δ-hGlu-Y-Glu and S-hGlu-5-hGlu.

In another embodiment, W is by three chains that the a- amino acid residues with 4-10 carbon atom are constituted respectively, the amino acid residue of the chain, which is selected from residue and side chain with neutral side chain, has the residue of carboxyl so that the chain contains the residue that at least one side chain has carboxyl.In one embodiment, the amino acid residue is the residue of codified.

In another embodiment, W is that have 4-10 carbon atom respectively by four, the chain of a- amino acid residues composition, the amino acid residue of the chain, which is selected from residue and side chain with neutral side chain, has the residue of carboxyl so that the chain contains the residue that at least one side chain has carboxyl.In one embodiment, the amino acid residue is the residue of codified.

In one embodiment, the W in-W-X-Y-Z can be connected to the epsilon-amino of lysine residue by urea derivative.X in side chain-W-X-Y-Z can be formula-￡ 0- group, pass through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and Ν-end of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in the side chain-W-X-Y-Z can be formula-CH (COOH) ￡ 0- group, pass through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in side chain-W-X-Y-Z can be formula-N (CH₂COOH)CH₂￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in side chain-W-X-Y-Z can be formula-N (CH₂CH₂COOH)CH₂￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in-W-X-Y-Z can be formula-N (CH₂COOH) CH₂CH₂￡ 0- group, passes through the formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in-W-X-Y-Z can be formula-N (CH₂COOH) CH₂CON(CH₂COOH)CH₂￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the a- amino of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in-W-X-Y-Z can be formula-N (CH₂C COOH) C CH 0- Group, passes through the amino formation amido link in the key and W for underlining carbonyl carbon；Or when W is covalent bond, X passes through the alpha-amido of the key from the carbonyl carbon underlined and the N- ends of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

In further embodiment, the X in-W-X-Y-Z can be formula-N (CH₂CH₂COOH) CH₂CH₂CON(CH₂CH₂COOH) CH₂CH₂￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through the alpha-amido of the key from the carbonyl carbon underlined and Ν-end of polypeptide compound or the epsilon-amino formation amido link with the lysine residue in polypeptide compound.

Υ in side chain-W-X-Y-Z can be formula-(CH₂)_mGroup, wherein m are 6-32,8-20,12-20 or 12-16 integer.

In another embodiment, the Y in-W-X-Y-Z be comprising 1,2 or 3-CH=CH- groups and multiple-CH₂The bivalent hydrocarbon chain of-group, the multiple-CH₂The total number of carbon atoms scope that the number of-group is met in hydrocarbon chain is 6-32,10-32,12-20 or 12-16.

In another embodiment, the Y in-W-X-Y-Z is formula-(CH₂)_VC₆H₄ (CH₂) _w- bivalent hydrocarbon chain, wherein v and w are integers, or one of them is zero so that the scope of V and w summations is 6-30,10-20 or 12-16.

In one embodiment, the Z in side chain-W-X-Y-Z is-COOH, and condition is that Z is not -COOH when W is covalent bond and X is-CO-.

In another embodiment, the Z in-W-X-Y-Z is-CO-Asp ,-CO-Glu ,-CO-Gly ,-CO-Sar ,-CH (COOH) 2 ,-N (CH₂COOH)₂、 -S0₃H or-P0₃H。

In further embodiment, the W in-W-X-Y-Z is a-Asp, p-Asp>A-Glu or γ-Glu;X is-CO- or-CH (COOH) CO-;Y is-(CH₂)_m, wherein m is 12-18 integer；Z be-COOH-,-CH (COOH)₂Or be not present.

In another embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, a-Glu or γ-Glu;- X-Y-Z is-CO (CH₂), by the amino formation amido link in key and W from the carbonyl carbon underlined, wherein n is the integer in 10-20.

In more specifically embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, a-Glu or γ-Glu;- X-Y-Z is

-CO(CH₂)₁₄。

In more specifically embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, a-Glu or γ-Glu;- X-Y-Z is-CO (CH₂)₁₆。

In more specifically embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, a-Glu or γ-Glu;- X-Y-Z is-CO (CH₂)₁₈。

In more specifically embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, a-Glu or γ-Glu;- X-Y-Z is cholesterol, bile acid（Such as cholic acid, chenodesoxycholic acid, liver and gall acid, taurocholate, deoxycholic acid, lithocholic acid）.

In above-claimed cpd structure, [1]-[6] represent the numbering of cysteine.The single chain compound of the present invention is in tertiary structure, and the disulfide bond formed with the frame mode of insulin in chain is specially：.And C_[4] form disulfide bond, C_[2] and C_[6]Formation Er Liu Key, 〇 [₃₁With_[5]Form two Kip keys.

In a kind of specific embodiment, the structure of the compound is：

X₃ooVNQHLC_[1]GSHLVEALYLVC_[2]GERGFFX₃₀₂X₃₀₃X₃o₄X30₅X306GX₃₀₇X3₀8X₃₀9X3i₀X3iiX3 ₁₂X₃₁₃X₃₁₄X₃i₅X₃i₆X₃₁₇GIVEQC_[3]C[₄〗TSIC[₅〗SLYQLENYC[₆₁NX₃₂₇, wherein,

X300 is phenylalanine or UL_ phenylalanines； X₃02 is tyrosine or missing； X_3Q3It is threonine or missing； X₃。₄It is proline or missing； X_3G5It is aspartic acid, proline, arginine, lysine or missing, or formula（I) structure； X₃₍₎₆It is Threonine, formula（I) structure or missing；X307 is lysine, serine, alanine or formula（I) structure； X₃o₈It is glycine or formula（I) structure； X_3Q9It is lysine, serine or formula（I) structure； X_31QIt is lysine, serine or formula（I) structure； X₃₁₁It is lysine, serine, alanine or formula（I) structure； x₃₁₂It is lysine, arginine, alanine, proline or formula（I) structure； x₃₁₃It is glycine, alanine, arginine, lysine, glutamine, proline or formula（I) structure； X₃₁₄It is arginine, alanine, proline, threonine or glutamine or formula（I) structure； x₃₁₅It is proline, glutamine, arginine or missing or formula（I) structure； x₃₁₆It is glutamine, threonine, arginine or missing or formula（I) structure； X₃₁₇It is threonine, arginine, lysine or missing； X₃₂₇It is formula（I) structure or missing； U_LAnd formula（I) structure is as defined herein.

In terms of this, the single chain compound of modification of the invention is selected from：

II -1 :

FV QHLCGSHLVEALYLVCGERGFFYTPTGK[N^£-( ^a-(HOOC(CH₂)₁₄CO)-Y-Glu)] GSSSRGRGIVEQCCTSICSLYQLENYCN;

II -2:

FVNQHLCGSHLVEALYLVCGERGFFYTPTGK[N^e-( ^a-(HOOC(CH₂)_I4CO)-Y-Glu)] GSSSAAAPQTGIVEQCCTSICSLYQLENYCN;

II -3:

FV QHLCGSHLVEALYLVCGERGFFYTPTGSGK[N^E-( ^a-(HOOC(CH₂),₄CO)-Y- Glu)]SSAAAPQTGIVEQCCTSICSLYQLENYCN;

II -4:

F(N^a-dPEG₁₂- maleimide-albumin）V QHLCGSHLVEALYLVCGERGFFYTPDTGSGSSS

AAAPQTGIVEQCCTSICSLYQLENYCN;

II -5:

FVNQHLCGSHLVEALYLVCGERGFFYTPPTGSGSSK[N^e-(N^a-(HOOC(CH₂)₁₄CO)-Y- Glu)] AAAPQTGIVEQCCTSICSLYQLENYCN;

II -6:

FVNQHLCGSHLVEALYLVCGERGFFYTPTGSGSSSAK[N^e-(N^a-(HOOC(CH₂)₁₄CO) -y-Glu)]APQTGIVEQCCTSICSLYQLENYCN;

II -7:

FV QHLCGSHLVEALYLVCGERGFFYTPRTGSGSSSK[N^E-(N^a-(HOOC(CH₂)i₄CO) -y-Glu)]AAPQTGIVEQCCTSICSLYQLENYCN;

II -8:

FV QHLCGSHLVEALYLVCGERGFFGSGSSSK[N^e-(N^a-(HOOC(CH₂)₁₄CO)-Y-Glu)] AAPQTGIVEQCCTSICSLYQLENYCN;

II -9:

FVNQHLCGSHLVEALYLVCGERGFFGSGSSSAK[N^£-(N^a-(HOOC(CH₂)_I4CO)-Y-Glu)] APQTGIVEQCCTSICSLYQLENYCN;

11 -10:

FVNQHLCGSHLVEALYLVCGERGFFGSGK[N^E-(N^a-(HOOC(CH₂)₁₄CO)-Y-Glu)]

SSAAAPQTGIVEQCCTSICSLYQLENYCN;

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■ Ζ -11 Ν Ren Ren Shang COi) 3AID Shang 0dWVSSSD [((niO- people)-N-niO- people-(OC^ H OOH NhN O Shang d Shang people;People lV3AlHSOCnH0NAd

： ε^-ιι

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HI

[(ni£>- people-(O3 " () OOH)-_DN)-₃N] ￡) Shang LL people iHEmaO Al enter lV3AlHS ￡) CnH0NAiI

HI

'•NDANH10AlSDIS13303AIO"a0dVSSSO [(nio- -(o3 (¾3)300H)-„N)-3N]¾OIdIAJJO¾303AlAlV3AlHSODlH0NAJ

■ z-n people N310A S:) IS Shang:The Shang dVVVSSSOSO of 3 Λ Ι of δ 0 [(η ￡)-- people-(03 " (:)) )OOH)-_DN)-₃N]:The ￡ of 5W Shang people Μ Ο 3) Λ enters lV3AlHSDCnH0NAd

:6ΐ-Π

A S IS Shang:) 03AIE)li)dVVVSSS£)S£MiI£)¾3£)3AlA V3AlHSD nH0NA(¾(^£)3I-„N)d

:81-11

：The AID10dWV i0 O3d- of N people N310A S IS Shang 03₃N)SSS£>SDl<I Shang people: IdCraSE AlA VSAlHSOCHH NAil ： L\- II

W people N3lt) AlS0IS Shang Shang 0d WVSSS0S0JJ0 H03AlAlV3AlHS031H NA [(ni0-l-^L-(03 (¾D)300H)-_I)N)-_I)N]j

:91-11

： [(ηιο-λ-(θ3^Η(¾3)300Η)-„Ν)-₃Μ]-

: -π

:Ν Α Ν 3) people IS IS Shang) 3 Λ Ι ￡) Shang 0d [(η Ο-^- (θ 3^Η(¾3)300Η)-„Ν)-₃Ν]¾88080λϋΟΉΗ03ΛΊΑΊν3ΛΊΗ803ΊΗ ΝΛί

W- ι ι people N3] i) A S:) ISl 03AIO Shang 0dVS [(ni ￡)-people-(0)^w(¾ ):)OOH)-_nN)-₃N] ￡) SD Shang people ^ ￡) 3 Ε Λ people lV3AlHS ￡ HHi) NAd

^:ei-n

' N enter the Λ Ι Ε) Shang 0dV of NT10A1S IS Shang 03 [(nio-X- (o3^ (D) 300H)-_DN)-₃N]¾SSOSOiIJO¾HODAlAlVHAlHS031H MAiI

[(niO-^-(03^,"(¾D)300H)-_I)N)-₃N]¾SOSOdJO¾30DAlAlV3AlHSODlH0NAd

Ll

00Z.100/Z10ZN3/X3d 98Z.980/CT0Z: OAV Wherein Ν^αRepresent the alpha-amido of amino acid or amino acid residue； Ν^εRepresent the epsilon-amino of amino acid or amino acid residue, the epsilon-amino of such as lysine side-chain.

The compound with hypoglycemic effect of the present invention can be provided with the composite form for the compound form or diction for being substantially free of zinc.When providing the zinc complexes of compound of the present invention, wherein the compound of the present invention can form six aggressiveness, each six aggressiveness can combine 2 Ζ η²⁺, 3 Ζ η²⁺Or 4 Ζ η²⁺.Pharmaceutical composition and purposes

In another aspect of the present invention, there is provided a kind of pharmaceutical composition, described pharmaceutical composition includes acceptable carrying agent in the compound and pharmaceutics according to the present invention of therapeutically effective amount, for treating 1 patients with type Ⅰ DM, diabetes B and causing other situations of hyperglycemia.It can be used for the preparation of the pharmaceutical composition for the other situations treated type 1 diabetes, diabetes B and cause hyperglycemia according to the insulin receptor combination analog of the present invention.

In another aspect of the present invention, there is provided a kind of pharmaceutical composition for the other situations treated type 1 diabetes, diabetes B and cause hyperglycemia, described pharmaceutical composition includes the compound according to the present invention of therapeutically effective amount, it is mixed with acceptable carrier and additive on the insulin with snap action effect or insulin analog, and pharmaceutics.

The routine techniques of pharmaceuticals industry can be used to prepare the Injectable composition of insulin analog of the present invention, including dissolved and mixed appropriate component and obtain required finished product.Therefore, according to a set of operating procedure, the insulin analog of the present invention is dissolved in a certain amount of water, its volume is slightly less than the final volume of composition to be prepared.If desired, adding preservative, isotonic agent and Slow electuaries.If it is necessary, adjusting the ρ Η of solution using sour (such as hydrochloric acid) or alkali (such as sodium hydroxide).It is final that required concentration is arrived into the volume regulation of solution with water.

In another embodiment of the present invention, Slow electuaries are selected from sodium acetate, sodium carbonate, twist lemon hydrochlorate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and three（Hydroxyl Yue bases）- amino Yue alkane, Ν-two（Ethoxy）Glycine, Ν-(hydroxyl Yue bases）Yue bases glycine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or its mixture.Each in these specific Slow electuaries constitutes alternative embodiment of the invention.

In another embodiment of the present invention, the preparation includes pharmaceutically acceptable preservative, and it is selected from phenol, o- Yue phenol, m- Yue phenol, p- Yue phenol, para hydroxybenzene Yue acid Yue esters, para hydroxybenzene Yue acetoacetic esters, para hydroxybenzene Yue propyl propionates, para hydroxybenzene Yue acid butyl esters, 2- phenoxetols, benzylalcohol, methaform, thimerosal, bronopol, benzoic acid, miaow urea, chlorhexidine, sodium dehydroacetate, chlorine Yue phenol, benzyl rope chloramines, Chlorphenesin or its mixture.In another embodiment of the present invention, the concentration of preservative is 0.1mg/mL-20mg/mL.In another embodiment of the present invention, the concentration of preservative is 0.1 mg/mL-5 mg/mL.In another embodiment of the present invention, the concentration of preservative is the alternative embodiment that each in 5mg/mL-10mg/mL. these specific preservatives constitutes the present invention.Preservative is applied to be well known to the skilled person in drug regimen.With reference to Remington:The Science and Practice of Pharmacy, the 19th edition, 1995.

In another embodiment of the present invention, the preparation further comprises isotonic agent, selected from salt（Such as sodium chloride）, sugar or sugar alcohol, amino acid, take sugar alcohol（Such as glycerine, propane diols, 1,3-PD, 1,3- butanediols）, polyethylene glycol（Such as PEG400) or its mixture.Any sugar, such as monose, disaccharides, polysaccharide or water-soluble dextran, including such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, general Shandong indigo plant, dextrin, cyclodextrin, soluble starch, HES and carboxylic Yue base celluloses-Na.In one embodiment, sugar additives are sucrose.Sugar alcohol is defined as the C4-C8 hydrocarbon with least one-OH group, including such as mannitol, sorbierite, inositol, galactitol, dulcitol, xylitol and arabite.In one embodiment, the sugar alcohol additive is mannitol.It is above-mentioned Carbohydrate or glycitols can be used alone or be applied in combination.To the unfixed limitation of consumption, as long as the sugar or sugar alcohol are dissolved in liquid preparation and will not produce harmful effect to the static stabilization obtained using the inventive method.In one embodiment, the concentration of sugar or sugar alcohol is 1 mg/mL-150 mg/mL.In another embodiment, the concentration of isotonic agent is lmg/mL-50mg/mL.In another embodiment, the concentration of isotonic agent is 1 mg/mL-7 mg/mL.In another embodiment, the concentration of isotonic agent is 8 mg/mL-24 mg/mL.In another embodiment, the concentration of isotonic agent is 25 mg/mL-50 mg/mL.Each in these specific isotonic agents constitutes alternative embodiment of the invention.Isotonic agent is applied to be that those skilled in the art are well-known in pharmaceutical composition.With reference to Remington:The Science and Practice of Pharmacy, the 19th edition, 1995.

Typical isotonic agent is sodium chloride, mannitol, two Yue sulfoxides and glycerine, and typical preservative is phenol, m- Yue phenol, para hydroxybenzene Yue acid Yue esters and benzylalcohol.

The example of surfactant includes sodium acetate, glycylglycine, ethoxy piperazine.Qin's ethyl sulfonic acid（) and sodium phosphate HEPES.Embodiment

Protection group：

Acm acetamidomethyl:Yi Ugly amine methyl；Alloc or AOC allyloxycarbonyl:Roach propylene carbonyl oxygen； Bom, benzyloxymethyl:Thousand oxygen Yue bases； 2-Br-Z, 2-bromobenzyloxycarbonyl:2- small stream section oxygen carbonyls； tBu, t-butyl:The tert-butyl group；Bz, benzoyl:Benzene Yue acyl groups； Bzl, benzyl:Benzyl； Boc:Tertbutyloxycarbonyl； CHO formyl:Yue acyl groups； cHx, cyclohexyl:Cyclohexyl；Cbz or Z benzyloxycarbonyl:Thousand oxygen are betrayed base； Cl-Z, 2-chlorobenzyloxycarbonyl :2- benzyloxycarbonylchloride bases； Fm, 9-fluorenylmethyl:9- fluorenyl Yue bases； Fmoc, 9-fluorenylmethoxycarbonyl:9- fluorenes Yue oxygen carbonyls；Mtt, 4-methyltrityl:4- Yue base triphen Yue bases； Npys, 3-nitro-2-pyridinesulfenyl:3- nitro -2- pyridine sulfenyls；Pmc, (2,2,5,7,8-pentametylchroman-6-sulphonyl:Yue base -6- the hydroxychromans of 2,2,5,7,8- five； Tos,4-toluenesulphonyl:To Yue benzene sulfonyls；Trt, tripheylmethyl:Triphen Yue bases；Xan, xanthyl:Ton base, oxygen is (miscellaneous）Anthryl.

Reagent and solvent：

ACN, acetonitrile：Acetonitrile； BOP, benzotriazol- 1 -yloxytris(dimethylamino) phosphonium hexafluorophosphate:BTA-three（Dimethylamino）- hexafluorophosphoric acid ester（The special condensing agent of card）； DCC, Ν,Ν'-Dicyclohexylcarbodiimide:Dicyclohexyl carbodiimide； DCM:Dichloro Yue alkane；DEPBT, 3- (Diethoxyphosphoryloxy)-l, 2,3-benzotriazin-4 (3H)-one:3- (diethoxy neighbour acyloxy) -1,2,3- phentriazine -4- ketone； DIC, N,N'-Diisopropylcarbodiimide:N, N'- DIC；DIPEA (or DIEA), diisopropylethylamine:Diisopropylethylamine； DMAP, 4-N,N-dimethylaminopyridine:The Yue aminopyridines of 4- Ν, Ν bis-； DMF:Ν, Ν-dimethyl Yue acid amides； DMSO:Two Yue sulfoxides； DTT, dithiothreitol:Dithiothreitol (DTT)；EDC or EDCI, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide:1-ethyl-(the Yue bases aminopropyls of 3- bis-) phosphinylidyne diimmonium salt hydrochlorate； EtOAc:Ethyl acetate；HBTU 0- (- the yl of 1 H-benzotriazole- 1)-N, N, N', N'-tetramethyluronium hexafluorophosphate BTAs-Ν, Ν, Ν ', Ν '-tetramethylurea hexafluorophosphate； ΗΟΒΤ l-hydroxybenzotriazole:L- hydroxyls-benzo-triazole；NMM, N-Methylmorpholine:N-methylmorpholine； NMP, N-methylpyrrolidinone:1-METHYLPYRROLIDONE； Piperidine:Piperazine.It is fixed； Su succinimide:Succinimide； TEA, triethylamine:Triethylamine；TFA, trifluoroacetic acid trifluoroacetic acids；TFE 2,2,2-Trifluoroethanol trifluoroethanols；THF tetrahydrofuran tetrahydrofurans；TIS triisopropylsilane triisopropyls baby cheats.

Chemiluminescent polypeptide synthetic method

Linear polypeptide uses Boc or Fmoc solid phase polypeptide synthesis.It is general to select Wang resins if the use of Fmoc chemical synthesis C- ends being the polypeptide of carboxyl；C- ends are that the polypeptide of acid amides generally selects Rink amide resins.If using Boc Chemical synthesis C- ends are the polypeptides of carboxyl, general to select Pam resins；C- ends are that the polypeptide of acid amides generally selects MBHA resins.Condensing agent and activator are DIC and HOBT, and other optional peptide bond condensing agents are including BOP, HBTU, DEPBT etc..5 times of excess of amino acid.The condensation time is 1 hour.Fmoc protection groups are removed with 50 °/A/DMF.Boc protection groups are removed with TFA.Peptide bond condensation reaction ninhydrin（Ninhydrin, 2,2-Dihydroxyindane-l, 3-dione) reagent monitoring.

During using Fmoc solid phase polypeptide synthesis, universal amino acid and protection group are as follows：

Fmoc-Cys(Trt)-OH、 Fmoc-Asp(OtBu)-OH、 Fmoc-Glu(OtBu)-OH、 Fmoc-His(Trt)-OH、 Fmoc-Lys(Boc)-OH 、 Fmoc-Asn(Trt)-OH 、 Fmoc-Gln(Trt)-OH 、 Fmoc-Arg(Pmc)-OH 、 Fmoc-Ser(tBu)-OH、 Fmoc-Thr(tBu)-OH> Boc-Trp(Boc)-OH、 Fmoc-Tyr(tBu)-OH。

After solid phase Fmoc chemistry synthesis polypeptide, conventional cutting reagent is TFA.Dried resin is placed in a shaking flask, appropriate amount TFA/TIS/H is added₂0 ( 95:2.5:2.5,10-25 mL/g resins）, close the lid, batch (-type) rotation concussion carried out at room temperature.Suction filtration resin after 2 hours, resin is cleaned 2-3 times with new TFA, and the ice ether of 8-10 times of volume is added dropwise in merging filtrate.Finally, the polypeptide crude product being precipitated out is collected by centrifugation.

During using Boc solid phase polypeptide synthesis, universal amino acid and protection group are as follows：

Boc-Cys(4-MeBzl)-OH >Boc-Asp (OcHx)-OH, Boc-Glu (OcHx)-OH, Boc-His (Bom)-OH, Boc-Lys (2-Cl-Z)-OH, Boc-Asn (Xan)-OH, Boc-Gln (Xan)-OH, Boc-Arg (Tos)-OH, Boc-Ser (Bzl)-OH, Boc-Thr (Bzl)-OH, Boc-Trp (CHO)-OH and Boc-Tyr (2-Br-Z)-OH.

After solid phase Boc chemically synthesized polypeptides, for PAM, mbha resin, general using HF cuttings, every 0.1 mM of resin adds 5 milliliters of HF, add simultaneously to Yue phenol, to reagents such as coloured glaze base phenol or methyl phenyl ethers anisoles, mixture is stirred 1 hour under condition of ice bath.After HF vacuum is drained, polypeptide water ether is precipitated, and precipitation is collected by centrifugation, is isolated and purified by HPLC, and freeze-drying obtains final product.The synthesis of single chain compound

Naturally it is connected chemically

Literature method (Kent, S.B.H. etc., " (IGF-1) and [Gly7D-Ala] IGF-l Prepared by Total Chemical Synthesis. of Comparative Properties of Insulin-like Growth Factor 1 " Angew. Chem. Int. Ed. 2008,47,1102-1106) it is partially improved according to amino acid sequence.

Single chain compound based on insulin is divided into two fragment synthesis.One section with Boc chemical synthesis insulin B chain fragments 1-18: EEEEEEM-[l-18]-COS- ( CH₂ ) ₂CO-(Arg)₄A.With S- trityl base propionic acid when thioesters residue is synthesized.Second segment include from B19Cys (correspond to formula in C [_2]) play the whole amino acid of B chains C-terminal, C chains and A chain amino acids.Two sections of polypeptide universal method synthesis in solid state, cut, purifying.

Naturally chemical coupled reaction is carried out in Slow fliud flushings.Slow fliud flushings contain 6 M guanidine hydrochlorides, 200 mM phosphate, 200 mM 4- carboxymethyl benzenethiols（MPAA, (4-carboxymethyl) thiophenol), (the 2- Yue acyl ethyls of 20 mM tri-）Phosphine（TCEP, tris- (2-carboxyethyl) phosphine), pH 6.9, polypeptide presses 1:1 mol ratio dissolves, the mM of concentration 2.Reaction is detected with HPLC, purified.

The IGF (SH) of purifying₆It is dissolved in 0.5 M guanidine hydrochlorides, 20 mM Tris, 8 mM cysteines, 1 mM cystine hydrochloride Slow fliud flushings, pH 7.8, the mg/mL of peptide concentration 0.5.After the completion of HPLC displays are folded, Slow fliud flushings are acidified to pH 3 with 0.1N hydrochloric acid.Polypeptide is purified with HPLC is prepared.

Compound I -2 synthesis：

I -2: YCN。

First fragment EEEEEEMFVNQHLCGSHLVEALYLV- (COS- C C-C0)-RRA Boc chemical syntheses, thick peptide is purified with RP-HPLC.Molecular weight calculation value 3419.9, mass spectrometric measurement molecular weight 3421.3.Second fragment CGERGFFYTPKTGSGSSSAAAPQTGIVEQCCTSICSLYQLENYCN is synthesized by universal method.Molecular weight calculation value 4773.4., mass spectrometric measurement molecular weight 4775.0.The mg of first fragment 34 (10 μ ι η ο) and the mg of the second fragment 48 (10 μ η ι ο) are dissolved in Slow fliud flushings（5 mL ).Slow fliud flushings include 6 M guanidine hydrochlorides, 200 mM phosphate, and 200 mM 4- carboxylic Yue bases benzene, which are dredged, expects, 20 mM tri- (2- Yue acyls ethyl) phosphines, pH 6.9.React and complete after 10 hours.Molecular weight calculation value 7703.6, mass spectrometric measurement molecular weight 7704.2.Mixture is transferred to size exclusion chromatography post, elution Slow fliud flushings are 0.5 M guanidine hydrochlorides, 20 mM Tris, pH 7.8.Collect and 8 mM cysteines are added after the part for including correct polypeptide molecular weight, merging, 1 mM cystine hydrochloride Slow fliud flushings, polypeptide is folded and finished after 2 hours.Slow fliud flushings are acidified to pH 3 with 0.1N hydrochloric acid, then purified with RP-HPLC.Molecular weight calculation value 7697.6, mass spectrometric measurement molecular weight 7699.5.

Polypeptide is dissolved in 70% Yue acid（Or 0.1 M hydrochloric acid）, add cyanogen bromide（30 times of amounts）, cultivate at room temperature.After the completion of HPLC detection reactions, major part Yue acid is volatilized with after cyanogen bromide with nitrogen, is diluted with 10% acetic acid, then RP-HPLC is purified.Finally, compound I -2 molecular weight calculation value 6791.7, mass spectrometric measurement molecular weight 6792.3.Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 2.

Other single chain compounds based on insulin are synthesized in the same way.

Further confirm that the structure of compound, the connected mode of especially three pairs disulfide bond, use document (Chance etc., " The production of human insulin using recombinant DNA technology and a new chain combination procedure ", Pept.:Synth., Struct., Funct., Proc. Am. Pept. Symp., 7th, 1981, Vol. 721, Issue 8, Page 721) in HPLC " fingerprints, analytic approach.In brief, 2 mg polypeptide samples are dissolved in 0.2 ml 0.01N hydrochloric acid, add the 0.05M NH that 0.8 ml contains 100 μ g S. aureus V8 protease₄HC0₃, pH is reached 7.9.Cultivated 24 hours in 37'C.Sample fraction adds DTT and cultivated 30 minutes.The structure of disulfide bond and compound is determined using the retention time (retention time) and molecular weight of LC-MS comparative samples and each fragment of standard items.Composite result:

The single chain compound based on insulin is synthesized using the above method, by the molecular weight of the general each compounds of detection of matter i, the structure of each compound is detected by the method for sequencing, it is as a result as follows：

1 -1 :Molecular weight calculation value 7038.0, mass spectrometric measurement molecular weight 7038.2;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 1 ;

1 -3:Molecular weight calculation value 6718.6, mass spectrometric measurement molecular weight 6719.7;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 3；

1 -4:Molecular weight calculation value 6846.8, mass spectrometric measurement molecular weight 6848.1;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 4;

1 -5:Molecular weight calculation value 6830.8, mass spectrometric measurement molecular weight 6829.9;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 5;

1 -6:Molecular weight calculation value 6775.7, matter language test molecule amount 6776.9;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 6;

1 -7:Molecular weight calculation value 6635.6, matter knows test molecule amount 6536.8 well;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 7；

1 -8:Molecular weight calculation value 6550.4, mass spectrometric measurement molecular weight 6551.5;Through sequencing, the amino acid sequence of the compound is It is SEQIDNO: 8;

1-9:Molecular weight calculation value 6621.5, mass spectrometric measurement molecular weight 6622.3;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 9;

I -10:Molecular weight calculation value 6536.4, mass spectrometric measurement molecular weight 6538.0;Through sequencing, the amino acid sequence of the compound is SEQIDNO: 10;

I -11:Molecular weight calculation value 6476.4, mass spectrometric measurement molecular weight 6477.6;Amino acid sequence through the compound is sequenced is SEQ ID Ν Ο:11;

I -12:Molecular weight calculation value 6476.4, mass spectrometric measurement molecular weight 6477.2;Amino acid sequence through the compound is sequenced is SEQ ID NO: 12;

I -13:Molecular weight calculation value 6465.3, mass spectrometric measurement molecular weight 6466.7;Amino acid sequence through the compound is sequenced is SEQIDNO:13;

I -14:Molecular weight calculation value 6465.3, mass spectrometric measurement molecular weight 6466.0;Amino acid sequence through the compound is sequenced is SEQ ID NO: 14;

I -15:Molecular weight calculation value 6495.4, mass spectrometric measurement molecular weight 6495.9;Amino acid sequence through the compound is sequenced is SEQIDNO:15;

I -16:Molecular weight calculation value 6405.3, mass spectrometric measurement molecular weight 6406.2;Amino acid sequence through the compound is sequenced is SEQ ID NO: 16;

I -17:Molecular weight calculation value 6405.3, mass spectrometric measurement molecular weight 6406.8;Amino acid sequence through the compound is sequenced is SEQ ID NO: 17;

I -18:Molecular weight calculation value 6435.3, mass spectrometric measurement molecular weight 6436.5;Amino acid sequence through the compound is sequenced is SEQ ID NO: 18;

1-19:Molecular weight calculation value 6435.3, mass spectrometric measurement molecular weight 6437.0;Amino acid sequence through the compound is sequenced is SEQ ID NO: 19;

I -20:Molecular weight calculation value 6408.3, mass spectrometric measurement molecular weight 6409.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:20;

I -21:Molecular weight calculation value 6362.3, mass spectrometric measurement molecular weight 6362.9;Amino acid sequence through the compound is sequenced is SEQIDNO:21;

I -22:Molecular weight calculation value 6378.3, mass spectrometric measurement molecular weight 6379.1;Amino acid sequence through the compound is sequenced is SEQ ID NO:22;

I -23:Molecular weight calculation value 6305.2, mass spectrometric measurement molecular weight 6306.5;Amino acid sequence through the compound is sequenced is SEQIDNO:23;

I -24:Molecular weight calculation value 6291.2, mass spectrometric measurement molecular weight 6292.6;Amino acid sequence through the compound is sequenced is SEQIDNO:24;

1-25:Molecular weight calculation value 6321.2, mass spectrometric measurement molecular weight 6422.4;Amino acid sequence through the compound is sequenced is SEQIDNO:25;

I -26:Molecular weight calculation value 6201.0, mass spectrometric measurement molecular weight 6202.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:26;

I -27 ：Molecular weight calculation value 6156.0:Mass spectrometric measurement molecular weight 6157.1;Amino acid sequence through the compound is sequenced is SEQ ID NO:27;

I -28:Molecular weight calculation value 6348.2, mass spectrometric measurement molecular weight 6348.7;Through the amino acid sequence that the compound is sequenced It is SEQ ID NO:28;

I -29:Molecular weight calculation value 6378.2, matter language test molecule amount 6379.5;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:29;

I -30:Molecular weight calculation value 6362.2, mass spectrometric measurement molecular weight 6363.4;Amino acid sequence through the compound is sequenced is SEQ ID NO:30;

1 -31 :Molecular weight calculation value 6281.1, mass spectrometric measurement molecular weight 6280.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:31 ;

I -32:Molecular weight calculation value 6295.1, mass spectrometric measurement molecular weight 6296.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:32;

I -33:Molecular weight calculation value 6548.4, mass spectrometric measurement molecular weight 6549.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:33;

1 -34:Molecular weight calculation value 6241.1, mass spectrometric measurement molecular weight 6242.3;Amino acid sequence through the compound is sequenced is SEQ ID NO:34;

I -35:Molecular weight calculation value 6777.7, mass spectrometric measurement molecular weight 6778.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:35;

I -36 ：The mass spectrometric measurement molecular weight 6795.0 of molecular weight calculation value 6793.7;It is SEQ ID NO through the amino ^^ of the compound is sequenced and arranges:36;

I -37:Molecular weight calculation value 6793.7, mass spectrometric measurement molecular weight 6793.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:37;

I -38:Molecular weight calculation value 6850.8, mass spectrometric measurement molecular weight 6851.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:38;

I -39:Molecular weight calculation value 6845.8, mass spectrometric measurement molecular weight 6846.6;Amino acid sequence through the compound is sequenced is SEQ ID NO:39;

I -40:Molecular weight calculation value 6902.9, mass spectrometric measurement molecular weight 6904.4;Amino acid sequence through the compound is sequenced is SEQ ID NO:40;

I -41 :Molecular weight calculation value 6916.7, matter knows test molecule amount 6917.6 well;It is SEQ ID NO through the amino ^^ of the compound is sequenced and arranges:41 ;

I -42:Molecular weight calculation value 6904.9, mass spectrometric measurement molecular weight 6905.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:42;

I -43:Molecular weight calculation value 6962.8, mass spectrometric measurement molecular weight 6964.3;Amino acid sequence through the compound is sequenced is SEQ ID NO:43;

I -44:Molecular weight calculation value 6856.8, mass spectrometric measurement molecular weight 6858.5;Amino acid sequence through the compound is sequenced is SEQ ID NO:44;

I -45 ：Molecular weight calculation value 6890.8, mass spectrometric measurement molecular weight 6891.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:45;

I -46:Molecular weight calculation value 6934.9, mass spectrometric measurement molecular weight 6936.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:46;

I -47:Molecular weight calculation value 6746.7, matter Fan test molecules amount 6747.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:47;

I -48:Molecular weight calculation value 7610.6, mass spectrometric measurement molecular weight 7612.0;Through the amino acid sequence that the compound is sequenced It is SEQ ID NO:48;

I -49:Molecular weight calculation value 6835.8, mass spectrometric measurement molecular weight 6837.1;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:49;

I -50:Molecular weight calculation value 6835.8, matter test molecule amount 6836.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:50;

I -51 :Molecular weight calculation value 6875.8, mass spectrometric measurement molecular weight 6876.6;Amino acid sequence through the compound is sequenced is SEQ ID NO:51 ;

I -52:Molecular weight calculation value 6859.8：Mass spectrometric measurement molecular weight 6860.4;Amino acid sequence through the compound is sequenced is SEQ ID NO:52;

I -53:Molecular weight calculation value 6972.0:Amino acid sequence of the mass spectrometric measurement molecular weight 6973.8 through the compound is sequenced is SEQ ID NO:53;

I -54 ：The mass spectrometric measurement molecular weight 7710.4 of molecular weight calculation value 7708.7 is SEQ ID NO through the amino ^^ of the compound is sequenced and arranges:54;

I -55:Molecular weight calculation value 6845.8:Amino acid sequence of the mass spectrometric measurement molecular weight 6846.9 through the compound is sequenced is SEQ ID NO:55;

I -56:Molecular weight calculation value 6875.8：Amino acid sequence of the matter language test molecule amount 6876.2 through the compound is sequenced is SEQ ID NO:56;

I -57:Molecular weight calculation value 6902.8_;Amino acid sequence of the mass spectrometric measurement molecular weight 6903.5 through the compound is sequenced is SEQ ID NO:57;

I -58:Molecular weight calculation value 6861.8, mass spectrometric measurement molecular weight 6863.1；Through sequencing, the amino acid sequence of the compound is SEQ ID NO:58;

I -59:Molecular weight calculation value 6930.9, mass spectrometric measurement molecular weight 6932.6;Amino acid sequence through the compound is sequenced is SEQ ID NO:59;

I -60:Molecular weight calculation value 8412.5, mass spectrometric measurement molecular weight 8413.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:60;

I -61 :Molecular weight calculation value 6858.8, matter language test molecule amount 6860.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:61 ;

I -62:Molecular weight calculation value 6817.7, mass spectrometric measurement molecular weight 6818.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:62;

I -63:Molecular weight calculation value 7315.3, matter test molecule amount 7316.5;Amino acid sequence through the compound is sequenced is SEQ ID NO:63;

I -64:Molecular weight calculation value 8745.7, matter language test molecule amount 8746.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:64;

I -65:Molecular weight calculation value 6933.9, mass spectrometric measurement molecular weight 6935.1;Amino acid sequence through the compound is sequenced is SEQ ID NO:65;

I -66:Molecular weight calculation value 6877.8, mass spectrometric measurement molecular weight 6879.3;Amino acid sequence through the compound is sequenced is SEQ ID NO:66;

I -67:Molecular weight calculation value 6930.9, mass spectrometric measurement molecular weight 6931.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:67;

I -68:Molecular weight calculation value 6922.9, mass spectrometric measurement molecular weight 6924.4;Through the amino acid sequence that the compound is sequenced It is SEQ ID NO:68;

I -69:Molecular weight calculation value 6975.9, mass spectrometric measurement molecular weight 6776.5;Amino acid sequence through the compound is sequenced is SEQ ID NO:69;

I -70:Molecular weight calculation value 6821.7, mass spectrometric measurement molecular weight 6823.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:70;

I -71 :Molecular weight calculation value 6809.7, mass spectrometric measurement molecular weight 6810.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:71 ;

I -72 ：Molecular weight calculation value 6848.8, mass spectrometric measurement molecular weight 6850.4;Through the amino ^ that the compound is sequenced!>Row are SEQ ID NO:72;

I -73:Molecular weight calculation value 8228.3, mass spectrometric measurement molecular weight 8230.1;It is SEQ ID NO through the amino ^^ of the compound is sequenced and arranges:73;

I -74:Molecular weight calculation value 6894.8, matter language test molecule amount 6896.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:74;

I -75:Molecular weight calculation value 7004.0, mass spectrometric measurement molecular weight 7005.5;It is SEQ ID NO through the amino ^^ of the compound is sequenced and arranges:75;

I -76:Molecular weight calculation value 7788.7, matter language test molecule amount 7790.2;Through sequencing, the amino acid sequence of compound is SEQ ID NO:76;

I -77:Molecular weight calculation value 7358.3, matter language test molecule amount 7360.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:77;

I -78:Molecular weight calculation value 7586.6, matter knows test molecule amount 7587.7 well;Amino acid sequence through the compound is sequenced is SEQ ID NO:78;

I -79:Molecular weight calculation value 7142.2, mass spectrometric measurement molecular weight 7143.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:79;

I -80:Molecular weight calculation value 6208.1, mass spectrometric measurement molecular weight 6209.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:80;

I -81 :Molecular weight calculation value 6756.7, mass spectrometric measurement molecular weight 6757.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:81 ;

I -82:Molecular weight calculation value 7296.3, mass spectrometric measurement molecular weight 7297.5;Amino acid sequence through the compound is sequenced is SEQ ID NO:82;

I -83:Molecular weight calculation value 7455.4, matter Pass test molecules amount 7457.6;Amino acid sequence through the compound is sequenced is SEQ ID NO:83;

I -84:Molecular weight calculation value 6812.6, mass spectrometric measurement molecular weight 6813.8;Amino acid sequence through the compound is sequenced is SEQ ID NO:84;

I -85:Molecular weight calculation value 6638.4, mass spectrometric measurement molecular weight 6639.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:85;

I -86:Molecular weight calculation value 6846.8, mass spectrometric measurement molecular weight 6848.2;Amino acid sequence through the compound is sequenced is SEQ ID NO: 86;

I -87:Molecular weight calculation value 7136.1, matter Pass test molecules amount 7137.9;Amino acid sequence through the compound is sequenced is SEQ ID NO:87;

I -88:Molecular weight calculation value 7472.6, mass spectrometric measurement molecular weight 7474.9;Through the amino acid sequence that the compound is sequenced It is SEQ ID NO:88;

I -89:Molecular weight calculation value 6605.5, mass spectrometric measurement molecular weight 6606.8;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:89;

I -90:Molecular weight calculation value 6837.8, mass spectrometric measurement molecular weight 6839.1;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:90;

I -91 :Molecular weight calculation value 6770.7, mass spectrometric measurement molecular weight 6772.4;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:91 ;

I -92:Molecular weight calculation value 6586.5, mass spectrometric measurement molecular weight 6587.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:92;

I -93:Molecular weight calculation value 6572.5, mass spectrometric measurement molecular weight 6573.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:93;

I -94:Molecular weight calculation value 7008.0, mass spectrometric measurement molecular weight 7009.2;Amino acid sequence through the compound is sequenced is SEQ ID NO:94;

I -95:Molecular weight calculation value 6607.5, matter Pan's test molecule amount 6608.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:95;

I -96:Molecular weight calculation value 7079.1, mass spectrometric measurement molecular weight 7080.6;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:96;

I -97:Molecular weight calculation value 6951.9, mass spectrometric measurement molecular weight 6953.4;Through sequencing：The amino acid sequence of the compound is SEQ ID NO:97;

I -98:Molecular weight calculation value 8239.3, mass spectrometric measurement molecular weight 8241.1;Amino acid sequence through the compound is sequenced is SEQ ID NO:98;

I -99:Molecular weight calculation value 6322.1, shield spectrum test molecule amount 6322.7;Amino acid sequence through the compound is sequenced is SEQ ID NO:99;

I -100:Molecular weight calculation value 7890.0, matter language test molecule amount 7891.4;Amino acid sequence through the compound is sequenced is SEQ ID NO: 100;

I -101 :Molecular weight calculation value 6709.5, mass spectrometric measurement molecular weight 6710.6;Amino acid sequence through the compound is sequenced is SEQ ID NO: 101 ;

I -102:Molecular weight calculation value 6235.0, mass spectrometric measurement molecular weight 6235.9;Amino acid sequence through the compound is sequenced is SEQ ID NO: 102;

I -103:Molecular weight calculation value 7371.3, matter Pass test molecules amount 7372.5;Amino acid sequence through the compound is sequenced is SEQ ID NO:103;

I -104:Molecular weight calculation value 7485.5, mass spectrometric measurement molecular weight 7487.0;Amino acid sequence through the compound is sequenced is SEQ ID NO:104;

I -105:Molecular weight calculation value 7752.7, mass spectrometric measurement molecular weight 7754.2;Amino acid sequence through the compound is sequenced is SEQ ID NO: 105;

I -106:Molecular weight calculation value 7387.3, mass spectrometric measurement molecular weight 7388.7;Amino acid sequence through the compound is sequenced is SEQ ID NO: 106；

1 -107:Molecular weight calculation value 7197.1, mass spectrometric measurement molecular weight 7198.6;Amino acid sequence through the compound is sequenced is SEQ ID NO: 107;

I -108:Molecular weight calculation value 7098.1, matter language test molecule amount 7099.0;Through the amino acid sequence that the compound is sequenced It is SEQ ID NO:108;

I -109:Molecular weight calculation value 7124.0, mass spectrometric measurement molecular weight 7124.8;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 109;

I -110:Molecular weight calculation value 7640.7, matter language test molecule amount 7641.4;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 110;

I -111 :Molecular weight calculation value 7936.9, mass spectrometric measurement molecular weight 7938.5;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:lll ;

I -112:Molecular weight calculation value 6902.8, mass spectrometric measurement molecular weight 6903.1;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 112;

1 -113:Molecular weight calculation value 7669.7, mass spectrometric measurement molecular weight 7671.1；Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 113;

I -114:Molecular weight calculation value 7529.6, mass spectrometric measurement molecular weight 7531.8;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 114;

I -115:Molecular weight calculation value 7041.0, mass spectrometric measurement molecular weight 7041.6;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 115;

I -116:Molecular weight calculation value 7007.9, shield spectrum test molecule amount 7009.2;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:116;

I -117:Molecular weight calculation value 7630.7, mass spectrometric measurement molecular weight 7631.9;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 117;

I -118:Molecular weight calculation value 7421.3, mass spectrometric measurement molecular weight 7423.3;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:118;

I -119:Molecular weight calculation value 6965.9, mass spectrometric measurement molecular weight 6967.0;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:119;

I -120:Molecular weight calculation value 6375.3, matter language test molecule amount 6376.7;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 120;

I -121 :Molecular weight calculation value 6304.2, mass spectrometric measurement molecular weight 6305.6;Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 121 ;

I -122:Molecular weight calculation value 6595.4, mass spectrometric measurement molecular weight 6597.8;Through sequencing, the amino acid sequence of the compound is SEQ ID NO:122;

I -123:Molecular weight calculation value 6932.9, mass spectrometric measurement molecular weight 6934.1；Through sequencing, the amino acid sequence of the compound is SEQ ID NO: 123.The material of compound

Stization is reacted

It is prepared by tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu

Hexadecandioic acid (hexadecane diacid)（5.72 g, 20 mmol) dry DMF (240 mL) is dissolved in, cooled down with water-bath.Gradually add 2- Yue base -2- propyl alcohol（ 1.48 g, 20mmol)、 DIC ( 2.7 g, 2.25 mL, 21.4 mmol), HOBT ( 2.88 g, 21.4 mmol). NMM ( 2.16g, 2.34 mL, 21.4 mmol) > DMAP ( 244 mg, 2 mmol).Mixture is stirred at room temperature overnight.80 mL water are added, pH 3 is acidified to, is extracted with ethyl acetate, organic layer is washed with 0.1 N HC1 and saturated common salt, after magnesium sulfate is dried, solvent under reduced pressure evaporation obtains the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one（3.32g, yield 47%).Nuclear magnetic resonance data is 'H-NMR(CDC1₃) δ:2.35 (t, 2H), 1.56-1.66 (m, 4H), 1.44 (s, 9H), 1.21-1.35 (m, 20H).

Fmoc-Glu-OtBu (4.25g, 10 mmol) it is dissolved in DCM (30 mL), power mouthful is to 3 grams of 2-CTC resins (2-chlorotrityl chloride resin, sub. lmmol/g), continuously add DIPEA (1.29g, 10 mmol, 1.74 mL).Mixture adds DIPEA (1.93g, 15 mmol, 2.6 mL) after shaking machine vibrates 5 minutes.Mixture high vibration 1 hour.

HPLC grades of methanol are added in order to seal in active triphen Yue trips base, resin（2.4 mL), mix 15 minutes.Resin filter, with DCM (mL of 3 X 30), DMF (mL of 2 X 30), DCM (mL of 3 X 30) after Yue alcohol (mL of 3 X 30) cleaning, are dried in a vacuum.

After piperidines removing Fmoc, 3 g resins (3mmol) and the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one（3.43 g, 10 mmol) add dry DMF (50 mL), gradually add DIC (1.35 g, 1.12 mL, 10.7 mmol), HOBT (1.44 g, 10.7 mmol), DIPEA (1.3 g, 10 mmol, 1.74 mL).After shaking at room temperature is stayed overnight, resin is cleaned with DMF (mL of 2 X 30) and DCM (mL of 2 X 30).

Prepare AcOH/TFE/DCM (1:1 :8) cutting liquid (20 mL/g resins）.Resin is suspended in the cutting liquid of half, places 30 minutes at room temperature.Resin is filtered, resin is washed with second half cutting liquid three times.The n-hexane that filtrate adds 15 times of volumes is mixed, revolving removes unnecessary acetic acid, obtains tert-butyl hexadecandioyl diacyl-L-GIu-OtBu.Nuclear magnetic resonance data is ' H-NMR (CDC1₃) δ:6.25 (d, lH), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m, 4H), 1.92 (m, 1H), 1.58 (m, 4H)_; 1.47(s, 9H), 1.22-1.43 (m, 18H)。

Tert-butyl hexadecandioyl diacyl-L-Glu-OtBu (lg, 1.9 mmol) is dissolved in dry DMF/DCM (1 mL:4 mL) adds DCC (0.412 g, 2 mmol) and N- hydroxysuccinimides（ 0.23 g, 2 mmol) .Mixture is stirred at room temperature overnight.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1 N HC1 and saturated common salt water washing, after magnesium sulfate is dried, reduction vaporization obtains tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu.Nuclear magnetic resonance data is： 1H-NMR(CDC1₃) δ:6.17 (d, lH), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m, 1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.20-1.33 (m, 20H).

Compound II -2 synthesis：

II -2： FVNQHLCGSHLVEALYLVCGERGFFYTPTGK[N^£-(N^a-(HOOC(CH₂)₁₄CO)-Y-Glu)] GSSSAAAPQTGIVEQCCTSICSLYQLENYCN。

First fragment EEEEEEMFVNQHLCGSHLVEALYLV- (COS- CH₂ CH₂- CO)-RRA Boc chemical syntheses.Molecular weight calculation value 3419.9, mass spectrometric measurement molecular weight 3420.6.Thick peptide is purified with RP-HPLC.Into molecular weight calculation value 4686.3, mass spectrometric measurement molecular weight 4687.5.Thick peptide is purified with RP-HPLC.The mg of first fragment 34 (Ι Ο μ η ι ο Ι) and the mg of the second fragment 47 (Ι Ο μ η ι ο Ι) are dissolved in Slow fliud flushings（5 mL ).Slow fliud flushings include 6 M guanidine hydrochlorides, 200 mM phosphate, the 200 stupid thiophenols of mM 4- carboxylic Yue bases, 20 mM tri- (2- carboxyethyls) phosphines, pH 6.9.Molecular weight calculation value 7913.9, mass spectrometric measurement molecular weight 7915.7.Mixture is transferred to size exclusion chromatography post, elution Slow fliud flushings are 0.5 M guanidine hydrochlorides, 20 mM Tris, pH 7.8.Collect and 8 mM cysteines are added after the part for including correct polypeptide molecular weight, merging, 1 mM cystine hydrochloride Slow fliud flushings.After polypeptide folding is finished, Slow fliud flushings are acidified to pH 2 with 0.1N hydrochloric acid, then RP-HPLC is purified.Molecular weight calculation value 7610.5, mass spectrometric measurement molecular weight 7611.2.

At room temperature by polypeptide（78 mg) it is dissolved in 100mM Na₂C0₃(2 mL, pH 10) solution.Tert-butyl hexadecandioyl diacyl-L-G】U (OSu)-OtBu (6.5 mg) is dissolved in acetonitrile（2 mL), add polypeptide solution.After stirring 30 minutes, 50% acidifying with acetic acid, upper RP-HPLC C5 posts purifying are used.Slow fliud flushings A:The 0.1%TFA aqueous solution, 10% acetonitrile；Slow fliud flushings B:The 0.1%TFA aqueous solution, 80% acetonitrile.Polypeptide after preliminary purification is lyophilized adds TFA/TIS/H₂0 ( 95:2.5:2.5,10mL) it is, true after 30 minutes Empty evaporation solvent, is dissolved in Slow fliud flushings A by crude product and freezes.Product is obtained using the purifying of RP-HPLC C5 posts.Molecular weight calculation value 8008.0, mass spectrometric measurement molecular weight 8010.1.

Polypeptide is dissolved in 70% Yue acid（Or 0.1 M hydrochloric acid）, add cyanogen bromide（30 times of amounts）, cultivate at room temperature.After the completion of HPLC detection reactions, after the most of formic acid of nitrogen volatilization and cyanogen bromide, diluted with 10% acetic acid, then RP-HPLC is purified.The final molecular weight calculation value 7102.1 for obtaining II -2, mass spectrometric measurement molecular weight 7103.5.

Minority specioz observes FVNQHLCGSHLVEALYLVCGER (calculating molecular weight 2487.9, matter language test molecule amount 2488.1), GFFYTPTGK [N after DTT reduction and trypsin degradation with liquid chromatogram-matter language combination^E-(N^a-(HOOC (CH₂)₁₄CO)-Y-G1U)] GSSSAAAPQTGIVEQCCTSICSLYQLENYCN (calculating molecular weight 4638.3, mass spectrometric measurement molecular weight 4639.6).Through analysis, the material of synthesis is 11-2.

OSu-CO-(CH₂CH₂0)₅-(CH₂)₂-NH-[N^a-(HOOC (CH₂)₁₆CO)-y-Glu] synthesis

Octadecane diacid（2.5g, 8.0 mmol) DCM (60 ml) is suspended in, add triethylamine（1.16 ml, 8.3 mmol) and be cooled with an ice bath.The sour benzyl esters of chlorine Yue are added dropwise in nitrogen environment（1.14 ml), add DMAP (0.097g, 0.8 mmol).After stirring 30 minutes, solvent is evaporated under reduced pressure, crude product is purified with silicagel column（Ethyl acetate：Heptane 1:7-1 :1) benzyl ester of octadecane diacid one, is obtained after evaporation solvent（U2g, 35%).

1H-NMR ( CDC1₃) δ:7.35 (m, 5H), 5.11 (s, 2H), 2.35 (t, 4H), 1.63 (t, 4H), 1.30-1.22 (m, 24).The benzyl ester of octadecane diacid one (0.8g, 2 mmol) is dissolved in DMF (3 ml) and DCM (3 ml), is cooled with an ice bath.Add DCC (0.408 g, 2 mmol) and N- hydroxysuccinimides（0.23 g, 2 mmol).Mixture is stirred at room temperature 24 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1 N HC1 and saturated common salt water washing, after magnesium sulfate is dried, reduction vaporization obtains the benzyl octadecane diacid ester of succinimido one.

1H-NMR ( CDC1₃) δ:7.35 (m, 5H), 5.11 (s, 2H), 2.83 (s, 4H), 2.60 (t, 2H), 2.35 (t, 2H),

1.80-1.60 (m, 4H), 1.40-1.20 (m, 24H).

Benzyl octadecane diacid ester (95 mg of succinimido one, 0.19 mmol) DMF (1.5 ml) is dissolved in, add L-Glu-OBzl (49 mg, 0.21 mmol) and DIEA (52 μ, 0.3 mmol), stir 16 hours.Solvent is evaporated under reduced pressure, ethyl acetate is added, with 0.1N HCK saturated common salt water washings, magnesium sulfate is evaporated under reduced pressure solvent after drying, obtains BzlO- octadecandioyls-L-Glu-OBzl.

1H-NMR ( CDCI3 ) δ:7.35 (m, 5H), 6.22 (d, 2H), 5.17 (s, 2H), 5.11 (s, 2H), 4.71 (m, 1H), 2.37 (m, 4H), 2.22 (m, 3H), 1.98 (m, 1H), 1.63 (m, 4H), 1.31-1.20 (m, 24H).

BzlO- octadecandioyls-L-Glu-OBzl (110 mg, 0.18 mmol) is dissolved in DMF (1 ml) and DCM (1 ml), is cooled down with water-bath.Add DCC (41 mg, 0.2 mmol) and N- hydroxysuccinimides (23 mg, 0.2 mmol).Mixture is stirred at room temperature 12 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1 N HC1 and saturated common salt water washing, after magnesium sulfate is dried, reduction vaporization obtains BzlO- octadecandioyls-L-Glu (OSu)-OBzl.

Ή-NMR ( CDC1₃) δ:7.36 (m, 5H), 6.40 (d, 2H), 5.19 (s, 2H), 5.11 (s, 2H), 4.75 (m, 1H), 2.82 (s, 4H), 2.68 (m, 1H), 2.59 (m, 1H), 2.35 (t, 2H), 2.19 (t, 2H), 1.62 (m, 4H), 1.32-1.21 (m, 24H).

BzlO- octadecandioyls-L-Glu (OSu)-OBzl (72mg, 0.1 mmol) and H₂N-(CH2)2-(OC¾CH2)₅COOH (31mg, O.lmmol) is dissolved in DMF/DCM (0,5 ml:1.5tnl), DIEA (26 L, 0.15 mmol) is added, is stirred 16 hours.Solvent is evaporated under reduced pressure, add ethyl acetate, use 0.1N HC1, saturated common salt water washing, solvent is evaporated under reduced pressure after drying in sulfuric acid lance, obtain 3- [2- [2- [2- [2- [2- [[5- benzyloxies -4- [(18- benzyloxies -18- oxos-octadecanoyl) amino] -5- oxos-valeryl] amino] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] propionic acid (3- [2- [2- [2- [2- [2- [[5-benzyloxy-4- [(18-benzyloxy-18-oxo-octadecanoyl) amino] -5-oxo- pentanoyl] amino] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] propanoi c acid).Calculate molecular weight 915.2, test molecule amount 916.5。

3- [2- [2- [2- [2- [2- [[5- benzyloxies -4- [(18- benzyloxies -18- oxos-octadecanoyl) amino] -5- oxo-pentanoyls] amino] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] propionic acid (91mg; O.lmmol DMF (1 ml) and DCM (1 ml)) are dissolved in, is cooled down with water-bath.DCC (24.7mg, 0.12 mmol) and N- hydroxysuccinimides (13.8 mg, 0.12 mmol) mixtures are added to be stirred at room temperature 12 hours.Filter mixture, filtrate is diluted with ethyl acetate, with 0.1 N HC1 and saturated common salt water washing, after magnesium sulfate is dried, reduction vaporization obtain benzene Yue bases 18- [[1- benzyloxycarbonyl groups -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -³- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino-oxo-butyl] amino]-I⁸- oxo-stearate（benzyl 18-[[ 1 -benzyloxycarbonyl- 4-[2- [2-[2- [2-[2-[3-(2,5-dioxopyrrolidin-l-yl) oxy-3 -oxo-propoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethylamino]-4-oxo-butyl]amino] - 18-oxo-octadec anoate ).Calculate molecular weight 1012.2, test molecule amount 1013.1.

Benzene Yue bases 18- [[1- benzyloxycarbonyl groups -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidins -1-yl) oxygen _³- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino]-4- oxo-butyls] amino]-18- oxos-stearate (5 lmg, 0.05mmol) it is dissolved in the TFA of Yue alcohol/acetone/0.1%, add Pd/C, it is stirred at room temperature in a nitrogen environment 5 hours, filtered by diatomite, precipitation and evaporation residue solvent from heptane, obtain 18- [[carboxyl-4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidins-yl) oxygen-3- oxos-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino] 4- oxo-butyls] amino]-18- oxos-stearic acid (18- [[l-carboxy-4- [2- [2- [2- [2- [2- [3- (2,-the yl of 5- dioxopyrrolidin- 1) oxy-3-oxo-propoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethyl amino]-4-oxo-butyl] amino]-18-oxo-octadecanoic acid).Calculate molecular weight 832.0, test molecule amount 833.4.Structure is as follows：

11-24 is synthesized

FV QHLCGSHLVEALYLVCGERGFFYTPTGKGSSSAAAPQTGIVEQC CTSICSLYQLENYCN are synthesized in aforementioned manners.Calculate molecular weight 6704.6, mass spectrometric measurement molecular weight 6706.3.At room temperature by polypeptide（67 mg, 10 μ η ι ο) it is dissolved in 100mM Na₂CO₃(1 ml, pH 10) solution.18- [[1- carboxyls -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -³- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino] oxo-butyl] amino]-I⁸- oxo-stearic acid (9.2 mg, 11 μ ι η ο) is dissolved in acetonitrile（0.5 ml), add polypeptide solution.Acidifying with acetic acid, upper RP-HPLC C5 posts purifying are used in stirring after 30 minutes.Slow fliud flushings A:The 0.1%TFA aqueous solution, 10% acetonitrile；Buffer B:The 0.1%TFA aqueous solution, 80% acetonitrile.Molecular weight calculation value 7421.5, mass spectrometric measurement molecular weight 7422.9.Through analysis, gained compound is 11-24.

Octadecandioyl-Glu (Glu (OSu)-OH)-OH preparation

Fmoc-Glu-OBzl (4.59g, 10 mmol) it is dissolved in DCM (30 mL), power mouthful is to 3 grams of 2-CTC resins (2-chlorotrityl chloride resin, sub. lmmol/g), continuously add DIPEA (1.29g, 10 mmol, 1.74 mL) mixtures shaking machine vibrate 5 minutes after, add DIPEA (1.93g, 15 mmol, 2.6 mL)₀Mixture high vibration 1 hour.

In order to seal HPLC grades of Yue alcohol of addition in active triphenylmethyl, resin（2.4 mL), mix 15 minutes.Resin filter, with DCM (mL of 3 X 30), DMF (mL of 2 X 30), DCM (mL of 3 X 30) after Yue alcohol (mL of 3 X 30) cleaning, are dried in a vacuum.

After piperidines removing Fmoc; 3 g resins (3mmol) and BzlO- octadecandioyl-L-Glu-OBzl (6.24g; 10 mmol) add dry DMF (50 mL); gradually add DIC (1.35 g; 1.12 mL; 10.7 mmol), HOBT (1.44 g, 10.7 mmol), DIPEA ( 1.3 g, 10 mmol, 1.74 mL).After shaking at room temperature is stayed overnight, resin is cleaned with DMF (mL of 2 X 30) and DCM (mL of 2 X 30).

Prepare AcOH/TFE/DCM (1:1:8) cutting liquid (20 mL/g resins）.Resin is suspended in the cutting liquid of half, places 30 minutes at room temperature.Resin is filtered, resin is washed with second half cutting liquid three times.The n-hexane that filtrate adds 15 times of volumes is mixed, revolving removes unnecessary acetic acid.BzlO- octadecandioyls-Glu (Glu-OBzl)-OBzl is dissolved in DMF (3 ml) and DCM (3 ml), is cooled with an ice bath.DCC (618 mg, 3 mmol) and N-hydroxysuccinimide (345 mg, 3 mmol) mixtures are added to be stirred at room temperature 12 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1 N HC1 and saturated common salt water washing, after bowl acid magnesium is dried, reduction vaporization obtains BzlO- octadecandioyls-Glu (Glu (OSu)-OBzl)-OBzl.

BzlO- octadecandioyls-Glu (Glu (OSu)-OBzl)-OBzl is dissolved in the TFA of methanol/acetone/0.1%; add Pd/C; it is stirred at room temperature in a nitrogen environment 5 hours; filtered by diatomite, simultaneously evaporation residue solvent obtains octadecandioyl-Glu (Glu (OSu)-OH)-OH to precipitation from heptane.Calculate molecular weight 669.8, mass spectrometric measurement molecular weight 670.3.

11-23 is synthesized

FVNQHLCGSHLVEALYLVCGERGFFYTPTGKGSSSAAAPQTGIVEQCC TSICSLYQLENYCN are synthesized in aforementioned manners.Calculate molecular weight 6704.6, mass spectrometric measurement molecular weight 6705.9.At room temperature by polypeptide（67 mg, 10 μ ι η ο) it is dissolved in lOOmM Na₂C0₃(1 ml, pH 10).Octadecandioyl-Glu (Glu (OSu)-OH)-OH is dissolved in acetonitrile（0.5 ml), add polypeptide solution.Acidifying with acetic acid, upper RP-HPLC C5 posts purifying are used in stirring after 30 minutes.Slow fliud flushings A:The 0.1%TFA aqueous solution, 10% acetonitrile Slow fliud flushings B:The 0.1%TFA aqueous solution, 80% acetonitrile.Molecular weight calculation value 7259.3, mass spectrometric measurement molecular weight 7260.2.Through analysis, gained compound is 11-23.Pegylation ^ Η Ψ methods（PEGylation)

A) reductive alkylation (reductive alkylation)

Polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH₃CN 1) is pressed:2:45 ratios are dissolved in the acetums of pH 4.3 (O.lM NaCl, 0.2 M CH₃COOH, 0.1M Na₂CO₃).Peptide concentration is 0.5-1 mg/mL.Reaction is detected and purified with HPLC.Yield about 55%.Polyethylene glycol can be selectively combined in B1 by reductive alkylation reaction.

11-18 synthesis.

GIVEQCCTSICSLYQLENYCN, and mPEG20K-CHO reductive alkylation reactions obtain product.With Mass Spectrometer Method, the compound molecular weight calculated value 26201.0, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 26206.3.Minority specioz is after DTT reduction and trypsin degradation, with liquid chromatograph mass spectrography it was observed that GFFGSGSSSAAAPQT GIVEQCCTSICSLYQLENYCN mass（Calculate molecular weight 3737.2, mass spectrometric measurement molecular weight 3738.5), FVNQHLCGSHLVEALYLVCGER mass is not observed（Calculate molecular weight 2487.9), but mass spectrometric measurement obtains a broad peak, intermediate molecular weight 22495.6, it was demonstrated that and PEG binding sites are Bl.Through analysis, the material of synthesis is 11-18.

B) NHS esters (N- hydroxysuccinimides) are acylated

Polypeptide and mPEG20K-NHS in molar ratio 2:1 is dissolved in 0.1N N, double (2- ethoxys) glycine solutions of N-（PH 10), peptide concentration 0.5mg/mL.Reaction is carried out 1 hour in room temperature, is purified with HPLC.Yield 56%.

II -17 is synthesized：

CN synthetic methods are with reference to I -2.Polypeptide obtains product after being reacted with mPEG20K-NHS by above-mentioned process for acylating.With Mass Spectrometer Method, molecular weight calculation value 26704.6, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 26710.7.Minority specioz through DTT reduction and trypsin degradation after, with liquid phase color i it is general-matter promise combination observe FVNQHLCGSHLVEA LYLVCGER (meter Calculate molecular weight 2487.9, mass spectrometric measurement molecular weight 2489.0), GFFYTPTGSGSSK (N^£- PEG 20K) AAAPQTGIVEQ CCTSICSLYQLENYCN (calculating molecular weight 24240.8, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 24247.6), molecular weight 22487.9 is not observed, it was demonstrated that PEG binding sites are C peptide lysine side-chains.Through analysis, the material of synthesis is 11-17.

II -4 is synthesized

More than YCN uses natural chemical connection process synthesis.Molecular weight calculation value 6778.6, mass spectrometric measurement molecular weight 6779.8.Polypeptide (68 mg) is dissolved in DMF (3 mL), adds Mal-dPEG₁₂- NHS (8.7 mg) (Quanta Biodesign) and triethylamine (30 L).Reaction is stirred at room temperature 2 hours.Depressurize after solvent flashing, crude product is dissolved in 0/ACN (3:1), purified with RP- HPLC.Maleimide polypeptide is dissolved in pure water, the mM of peptide concentration 10.Add human albumin（665 mg), cultivated 30 minutes at 37 °C.Then it is diluted to 5% human albumin with the 20 mM sodium radio-phosphate,P-32 solutions containing 5 mM Sodium Caprylates and 750 mM ammonium sulfate.Unreacted reagent is removed with gel-filtration chromatography, 0.05M ammonium bicarbonate aqueous solutions are used as eluent.Sterling is obtained after vacuum freeze drying.Through analyzing, molecular weight calculation value 74001.7, mass spectrometric measurement molecular weight 74003.8, the compound of gained is II -4.

Composite result:

The compound of the respectively modification based on insulin is respectively synthesized according to the method described above, it is as a result as follows by the structure of the general each compounds of detection of shield i：

II -1 :Molecular weight calculation value 6932.0, mass spectrometric measurement molecular weight 6933.8, through analysis, the material of synthesis is II -1;

II -3:Molecular weight calculation value 7102.1, matter Pass test molecules amount 7103.4, through analysis, the material of synthesis is II -3;

II -5:Molecular weight calculation value 7199.2, mass spectrometric measurement molecular weight 7200.9, through analysis, the material of synthesis is II -5;

II -6:Molecular weight calculation value 7118.1, mass spectrometric measurement molecular weight 7120.2, through analysis, the material of synthesis is II -6;

II -7:Molecular weight calculation value 7274.3, mass spectrometric measurement molecular weight 7275.5, through analysis, the material of synthesis is II -7;

II -8:The , Zhi Wrong test molecules amount 6656.7 of molecular weight calculation value 6655.6, through analysis, the material of synthesis is Π -8;

II -9:Molecular weight calculation value 6655.6, matter language test molecule amount 6657.3, through analysis, the material of synthesis is II -9;

II -10 ：The matter Pan's test molecule amount 6640.4 of molecular weight calculation value 6639.6, the material through analysis synthesis is 11-10;

II -11 ：Molecular weight calculation value 6639.6, matter language test molecule amount 6741.1, through analysis, the material of synthesis is 11-11;

II -12 ：The matter language test molecule amount 6740.8 of molecular weight calculation value 6639.6, through analysis, the material of synthesis is Π -12;

II -13 ：The mass spectrometric measurement molecular weight 6676.0 of molecular weight calculation value 6674.7, through analysis, the material of synthesis is 11-13;

II -14 ：The mass spectrometric measurement molecular weight 6732.5 of molecular weight calculation value 6731.7, through analysis, the material of synthesis is II -14;

II -15 ：The mass spectrometric measurement molecular weight 6728.2 of molecular weight calculation value 6726.7, through analysis, the material of synthesis is II -15;

II -16 ：The matter i general surveys of molecular weight calculation value 6598.5 try molecular weight 6599.6, and through analysis, the material of synthesis is Π -16;

II -19 ：The mass spectrometric measurement molecular weight 7089.7 of molecular weight calculation value 7088.1, through analysis, the material of synthesis is II -19;

II -20 ：The mass spectrometric measurement molecular weight 6961.8 of molecular weight calculation value 6960.0, through analysis, the material of synthesis is II -20;

II -21 ：The mass spectrometric measurement molecular weight 7086.9 of molecular weight calculation value 7086.1, through analysis, the material of synthesis is II -21;

II -22 ：Molecular weight calculation value 6944.0, mass spectrometric measurement molecular weight 6945.1, through analysis, the material of synthesis is II -22.Receptor binding assay

1、 ¹²⁵I-IGF-1 and¹²⁵The preparation of 1- insulin

Literature method (Cresto etc., " Preparation of biologically active mono-¹²⁵I-insulin of high specific activity" , Acta Physiol Lat Am. 1981 , 31(l):13-24 ) 2nd, the receptor binding assay of compound

Literature method (E. K. Frandsen and R. A. Bacchus. " New; simple insulin-receptor assay with universal application to solubilized insulin receptors and receptors in broken and intact cells. " Diabetes, 1987,36,3:335-340) or one of following methods.Unless otherwise instructed, by preparation also such as literature method, user's placental membrane.Generally, insulin receptor Binding experiment uses 0.025 milligram of placental membrane；IGF-1 Receptor Binding Assays use 0.2 milligram of placental membrane.

In the experiment of insulin receptor binding analysis, the initial concentration of insulin standard and the compounds of this invention is Ι Ο Ο η Μ, then by the 3 times of dilutions of insulin and the compounds of this invention series, respectively obtains control and the compound solution of 7 various concentrations（ 100nM、 33.33nM、 l l.llnM, 3.70nM、 1.23nM、 0.41nM、 0.13nM、 0.04nM ).Activity for its insulin receptor in the present invention is less than the compound of actrapid monotard's standard 10%, and compound initial concentration is 500nM.In the experiment of IGF-1 receptor binding assays, the initial concentration of IGF-1 standards is Ι Ο Ο η Μ, the compounds of this invention initial concentration is Ι Ο Ο Ο η Μ, then by 3 times of dilutions of IGF-1 and the compounds of this invention series, respectively obtains control and the compound solution of 7 various concentrations（ 1000nM、 333.33nM、 l l l.l lnM, 37.04nM、 12.35nM、 4.12nM、 1.37nM、 0.46nM ).Activity for its IGF-1 acceptor in the present invention is less than the compound of IGF-1 standards 1%, and compound initial concentration is 5000nM.Receptor binding assay (1)

The water-soluble acceptor blocked

IGF-1 or insulin receptor,¹²⁵I-IGF-1 (3-10 pM) or¹²⁵1- insulin（3 pM) and the polypeptide of 3 times of dilutions of series add Slow fliud flushings [100 mM Hepes, pH 8.0,100 mM NaCl, 10 mM MgCl₂, 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X-100], the μ Ι ^ of cumulative volume 200,4.C is cultivated 48 hours.Acceptor and the polypeptide and part that are combined with acceptor are precipitated with 0.2 % γ-globulin and 500 μ 25 % (w/v) PEG 8000, the radioactivity in measurement precipitation.The concentration of acceptor will adjust the binding of receptor and ligand for having 15-20% when polypeptide is not added with.

Membrane-bound receptor

The membrane-bound receptor that receptor binding assay is used reaches the bhk cell of total length insulin or IGF-1 acceptors from altimeter.The transfection bhk cell (2000-5000) of equivalent is evenly distributed on each hole of 96 orifice plates, in the Eagle's culture mediums of the Dulbecco's improvement comprising 10% (v/v) hyclone（DMEM culture carries out receptor binding assay again after 24 hours in).Cell is first washed one time with combination buffer (DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8), adds 400 L¹²⁵I-IGF-l (6.5 pM) or¹²⁵1- insulin (6.5 pM) and the polypeptide for being dissolved in 3 times of dilutions of series with reference to Slow fliud flushings.16.C is cultivated 3 hours, and uncombined polypeptide is suctioned out with attractor, is washed one time with 1.2 ml combination Slow fliud flushings.Cell is dissolved in 500 μ L 1% (w/v) SDS, 100 mM NaCl, 25 mM Hepes (pH 7.8), then measures.Cell quantity will be adjusted to the binding of receptor and ligand for having 16-28 % when not adding peptide.

Receptor binding assay（ 2 )

IGF-1 acceptors： [Thr⁵⁹] IGF-l for tyrosine fine jadeization react（ iodination ).¹²⁵I-IGF-1 (50-80 Ci/g, 50 fmol), Human plactnta film（0.2 mg) and series 3 times dilution polypeptide add 0.2 milliliter of 0.1 M Hepes Slow fliud flushing, pH 8, comprising 120mM sodium chloride, 5mM potassium chloride, 0.12mM magnesium sulfate and 0.1% bovine albumin, 20.C is cultivated 1 hour.Sample is filtered with Whatman GF/F filters to be combined and uncombined polypeptide compound with separating.Filter is handled with 0.1 % polyethyleneimines in advance.Culture tube and filter are washed 4 times with 2.5 milliliters of cold Slow fliud flushings without bovine albumin.In the case of not having placental membrane, the polypeptide compound attachment less than 5% is on the filter.Under conditions of no polypeptide is striven unexpectedly, placental membrane combines about 38% part.Can be by the non-iodate [Thr of excessive addition to placental membrane non-specific binding⁵⁹] IGF-1 (0.3 μ Μ) measures to culture mix.Non-specific binding generally accounts for the 5% of part and placental membrane combination total amount. Insulin receptor：¹²⁵1- insulin（30 nCi), series 3 times dilution polypeptide and placental membrane（0.025 mg) in 0.05 milliliter of above-mentioned Slow fliud flushing, 20 °C are cultivated 1 hour.Sample is filtered with EHWP filters, and culture tube and filter are washed 4 times with 2.5 milliliters of cold Slow fliud flushings without bovine albumin.In the case of not having placental membrane, the polypeptide compound attachment less than 5% is on the filter.Can be by the non-Iodinated Insulin-125I of excessive addition to placental membrane non-specific binding（1 μ Μ) measured to culture mix.Non-specific binding generally accounts for less than the 1% of part and placental membrane combination total amount.

Specific bond percentage=(with reference to exit dose-non-specific binding exit dose I all with reference to exit dose-non-specific binding exit dose) χ 100.It is all the radiation total amount measured when being not added with polypeptide with reference to exit dose.It is to add the exit dose that measures after polypeptide with reference to exit dose.The IC of polypeptide compound₅₀Calculated using Origin softwares (OriginLab, Northampton, MA).Polypeptide is relative to actrapid monotard or activity=IC of IGF-1 standards₅₀Insulin or IGF-1 standards/IC₅₀Polypeptide.

3rd, zoopery

7-9 week old C57BL/6 male mices, average weight 20-25g, 6 are divided into one group, the fasting in 4 hours before experiment starts.Measurement blood glucose before experiment starts, and each specifies point in time sampling measurement blood glucose afterwards.Control group is physiological saline, and polypeptide is dissolved in physiological saline, is subcutaneously injected.Reaction of the mouse in experiment overall process is observed, any abnormal behaviour is recorded.

Experimental result：

Tested to detect the bioactivity of these compounds by the compound based on insulin and the adhesion of insulin receptor to the present invention, the benchmark of combination rate of insulin receptor is used as using natural insulin（100%) with benchmark of the natural IGF-1 as IGF-1 acceptor binding forces（100% ).Obtained result is shown in Table 2 and table 3 respectively.

The single chain compound based on insulin constituted with INSULIN A and B chains and junction fragment, with the adhesion of insulin receptor close to insulin level, but because IGF-1C peptides are the critical sequences that are combined with IGF-1 acceptors, also cause adhesion of the single chain polypeptide on IGF-1 acceptors to be far above natural insulin.C2Tyr is a very prominent amino acid residue in IGF-1 crystal structures, and display is probably the key combined with IGF-1 acceptors.After tyrosine is replaced by serine or alanine, insulin receptor activity retains substantially, but considerably reduces IGF-1 receptor actives, gets a desired effect.In addition, result is shown, the length of C peptides is not necessarily required to 12 amino acid residues.Former IGF-1 C-terminals 3,4,5,6 amino acid residues all can lack or be replaced.Especially IGF-1 C-terminals remove activity not only to insulin receptor of 3 amino acid residue PQT and 4 residue A PQT and had no adverse effect, and its IGF-1 receptor active is reduced to the level of insulin.Further investigation revealed that, in the case where the length for retaining C peptides is 12 amino acid residues, 5 amino acid residue X111X112X113X114X115 of insulin B chain C-terminal can have 1,2,3,4 or all missing, without reducing combination rate of insulin receptor.Further study showed that, IGF-1 C peptides are only a selection in a variety of junction fragments.These data illustrate that the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of appropriate C chain lengths, flexible C chain spaces conformation and specific site is to improve insulin receptor activity, reduces the key factor of IGF-1 acceptor binding forces.

Table 2:The bioactivity result of the unmodified single chain compound based on insulin

IGF-1R IGF-1R IGF-1R compounds IR (%) compound IR (%) compound IR (%)

( % ) ( % ) ( % )

I -1 114 29.0 I -42 59 0.4 I -83 73 0.3

I -2 96 1.5 I -43 54 0.6 I -84 64 <0.1

I -3 107 0.9 I -44 53 0.5 I -85 60 <0.1

1 -4 103 1.4 1 -45 56 0.7 I -86 87 <0.1

I -5 92 1.7 I -46 48 0.3 I -87 76 0.2

I -6 95 1.3 1 -47 51 0.3 I -88 46 <0.1

1 -7 90 1.6 I -48 85 0.4 1 I -89 91 <0.1 I -8 89 0.9 I -49 57 0.7 I -90 63 0.2

I -9 85 1.0 I -50 65 0.8 I -91 82 <0.1

I -10 76 1.0 I -51 49 0.6 I -92 54 0.3

I -11 72 0.8 I -52 56 0.4 I I -93 57 0.4

I -12 66 0.7 I -53 51 0.5 I -94 69 0.2

I -13 85 0.7 I -54 89 0.3 I -95 92 <0.1

I -14 73 0.6 I -55 52 0.4 I -96 81 0.2

I -15 68 0.4 I -56 58 0.3 1 I -97 75 <0.1

I -16 53 0.6 I -57 53 0.5 I -98 73 <0.1

I -17 60 0.4 I -58 66 0.4 I -99 75 <0.1

I -18 59 0.5 I -59 49 0.5 I -100 86 0.2

I -19 47 0.3 I -60 55 0.2 I -101 83 <0.1

I -20 52 0.3 I -61 46 0.1 I -102 85 0.2

I -21 44 0.2 I -62 50 0.3 1 I -103 76 <0.1

I -22 65 0.3 I -63 88 0.3 I -104 78 <0.1

I -23 53 0.2 I -64 63 0.2 I -105 61 <0.1

I -24 41 0.2 I -65 59 0.3 I -106 90 <0.1

I -25 72 0.3 I -66 91 0.3 I -107 77 0.2

I -26 97 0.7 I -67 76 0.4 I -108 86 <0.1

I -27 102 0.4 I -68 78 0.2 I -109 47 <0.1

I -28 99 0.6 I -69 54 0.2 I I -110 44 <0.1

I -29 90 0.6 I -70 60 0.6 I -111 49 <0.1

I -30 93 0.5 I -71 58 0.5 I -112 91 <0.1

I -31 98 0.8 I -72 69 0.5 1 I -113 60 <0.1

I -32 95 1.0 I -73 86 0.2 I -114 62 <0.1

I -33 82 0.9 I -74 85 0.2 I -115 85 <0.1

I -34 86 1.1 I I -75 77 0.3 I -116 73 <0.1

I -35 87 1.2 I -76 70 0.1 I -117 48 <0.1

I -36 89 0.7 I -77 83 0.2 I -118 81 <0.1

I -37 97 0.8 I -78 42 0.3 I -119 76 0.2

I -38 92 0.5 I -79 61 0.2 I -120 83 0.2

I -39 61 0.4 I -80 53 0.4 I -121 57 0.1

I -40 65 0.4 I -81 57 0.4 I -122 85 <0.1

I -41 58 0.3 I -82 85 <Wherein, IR represents insulin receptor to 0.1 I -123 79 0.3, and IGF-1R represents IGF-1 acceptors.

Pegylation and fatty-acylation are the common methods for extending action time in polypeptide body, but bioactivity is typically greatly lowered in Pegylation and fatty-acylation.Therefore finding the lysine etc. that can introduce new outside actrapid monotard B29 has the acylation sites of amino, and acylate has enough bioactivity significant for exploitation long-acting polypeptides.

It is demonstrated experimentally that all remaining suitable insulin receptor activity after the junction fragment of Bl, single chain compound, A22 modification. Due to use bovine albumin in Receptor Binding Assay to reduce albumen non-specific binding, but aliphatic acid on acyl polypeptide can be with albumin combination（This is also the mechanism of fatty acid prolonging action time）, so that polypeptide valid density in experiment is reduced, therefore the Receptor-Binding Data of acyl polypeptide seems relatively low.A large amount of zooperies of Past 30 Years show that external activity and the activity in vivo of insulin analog do not have clear and definite corresponding relation.External activity be substantially less than the analog of actrapid monotard in vivo with the work of actrapid monotard ' f gives birth to basic mesh when (with reference to " In vitro and in vivo potency of insulin analogs designed for clinical use " such as Volund, Diabetic Medicine, 1991,8:839-47;Ribel etc. " Equivalent in vivo biological activity of insulin analogs and human insulin despite different in vitro potencies ", Diabetes, 39,1033-39,1990).The animal experimental data of inventor also indicates that the internal substantial activity of Pegylation and fatty-acylation polypeptide is better than insulin control.

Table 3:The bioactivity of the single chain compound based on insulin of modification

Wherein, IR is insulin receptor.

The delay effect of the insulin analog of the present invention is given in experimental result.Tested respectively with actrapid monotard, compound Π -2 and negative control, insulin and II -2 consumption are respectively 70nmol/kg.The blood sugar concentration of mouse is monitored, Fig. 1 is as a result seen.Still there is obvious suppression hypoglycemic effect in the 5-10 hours of Π -2 upon administration, but insulin has lost suppression blood glucose ability gradually within this time.Actrapid monotard shows the hypoglycemic curve of typical V-shape in an experiment.It is too fast that the shortcoming of this hypoglycemic curve is that blood glucose at initial stage declines, and easily causes hypoglycemia, and later stage uncontrollable blood glucose.Π -2 shows the hypoglycemic curve of L-type, and glycemic control is steady and lasting, and effect is significantly better than actrapid monotard.

Blood sugar concentration change is monitored in another experiment, after the II -17 of 3 kinds of dosage of mouse subcutaneous injection, Fig. 2 is as a result seen.

11-17 dosage as little as 25nmol/kg are just enough steadily to control blood glucose at least 24 hours.And after dosage is increased to 90nmol/kg, there is not blood glucose low ebb yet in mouse.Therefore, II -17 is better than actrapid monotard in terms of control blood glucose and security.

Detect that blood glucose changes over time value in another experiment, after mouse subcutaneous injection physiological saline, actrapid monotard and II -11, as a result see Fig. 3.Similar with Fig. 1, actrapid monotard shows the hypoglycemic curve of typical V-shape, and II -11 shows the hypoglycemic curve of L-type, steady control blood glucose at least 24 hours, and effect is significantly better than actrapid monotard.

Three above is it is demonstrated experimentally that with the insulin analog in polyethylene glycol or the fatty acid modifying present invention, can improve the therapeutic effect and pharmacokinetic characteristic of insulin analog.

Claims

1st, a kind of single chain compound with blood sugar decreasing effect, the structure of the compound is： Xi^HLCnjGSXjogLVEALYLVCpjGEX^gGFXnoXniXmXinXiHXiis-CL-GIVEQC^jC^XneSICfs ]SLYQLENYC_[6]X₁₁₇X₁₁₈, wherein,

X₁₀₇It is phenylalanine-valine-asparagine-glutamin tetrapeptide, valine-asparagine-glutamin tripeptides, asparagine-glutamin dipeptides or glutamine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； X₁₍₎₈It is histidine, phenylalanine, arginine or grain weevil acid amides； X₁₍₎₉It is arginine, alanine, glutamic acid or aspartic acid； X₁₁₍₎It is phenylalanine, tyrosine or histidine； X_U1It is tyrosine, phenylalanine or missing； X₁₁₂It is threonine, asparagine, glutamic acid, aspartic acid or missing； X₁₁₃It is proline, lysine, glutamic acid, aspartic acid or missing；X is lysine, proline, arginine, glutamic acid, aspartic acid or missing； x₁₁₅It is threonine or missing； x₁₁₆It is threonine, histidine or arginine； x₁₁₇It is alanine, glycine or asparagine； X₁₁₈It is lysine, arginine-lysine dipeptides or missing；CL is junction fragment.

2nd, compound according to claim 1, it is characterised in that the structure of the compound is：

FVNQHLCGSHLVEALYLVCGERGFFXmX X XiwX-CL-GIVEQCCTSICSLYQLENY CN, wherein,

Xm is tyrosine or missing；X is threonine or missing； Χ₁₁₃It is proline or missing； X₁₁₄It is lysine or missing；X is threonine or missing；CL is junction fragment.

3rd, compound according to claim 2, it is characterised in that the structure of the compound is：

FVNQHLCGSHLVEALYLVCGERGFFYTPKT-CL-GIVEQCCTSICSLYQLENYCN。

4th, the compound according to claim any one of 1-3, it is characterised in that the junction fragment is the peptide sequence of 6-60 amino acid, each of which amino acid is all independently selected from glycine, alanine, serine, threonine, proline.

5th, compound according to claim 4, it is characterised in that the junction fragment C_LInclude 1 or more than 1 aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine.

6th, compound according to claim 5, it is characterised in that the junction fragment C_LIt is all or part of sequence of following polypeptide fragment, or have 1 with following polypeptide fragment, the difference of 2 or 3 amino acid residues, or have 70%, 80%, 90% similar with following polypeptide fragment, or following polypeptide fragment all or part of sequence 1,2,3,4 or 5 repetitive sequences：

(GASPGGSSGS) GR, wherein n are 1,2,3,4 or 5; GSSGSSGPGSSR; GSSGSGSSAPQT; GSGGAPSRSGSSR; GSPAGSPTSTGR; GGSGGSGGR; GSSPATSGSPQR; GASSSATPSPQR;

GSGSSSRAPPSAPSPQR； GSSSESPSGAPQT； GAGTPASGSAPGR； GSSPSGGSSAPQT；

GSTSSTARSPGR； GAGPSGTASPSR； GSSTPSGAPQT； SSSSAPPPSAPSPSRAPQR；

GSGSSSAAAPQT ; GSGSSSAAPQT ; GASPGTSSTSGR; GSGSSSAPQT ; GSGSSSRRA;

GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT; GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR;

GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT;

GASTSSRGAPSR; GSGSSSAGR; GPSGTSTSAPGR; GAGSSSAPQT; SSSSAPSAPSPSRPQR;

GSGASSPTSPQR; GAGGSGSGR; GSSPATSATPQT; GAGSSSAPPPSAPSPSRAPGPSAPQR;

GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR; GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSGRSSSPGR; GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR； RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; SSSAPPPSAPSPSRAPGPSPQ ； SAASSSASSSSASSASAG ； GAGGPSSGAPPPSPQT； GSGSSGGR; GAGSPAAPASPAPAPS AGR； SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR ; GSGSSSRRAPQT； SSSSAASAASASSSASGR； SSSRAPPSAPSPQR； GGPSSGAPPPSR； SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT; GGPSSGAPPPSPQT; SSSAPPPSAPSPSRAPQT; GAGPSSGAPPPSPQT; GGGGAPQT; GAGGPSSGAPPPQT; GGPSSGAPPPSPSPSRPGPSPQR; SSASSASSSSAGR; GAGSSR; SSASSSAASSSASSSASGR ; SSSGAPPPSPSRAPGPSPQR; GSGSASRGR ； SSSSAASSASGR ； SASASASASSASSGR ； SASSPSPSAPSSPSPAS ； GPSSPSPSAPSSPSPASPSSGR ； SSSAPPPASPSPSRAPGPQR ； SASASASASASSAGR ； GSGASSRGR ; GSGAAPASPAAPAPSAGR； GGPSSGAPPPSGR ; SSPSASPSSPASPSSGR ; GAPASPAPSAPAPAAPSGR； GPSSPSPSAPSSPSPASPSSAPQT； SSASSASSSSSASAGR； SAPSSPSPSAPSSPSASPSGR ； SSSAPPPSAPSPSAPQR ； GASSPSPSAPSSPSPASGR ； SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT ； GGGGAPQR ； GSGSSSAAR ； GSGSSAAPQR ； SSSSRRAPQR ； SSSGSGSSAPQR； SSGSGSSSAPQR； GSGSSSRS； SSSSRAPQR； GASPGGSSGSGR； GSGSSSAAAPQR; GAGSSSAAAPQR; GAGSSSAAAPQT; GSSGGSGR; GAGGGSSGR; GSGSSGSR ； GSGSSSSR ； GSGSGGGR ； GAGSSGR ； GSGSSGR ； SSSSRAPPPSAPSPSRAPGPSAPQR； GGGSSR； GSGSSSAAPQR； GASPGGSSGSSR； GSGSSSRSGR ； GTGPSSATPASR ； GAGPSGTASPSS ； SSSSAPSAPSPSRAPQR ； GSPSSPTRGSAT ； GPETPSGPSSAT ； GSSPATSGTPQT ；

GSGSSSRAPPPSAPSPSRAPGPSPAPQR; GSSTPSGAGPQT; GSGSSSRAPPPSAPSPSRAPQR; GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPAR; GAGSSSRAPPPSAPSPSRAPGPSPQR; GSGSSSRAPPSAPSAPQR; GSTAGSRTSTAR; GSSPSGRSSSPAR; SSASSASSSSSAASAGR; GSSSGSSGSPSAR ； SSSAPSPSRAPGPSPQR ； GAGSSSRAPPPSAPSPSRAPQR ； GSPAAPAPASPAAPAPSAGR; SSSAPSAPSPSAPQR; GGPSSGAPPPSPSPSRPGPSDTPPQR; SASASASASASASSASSGR ； SASSPSPSAPSSPSPASGR ； SASASASASASASSAGR ； SSPSASPSSPASPSPSSGR; GAPASPAPAAPSAPAPAAPSGR; GAGSPAAPAPASPAPAPSAGR; SSS APPPSAPSPSAPQT； GASPAAPSAPPAASPAAPSAGR； SSSAPPPSPSRAPGPSPQR； SSPSAPSPSSPASPSPSSGR; SSSSGPSSGAPPPSGR; GSSSRSPSGSPR; GGGPGAGSSPQR.

7th, compound according to claim 1, it is characterised in that the compound is selected from SEQ ID NO:Foretell SEQ ID NO:Compound shown in 123.

8th, a kind of compound with hypoglycemic effect, the structure of the compound is：

X3ooVNQHLC_[1]GSHLVEALYLVC_[2]GERGFX₃₀iX3₀₂X₃₀₃X3₀4X3₀5X3₀6GX₃₀₇X₃₀8X309X3ioX3i i

X312X313X314 315X316 317GIVEQC[₃]C[4] X₃₁₈X₃₁9X32oC[5〗X₃₂₁LX₃₂₂X₃₂₃LX3₂₄X₃25YC[₆]X₃₂₆X₃27, wherein,

X₃₀O is phenylalanine or UL- phenylalanines； X_{3( 1}It is phenylalanine, histidine or tyrosine； X₃o₂It is tyrosine, phenylalanine or missing； X₃C3 is threonine, asparagine, glutamic acid, aspartic acid or missing； X_3G4It is proline, relies ammonia Acid, glutamic acid, aspartic acid or missing； X_3Q5It is aspartic acid, glutamic acid, proline, arginine, lysine or missing, or formula（I) structure； X₃。₆It is threonine, formula（I) structure or missing； X₃。₇It is lysine, serine, alanine, glycine, formula（I) structure or missing； Χ_3ΜIt is glycine, formula（I) structure or missing； Χ₃ο₉It is lysine, glycine, serine, formula（I) structure or missing； X_31QIt is lysine, glycine, serine, formula（I) structure or missing； X_3UIt is lysine, glycine, serine, alanine, formula（I) structure or missing； X₃₁₂It is lysine, arginine, alanine, proline, glycine, formula（I) structure or missing； x₃₁₃It is glycine, alanine, arginine, lysine, glutamine, proline, formula（I) structure or missing； x₃₁₄It is arginine, alanine, proline, threonine, glutamine, glycine, formula（1) structure or missing； x₃₁₅It is proline, glutamine, arginine, glycine or missing or formula（I) structure； X₃₁₆It is glutamine, threonine, arginine, glycine or missing or formula（I) structure； x₃₁₇It is threonine, arginine, lysine or missing； X_31SIt is threonine, histidine, arginine or formula（I) structure； x₃₁₉It is serine or formula（I) structure； X_32QIt is isoleucine or formula（I) structure； X₃₂₁It is serine or formula（I) structure； x₃₂₂It is tyrosine or formula（I) structure； x₃₂₃It is glutamine or formula（I) structure； x₃₂₄It is glutamic acid or formula

(I) structure； X₃₂₅It is asparagine or formula（I) structure； x₃₂₆It is alanine, glycine or asparagine； ¾₂₇It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；Work as x₃₂₇During for dipeptides, one of amino acid is formula（I) structure；

Wherein, the formula（I) structure is：

U_LIt is-W-X-Y-Z structures, aliphatic acid, polyethylene glycol, albumin, L-M_LStructure, hydrogen atom or N^a-(N^a-(HOOC(CH₂)_nCO)-y-Glu)- N^a-( ^a-(CH₃(CH₂)_nCO)-y-Glu)-, wherein η is 8-20 integer, such as 8,10,12,14,16,18 or 20, Ν^αThe a- amino of amino acid or amino acid residue is represented, or is formula（II) structure.The formula（II) structure is：

J is-W-X-Y-Z structures, L_n-M_LStructure or hydrogen atom.

9th, compound according to claim 8, it is characterised in that the structure of the compound is：

X₃₀₀VNQHLC_[1]GSHLVEALYLVC_[2]GERGFFX₃₀₂X₃₀3X₃o4X₃₀5X3₀6GX₃₀₇X₃o8X₃₀9X3ioX3iiX3 i₂X₃₁3X₃₁₄X₃₁₅X₃₁₆X₃nGIVEQC_[3〗C_[4]TSIC_[5〗SLYQLENYC_[6iNX₃₂₇, wherein,

X₃₀0 is phenylalanine or U_L- phenylalanine； X₃o₂It is tyrosine or missing；X303 is threonine or missing；04 is proline or missing； X_3Q5It is aspartic acid, proline, arginine, lysine or missing, or formula（I) structure； X₃₀₆It is threonine, formula（I) structure or missing； X₃o₇It is lysine, serine, alanine or formula（I) structure； X₃o₈It is glycine or formula（I) structure； X_3Q9It is lysine, serine or formula（I) structure； X₃₁O is that lysine, silk atmosphere are sour or logical Formula（I) structure； X₃₁₁It is lysine, serine, alanine or formula（I) structure； x₃₁₂It is lysine, arginine, alanine, proline or formula（I) structure； x₃₁₃It is glycine, alanine, arginine, lysine, glutamine, proline or formula（I) structure； X₃₁₄It is arginine, alanine, proline, threonine or glutamine or formula（I) structure； x₃₁₅It is proline, glutamine, arginine or missing or formula（I) structure； x₃₁₆It is glutamine, threonine, arginine or missing or formula（I) structure； X₃₁₇It is threonine, arginine, lysine or missing； X₃₂₇It is formula（I) structure or missing, wherein, U_LAnd formula（I) as defined in claim 8.

10th, compound according to claim 8, it is characterised in that the compound is selected from：

II -1 :

FVNQHLCGSHLVEALYLVCGERGFFYTPTGK[N^E-(N^a-(HOOC(CH₂)i4CO)-Y-Glu)] GSSSRGRGIVEQCCTSICSLYQLENYCN;

II -2:

FV QHLCGSHLVEALYLVCGERGFFYTPTGK[N^£-( ^a-(HOOC(CH₂)₁₄CO)-y-Glu)] GSSSAAAPQTGIVEQCCTSICSLYQLENYCN;

II -3:

FVNQHLCGSHLVEALYLVCGERGFFYTPTGSGK[N^E-(N^a-(HOOC(CH₂)₁₄CO)-Y- Glu)]SSAAAPQTGIVEQCCTSICSLYQLENYCN;

II -4:

F(N^a-dPEG,₂- maleimide-albumin）VNQHLCGSHLVEALYLVCGERGFFYTPDTGSGSSS AAAPQTGIVEQCCTSICSLYQLENYCN;

II -5:

FVNQHLCGSHLVEALYLVCGERGFFYTPPTGSGSSK[N^£-(N^a-(HOOC(CH₂)i₄CO)-Y- Glu)]AAAPQTGIVEQCCTSICSLYQLENYCN;

II -6:

FV QHLCGSHLVEALYLVCGERGFFYTPTGSGSSSAK[N⁶~(N^a-(HOOC(CH₂)₁₄CO) -y-Glu)]APQTGIVEQCCTSICSLYQLENYCN;

II -7:

FVNQHLCGSHLVEALYLVCGERGFFYTPRTGSGSSSK[N^E-(N^a-(HOOC(CH₂)i₄CO) -Y-Glu)]AAPQTGIVEQCCTSICSLYQLENYCN;

II -8:

FVNQHLCGSHLVEALYLVCGERGFFGSGSSSK[N^E-(N^a-(HOOC(CH₂)i₄CO)-Y-Glu)] AAPQTGIVEQCCTSICSLYQLENYCN;

II -9:

FVNQHLCGSHLVEALYLVCGERGFFGSGSSSAK[N^E-( ^a-(HOOC(CH₂)i₄CO)-Y-Glu)] APQTGIVEQCCTSICSLYQLENYCN;

11 -10：

FV QHLCGSHLVEALYLVCGERGFFGSGK[N^E-( ^a-(HOOC(CH₂)₁₄CO)-y-Glu)]

SSAAAPQTGIVEQCCTSICSLYQLENYCN;

11 -11 :

FV QHLCGSHLVEALYLVCGERGFFGSGSK[N^£-(N^a-(HOOC(CH₂)₁₄CO)-Y-Glu)]

S AAAPQTGIVEQCCTSICSLYQLENYCN; Enter Λ Ι Ο Shang CWVWSSSD [(ni the ￡)-people-(00 of people 1S IS Shang 03^9,(¾3) DOOH) -_ηΝ)-Η ( :) (0¾ ¾ )-0 -₃Μ £)1<Ι Shang people ￡) 3 ￡ Λ enter lV3AlHSDCnH0NAd

■ Z-ll

：N people N310A1S IS Shang C 03AID Shang 0<IVVVSSSO [((ni ￡)-people）ζ-Ν-η Ο-people-(OC^'HC OOH Nh biO Shang d Shang people iWCm3COAlAlV3AlHS ￡):yTH0NAJ

'•£2-11

：M enters Mai A!S IS Shang) 03AI0I.DSSS ￡) [HO. people-(CO " (:)) )OOH)-_DN)-₃N] 3 ￡ OAlA of C) Shang d Shang people JJD!V3AlHS{) nH0NAJ

■ZZ- II 0£ ：N enters N3 0AlS ISlCO03AID 0d SSS ￡) [(W9- people-(O W HD OOH NhM^SE) Shang LL enter i!0 3COAlAlV3A HS0JlHiNAJ

HI

:Enter NEH AlS IS Shang: Oi)3AID¾0dVSSS£) [(nio^-(03^tI(¾D)DOOH)-_DN)-₃N]¾9IdXAJJO¾HODAlAlVHAlHSODlH0NAiI 1

HI

People M3 0A1S IS Shang:) 3 Λ Ι ￡) Shang i VWSSSOSO

[(WD-T people-(o w (D) OOH)-_DN)-₃N] Ll people JI9 3D AlA V3AlHS0:nHi)NAd

:6 Π people 1S IS Shang:The Λ Ι ￡ Shang 0dVWSSS9S of δ 3) iKm30:DAlAlV3AlHS£)CnH0NA ( SOK d—DN):!

^:81-Π

:N enters N310A S IS Shang:

AIOliMVW( (^03i-₃N) SSE) S ￡) Shang《I Shang people: Μ£Η30:) Λ enters the ￡ Η of 3 Λ Η of ν 5) Λ:Ι ： LV Π

Ν enters the ζ of Ν 3) people 1SOIS Shang δ Η Λ α SI WVSSSDSOJJO 303AlAlV3AlHS031H A [(niO-l-^ (03^H(¾3)300H)-_I)N)-_DM]j

:91-11

'.[(η₁ο-λ-(θ3 (¾3)300Η)-„Ν)-₃Ν]-

：People N3li) 0 03 Λ Ι Ο Shang i of AlS IS Shang) dW

[Ο-people-(and 0 " () OOH)-_DN)-₃N] SSE>SE) people ￡>Ή3ΕΌΛΊΑ ν3Λ1Η5Ε>:Π Η) Ν Λ Ι enter l^I^mS IS Shang

Shang 0dVS [(niO- people-(0;D^w(¾ ) OOH)-_DN)-₃N] 3 ￡) 3 Κ ^ of S9 Shang people, 3 ￡ Λ people lV3AlHS ￡>CHHi)MAd

•·ει-π

：N enters the Λ Ι Ο Shang tWWV of N310A1S IS Shang 0 03

[(nio-A-(OD (¾D)300H)-_DN)-₃N]¾SSOSOJJ9¾HDDAlAlV3AlHS031H0NAj is

00Z.100/J10ZM3/X3d 98Z.980/£10Z OAV 11st, acceptable carrier in a kind of pharmaceutical composition, including compound and pharmaceutics described in claim any one of 1-10.

12nd, the pharmaceutical composition according to claim 11, further comprises Semilente Insulin or insulin analog and/or adds power mouthful agent.

13rd, application of the compound according to claim any one of 1-10 in the medicine for the treatment of diabetes or hyperglycemia is prepared.

14th, a kind of method for treating diabetes or hyperglycemia, including the compound according to claim any one of 1-10 or the pharmaceutical composition described in claim any one of 11-12 are applied to the sufferer of needs.