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CN104011070A - Compound And Composition Having Hypoglycemic Effect And Use Thereof - Google Patents

Compound And Composition Having Hypoglycemic Effect And Use Thereof Download PDF

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CN104011070A
CN104011070A CN201280061410.6A CN201280061410A CN104011070A CN 104011070 A CN104011070 A CN 104011070A CN 201280061410 A CN201280061410 A CN 201280061410A CN 104011070 A CN104011070 A CN 104011070A
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秦树林
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Adedian Beijing Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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 the effect of p bars blood glucose 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 by the insulin to people and obtain, including Semilente Insulin and protamine zine insulin.
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 go out with zinc ion or ^ nucleoprotamine formation crystal suspension, 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 is connected to B29 lysines.It is slow that insulin detemir absorbs Slow after injection.Extinction time T at injection50%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 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 all 4 is blunt 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.It is disputable always that sweet smart pancreas is used for a long time because the generation of insulin-like growth factor-1 receptor and kinds cancer is closely related Whether island element can increase the cancered risk of patient.Bioactivity of the insulin detemir in human body is about the 20% of natural human insulin, therefore its dosage is 5 times of insulin regular dosage, and 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）, the kind water solubility of text, individual difference when 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.
First purpose of the present invention is to provide a kind of compound with hypoglycemic effect, and the compound includes A chains and B chains, wherein,
The amino acid sequence of A chains is： X_1 GIVDX5C[3]C[4]X8X9X10Ct5]X12LRRLEX18YC[6]X21X22, B chain amino acid sequences are：
23-26X27LC [！] GAX32LVDALX38X3 VC[2]GDX44GFX47X48X4 50X5lX52 53 ' wherein,
I is lysine, arginine or missing；It is lysine, arginine or missing； X5It is glutamic acid, asparagine, glutamine or serine； X8It is histidine, arginine, phenylalanine or threonine； X9It is arginine or serine； X10It is serine or isoleucine； X12It is aspartic acid, serine, paddy ammonia barefoot amine or asparagine； X】8It is asparagine, Yue methyllanthionines or threonine； X21It is asparagine, alanine or glycine； X22It is lysine, arginine-lysine dipeptides or missing； X23_26 is phenylalanine-valine-asparagine-glutamin tetrapeptide, or GPE, valine-asparagine-glutamin tripeptides, or proline-glutamicacid, asparagine-glutamin dipeptides, or glutamic acid, glutamine, lysine or arginine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； X27It is histidine or threonine； X32It is histidine, glutamic acid, glutamine, arginine or phenylalanine； X38It is phenylalanine or tryptophan； X39It is phenylalanine or tryptophan；4It is arginine, glutamic acid, aspartic acid or alanine；7 be phenylalanine, tyrosine or histidine；8 be-NH2、 dA-NH2, phenylalanine or tyrosine or missing；X49 is asparagine, aspartic acid, glutamic acid, threonine or missing； X5GIt is lysine, proline, arginine, aspartic acid, glutamic acid or missing； X51It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X52It is threonine or missing； X53Be glutamic acid, aspartic acid, Glu-Glu, Asp-Asp dipeptides or Missing；
In the compound, [1]-[6] represent the numbering of cysteine；Key is dredged by 63 pair two of cysteine formation in the compound, wherein A chains and B chains is connected by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, and the particular location of three pairs of disulfide bond is： C]And C[4]Form two ^ ^ keys, C [2]And C[6]Form disulfide bond, C[3]And C[5]Form disulfide bond.
In second aspect, the present invention provide it is a kind of it is single-stranded, can be combined with insulin receptor, the compound with hypoglycemic effect, the amino acid sequence structure of the compound is：
XioiaLC [！] GAX1o1bL DALXlolcX1oid C[2]GDRGFX1oie io2Xio3Xio4X[05Xio6-CL-GIVDQC[3] CwX^RSCwSLRRLENYCwX X, wherein,
Xioia is GPE-threonine, GPE-histidine tetrapeptide or benzene Two propylhomoserins-valine-asparagine-glutamin-histidine pentapeptide, or with the sequence after any of the GPE or phenylalanine-valine-asparagine-glutamin in lysine or the above-mentioned tetrapeptide of arginine substitution or pentapeptide amino acid residue；Xioib is histidine, glutamic acid, glutamine, arginine or phenylalanine； X101cIt is phenylalanine or tryptophan； X101dIt is phenylalanine or tryptophan； Xl()leIt is phenylalanine, tyrosine or histidine； X102It is phenylalanine or missing； X1MIt is asparagine or missing； X1MIt is lysine, proline or missing； X1Q5It is proline, lysine or missing； X1()6It is threonine or missing； X1()7It is phenylalanine, arginine or histidine； X1Q8It is alanine, glycine or asparagine； X109It is lysine, arginine-lysine dipeptides or missing；CL is the junction fragment being defined herein.
At the 3rd 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 alpha-amido of Ν-terminal amino acid residue of the alpha-amido for modifying the -terminal amino acid residue for being the B chains that modification side chain is connected to the double chain compound of the present invention or single chain compound, or it is connected to the epsilon-amino of lysine present in the double-strand or single chain compound of the present invention.
In one embodiment, the compound includes Α chains and Β chains, wherein,
The amino acid sequence of A chains is：
X399X400GIVDX405C[3]C[4]X408X409 410C[5]X412LX414X415LX417X418YC[6]X421X422)
The amino acid sequence of B chains is：
X423-426 427LC[1]GAHLVDALX438X439 C[2]GDRGFX447X448X449X450X45lX452X453X454X45 ;In the compound, [1]-[6] represent the numbering of cysteine；The compound is connected by 6 cysteine 3 pairs of disulfide bond of formation, wherein A chains and B chains by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is：C [,] and C[4]Formation disulfide bond, C [2] and C[6]Form two and dredge key, C[3] and C[5]Form Er Liu Key.Wherein,
X399 be lysine, arginine or missing；X4CG is lysine, arginine or missing；05 is glutamic acid, asparagine, glutamine or serine； 08It is histidine, arginine, phenylalanine, threonine or formula（I) structure；09It is arginine, serine or formula（I) structure；It is serine, isoleucine or formula（I) structure；12It is aspartic acid, serine, glutamine, asparagine or formula（I) structure；14It is arginine or formula（I) structure；15It is arginine or formula（I) structure；17It is glutamic acid or formula（I) structure；18It is asparagine or formula（I) structure；21 be alanine, glycine or asparagine；22It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；When22During for dipeptides, one of amino acid is formula（I) structure；23_ 426 be Gly-Pro
- glutamic acid tripeptides, UL- GPE, phenylalanine-valine-asparagine-glutamin tetrapeptide or UL- phenylalanine-valine-asparagine-glutamin；27 be histidine or threonine；38It is phenylalanine or tryptophan；39 be phenylalanine or tryptophan；47It is phenylalanine, histidine or tyrosine；48It is-NH2, phenylalanine, tyrosine or missing；49 be asparagine, threonine, glutamic acid, aspartic acid or missing；50 be lysine, arginine, paddy Propylhomoserin, aspartic acid, proline or missing；51It is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or is formula（I) structure；52It is threonine, lysine or missing, or is formula（I) structure；53It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；54It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；55It is lysine or missing, or is formula（I) structure； ULAnd formula（I) the structure such as present invention is as defined below.
In another embodiment, the compound is single-stranded structure, and amino acid sequence structure is：
X201aLC [！] GAX201 bLVDALX201 CX201 dVC[2]GDRGFX201 eX202 2o3X204 205 206GX207X2o7a 208X
209X210X2uX212 213X21 215 216GIVDQC[3]C[4]X2i7RSC[5]X2i8LX2i9X220LX22iX222YC [6] X223X224 ' wherein,
X201aBe GPE-Soviet Union's ammonia look askance at, GPE-Group propylhomoserins, phenylalanine-valine
- asparagine-glutamin-histidine, UL- GPEs-threonine, UL- GPEs-histidine or UL- phenylalanines-valine-asparagine-glutamin-histidine； X2QIb is histidine, glutamic acid, glutamine, arginine or phenylalanine； X20k is phenylalanine or tryptophan； X201D is phenylalanine or tryptophan； X201e is phenylalanine, tyrosine or histidine； X2o2It is phenylalanine, tyrosine or missing； Χ3It is asparagine, threonine, aspartic acid, glutamic acid or missing； X2Q4It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X2Q5It is proline, lysine, arginine, aspartic acid, glutamic acid or missing or formula（I) structure； X2.6 be threonine, lysine or missing or formula（I) structure； X2Q7It is serine, alanine, glycine, formula（I) structure or missing； X207aIt is serine, alanine, glycine, formula（I) structure or missing； X2。8It is serine, formula（I) structure or missing； X209It is serine, formula（I) structure or missing； Χ21()It is serine, formula（I) structure or missing； X211It is arginine, alanine, glycine, formula（I) structure or missing； X212It is arginine, alanine, glycine, formula（I) structure or missing； X213It is alanine, proline, arginine, glycine, formula（I) structure or missing； x214It is proline, glutamine, glycine, formula（I) structure or missing； X215It is glutamine, threonine, glycine, formula（I) structure or missing； X216It is threonine, arginine, lysine, formula（I) structure or missing； x217It is phenylalanine, arginine, histidine or formula（I) structure； X218It is aspartic acid, serine, glutamine, asparagine or formula（I) structure； X2I9 is arginine or formula（I) structure； x22QIt is arginine or formula（I) structure； x221It is glutamic acid or formula（I) structure； X222It is asparagine or formula（I) structure； X223It is alanine, glycine or asparagine； X224 be lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；When22During for dipeptides, one of amino acid is logical formula (I) structure； ULAnd formula（I) structure is as the present invention is defined hereinafter.
The fourth 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, described pharmaceutical composition further comprises Insulin Asp；The Insulin Asp can be AspB28Actrapid monotard, LysB28Pro B29Actrapid monotard or LysB3GluB29Actrapid monotard.
The fifth 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 sixth 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, the high activity with bound insulin acceptor, low to the toxic action of individual, 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 three kinds of various doses III-1 compounds of the invention after blood glucose change over time value；
Fig. 2 be mouse subcutaneous injection physiological saline, actrapid monotard and the present invention III -12 compounds after blood glucose change over time value；Fig. 3 be mouse subcutaneous injection physiological saline, actrapid monotard and the present invention Π Ι -7 compounds after blood glucose change over time value.Embodiment
Determine 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 ot- amino acid are connected on same carbon atom（α carbon).α carbon can have 1-2 organic substituent.Amino acid includes L and dexiroisomer and racemic mixture.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 amino 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；
α-hGlu are-HNCH (CO-) CH2CH2CH2COOH L isomers；
δ-hGlu are a HNCH (COOH) CH2CH2C CO- L isomers；
A-Asp for-HNCH (CO-) C COOH L isomers；
β-Asp are-HNCH (COOH) CH2CO- L isomers；
A-Glu is a 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 write a Chinese character in simplified form
" natural insulin " refers to the mammalian islet element (such as actrapid monotard, bovine insulin, pork insulin) 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 Al l.B7, A7 refer to the amino acid residue of natural insulin B chains position 7 (from N-terminal number) and the amino acid residue of INSULIN A chain position 7 (from N-terminal number).
" 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 " has part, whole or the enhancing activity of natural insulin, or the polypeptide of the part with natural insulin, whole or enhancing activity can be converted into 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.
IGF refers to IGF（Insulin-like growth factor), including insulin-like growth factor-i (IGF-1) and insulin-like growth factor-2 (IGF-2).
The sequence of people IGF-1 A chains is SEQ ID NO:Sequence shown in 1, people IGF-1 B chain-orderings are SEQ ID NO:Sequence shown in 2.IGF-1 analogs have one or more amino acid mutation, substitution, missing relative to natural human IGF-1 molecules or added.IGF-1 analogs include double-strand and single-stranded IGF-1 analogs.
IGF-2 analogs have the sour residue mutations of one or more multiple atmosphere bases, substitution, missing relative to natural human IGF-2 molecules or added.IGF-2 analogs include view chain and single-stranded IGF-2 analogs.
Unless otherwise specified, the insulin being related in the application refers to actrapid monotard, and IGF-1 refers to the IGF-1 of people.
The amino acid number rule of compound:
In present specification, unless specifically indicated, when being related to double chain compound, the A chains of double chain compound and the numbering of B chains defer to following principle：
The A chains of the compound are from (the X of numbering 1！Or) start to (the X of numbering 2222Or22), in addition to the site changed in the application, preceding 21 amino acids of compound A chains are corresponding with the amino acid of people IGF-1 A chains, particularly (the X of compound A chains the 6th6Or06 )、 7 ( 7Or07), 11 (X " oru) and 20 (Χ2.Or20) position is cysteine.If the N-terminal in X adds amino acid residue, then the numbering of amino acid residue is：With；If01's>Add amino acid residue in 1 end, then the numbering of amino acid residue is X399With00。
The B chains of the compound are from (the X of numbering 2323Or23) start, in addition to the site changed in the application, (the X of numbering 28 of compound B chains28Or28) to numbering 47 (X47 or 47) amino acid it is corresponding with the amino acid of 5 to 24 of people IGF-1 B chains, be particularly compound B chains the (X of numbering 2929Or 29), 41 (X4i or X441) positions be cysteine.
When individually referring to some amino acid, it can be represented with such as A1G, BIG or B9H, it is 0, G, H respectively that it refers in first amino acid of A chains, the first of B chains and the 9th amino acid residue respectively.
The numbering of single chain compound is defined according to the explanation of each compound.
Single chain compound is referred to general structure B chains-CLThe peptide sequence of-A chains or the peptide sequence of modification, wherein B chains are IGF-1 B chains or the like, and A chains are IGF-1 A chains or the like, CLIt is the peptide chain for connecting B chain C-terminal amino acid residues and A chain N-terminals.
Unless otherwise specified, with A chains or the amino acid of B chain position descriptions in the application, such as A14, B28 represent and IGF-1 A chains or the amino acid of B chain opposite positions or its change that wherein IGF-1 A chains or the numbering of B chains are since 1.
The numbering of cysteine in compound:
For convenience of describing, the cysteine in each compound in the present invention is numbered, respectively Cn] ~C[61, its correspondence Be：
When the compound is double-strand, Cn] and.[2]Correspond to respectively in the B chains of double chain compound from N-terminal to the two of C-terminal cysteines；3]~ [6]Be corresponding in turn in A chains from N-terminal to the 4 of C-terminal cysteines, i.e., respectively corresponding A chain numbering be 6, the cysteine of 7,11 and 20；
When the compound is single-stranded, Cn]~ 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 IGF-1, and IGF-1 and IGF-2 forms disulfide bond in an identical manner with insulin.Disulfide bond is all included in the tertiary structure of the compound of any one in the present invention, all single-stranded IGF-1 analogs and double-strand IGF-1 analogs have three pairs of disulfide bond that mode is equally constituted with insulin.Therefore, 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 "
IGF-1 analogs can include one or more modification groups.Modification group can provide the feature of IGF-1 analogs needs.For example, modification group can reduce IGF-1 analogs under circumstances（Such as alimentary canal, blood）Degradation rate.It is preferred that modification group be that those allow IGF-1 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 IGF-1 analogs than the non-modified lower lipophilicity of analog, the group of more highly-water-soluble.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.
PAG（Polyalkylene Glycol, PAG) group 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-(CH2C¾0)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- Yue base -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 the peg molecule that molecular weight is 20,000.
The PEG other ends are typically activating functional group or are easily formed the functional group of covalent bond, such as amino, carboxyl, hydroxyl, sulfydryl, aldehyde.PEG- maleimides, PEG- vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan)s and PEG- iodoacteyls（CO-CH2- I) etc. can react to form stable covalent bond with the coloured glaze base-SH of cysteine side chain；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 season Penta tetrol, 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 is usually polydispersion, and polydispersity index is less than 1.05.PEG group can also be single ^ ^.Single ^^, 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, such as lactic-co-glycolic acid are manifested during degraded（Poly (lactide- Co- glycolide)), polyanhydride, poe be also all bioadhesive polymer.These polymer can deliver IGF-1 analogs and arrive intestines and stomach.The carboxyl being exposed during depolymerization can be firmly attached to intestines and stomach, assist to deliver IGF-1 analogs.
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 are not limited to：Alkyl, alkenyl, alkynyl, aryl, aryl alkyl, alkylaryl, aliphatic acid, cholesterine and lipophilicity 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 it is) tactful
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 IGF-1 analogs 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, IGF-1 conjugates are unijunction compounds.In another embodiment, IGF-1 conjugates are many conjugates.Another In individual embodiment, IGF-1 conjugates are the mixtures of unijunction compound, binode compound, three conjugates, four conjugates etc..Modification group can be with identical, can also be different.When IGF-1 conjugates have multiple modification groups, one or more modification groups are connected preferably by hydrolyzable bond with IGF-1 and other one or more modification groups are connected preferably by non-hydrolysable key with IGF-1 conjugates.Or, all modification groups are all connected by hydrolyzable bond with IGF-1, but the hydrolysis rate faster or slower of each modification group in vivo.
It is preferably to make conjugate that there is the part or all of bioactivity of original IGF-1 analogs with reference to strategy.It is preferred that the B 1-N ends of binding site including double-chain polypeptides, B chains lysine side chain amino groups or single-stranded N-terminal, lysine side chain amino groups etc..B1 unijunctions compound and B chain binode compounds are the most frequently used.It can introduce the natural or alpha-non-natural amino acid with amino or coloured glaze base to create other binding sites by the junction fragment in single chain compound or double-chain polypeptides A chains, B chains in addition.
Modification group can pass through hydrolyzable bond with IGF-1 analogs（Such as ester, carbonic acid, hydrolyzable amino Yue acid esters）With reference to.Hydrolyzable bond makes IGF-1 conjugates have the effect of prodrug.If it is desired to modification group and the no activity of IGF-1 conjugates, such as the binding site of modification group is in IGF-1 analogs 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 IGF-1 conjugates, so as to discharge active IGF-1 analogs, the effect that delay release or Slow are released can be provided using hydrolyzable bond.
In one embodiment, IGF-1 analogs pass through non-hydrolytic key（Such as amido link , Mi keys）It is connected with modification group.If necessary, non-hydrolytic key helps to extend circulation time of the IGF-1 conjugates in blood plasma.
IGF-1 homologues 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 IGF-1 analog A chains, B chain N- ends all have nucleophilic amino.IGF-1 homologues 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 low Kidney clearance rates can be dropped with aobvious write by removing critical point more than kidney, 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 coloured glaze base albumin in blood vessel（Mercaptalbumin), the side chain thiol of its cysteine -34 is activity most strong coloured glaze base in blood plasma.IGF-1 analogs can react generation IGF-1-albumin conjugates by linker of the one end with activating functional groups such as maleimides.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 IGF-1 analogs 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 SrtAStAph) 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.
It is another related<»re/tococa« ;7j oge " es sortase can receive the nucleophilic group being made up of two alanine, but ＆ aureus enzymes can not.This sortase (SrtAstrep) cut-out LPXTA motifs in peptide bond Thr-Ala, it is allowed to the nucleophilic group based on alanine.SrtAstep can also recognize LPXTG motifs, but activity is relatively low.LPXTA motifs will not be by SrtAStaphCut-out.For the sake of simplicity, following methods are discussed with SrtAStaphExemplified by, but SrtAstrepDeng isotype can also with identical or Similar method.
SrtA is repeated to the glycine of LPXTG motifs and N-terminal with free amino group（ a-Glyn) 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 real face, 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-GlynThe molecule of fusion can turn into SrtA substrate.How part is referred to pertinent literature (such as Tsukiji, " Sortase-Mediated Ligation for specific method and reaction: 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 IGF-1 analogs, and specific plan is occupied depending on the structure of IGF-1 analogs.For double-strand IGF-1 analogs, the binding site of general 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, then the N-terminal of IGF-1 B chains is preferably introduced multiple glycine, such as GGGGG-IGF-1 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, IGF-1 A-LPATGGGGG or IGF-1 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-stranded IGF-1 analogs, 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.Double chain compound
Insulin-like growth factor-i (IGF-1) is mainly generated by liver, the single chain protein being made up of 70 amino acid, with four domains（Claim in some documents " chain,：).The A chains and B chain structures in wherein A and B domains and insulin are homologous；And C domains connect A and B domains, the C peptides with proinsulin are corresponding；D domains are the C-terminal extensions in A domains.IGF-1 three-dimensional structure has had the report of NMR and X-ray method for crystallising.The NMR structures of its B domain and the structure in A domains and the crystalline texture of insulin and proinsulin are closely similar.
Insulin Receptor Family includes insulin receptor（IR), insulin-like growth factor-i acceptor（IGF-1R) and insulin receptor associated receptor, they are all receptor tyrosine kinases, and signal is transmitted by increasing phosphoric acid molecules on specific tyrosine.The member of Insulin Receptor Family is made up of two acceptor portions, and each includes the β subunits of extracellular α subunits and cross-film, passes through disulfide bond.When not combining aglucon, this receptor exists with dimeric forms, forms the beta 2 receptors of α 2 together by the disulfide-bonded between two α subunits.Depending on the region being compared, the sequence similarity of insulin receptor and IGF-1 acceptors is 41-84%.
The ability of IGF-1 bound insulin acceptors is lower than insulin 100 times, is not inconsistent with the practical capacity of IGF-1 induced insulin receptor phosphorylation and hypoglycemia in vivo（IGF-1 activation insulin receptor ability be about insulin 10%).Therefore part signal conduction may be by IGF-1 acceptors/insulin receptor heterodimer.
Almost each cell in human body is influenceed by IGF-1, and particularly muscle, cartilage, bone, liver, Kidney be dirty, neural, cell in skin and lung.Except insulin-like effects, IGF-1 can equally adjust cell growth and development, particularly nerve cell, and the DNA of cell is synthesized.
About 98% IGF-1 always with six kinds of IGF-1 associated proteins（One of) IGF-1-BP combine.Wherein IGF-1 Β Ρ 3 Quantity is maximum, and the combination mol ratio for combining 80% IGF-U IGF-1 and IGF-1 BP-3 is 1:1.
IGF-1 is similar with the metabolic response of insulin：Two kinds of hormones increase glucose absorption and oxidation in a very similar way, suppress glucose production, free fatty acids level and fat oxidation speed.
Insulin resistance is made up of many common and several uncommon clinical symptoms.The patient for having mutation in insulin receptor gene or the gene related from signal transduction path has different phenotypes, including lipodystrophy, partial lipodystrophy, A pattern synthesis disease, false acromegaloidism, short evil spirit's looks syndrome and the Rabson-Mendenhall syndromes of the insulin resistance being mutated with insulin receptor gene.In many such sufferers, hyperglycemia is quite difficult to treat, because insulin is invalid.
Insulin resistance is same common in the sufferer of adult-onset diabetes.Human body overcomes this problem by increasing insulin secretion, but causes the Expression of Insulin Receptor on target tissue to reduce, is degrading insulin resistance.Hypertriglyceridemia is that cometabolism is abnormal in these sufferers.Therefore, the treatment specific to insulin resistance can be obviously improved treating diabetes effect.
IGF-1 has been proposed for the treatment of severe insulin Antagonism.Because its biological action and insulin type are seemingly, it is possible to avoid the physical function defect for hindering insulin to play a role.Have found intravenous injection restructuring IGF-1 reduce two have insulin resistance A pattern synthesis diseases sufferer and one have Rabson-Mendenhall syndromes child blood glucose and serum insulin concentration.In the research of the sufferer of several severe insulin Antagonisms with different phenotypes, fasting and 24 hourly average serum insulin concentrations are reduced using IGF-1, glucose tolerance is improved, increases insulin sensitivity and reduces fasting serum triglyceride concentrations.
About 60% diabetic can cause hand or pin stolidity or having tingle by neurotrosis in life.Similar neurotrosis may weaken control of the human body to blood pressure, cause incontinence or impotence, or trigger diarrhoea or constipation.Diabetes rat research shows that neurotrosis caused by this paradiabetes can be treated by IGF-1 to recover.According to conjecture, diabetes prevent nerve cell from being unable to normal development, and IGF-1 is allowed to recover normal.
But, IGF-1 beneficial effect has cost.IGF-1 may cause cardiovascular vigorous reaction, including the rhythm of the heart to stop and low blood pressure.Some reactions are probably because IGF-1 causes blood phosphate drastically very few.IGF-1, which is subcutaneously injected, in long-term, high-dose can produce uncomfortable temporomandibular joint, face and hand edema, increased weight, expiratory dyspnea, sinus tachycardia.But studies have shown that most recently can be resistant to very well and effective dosage can be accomplished.But the best way is exactly to change molecular structure, adhesion of the IGF-1 analogs in insulin receptor is improved, while reducing its activity on IGF-1 acceptors.
The further advantage of IGF-1 analogs includes, but are not limited to, water solubility more more preferable than actrapid monotard, and higher yield develops potentiality of double agonists of insulin receptor and IGF-1 acceptors etc..
Inventor is through the discovery that studies for a long period of time, IGF-1 A chains and B chains are connected in the way of insulin is connected, and replace with the B15 amino acids residue of B chains after W or F by Q, such IGF-1 analogs are suitable with insulin receptor binding ability and actrapid monotard.
Further, the IGF-1 analogs of the invention highly combined with insulin receptor are that amino acid residue of the IGF-1 B chains on two positions of B15 and B16 is substituted.After the QF on former B15 and B16 positions is replaced by FF, WF or WW, the IGF-1 analogs newly obtained also show the performance combined with insulin receptor suitable with natural insulin.The ability that part is combined with IGF-1 associated proteins is also maintained by FF, WF or WW IGF-1 analogs replaced.The protein-bonded this abilities of IGF-1 analog combinations IGF-1 are believed to be helpful in the time that extension IGF-1 analogs are circulated and acted in serum.
Two chain analogs including people's IGF-1 A chains and actrapid monotard's B chains are tested in insulin-like activity（Such as fat generation）In show equivalent to insulin ability about 40%, but growth factor experiment（Such as thymidine incorporation）In, activity is substantially higher than insulin, equivalent to about 730%.But, the compound is than IGF-1 poorly efficient growth factors in itself, equivalent to IGF-1 About 26.5% although it have been demonstrated that IGF-1 C domains are the principal elements of IGF-1 acceptor binding forces, but the structural property that the two chain analogs including people's IGF-1 A chains and actrapid monotard's B chains show to include in IGF-1 acceptor binding forces weaken but still obvious, the A domains for showing IGF-1 causes elevated growth promotion ability.And multiple studies have shown that, too active IGF-1 acceptors may cause cancer.
Insulin receptor has two hypotypes：IR-A and IR-B.IR-A has the growth promoting function similar to IGF-1 acceptors, and IR-B major function is Metabolism regulation（Such as glycometabolism).Natural human insulin is substantially suitable with the binding ability of two receptor subtypes.And IGF-1 and IGF-2 is significantly higher than the binding ability to IR-B to IR-A binding ability.IGF-1 analogs still partly maintain this selectivity.And preferably IGF-1 analogs should be able to highly be combined with insulin receptor, but the ability combined with IGF-1 acceptors is relatively low.In addition, IGF-1 analogs should have bioactivity in a balanced way on insulin receptor two hypotypes IR-A and IR-B.
IGF-1 A chains and INSULIN A chain have the sequence homology of height.Two significant differences in the sequence of INSULIN A chain and IGF-1 A chains are A5Gln-A5Glu and A12Ser-A12Asp.These residues are conservative in IGF-1 and IGF-2.These changes in sequence are related to the change between the acidic residues in tral residue and IGF-1 in insulin.Inventor has found that A5 glutamic acid of A chains and the Asp side chain carboxyl of A12 are the keys for determining receptor-selective.A5 glutamic acid are converted into glutamine, asparagine or serine, A12 aspartic acids are converted into serine, glutamine or asparagine etc. approximate size and polarity, but the amino acid residue of the side chain without negatively charged functional group, thus obtained analog has more preferable receptor-selective.A5 and A10 replaces the single substitution effect than alternative one to become apparent from simultaneously.
Based on above inventive concept, the present invention provides a kind of compound with hypoglycemic effect, and the compound includes A chains and B chains, wherein,
The amino of A chains is classified as： X^XoGrVDXsCCTCwXsXsXioCwXnLRRLEXwYCwX Xss ,
B chain amino acid sequences are：
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFX47X48 X49 X50 X51 X52 X53, wherein,！It is lysine, arginine or missing；It is lysine, arginine or missing； X5It is glutamic acid, asparagine, glutamine or serine； X8It is histidine, arginine, phenylalanine or threonine； X9It is arginine or serine； X10It is serine or isoleucine； X12It is aspartic acid, serine, glutamine or asparagine； X18It is asparagine, Yue methyllanthionines or threonine； X21It is asparagine, alanine or glycine； X22It is lysine, arginine-lysine dipeptides or missing；X23-26 is phenylalanine-valine-asparagine-glutamin tetrapeptide, or GPE, valine-asparagine-glutamin tripeptides, or proline-glutamicacid, asparagine-glutamin dipeptides, or glutamic acid, glutamine, lysine or arginine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； X27It is histidine or threonine； X32It is histidine, glutamic acid, glutamine, arginine or phenylalanine； x38It is phenylalanine or tryptophan； x39It is phenylalanine or tryptophan；4It is arginine, glutamic acid, aspartic acid or alanine；7It is phenylalanine, tyrosine or histidine；8 -NH2、 dA-NH2, phenylalanine or tyrosine or missing；9It is asparagine, aspartic acid, glutamic acid, threonine or missing； X5GIt is lysine, proline, arginine, glutamic acid, aspartic acid or missing； X51It is proline, lysine, arginine, glutamic acid, aspartic acid or missing； X52It is threonine or missing； X53It is glutamic acid, aspartic acid, Glu-Glu, Asp-Asp dipeptides or missing；
In the compound, [1]-[6] represent the numbering of cysteine；By 6 cysteine 3 pairs of disulfide bond of formation in the compound, wherein A chains and B chains is connected by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is： Cfl]And C[4]Form disulfide bond, C[2]And C[61Form disulfide bond, C[3] and C[5]Form disulfide bond.
It is important to note that X32、 4、 9、 X50、 X51Amino acid residue whether be related to compound as people's pancreas islet Element is equally produced from connection（ 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 (" Monomeric insulins and their experimental and clinical implications " Diabetes Care such as reference Brange after hypodermic injection, the No.9 of Vol 13,923-54,1990).If X32It is histidine, then be conducive to compound to form six dimeric structures under the assistance of zinc ion.If X32It is the amino acid residues such as aspartic acid, glutamic acid, phenylalanine, glutamine, arginine, then can not forms six stable dimeric structures.If4、 X5o、 51Amino acid residue Deng site is aspartic acid or glutamic acid, is not easy to form stable connection certainly.Therefore, if X32It is non-histidine amino acid residue, or X44, X50、 X51One or more sites Deng the amino acid residue in site are aspartic acid or glutamic acid, then respective compound is easier to, with dimer or monomeric form presence, blood be rapidly entered after hypodermic injection, can reach reduces the effect of blood glucose in a short time.
In a preferred embodiment of present aspect, the compound with hypoglycemic effect includes A chains and B chains, wherein, the amino ^^ of A chains is classified as： GIVDX5C[3]C[4]X8RSC[51X12L RLEX18YC[6]X21X22,
B chain amino acid sequences are：
X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFYX48X49 X50 X51 X52, wherein,
X5It is glutamic acid, asparagine, glutamine or serine； X8It is histidine, arginine or phenylalanine；12It is aspartic acid, serine, glutamine or asparagine； x18It is asparagine, Yue methyllanthionines or threonine； x21It is asparagine, alanine or glycine； x22It is lysine, arginine-lysine dipeptides or missing； X2 6It is Gly-Pro-glutamic acid tripeptides or phenylalanine-valine-asparagine-glutamin tetrapeptide； x27It is histidine or threonine； x32It is histidine, glutamic acid, glutamine, arginine or phenylalanine； x38It is phenylalanine or tryptophan； x39It is phenylalanine or tryptophan；4It is arginine, glutamic acid, aspartic acid or alanine；8It is-NH2, dA-N or phenylalanine；9 be asparagine or missing； X5QIt is lysine, proline or missing； x51It is proline, lysine or missing； x52It is threonine or missing；
In the compound, [1]-[6] represent the numbering of cysteine；By 6 cysteine 3 pairs of disulfide bond of formation in the compound, wherein A chains and B chains is connected by two pairs of whetstone keys of interchain two, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is：And C[4] form disulfide bond, C[2] and C[6]Form disulfide bond, C[3]And C[5]Form disulfide bond.
In further preferred embodiment, the sequence of the B chains is：
GPEX27LCGAX32LVDALX38X39VCGDX44GFY-NH2;
In further preferred embodiment, the sequence of the B chains is：
GPEX27LCGAX32LVDALX38X39VCGDX44GFYFNKPT;
In further preferred embodiment, the sequence of the B chains is：
GPEX27LCGAX32LVDALX38X39VCGDX44GFYdA-NH2;
In further preferred embodiment, the sequence of the B chains is：
FVNQX27LCGAX32LVDALX3gX39VCGDX44GFYFNKPT;
In further preferred embodiment, the sequence of the A chains is：
GIYDX5CCX8RSCX12LRRLEX18YCA;
In further preferred embodiment, the sequence of the A chains is：
GIVDX5CCX8RSCXi2LRRLEXi8YCN;
In these further apply mode, the X in A chains5It is glutamic acid, asparagine, glutamine or serine； X8It is histidine, arginine or phenylalanine； X12It is aspartic acid, serine, glutamine or asparagine；18It is asparagine, Yue methyllanthionines or threonine；X in B chains27It is histidine or threonine； X32Be histidine, glutamic acid, paddy ammonia barefoot amine, Arginine or phenylalanine； X38It is phenylalanine or tryptophan； X39It is phenylalanine or tryptophan；4It is arginine, glutamic acid, aspartic acid or alanine.
In this regard, the compound with insulin receptor with high binding capacity includes A chains and B chains, and A chains and B chains are connected by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, is specially： CwAnd C[4]Form two υ keys, C[2]With.[6]Form disulfide bond, C[3]And C[5]Form two stones and fill key；The compound is selected from following double-chain polypeptides：
1-1 :Wherein A chain-orderings are GIVDECCFRSCDLRRLEMYCA (SEQ ID NO: 1 );The sequence of B chains is GPETLCGAELVDALFFVCGDRGFY-NHz (SEQ ID NO:4 );
1-2：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 1；The sequence of B chains is GPETLCGAELVDALWFVCGDRGFY-NH2 ( SEQ ID NO:5 );
1-3 ：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 1；The sequence of B chains is
GPETLCGAELVDALWWVCGDRGFY-NH2 ( SEQ ID NO:6 );
1-4:Wherein A chain-orderings are GIVDECCFRSCDLRRLEMYCN (SEQ ID NO: 7 )；The sequence of B chains is GPETLCGAELVDALFFVCGDRGFYFNKPT (SEQ ID NO:3 );
1-5:Wherein A chain-orderings are GIVDECCFRSCDLR LENYCA (SEQ ID NO: 8 )；The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-6:Wherein A chain-orderings are GIVDECCFRSCDLRRLETYCA (SEQ ID NO:9 )；The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-7:Wherein A chain-orderings are GIVDECCRRSCDLRRLENYCN (SEQ ID NO: 10 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-8:Wherein A chain-orderings are GIVDECCHRSCDLRRLENYCN (SEQ ID NO: 11 );The sequence of B chains is
SEQ ID NO:Sequence shown in 3；
1-9:Wherein A chain-orderings are GIVDQCCFRSCDLRRLENYCA (SEQ ID NO: 12 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-10:Wherein A chain-orderings are GIVDNCCFRSCDLRRLENYCA (SEQ ID NO: 13 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-11 :Wherein A chain-orderings are GIVDECCFRSCSLRRLENYCA (SEQ ID NO: 14 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-12:Wherein A chain-orderings are GIVDECCFRSCNLRRLENYCA (SEQ ID NO: 15 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-13:Wherein A chain-orderings are GIVDECCFRSCQLRRLENYCA (SEQ ID NO: 16 );The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-14:Wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 17 )；The sequence of B chains is SEQ ID NO:Sequence shown in 3；
1-15：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPETLCGAHLVDALFFVCGDRGFYF KPT (SEQ ID NO: 18 );
1-16:Wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCAK (SEQ ID NO: 19 );The sequence of B chains is SEQ ID NO:Sequence shown in 18；
1-17:Wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCARK (SEQ ID NO: 20 );The sequence of B chains is SEQ ID NO:Sequence shown in 18；
1-18：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPETLCGAHLVDALFFVCGDRGFYdA-NH2 ( SEQ ID NO:21 );
1-19:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPEHLCGARLVDALFFVCGDRGFYFNKPT (SEQ ID NO:22 );
1-20:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPEHLCGAFLVDALFFVCGDRGFYFNKPT (SEQ ID NO:23 );
1-21 :Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPEHLCGAHLVDALFFVCGDEGFYFNKPT (SEQ ID NO:24 );
1-22:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 8；The sequence of B chains is FVNQHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO:25 );
1-23:Wherein A chain-orderings are GIVDQCCHRSCSLRRLENYCA (SEQ ID NO:26 );The sequence of B chains is SEQ ID NO:Sequence shown in 25；
1-24:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17;The sequence of B chains is SEQ ID NO:Sequence shown in 25;
1-25:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPEHLCGAHLVDALFFVCGDAGFYFNKPT (SEQ ID NO:27 );
1-26:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is GPEHLCGAQLVDALFFVCGDRGFYFNKPT (SEQ ID NO:28 );
1-27:Wherein A chain-orderings are KGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 151 );The sequence of B chains is GPEHLCGAHLVDALFFVCGDRGFYFNKPT (SEQ ID NO: 152 );
1-28:Wherein A chain-orderings are RGIVDQCCFRSCSLRRLENYCA (SEQ ID NO: 153)；The sequence of B chains is SEQ ID NO:Sequence shown in 152；
1-29:Wherein A chain-orderings are KGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:154);The sequence of B chains is GPEHLCGAHLVDALFFVCGDRGFYFNPKT (SEQ ID NO: 155 );
1-30:Wherein A chain-orderings are RGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 156 )；The sequence of B chains is SEQ ID NO:Sequence shown in 155；For SEQ ID NO:Sequence shown in 155；
1-32:Wherein A chain-orderings are KKGIVDQCCHRSCSLRRLENYCN (SEQ ID NO: 158);The sequence of B chains is SEQ ID NO:Sequence shown in 155；
1-33:Wherein A chain-orderings are GIVDQCCHRSCSLRRLENYCN (SEQ ID NO:159);The sequence of B chains is GPEHLCGAHLVDALFFVCGDRGFYFNPKTE (SEQ ID NO: 160 );
1-34:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 159；The sequence of B chains is SEQ ID NO:Sequence shown in 155.Single chain compound
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 ￡.These changes of proinsulin remove C peptides.Remaining B chains and A chains are by together with disulfide-bonded.
The duplex structure of insulin causes insulin to have a variety of conformations.Insulin has the potential of sizable conformation change, right The limitation of these changes significantly reduces affinity of the insulin receptor to part.Closing GlyAl aminoterminal equally weakens 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.Solution is used to cause insulin molecule to be exposed to many factors in insulin administration, 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 to reduce molecular flexibility and be inclined to while reducing fibrillation, limitation or modification pH decanting zones by introducing C peptides between B and A chains.In addition, the main method of the insulin of genetic engineering production at present is produced first by insulin B chain and A chains by the single-chain insulin precursor of small peptide head and the tail connection, two-chain insulin is generated after then insulin precurosor is 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 pancreas element 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.
In the present invention, inventor further design, synthesize and characterize can be combined with insulin receptor, the single-stranded IGF-1 analogs with hypoglycemic effect.6-12 or the junction fragment of 8-12 amino acid are used in the IGF-1 analogs（C chains）The A1 positions of the C-terminal of IGF-1 B chain analogs and IGF-1 A chain analogs are connected, the single-stranded IGF-1 analogs of gained are expressed as B chain-C chain-A chains.Preferable single-stranded IGF-1 analogs should have height adhesion to insulin receptor, realize electrostatic equilibrium；There are excellent thermodynamic stability, no self assembly.
Further, in these IGF-1 analogs, the A chains and B chains are IGF-1 A chains and B chains, or their analog.Based on the Part I of the present invention, the B chains are the amino acid Q (glutamine of B15）It is replaced by F (phenylalanines）Or W (tryptophans）IGF-1 B chain variant.It can strengthen combination rate of insulin receptor in double-strand IGF-1 analogs, improve receptor-selective, increase water-soluble and stability the various amino acid of polypeptide to change, the change in the site such as A5, A12, A18, A22, B9 is also applied for single-stranded IGF-1 analogs.It is used in double-strand IGF-1 analogs Change compound from ability is joined, be allowed to the selection and change of amino acid mainly existed with dimer or monomer, be also applied for single-stranded IGF-1 analogs.
Junction fragment
Inventor is had found under study for action, and the double-strand of IGF-1 molecules 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 CLIt is the peptide sequence of 6-60 amino acid, each of which amino acid residue is all independently selected from glycine, alanine, serine, threonine, proline.Applicable junction fragment CLWith 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.When junction fragment is shorter in length than fan's amino acid number or is 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 methodLIt can be replaced by amino acid residue and inserted comprising 1 or more than 1 aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine. CLCan 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 IGF-1 molecules after modification have different physics, chemistry and biological nature.
According to a kind of embodiment, CLC-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, CLC end amino acids be selected from lysine or arginine.
In a particular embodiment, cLIt 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；
GASPGTSSTSGR ； GSGSSSAAAPQT; GSGSSSAAPQT; GSGSSSAPQT; GSGSSSRRA;
GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR;
GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR ； GPSGTSTSAPGR ； GAGSSSAPQT; SSSSAPSAPSPSRPQR ；
GSGASSPTSPQR ； GSSPATSATPQT ； GAGSSSAPPPSAPSPSRAPGPSAPQR ；
GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR;
GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQ ; GSSPSGRSSSPGR;
GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR； RREAEDGGGPGAGSSQRK； GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK ； SSSAPPPSAPSPSRAPGPSPQR ； SAASSSASSSSASSASAGR ； GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR ; GSGSSSRRAPQT; SSSSAASAASASSSASGR； SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT ; GGPSSGAPPPSPQT ; SSSAPPPSAPSPSRAPQT； GAGPSSGAPPPSPQT； GGGGAPQT; GAGGPSSGAPPPQT； GGPSSGAPPPSPSPSRPGPSPQR ； SSASSASSSSAGR ； SSASSSAASSSASSSASGR ； SSSGAPPPSPSRAPGPSPQR； GSGSASRGR; SSSSAASSASGR； SASASASASSASSGR； SASSPSPSAPSSPSPAS ； GPSSPSPSAPSSPSPASPSSGR； SSSAPPPASPSPSRAPGPQR； SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; SSPSASPSSPASPSSGR; GAPASPAPSAPAPAAPSGR； GPSSPSPSAPSSPSPASPSSAPQT； SSASSASSSSSASAGR； SAPSSPSPSAPSSPSASPSGR ； SSSAPPPSAPSPSAPQR ； GASSPSPSAPSSPSPASGR ； SSPSAPSPSSPASPSSGR; GAGPAAPSAPPAASPAAPSAGR; SSSSPSAPSPSSPASPSPSSAPQR; GSGSSR; GSGSSSAR; GSGSSSGR; GSGAPQR; SSSSAPSAPSPSRAPGPSPAPQR; GSGSSSR; GSGSSAPQT ； GGGGAPQR ； GSGSSSAAR ； GSGSSAAPQ ； SSSSRRAPQR ； SSSGSGSSAPQR; SSGSGSSSAPQR; GSGSSSRS; SSSSRAPQR; GASPGGSSGSGR.
Based on the studies above result, the present invention further provides a kind of single-stranded compound with blood sugar decreasing effect, structure of the compound based on people IGF-1 is transformed, and the structure of the compound is：
XioiaLC [！] GAX101bLVDALXlolcX1o1d C[2]GDRGFX1oleXio2Xi03 i04 i05 io6-CL-Gr DQC[3] C[4]X107RSC[5]SLRRLENYC[6]X108 X109, wherein,
X1()laIt is GPE-threonine, GPE-histidine tetrapeptide or phenylamino acid
- valine-asparagine-glutamin-histidine pentapeptide, or with the sequence after any of the GPE or phenylalanine-valine-asparagine-glutamin in lysine or the above-mentioned tetrapeptide of arginine substitution or pentapeptide amino acid residue； X101bIt is histidine, glutamic acid, glutamine, arginine or phenylalanine； X101cIt is phenylalanine or tryptophan； X101dIt is phenylalanine or tryptophan；Xnne is phenylalanine, tyrosine or histidine； X1()2It is phenylalanine or missing；1()3It is asparagine or missing； X104It is lysine, proline or missing； X1Q5It is proline, lysine or missing； X106It is threonine or missing； X1()7It is phenylalanine, arginine or histidine； X1()8It is alanine, glycine or asparagine； X109It is lysine, arginine-lysine dipeptides or missing；It is to be selected from above-mentioned junction fragment.
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： CwAnd C[4]Form disulfide bond, C[2]With C [6] form disulfide bond, C[3] and C [5] formed two stones fill key.
In a preferred embodiment of present aspect, the structure of the single chain compound is：
X!O!aLCnjGAHLVDALFFVC^!GDRGFYX!o^!oaXi^XiosXioe-CL-GIVDQC^jC^FRSCisjSL RRLENYC[6]A X109, wherein,
X10La is GPE-threonine tetrapeptide or phenylalanine-valine-asparagine-glutamin-histidine pentapeptide； X1() 2 it is phenylalanine or missing； X1Q3It is asparagine or missing； X1Q4It is lysine, proline or missing； Χι05It is proline, lysine or missing； X1Q6It is threonine or missing； X1G9It is lysine, arginine-lysine dipeptides or missing；It is to be selected from above-mentioned junction fragment.
In further embodiment, the structure of the single chain compound is：
GPETLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;
In further embodiment, the structure of the single chain compound is： :π-π
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GPETLCGAHLVDALFFVCGDRGFYSSSSPSAPSPSSPASPSPSSAPQRGIVDQCCFRSCSL RRLENYCA(SEQ ID NO: 146);
11 -119:
GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSRGIVDQCCFRSCSLRRLENYCA
(SEQ ID NO: 147);
11 -120:
GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSARGIVDQCCFRSCSLRRLENYCA
(SEQ ID NO: 148 );
11 -121 :
(SEQ ID NO: 149 )；
11 -122: EQ ID NO: 150 )；
11 -123:
GPEHLCGAHLVDALFFVCGDRGFYFNKGGGGAPQTGIVDQCCHRSCSLRRLENYCN(S EQ ID NO: 161);
11 -124:
NYCN (SEQ ID NO: 162).Modified compound
Chemiluminescent polypeptide man is used for several method to solve the dirty problems quickly removed of drug molecule Bei Kidney that blood plasma middle-molecular-weihydroxyethyl is less than 67kDa.1st, injection site is built in " storehouse "（ depot );2nd, combine to prevent glomerular filtration with non-covalent bond with the carrier protein in blood plasma；3rd, it is connected with carrying agent albumen with covalent bond；4th, combined with macromolecule modification group, such as macromolecule PEG, polysaccharide（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）.Carrier protein molecule amount great Yu Kidney glomerular filtration maximum molecular weights, therefore the dirty removings of Bu Yi Bei Kidney, can be circulated many days in blood plasma.Therefore, the polypeptide combined with carrier protein is difficult by glomerular filtration 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-albumin macromolecule conjugate release Slow of formation is slow；Second, the macromolecule of polypeptide-aliphatic acid-albumin conjugates reduces renal clearance；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 t1/2=14 hours）.And it is unmodified Action time of insulin only have 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 the alpha-amido of -terminal amino acid residue or the alpha-amido for the Ν-terminal amino acid residue for being connected to single chain compound of the invention or the epsilon-amino for being connected to lysine present in the double-strand or single chain compound of the present invention that side chain is connected to the B chains of silent chain compound of the invention.
In one embodiment, the compound is transformed based on IGF-1 analogs, and the compound includes Α chains and Β chains, wherein,
X423-426X427LC[1]GAHLVDALX438X439 C[2]GDRGFX447X448X49 450In compound described in the 55' of X45lX452X453 54, [1]-[6] represent the numbering of cysteine；The compound is connected by 6 cysteine 3 pairs of disulfide bond of formation, wherein A chains and B chains by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is： Ctl]And C[4]Form disulfide bond,.[21With.[6]Disulfide bond is formed, [3]With.[51Disulfide bond is formed, wherein,
X399 be arginine, lysine or missing；X4 is arginine, lysine or missing； 05It is glutamic acid, asparagine, glutamine or serine； 08It is histidine, arginine, phenylalanine, threonine or formula（I) structure, the formula（I) structure is：
09 is arginine, serine or formula（I) structure；10 be histidine, arginine, phenylalanine or formula（I) structure；12It is serine, isoleucine or formula（I) structure；14It is arginine or formula（I) structure；15 be arginine or formula（I) structure；17It is glutamic acid or formula（I) structure；18It is asparagine or formula（I) structure； 21It is alanine, glycine or asparagine；22It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；When22During for dipeptides, one of amino acid is formula（I) structure；23_426It is GPE tripeptides, UL- GPE, phenylalanine-valine-asparagine-glutamin tetrapeptide or UL- phenylalanine-valine-asparagine-glutamin； X427It is histidine or threonine；38 be phenylalanine or tryptophan；39It is phenylalanine or tryptophan；47It is phenylalanine, histidine or tyrosine；X448 is-N, phenylalanine, tyrosine or missing；49 be asparagine, threonine, glutamic acid, aspartic acid or missing； X45GIt is lysine, arginine, glutamic acid, aspartic acid, proline or missing；51It is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or is formula（I) structure；52It is threonine, lysine or missing, or is formula（I) structure；53It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；54It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；55It is lysine or missing, or is formula（I) structure；
ULIt is-W-X-Y-Z structures, aliphatic acid, polyethylene glycol, albumin, Ln-MLStructure, hydrogen atom or Na-( a-(HOOC(CH2)nCO)-Y-Glu)- , Na-(Na-(CH3(CH2) CO)-Y-Glu)-, wherein η is 8-20 integer, such as 8、 10、 12、 14、 16、 20, ΝαThe alpha-amido of amino acid or amino acid residue is represented, or is formula（Π) structure, the formula（Π) structure is：
J is-W-X-Y-Z structures, Ln-MLStructure or hydrogen atom.
Wherein Ln-MLIn structure, MLIt is modification group, including but not limited to-W-X-Y-Z, aliphatic acid, polyethylene glycol, albumin, IgG Fc, glycosyl group etc..
L is optional linker, covalent bond or is not present.Optional linker includes but is not limited to, and 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.Can be-NH- (CH2)n-£0-、 -NH-(CH2CH20)n-CH2-CO- , -NH-(CH2CH20)n-(CH2)r- CO-, n are 1-20 integers, and r is 1-10 integer.In one embodiment, 1^ be->¾-(.¾〇¾0)2-。¾-。0 11-(〇 0120)2- CH2-C0-oIn one embodiment, be-NPHCH m-CKCi^CHzC nHCH na- O-, nl, n2, n3 be respectively 1-16 integer.In one embodiment, Nl, n2 are 1-16 integer respectively.In embodiment of above, the amino formation amido link that L passes through the key from the Suan base carbon underlined and polypeptide compound.The other end and MLForm covalent bond.In one embodiment, L with the amino of polypeptide compound by the key from the base carbon of betraing underlined with forming amido link, and the other end forms amido link with-W-X-Y-Z.
In the present invention ,-W-X-Y-Z structures are：
W is the a-amino acid residue that side chain has carboxyl, the residue with the alpha-amido of a carboxyl and Ν-terminal amino acid residue of the Β chains of double center chain compound of the present invention or with the alpha-amido of Ν-terminal amino acid residue of single chain compound or with it is single-stranded or double-stranded on lysine residue epsilon-amino together with form amide groups；
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 alpha-amido of Ν-terminal amino acid residue of the Β chains of double chain compound by amido link or is connected to the alpha-amido of Ν-terminal amino acid residue of single chain compound or the epsilon-amino for the lysine residue being connected in single-stranded or double-stranded compound, W amino acid residue, which is selected from amino acid residue and side chain with neutral side chain, has the amino acid residue of carboxyl so that W contains the amino acid residue that at least one has carboxyl in side chain；
Or W be Ν-end amino acid of the Β chains from X to double chain compound alpha-amido or to single chain compound Ν-terminal amino acid residue alpha-amido or to double-strand or the covalent bond of the epsilon-amino of the lysine residue of single chain compound；
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, above-mentioned X passes through the key from the carbonyl carbon underlined and the N- terminal aaminos of the B chains of double chain compound or the alpha-amido with the -terminal amino acid residue of single chain compound or residual with the lysine of double-strand or single chain compound The epsilon-amino formation amido link of base；
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 sour residue of the α-atmosphere base encoded by genetic codon.For example, W can be selected from a-Asp, β-Asp, α-Glu or γ-Glu;W other selections are, for example, a-hGlu or S-hGlu.
In another embodiment, the chain that W is made up of two a- amino acid residues, one of a- amino acid residues have 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 residues without free carboxyl group can be the a- amino acid residues of neutral codified.It is according to the W of this embodiment example：A-Asp-Gly, Gly-a-Asp, Jie-Asp-Gly, Gly-p-Asp a-Glu-Gly, Gly-a-Glu, γ-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 residues of codified.Example according to the W of this embodiment is： a-Asp-a-Asp , a-Asp-a-Glu . a-Asp-a-hGlu > a-Asp- -Asp、 a-Asp-y-Glu、 a-Asp-6-hGlu、 β-Asp-a-Asp、 β-Asp-a-Glu、 β-Asp-a-hGlu、 β-Asp-P-Asp、 P-Asp-y-Glu、 P-Asp-5-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、 y-Glu-a-Asp、 γ-Glu-a-Glu» γ-Glu-a-hGlu. y-Glu-P-Asp> y-Glu-y-Glu^ Y-Glu-6-hGlu» a-hGlu-a- Asp、 a-hGlu-a-Glu、 a-hGlu-a-hGlu> a- hGlu-P_Asp、 a-hGlu-y-Glu, a-hGlu-5-hGlu>S-hGlu-a-Asp, δ-hGlu-a-Glu, δ-hGlu-a-hGlu, δ-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, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the a- amino of Ν-end of the Β chains of double chain compound or the a- amino with the N- ends of single chain compound or with the lysine residue in single-stranded or double-stranded compound.
In further embodiment, the X in the side chain-W-X-Y-Z can be formula-CH (COOH) ￡ 0- group, pass through the elephant base formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the a- amino of the N- ends of the B chains of double chain compound or the a- amino with the N- ends of single chain compound or with the lysine residue in double-strand or single chain compound. In further embodiment, the X in Side chains-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, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the alpha-amido of the N- ends of the B chains of double chain compound or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain compound.
In further embodiment, the X in side chain-W-X-Y-Z can be formula-N (CH2CH2COOH) C ￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the alpha-amido of the N- ends of the B chains of double chain compound or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain compound.
In further embodiment, the X in-W-X-Y-Z can be formula-N (CH2COOH) 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, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the alpha-amido of the N- ends of the B chains of double chain compound or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain 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, ε-amino formation amido links of the X by key and the alpha-amido of N- ends of the B chains of double chain compound from the base carbon of betraing underlined or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain compound.
In further embodiment, the X in-W-X-Y-Z can be formula-N (CH2C¾COOH) CH2CH 0- group, passes through the amino formation amido link in the key and W for underlining carbonyl carbon；Or when W is covalent bond, epsilon-amino formation amido links of the X by key and the alpha-amido of N- ends of the B chains of double chain compound from the Talk base carbon underlined or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain compound.
In further embodiment, the X in-W-X-Y-Z can be formula-N (CH2C COOH) CH2CH2CON(CH2CH2COOH) C¾CH2￡ 0- group, passes through the amino formation amido link in key and W from the carbonyl carbon underlined；Or when W is covalent bond, epsilon-amino formation amido links of the X by the key from the carbonyl carbon underlined and the alpha-amido of the N- ends of the B chains of double chain compound or the alpha-amido with Ν-end of single chain compound or with the lysine residue in double-strand or single chain 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 (C COOH)2、 -S03H or-P03H。
In further embodiment, the W in-W-X-Y-Z is a-Asp, β-Asp, α-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-GIu or γ-GIu;- X-Y-Z is-CO (CH2)n, by the formation amido link in the key and W of betraing base carbon underlined, wherein n is the integer in 10-18.
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-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-Α β ρ>A-Glu or γ-Glu;- X-Y-Z is-CO (C)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, the horizontal cholic acid of ox, deoxycholic acid, lithocholic acid）；
In a preferred embodiment, the compound includes A chains and B chains, wherein,
The amino acid sequence of A chains is：
GrVDQC[3]C[4]FRSC[5]X4i2LX414X415LX4i7X4i8Y [6]AX422)
B chain amino acid sequences are：
X423-426 427LC[i]GAHLVDALFFVC[2]GDRGFYX448X44 X45oX45lX452¾53X454X455 '
In the compound, [1]-[6] represent the numbering of cysteine；The compound is connected by 6 cysteine 3 pairs of disulfide bond of formation, wherein A chains and B chains by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is： Cn]With.[4]Formation disulfide bond, C [2]With C [6] disulfide bond is formed, [3]With.[5]Form disulfide bond；
Wherein12For serine or formula（I) structure；14For arginine or formula（I) structure；15It is arginine or formula（I) structure；17For glutamic acid or formula（I) structure；18For asparagine or formula（I) structure；22For lysine, arginine-lysine dipeptides or missing, or it is formula（I) structure；When22During for dipeptides, one of amino acid is formula（I) structure；X42W26 is GPE tripeptides, UL- GPE, phenylalanine-Valerian propylhomoserins-asparagine-glutamin tetrapeptide or Ui phenylalanines-valine-asparagine-glutamin；27 be histidine or threonine；48It is-NH2, phenylalanine or missing；X449 is asparagine or missing；50 is lysine, arginine, glutamic acid, aspartic acid, proline or missing；51It is proline, lysine or missing, or is formula（I) structure；52It is threonine, lysine or missing, or is formula（I) structure；53It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；54It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；55It is lysine or missing, or is formula（I) structure； ULAnd formula（I) structure is as defined in the present invention.
In another embodiment, the compound is to be based on IGF-1Zhang single-stranded structure made, the structure of the compound is：
X2o 1 aLC[！ ]G AX2o njLVD ALX2o 10X201 d VC[2]GDRGFX201 eX202X2o3X204 205X206G¾07X207aX208
209X210X21l 212¾13X214X215X216GIVDQC[3]C[4]X2i7RSC[5]X218LX21 X220LX22iX222YC[6]X223X224, wherein,
X2oiaIt is GPE-threonine, GPE-histidine, 1 propylhomoserin of benzene-valine-asparagine-glutamin-histidine, UL- GPEs-threonine, UL- GPEs-histidine or UL- phenylalanines-valine-asparagine-glutamin-histidine； X2。lbIt is histidine, glutamic acid, glutamine, arginine or phenylalanine； X2olcIt is phenylalanine or tryptophan； X2Oid is phenylalanine or tryptophan； X201eIt is phenylalanine, tyrosine or histidine； X2o2It is phenylalanine, tyrosine or missing； X2o3It is asparagine, threonine, aspartic acid, glutamic acid or missing； X2() 4 is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X2Q5 is proline, lysine, arginine, asparagus fern atmosphere acid, glutamic acid or missing or formula（I) structure； X2o6It is threonine, relies ammonia Acid or missing or formula（I) structure； X2Q7It is serine, alanine, glycine, formula（I) structure or missing； x2()7aIt is serine, alanine, glycine, formula（I) structure or missing； X2G8It is serine, formula（I) structure or missing； x209It is serine, formula（I) structure or missing； x21QIt is serine, formula（I) structure or missing； x211It is arginine, alanine, glycine, formula（I) structure or missing； X212It is arginine, alanine, glycine, formula（I) structure or missing； X213It is alanine, proline, arginine, glycine, formula（I) structure or missing； X214It is proline, glutamine, glycine, formula（I) structure or missing； X215It is glutamine, threonine, glycine, formula（I) structure or missing； X216It is threonine, arginine, lysine, formula（I) structure or missing； x217It is phenylalanine, arginine, histidine or formula（I) structure； X218It is aspartic acid, serine, glutamine, asparagine or formula（I) structure； x219It is arginine or formula（I) structure； X22GIt is arginine or formula（I) structure； X221It is glutamic acid or formula（I) structure； X222It is asparagine or formula（I) structure； x223It is alanine, glycine or asparagine； x224It is lysine, smart atmosphere acid-lysine dipeptides or missing, or is formula（I) structure, works as X224During for dipeptides, one of amino acid is formula
(I) structure； ULAnd formula（I) structure is as defined in the present invention；
In above-claimed cpd structure, [1]-[6] represent the numbering of cysteine.The single chain compound of the present invention dredges key with two in the frame mode formation chain of insulin, is specially in tertiary structure： Cn]And C[4]Form disulfide bond, C[2]And C[6]Form disulfide bond, C[3]And C[5]Form disulfide bond.
In a preferred embodiment, the structure of the single chain compound is：
X201aLC[ I ] G AHLVD ALFF VC[2] GDRGFYX2o2X203 204X205 206GX2o7GX208X2o9X210X2 πΧ2ι2Χ 213X2"X2i5X2i6GrVDQC[3]C[4]FRSC[5]X218LX219X220LX221X222YC[6] A, wherein,
X201aIt is GPE-threonine, phenylalanine-valine-asparagine-glutamin-histidine, 1-GPE-threonine or UL- phenylalanine-valine-asparagine-glutamin-histidine； X2O2 is phenylalanine or missing； X2Q3 is asparagine or missing； X2Q4It is proline, lysine, arginine, aspartic acid or missing； X2Q5It is proline, lysine or formula（I) structure； X2Q6It is threonine, lysine or formula（I) structure； X2O7 is serine, alanine or formula（I) structure； X2()8It is serine or formula（I) structure； X2Q9It is serine or formula（I) structure； X21DIt is serine or formula（I) structure； X2UIt is arginine, alanine, formula（I) structure or missing； x212It is arginine, alanine, glycine, formula（I) structure or missing； X213It is alanine, proline, arginine, logical formula (I) structure or missing； X2I4 is proline, glutamine, formula（I) structure or missing； X215It is glutamine, threonine, formula（I) structure or missing； X216It is threonine, arginine, lysine, formula（I) structure or missing； x218It is serine or formula（I) structure；219It is arginine or formula（I) structure； X22QIt is arginine or formula（I) structure； 221It is glutamic acid or formula（I) structure； X222It is asparagine or formula（I) structure； ULAnd formula（I) structure is as defined herein.
In terms of the compound for the modification transformed based on IGF-1, the compound of modification of the invention is selected from：
III- 1:Duplex structure, including A chains and B chains, wherein, A chain-orderings are SEQ ID NO:Sequence shown in 17；B chain-orderings are G (Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT;
III-2:Duplex structure, including A chains and B chains, wherein, A chain-orderings are SEQ ID NO:Sequence shown in 17；B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPK (NE-PEG 20K);
III-3:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGK(Ne-PEG20K)GSSSR APQTGIVDQCCFR SCSLRRLENYCA;
III-4:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPPTGK(NE-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
III-5:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N£-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
111-6:Duplex structure, including A chains and B chains, wherein, A chain-orderings are： SEQ ID NO:Sequence shown in 17；B chain-orderings are： G(a-CO(C¾)14COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT;
III-7:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-( a- (HOOC(CH2)14CO)-y-Glu)]；
III-8:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[Ns-(Na- (HOOC(CH2)16CO)-y-Glu)];
III-9:Duplex structure, including Α chains and Β chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)12CO)-y-Glu)]；
111-10:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK{NE-[Na- (HOOC(C¾)iiNHCO(CH2)3CO) -γ-Glu]};
III-ll :Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu -N-(Y-G1U)];
III-12:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGK[N£-(Na-(HOOC(CH2)14CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA;
111-13:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[NE-(Na-(HOOC(CH2)14CO)-y-Glu)]SS AAPQTGIVDQCCFRSCSLRRLENYCA;
III -14：Single-stranded structure, its sequence is： GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK
[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)]AAPQTGIVDQCCFRSCSLRRLENYCA;
111-15:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA;
111-16:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[NE-( a-(HOOC(CH2)14 CO)-y-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
III-17:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[NE-(Na-(HOOC (CH2)14CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
111-18:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGD GFYFNPTGSGK[Ne-(Na-(HOOC(CH2)I4CO)-y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA;
III- 19:Joyous chain structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPK [NS- (Na-(HOOC (CH2)14CO)-y-Glu)]； 111-20:Single-stranded structure, its sequence is：
GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA;
III -21 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are： GIVDQCCFRSCSLK[Ne-(Na-(HOOC(CH2)14CO)-y-Glu)]RLENYCA ；B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 163 );
III -22 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are： GIVDQCCFRSCSLRK[NE-(Na-(HOOC(CH2)i4CO)-y-Glu)]LENYCA ；B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 164 );
III -23 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are
GIVDQCCFRSCSLRRLENYCAK[NE-( a-(HOOC(CH2)14CO)-Y-Glu)] ;B chain-orderings are SEQ ID NO:Sequence shown in 164；
III -24 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCARK [N£-(Na-(HOOC(CH2)i4CO)-Y-Glu)], B chain-orderings are SEQ ID NO:Sequence shown in 164；
ΠΙ-25:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK [Ne- (Na-(HOOC(CH2)14CO)-Y-Glu)];
111-26:Duplex structure, including Α chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [Ne-(Na-(HOOC(CH2)i4CO)-Y-Glu)];
111-27:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings be FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK [N (a-(HOOC(CH2)i4CO)-Y-Glu)]；
111-28:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain sequences are bad ' J is GPETLCGAHLVDALFFVCGDRGFYFNPK [NE- (NA- (HOOC(CH2)14CO)-Y-G1U-N-
(γ-Qiu))];
111-29:Duplex structure, including Α chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are G [Na-(Na-(CH3(C¾)14CO)-Y-L-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT;
111-30:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings be G (a-dPEG12- maleimide-albumin） PETLCGAHLVDALFFVCGDRGFYFNKPT;
111-31 :Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPK [NS- (Na-(HOOC(CH2)14CO)-Y-Glu)]；
111-32:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTK [NE- (Na-(HOOC(CH2)14CO)-y-Glu)];
III-33 :Duplex structure, including Α chains and Β chains, wherein A chain-orderings are sequence shown in SEQ ID NO 17, and B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTEK [N£-(Na-(HOOC(CH2)14CO)-Y-Glu)]；
111-34:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK [N (Na-(HOOC(CH2) i4CO)-y-Glu)]；
ΠΙ-35:Single-stranded structure G (Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA;
III-36:Duplex structure, including Α chains and B chains, wherein A chain-orderings are GIVDQCCHRSCSLRRLENYCA, and B chain-orderings are GPEHLCGAHLVDALFFVCGDRGFYFNPK [NE-CO-(CH2CH20)5-(CH2)2- NH- (N"-(HOOC
111-37:Single-stranded structure GPEHLCGAHLVDALFFVCGDRGFYFNPTGK [N£-CO-(CH2CH20)5- (CH2)2-NH-(Na-(HOOC (CH2)16 CO)-y-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA;
NaRepresent the a- amino of amino acid or amino acid residue； ΝεRepresent the epsilon-amino of amino acid or amino acid residue, the epsilon-amino of such as lysine side-chain.
It is specially with the disulfide bond in the frame mode formation chain of insulin in above-mentioned double-strand or the tertiary structure of single chain compound： C[uWith C [41Form two stones and fill key, C[2] and C [6]The thin keys of formation two, C [3][5]Form the numbering of disulfide bond cysteine as defined herein.
The compound with hypoglycemic effect of the present invention can be provided with the composite form for the compound form or zinc 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 Zn2+3 Zn2+Or 4 Zn2+Pharmaceutical composition and purposes
In another aspect of the present invention, there is provided a kind of pharmaceutical composition, described pharmaceutical composition includes acceptable carrier in the compound and pharmaceutics according to the present invention of therapeutically effective amount, for treating type 1 diabetes, diabetes B and causing other situations of hyperglycemia.The pharmaceutical composition for the other situations that can be used for preparing treatment 1 patients with type Ⅰ DM, diabetes B and causing hyperglycemia according to the compound of the present invention.
In another aspect of the present invention, there is provided a kind of pharmaceutical composition for treating 1 patients with type Ⅰ DM, diabetes B and the other situations for causing 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 IGF-1 analogs of the present invention, including dissolved and mixed appropriate component and obtain required finished product.Therefore, according to a set of operating procedure, the IGF-1 analogs of the present invention are 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 pH 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（Methylol）- amino Yue alkane, N- bis-（Ethoxy）Glycine, N- (hydroxyl Yue bases）Methylglycine, 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-Cresol, para hydroxybenzene Yue acid Yue esters, ethyl-para-hydroxybenzoate, para hydroxybenzene Yue propyl propionates, butyl p-hydroxybenzoate, 2- phenoxetols, benzylalcohol, methaform, thimerosal, bronopol, benzoic acid, miaow urea, chlorhexidine, sodium dehydroacetate, chloreresol, benzyl rope chloramines, chlorine Ben Gan Mi 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 5mg/mL-10mg/mLoEach in these specific preservatives constitutes alternative embodiment of the 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, alditol（Such as glycerine, propane diols, 1,3-PD, 1,3-BDO）, 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.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 be 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, hydroxyethyl piperazineethanesulfonic acid（) and sodium phosphate HEPES.Embodiment
Protection group：
Acm acetamidomethyl:Yi Ugly amine methyl；Alloc or AOC allyloxycarbonyl:The oxygen of marine crab third is betrayed base； Bom, benzyloxymethyl:Benzyloxymethyl； 2-Br-Z, 2-bromobenzyloxycarbonyl:2- bromo-benzyloxycarbonyls； tBu, t-butyl:The tert-butyl group；Bz, benzoyl:Benzene Yue barefoot bases；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- chlorine grandfather's oxygen carbonyls； Fm, 9-fluorenylmethyl:9- medicine base Yue bases； Fmoc, 9-fluorenylmethoxycarbonyl:9- medicine Yue oxygen is betrayed base； 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:2,2,5,7,8- pentamethyl -6- hydroxychromans；Tos, 4-toluenesulphonyl:To the stupid sulphonyl of first； 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 -1- three（Dimethylamino）- hexafluorophosphoric acid ester（The special condensing agent of card）； DCC, Ν,Ν'-Dicyclohexylcarbodiimide:Dicyclohexyl carbodiimide； DCM:Dichloromethane protective embankment； DEPBT, 3-(Diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one:3- (diethoxy neighbour acyloxy) -1,2,3- benzos three.The Qin's -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-two Yue base Yue acid amides； DMSO:Two Yue sulfoxides； DTT, dithiothreitol:Dithiothreitol (DTT)；EDC or EDCI, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide:1- ethyls-(the Yue bases aminopropyls of 3- bis-) phosphinylidyne diimmonium salt hydrochlorate； EtOAc:Ethyl acetate；HBTU 0- (lH-benzotriazole-l-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate BTAs-Ν, Ν, Ν ', Ν '-tetramethylurea hexafluorophosphate； Η0ΒΤ l-hydroxybenzotriazole:L- hydroxyls-benzo-triazole；NMM, N-Methylmorpholine:N- Yue bases drench； NMP, N-methylpyrrolidinone:N- Yue base pyrrolidones； Piperidine：Pyridine； Su succinimide:Succinimide；
TEA, triethylamine:Triethylamine；TFA, trifluoroacetic acid trifluoroacetic acids；TFE 2,2,2-Trifluoroethanol trifluoroethanols；THF tetrahydrofuran tetrahydrofurans；TIS triisopropylsilane tri isopropyl silanes.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.It is general to select Pam resins if the use of Boc chemical synthesis C- ends being the polypeptide of carboxyl；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 include BOP, HBTU, DEPBT etc..5 times of excess of amino acid.The condensation time is 1 hour.50% piperazine of Fmoc protection groups.^/DMF is removed.Boc protection groups are removed with TFA.Peptide bond condensation reaction ninhydrin（Ninhydrin, 2,2-Dihydroxyindane- 1,3-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
If the side-chain amino group of lysine is used for acylation reaction, the side-chain amino group of lysine can use allyloxycarbonyl（Aloe) protect.Peptide chain synthesis is finished, and removing allyloxycarbonyl can use tetrakis triphenylphosphine palladium (0) and 37:2:DCM, glacial acetic acid and NMM (the 15 mL/g resins of 1 ratio）Stirred 2 hours under ar gas environment, room temperature condition.Resin is needed with 0.5% DIPEA/DMF (lOmL), 0.5% 3 hydration diethyl-dithio sodium carbonate/DMF (mL of 3 X 10), 1 after reaction:1 DCM:DMF (5 XlO mL) is cleaned.The side-chain amino group of lysine can also use 4- methyl triphen Yue bases（Mtt) protect.Resin is suspended in DCM, adds TFA/TIS/DCM (1:2:97), shake 10 minutes.After repeating 2 times, resin is washed with DCM, DMF and isopropanol.
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 m!Jg 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 Bing Yi Mi of 8-10 times of volume are 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.
If the side-chain amino group of lysine is used for acylation reaction, the side-chain amino group of lysine can use allyl oxygen phlegm base（Aloe) protection or Fmoc protections.If the side chain carboxyl group of aspartic acid or glutamic acid is used for acylation reaction, carboxyl should be converted into allyl ester (allyl ester) or 9- fluorenyl Yue bases are protected.
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 toluene is expected, the reagent such as p-mercaptophenol or stupid Yue ethers, mixture stirs 1 hour under condition of ice bath.After HF vacuum is drained, polypeptide is used;Shui Yi Mi are precipitated, and precipitation is collected by centrifugation, is isolated and purified by HPLC, and freezing thousand is dry to obtain final product.
The synthesis of double chain compound
Method 1:
Sulfydryl sulfonating reaction (Sulfitolysis)
Reaction dissolvent composition is as follows：
6M guanidine hydrochlorides（MW:95.53) 86 grams/150 milliliters
O.lM Tris ( MW:121.1) 1.8 grams/150 milliliters
0.28 M Na2S03(MW:126) 5.2 grams/150 milliliters
0.082 M Na2S406(MW:306.2) 3.75 grams/150 milliliters
0.5mM IGF-1A chains or B chains are dissolved in above-mentioned reaction dissolvent, and 8.5-8.7 is arrived in pH value regulation.Mixture is stirred vigorously 1-1.5 hours in room temperature, then using G10 or G25 post desalinations.Slow fliud flushings A is 0.05M ammonium bicarbonate aqueous solutions, and Slow fliud flushings B is the acetonitrile solution of 0.05M ammonium hydrogen carbonate/50%.Sterling low-pressure refrigeration after desalination is dried（ lyophilization ).
The connection of A chains and B chains
Literature method (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).The A chain S sulphonic acid esters and B chain S sulphonic acid esters obtained by coloured glaze base sulfonating reaction is by 2:1 weight is dissolved in 0.1 M glycine solutions (pH 10.5) than mixing, and peptide concentration is 5-10 mg/mls.DTT is added, makes SH:S sulphonic acid ester ratios are 1.2.Reaction is stirred 12 hours under 4 °C.Reaction gained mixture is purified using RP-HPLC.
1-15 synthesis：
A chains and the Fmoc chemical syntheses of B chains.A chains calculate molecular weight 2436.9, mass spectrometric measurement molecular weight 2437.6;B chains calculate molecular weight 3175.7, mass spectrometric measurement molecular weight 3176.9.The calculating molecular weight 5606.5 of final product, mass spectrometric measurement molecular weight 56073.
Method 2:
The connection of A chains and B chains
Literature method (Han etc., " Insulin chemical synthesis using a two-step orthogonal formation of the three American Peptide Society Symposium, 2009 ).A chains and B chains are synthesized with Fmoc or Boc chemical synthesis process, and A7, B6 (correspond to X in formula29Or 29) the general protection group of cysteine, but A6, All, A20 and B18 (correspond in formula i or41) the side chain coloured glaze base of cysteine protected with Acm.Synthetic A chains and B chains becomes A- (SH) after getting off from resin cleavage7 ( S-Acm ) 6' 20With B- (SH)6 ( S-Acm ) 18.B chains are dissolved in DMF or DMSO, add equimolar 2, and the sulphur of 2'- bis- is double (5- nitropyridines).Reaction is detected and purified with HPLC, obtains B- (S-Npys)6 ( S-Acm ) 18.Equimolar A- (SH)7 ( S-Acm ) 6' 20With B- (S-Npys)6 ( S-Acm ) 18It is dissolved in DMSO, peptide concentration 15mg/mL.After A7-B6 disulfide formations, 80% aqueous acetic acid is added, peptide concentration is diluted to l mg/mL.Add 40 times 12.Reaction was stirred at room temperature after 1 hour, added aqueous ascorbic acid terminating reaction.Mixture is purified with HPLC, and end-product is confirmed with matter language.
1-24 synthesis A chains and the Fmoc chemical syntheses of B chains. A- ( SH )7 ( S-Acm ) 6' 20Calculate molecular weight 2650.1, mass spectrometric measurement molecular weight 2651.3; Β- ( SH )7 ( S-Acm ) 19Calculate molecular weight 3246.7, mass spectrometric measurement molecular weight 3247.5.The calculating molecular weight 5847.8 of final product, matter Fan test molecules amount 5849.0.
Other double chain compounds are synthesized with same method.
The connected mode of pair disulfide bond of the structure of compound, especially three is further confirmed that, the HPLC in Chance documents is used
" referring to township zhang " analytic approach (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.For cylinder it, 2 mg polypeptide samples are dissolved in 0.2 ml 0.01N hydrochloric acid, add 0.8 ml and contain the liquor-saturated 0.05M NH of 100 μ g S. aureus V8 albumen4HC03, H is reached 7.9.Cultivated 24 hours at 37 °C.Sample fraction adds DTT and cultivated 30 minutes.Just can be with disulfide bond and the structure of compound using the retention time (retention time) and molecular weight of LC-MS comparative samples and each fragment of standard items.This analysis method is generally applicable to the single-stranded or double-stranded polypeptide of various method synthesis in the present invention.
The composite result of double chain compound:
Double chain compound is respectively synthesized using the above-mentioned synthetic method of the present invention, these compounds are tested analysis using the method for sequencing and mass spectrometric measurement, sequencing result shows that the amino acid sequence of compound is correct, mass spectral results show that the structure of compound is consistent with former design structure, that is, the compound synthesized is desired compound.Data result is as follows：
1-1 :Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds：Qi] and C[4] between and C [2]And C[6]Between form interchain disulfide bond, C[3] and C[51Between form intrachain disulfide bond；Molecular weight calculation value 5055.9, mass spectrometric measurement molecular weight 5057.3;
1-2:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Ctl]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond,.[3]And C[5Intrachain disulfide bond is formed between j；Molecular weight calculation value 5094.9, mass spectrometric measurement molecular weight 5095.7;
1-3:Matter Pass detection displays, A chains and B chains are dredged key by two pairs of intermolecular disulfide bonds and a pair of intramoleculars two and connected： CmWith 〇 [4]Between and.[2]With.[6]Between form interchain disulfide bond,3]With.[5]Between form intrachain disulfide bond；Molecular weight calculation value 5133.9, mass spectrometric measurement molecular weight 5135.1;
1-4:The detection display of matter blind, Α chains and Β chains are connected by two pairs of intermolecular disulfide bonds and a pair of whetstone keys of intramolecular two： Ctl]With C [4] between and.[2]And C[6]Between form interchain disulfide bond, C [3] and C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5687.6, matter knows test molecule amount 5689.3 well;
1-5:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： C[nAnd C[4]Between and C[2] and C[6]Between form interchain disulfide bond, [3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5627.4, mass spectrometric measurement molecular weight 5628.5;
1-6:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of hard and infertile keys of intramolecular two： CmAnd C[4]Between and C[2] and C[6]Between form interchain disulfide bond, C [3] and C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5614.4, mass spectrometric measurement molecular weight 5615.9;
1-7:Matter Fan detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds：Qi] and.[4]Between and.[2]With C [6]Between form interchain disulfide bond, 0 [3]With C [5]Between form intrachain disulfide bond；Molecular weight calculation value 5636.5, mass spectrometric measurement molecular weight 5636.7;
1-8:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular two bowls of keys and a pair of intramolecular disulfide bonds： CmAnd C[4]Between and C[2]And C[6]Between formed the ^ keys of interchain two, [3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5660.4, mass spectrometric measurement molecular weight 5661.2; 1-9:The general detection displays of matter i, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CmAnd C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5626.5, matter praseodymium test molecule amount 5627.6;
1-10:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CtI]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5612.4, matter knows test molecule amount 5614.1 well;
1-11 :The general detection displays of shield i, A chains and B chains dredge key by two pairs intermolecular two and a pair of intramolecular disulfide bonds are connected： Cn]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5599.4, mass spectrometric measurement molecular weight 5601.7;
1-12:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CmAnd C[4] between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5626.5, matter consults test molecule amount 5627.3;
1-13:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]With.[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5640.5, all test molecule amounts 5641.2 of matter;
1-14:The all detection displays of matter, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Ctl]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5598.5, matter imperial mandate test molecule amount 5599.1;
1-16:Matter Pan detection display, A chains and B chains are dredged key by two pairs of intermolecular disulfide bonds and a pair of intramoleculars two and connected： CmAnd C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5】Between form intrachain disulfide bond；Molecular weight calculation value 5734.7, mass spectrometric measurement molecular weight 5735.4;
1-17:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cfl]And C[4] between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5890.8, mass spectrometric measurement molecular weight 5992.2;
1-18:The detection display of matter language, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]And C[4]Between and C [2] and CwBetween form interchain disulfide bond,.[3]With C [5]Between form intrachain disulfide bond；Molecular weight calculation value 5088.9, matter knows test molecule amount 5090.5 well;
1-19:The detection display of matter language, A chains and B chains are dredged key by two pairs of intermolecular disulfide bonds and a pair of intramoleculars two and connected： Cu]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5661.6, matter language test molecule amount 5673.2;
1-20:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5652.6, mass spectrometric measurement molecular weight 5652.9;
1-21:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]And C[4]Between and C[21And C[6]Between form interchain disulfide bond, C[3]^。[51Between form intrachain disulfide bond；Molecular weight calculation value 5642.5, matter language test molecule amount 5643.3;
1-22:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular two bowls of keys and a pair of intramolecular disulfide bonds： C[uAnd C[4] between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5876.8, mass spectrometric measurement molecular weight 5877.6;
1-23:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Ctl] And C[4] between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5837.7, mass spectrometric measurement molecular weight 5839.0;
1-25:Matter borrows detection to show, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Ctl]And C[4" between and C [2] and C[6]Between form interchain disulfide bond, C[3] and C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5557.4, matter knows test molecule amount 5558.7 well;
1-26:The detection display of matter language, A chains and B chains are dredged key by two pairs of intermolecular disulfide bonds and a pair of intramoleculars two and connected： Cfl]And C[4]Between and 〇[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5633.5, matter says test molecule amount 5634.9;
1-27:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cfl]And C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3】With C [5]Between form intrachain disulfide bond；Molecular weight calculation value 5770.7, mass spectrometric measurement molecular weight 5772.1;
1-28:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]And C[4]Between and C[2]With.[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5798.7, matter knows test molecule amount 5799.6 well;
1-29:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CmAnd C[4]Between and C[2] and C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5803.7, mass spectrometric measurement molecular weight 5805.3;
1-30:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CtI]And C[4]Between and C [2][6]Between formed the key of interchain two, CCTAnd C[5】Between form intrachain disulfide bond；Molecular weight calculation value 5831.7, mass spectrometric measurement molecular weight 5832.8;
1-31:Mass Spectrometer Method shows that A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]With C [4]Between and.[2]And C[6]Between form interchain disulfide bond, C[3]And C[51Between form intrachain disulfide bond；Molecular weight calculation value 5987.9, mass spectrometric measurement molecular weight 5990.4;
1-32:The detection display of matter language, A chains and B chains are connected by two bowls of keys of two pairs of intermolecular disulfide bonds and a pair of intramoleculars： C]And C[4] between and C[2]And C[6]Between form interchain disulfide bond,.[3]With.[5]Between formed chain in two υ keys；Molecular weight calculation value 5931.9, matter knows test molecule amount 5932.5 well;
1-33:The detection display of matter language, Α chains and Β chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： Cn]With C [4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5804.6, matter knows test molecule amount 5805.9 well;
1-34:Matter Pass detection displays, A chains and B chains are connected by two pairs of intermolecular disulfide bonds and a pair of intramolecular disulfide bonds： CtI] and C[4]Between and C[2]And C[6]Between form interchain disulfide bond, C[3]And C[5]Between form intrachain disulfide bond；Molecular weight calculation value 5675.5, mass spectrometric measurement molecular weight 5676.8.The synthesis of single chain compound
Naturally it is connected chemically (Native Chemical Ligation)
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.
IGF-1 analogs are divided into two fragment synthesis.One section is to use Boc chemical synthesis IGF-1 B chain [l-17]-COS- (CH2 ) 2 CO-(Arg)4A.With S- triphen Yue base -3- coloured glaze base propionic acid when thioesters residue is synthesized.Second segment includes (corresponding to logical from B18Cys X in formula41Or X44l) play the whole amino acid of B chains C-terminal, C peptides 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- carboxylic Yue base ^^ phenol（MPAA), 20 mM tri- (2- Yue acyls ethyl) phosphine（), TCEP pH 6.9, polypeptide presses 1:1 mol ratio dissolves, the mM of concentration 2.Reaction is detected with HPLC, purified.
The IGF-1 (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 pH3 with 0.1N hydrochloric acid.Polypeptide is purified with RP-HPLC is prepared.
II -3 preparation.
RPUPLC is purified.Molecular weight calculation value 2573.1, mass spectrometric measurement molecular weight 2573.8.
Second fragment CGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA is synthesized by universal method, and thick peptide is purified with RP-HPLC.Molecular weight calculation value 4825.4, mass spectrometric measurement molecular weight 4827.0.
The mg of first fragment 25.7 (10 μ η ι ο) and the mg of the second fragment 48.3 (10 μ ι η ο) are dissolved in Slow fliud flushings（5 mL ).Slow fliud flushings include 6 M guanidine hydrochlorides, 200 mM phosphate, 200 mM 4- carboxylic Yue base benzenethiols, 20 mM tri- (2- Yue acyls ethyl) phosphines, pH 6.9.React and complete after 10 hours.Molecular weight calculation value 6596.5, mass spectrometric measurement molecular weight 6597.1.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 6590.5, matter language test molecule amount 6591.6, sequencing result and SEQ ID NO:31 is consistent.
Method described above prepares the single chain compound based on IGF-1, using the molecular weight of matter language detection molecules, detects the structure of the single chain polypeptide prepared by being sequenced to verify synthesized compound, its result is：
1-1 molecular weight calculation value 6798.8, mass spectrometric measurement molecular weight 6799.3, sequencing result and SEQ ID NO:29 is consistent
1-2 molecular weight calculation value 6760.7, mass spectrometric measurement molecular weight 6763.4, sequencing result and SEQ ID NO:30-cause
1-4 molecular weight calculations value 6519.4, mass spectrometric measurement molecular weight 6521.5, sequencing result and SEQ ID NO:32 is consistent
1-5 molecular weight calculation value 6448.3, mass spectrometric measurement molecular weight 6450.1, sequencing result and SEQ ID NO:33-cause
1-6 molecular weight calculation value 6434.3, matter Pass test molecules amount 6436.0, sequencing result and SEQ ID NO:34 is consistent
1-7 molecular weight calculation value 6420.3, mass spectrometric measurement molecular weight 6421.9, sequencing result is consistent with SEQ ID NO-.35
1-8 molecular weight calculation value 6404.3, mass spectrometric measurement molecular weight 6406.2, sequencing result and SEQ ID NO:36 is consistent
1-9 molecular weight calculation value 6404.3, mass spectrometric measurement molecular weight 6405.6, sequencing result and SEQ ID NO:37 is consistent
1-10 molecular weight calculation value 6248.1, mass spectrometric measurement molecular weight 6250.8, sequencing result and SEQ ID NO:38-cause
1-11 molecular weight calculation value 6234.1, mass spectrometric measurement molecular weight 6235.7, sequencing result and SEQ ID NO:39 is consistent
1-12 molecular weight calculation value 6120.0, mass spectrometric measurement molecular weight 6121.4, sequencing result and SEQ ID NO:40 is consistent
1-13 molecular weight calculation value 6207.1, mass spectrometric measurement molecular weight 6208.0, sequencing result and SEQ ID NO:41-cause
1-14 molecular weight calculation value 6174.0, mass spectrometric measurement molecular weight 6175.5, sequencing result and SEQ ID NO:42-cause
1-15 molecular weight calculation value 6234.1, matter Venezuelan test molecule amount 6235.4, sequencing result and SEQ ID NO:43-cause
1-16 molecular weight calculation value 6261.1, mass spectrometric measurement molecular weight 6261.6, sequencing result and SEQ ID NO:44 is consistent
1-17 molecular weight calculation value 6432.3, mass spectrometric measurement molecular weight 6433.5, sequencing result and SEQ ID NO:45 is consistent
1-18 molecular weight calculation value 6002.8, matter language test molecule amount 6004.1, sequencing result and SEQ ID NO:46 is consistent
1-19 molecular weight calculation value 6078.9, mass spectrometric measurement molecular weight 6080.2, sequencing result and SEQ ID NO:47 is consistent The sequencing result of 6121.9 matter language test molecule amount of -20 molecular weight calculation value 6122.7 and SEQ ID NO:The 48 consistent sequencing results of the matter of -21 molecular weight calculation value 6163.0 scheme test molecule amount 6164.4 and SEQ ID NO:The 49 consistent sequencing results of 6071.0 matter imperial mandate test molecule amount of -22 molecular weight calculation value 6072.8 and SEQ ID NO:The 50 consistent sequencing results of 6590.5 matter imperial mandate test molecule amount of -23 molecular weight calculation value 6592.3 and SEQ ID NO:The 51 consistent sequencing results of 6505.4 matter language test molecule amount of -24 molecular weight calculation value 6506.0 and SEQ ID NO:The sequencing result of 52 6831.8 matter test molecule amount of consistent -25 molecular weight calculation value 6833.7 and SEQ ID NO:The 53 consistent matter of -26 molecular weight calculation value 5899.7 know the sequencing result of test molecule amount 5900.6 matter of -27 molecular weight calculation value 6114.0 consistent with SEQ ID N0.54 well and know test molecule amount 6115.9 well:Sequencing result and SEQ ID NO:55 is consistent;The sequencing result of 6089.9 matter language test molecule amount of -28 molecular weight calculation value 6091.5 sequencing result of 6089.9 matter language test molecule amount of -29 molecular weight calculation value 6090.7 consistent with SEQ ID N0.56 and SEQ ID NO:57-cause all sequencing results of test molecule amount 6162.4 of the matter of-30 molecular weight calculation value 6161.0 and SEQ ID NO:The sequencing result of 58 6066.9 matter test molecule amount of consistent -31 molecular weight calculation value 6068.1 all sequencing results of test molecule amount 6095.6 of the matter of -32 molecular weight calculation value 6093.9 consistent with SEQ ID N0.59 and SEQ ID NO:The 60 all sequencing results of test molecule amount 6336.0 of the consistent matter of -33 molecular weight calculation value 6334.2 matter of -34 molecular weight calculation value 7159.1 consistent with SEQ ID N0.61 knows the sequencing result of test molecule amount 7161.2 and SEQ ID NO well:The 62 consistent matter of -35 molecular weight calculation value 6546.4 say the sequencing result of test molecule amount 6546.0 and SEQ ID NO:The 63 all sequencing results of test molecule amount 6593.8 of the consistent matter of -36 molecular weight calculation value 6592.5 and SEQ ID NO:The 64 consistent matter of -37 molecular weight calculation value 6592.4 say the sequencing result of test molecule amount 6594.1 and SEQ ID NO:The 65 all sequencing results of test molecule amount 6733.4 of the consistent matter of -38 molecular weight calculation value 6732.6 and SEQ ID NO:The sequencing result of 66 6644.6 mass spectrometric measurement molecular weight of consistent -39 molecular weight calculation value 6646.7 and SEQ ID O:The 67 consistent matter of -40 molecular weight calculation value 6701.6 know the sequencing result of test molecule amount 6702.9 and SEQ ID NO well:The 68 consistent sequencing results of 6715.7 matter Pan's test molecule amount of -41 molecular weight calculation value 6716.6 and SEQ ID NO:The 69 all sequencing results of test molecule amount 6609.7 of the consistent matter of -42 molecular weight calculation value 6608.5 and SEQ ID NO:The 70 modest sequencing results of test molecule amount 6762.5 of the consistent matter of -43 molecular weight calculation value 6761.6 and SEQ ID NO:71-cause all sequencing results of test molecule amount 6660.4 of the matter of-44 molecular weight calculation value 6659.6 and SEQ ID NO:The 72 all sequencing results of test molecule amount 6691.2 of the consistent matter of -45 molecular weight calculation value 6689.6 and SEQ ID NO:73-cause the matter of-46 molecular weight calculation value 6733.7 to know the sequencing result of test molecule amount 6734.1 and SEQ ID NO well:The 74 consistent matter of -47 molecular weight calculation value 6614.5 know the sequencing result of test molecule amount 6615.8 and SEQ ID NO well:The 75 consistent matter of -48 molecular weight calculation value 6559.5 know the sequencing result of test molecule amount 6560.7 and SEQ ID NO well:The sequencing result of 76 6846.8 matter Venezuelan test molecule amount of consistent -49 molecular weight calculation value 6848.1 and SEQ ID NO:The 77 consistent sequencing results of 6634.5 matter language test molecule amount of -50 molecular weight calculation value 6636.0 and SEQ ID NO:78-cause the sequencing result of 6774.6 matter Venezuelan test molecule amount of-51 molecular weight calculation value 6675.9 and SEQ ID NO:The 79 consistent matter of -52 molecular weight calculation value 6644.6 know the sequencing result of test molecule amount 6645.3 and SEQ ID NO well:The 80 modest sequencing results of test molecule amount 6772.2 of the consistent matter of -53 molecular weight calculation value 6770.7 matter of -54 molecular weight calculation value 6882.8 consistent with SEQ ID NO-.81 consults the sequencing result of test molecule amount 6884.1 and SEQ ID NO:The 82 consistent matter of -55 molecular weight calculation value 8098.2 know the sequencing result of test molecule amount 8099.6 and SEQ ID NO well:The 83 modest sequencing results of test molecule amount 6731.5 of the consistent matter of -56 molecular weight calculation value 6729.7 and SEQ ID NO:84 is consistent:The matter of -57 molecular weight calculation value 6679.5 knows the sequencing result of test molecule amount 6680.2 and SEQ ID NO well:85 is consistent:The sequencing result of 6960.9 mass spectrometric measurement molecular weight of -58 molecular weight calculation value 6961.6 and SEQ ID NO:86 is consistent:The matter of -59 molecular weight calculation value 7160.1 knows the sequencing result of test molecule amount 7161.5 and SEQ ID NO well:87-cause: - 60 molecular weight calculation values 6899.8, matter language test molecule amount 6901.6, sequencing result and SEQ ID NO:88 is consistent;- 61 molecular weight calculation values 6703.6, all test molecule amounts 6704.3 of matter, sequencing result and SEQ ID NO:89 is consistent;- 62 molecular weight calculation values 6644.6, matter language test molecule amount 6645.8, sequencing result and SEQ ID NO:90 is consistent;- 63 molecular weight calculation values 6581.5, matter Fan test molecules amount 6583.3, sequencing result and SEQ ID NO:91 is consistent;- 64 molecular weight calculation values 6701.6, matter knows test molecule amount 6702.4, sequencing result and SEQ ID NO well:92 is consistent;- 65 molecular weight calculation values 6674.6, mass spectrometric measurement molecular weight 6675.7, sequencing result and SEQ ID NO:93 is consistent;- 66 molecular weight calculation values 7354.3, all test molecule amounts 7356.2 of matter, sequencing result and SEQ ID NO:94 is consistent;- 67 molecular weight calculation values 6729.7, all test molecule amounts 6730.2 of matter, sequencing result and SEQ ID NO:95 is consistent;- 68 molecular weight calculation values 6707.6, matter language test molecule amount 6709.0, sequencing result and SEQ ID NO:96 is consistent;- 69 molecular weight calculation values 6774.7, all test molecule amounts 6775.4 of matter, sequencing result and SEQ ID NO:97 is consistent;- 70 molecular weight calculation values 6620.5, matter language test molecule amount 6621.1, sequencing result and SEQ ID NO:98 is consistent;- 71 molecular weight calculation values 6608.5, mass spectrometric measurement molecular weight 6609.7, sequencing result is consistent with SEQ ID N0.99;- 72 molecular weight calculation values 6647.6, matter language test molecule amount 6648.6, sequencing result and SEQ ID NO:100 consistent -73 molecular weight calculation values 6691.6, mass spectrometric measurement molecular weight 6693.0, sequencing result and SEQ ID NO:101 consistent -74 molecular weight calculation values 7256.2, mass spectrometric measurement molecular weight 7257.9, sequencing result and SEQ ID NO:102 consistent -75 molecular weight calculation values 6788.7, matter language test molecule amount 6790.9, sequencing result and SEQ ID NO:103 consistent -76 molecular weight calculation values 7587.5, matter language test molecule amount 7588.3, sequencing result and SEQ ID NO:104 consistent -77 molecular weight calculation values 8530.5, mass spectrometric measurement molecular weight 8532.8, sequencing result and SEQ ID NO:105 consistent -78 molecular weight calculation values 7385.4, mass spectrometric measurement molecular weight 7386.0, sequencing result and SEQ ID NO:106-cause-79 molecular weight calculation values 6941.0, mass spectrometric measurement molecular weight 6941.5, sequencing result and SEQ ID NO:107 consistent -80 molecular weight calculation values 6009.8, mass spectrometric measurement molecular weight 6011.4, sequencing result and SEQ ID NO:108 consistent -81 molecular weight calculation values 6555.5, matter language test molecule amount 6556.6, sequencing result and SEQ ID NO:109-cause-82 molecular weight calculation values 7098.1, mass spectrometric measurement molecular weight 7100.2, sequencing result and SEQ ID NO:110-ft-83 molecular weight calculations value 7186.0, mass spectrometric measurement molecular weight 7187.1, sequencing result and SEQ ID NO:L ll-the molecular weight calculation of cause -84 value 6934.9, mass spectrometric measurement molecular weight 6935.6, sequencing result and SEQ ID NO:112-cause-85 molecular weight calculation values 7160.2, matter language test molecule amount 7161.9, sequencing result and SEQ ID NO:113-cause-86 molecular weight calculation values 6664.6, matter language test molecule amount 6665.1, sequencing result and SEQ ID NO:114 consistent -87 molecular weight calculation values 6338.2, mass spectrometric measurement molecular weight 6339.8, sequencing result and SEQ ID NO:115-cause-88 molecular weight calculation values 6511.3, mass spectrometric measurement molecular weight 6512.4, sequencing result and SEQ ID NO:116-cause-89 molecular weight calculation values 6407.3, mass spectrometric measurement molecular weight 6408.2, sequencing result and SEQ ID NO:117-cause-90 molecular weight calculation values 6636.6, mass spectrometric measurement molecular weight 6637.5, sequencing result and SEQ ID NO:118-cause-91 molecular weight calculation values 6569.5, mass spectrometric measurement molecular weight 6570.9, sequencing result and SEQ ID NO:119 consistent -92 molecular weight calculation values 6385.3, mass spectrometric measurement molecular weight 6387.0, sequencing result and SEQ ID NO:120 consistent -93 molecular weight calculation values 6371.3, mass spectrometric measurement molecular weight 6372.3, sequencing result and SEQ ID NO:121-cause-94 molecular weight calculation values 6806.8, mass spectrometric measurement molecular weight 6808.1, sequencing result and SEQ ID NO:122 consistent -95 molecular weight calculation values 6704.6, matter says test molecule amount 6705.9, sequencing result and SEQ ID NO:123 consistent -96 molecular weight calculation values 6877.8, mass spectrometric measurement molecular weight 6878.7, sequencing result and SEQ ID NO:124 consistent -97 molecular weight calculation values 6750.7, mass spectrometric measurement molecular weight 6771.9, sequencing result and SEQ ID NO:125 consistent -98 molecular weight calculation values 7724.8, mass spectrometric measurement molecular weight 7726.2, sequencing result and SEQ ID NO:126-cause-99 molecular weight calculation values 6123.9, matter language test molecule amount 6125.8, sequencing result and SEQ ID NO:127 is consistent: II -100:Molecular weight calculation value 6614.4, the sequencing result of mass spectrometric measurement molecular weight 6615.3 is consistent with SEQ ID NO 128;
II -101:Molecular weight calculation value 6899.8, the sequencing result of mass spectrometric measurement molecular weight 6901.4 is consistent with SEQ ID NO 129;
II -102:Molecular weight calculation value 6036.8, the sequencing result of mass spectrometric measurement molecular weight 6038.0 is consistent with SEQ ID NO 130;
Π-103:Molecular weight calculation value 6853.7, it is consistent with SEQ ID NO 131 that matter knows the sequencing result of test molecule amount 6854.5 well;
II -104:Molecular weight calculation value 7284.2, the sequencing result of matter language test molecule amount 7285.6 is consistent with SEQ ID NO 132;
II -105:Molecular weight calculation value 7551.5, the sequencing result of matter language test molecule amount 7552.9 is consistent with SEQ ID NO 133;
II -106:Molecular weight calculation value 6913.9, matter says that the sequencing result of test molecule amount 6915.3 is consistent with SEQ ID NO'134;
II -107:Molecular weight calculation value 6837.7, the sequencing result of matter Pan's test molecule amount 6839.3 is consistent with SEQ ID NO 135;
Π-108:Molecular weight calculation value 7063.1, the sequencing result of matter language test molecule amount 7064.7 is consistent with SEQ ID NO 136;
II -109:Molecular weight calculation value 6528.4, the sequencing result of mass spectrometric measurement molecular weight 6529.8 is consistent with SEQ ID NO 137;
Π -110:Molecular weight calculation value 7200.3, it is consistent with SEQ ID NO 138 that matter knows the sequencing result of test molecule amount 7201.4 well;
II -ll l :Molecular weight calculation value 7735.7, the sequencing result of matter Fan test molecules amount 7737.0 is consistent with SEQ ID NO 139;
Π -112:Molecular weight calculation value 6369.1, the sequencing result of mass spectrometric measurement molecular weight 6370.6 is consistent with SEQ ID NO 140;
II -113:Molecular weight calculation value 7468.4, it is consistent with SEQ ID NO 141 that matter teaches the sequencing result of test molecule amount 7470.2;
11 -114:Molecular weight calculation value 6603.5, the sequencing result of mass spectrometric measurement molecular weight 6604.7 is consistent with SEQ ID NO 142;
Π -115:Molecular weight calculation value 7254.2, matter borrows the sequencing result of test molecule amount 7255.5 consistent with SEQ ID NO. 143;
II -116:Molecular weight calculation value 6625.5, matter says that the sequencing result of test molecule amount 6627.1 is consistent with SEQ ID NO 144;
Π -117:Molecular weight calculation value 7399.5, the modest sequencing result of test molecule amount 7401.3 of matter and SEQ ID NO:145 is consistent;
Π -118:Molecular weight calculation value 7223.1, matter says the sequencing result of test molecule amount 7224.4 and SEQ ID NO:146 is consistent;
II -119:Molecular weight calculation value 6120.0, the sequencing result of matter imperial mandate test molecule amount 6121.6 is consistent with SEQ ID NO- 147;
II -120:Molecular weight calculation value 6278.2, all sequencing results of test molecule amount 6277.9 of matter and SEQ ID NO:148 is consistent;
Π -121:Molecular weight calculation value 6264.1, the sequencing result of mass spectrometric measurement molecular weight 6265.8 is consistent with SEQ ID NO- 149;
II -122:Molecular weight calculation value 6242.2, all sequencing results of test molecule amount 6243.4 of matter and SEQ ID NO:150 is consistent;
II -123:Molecular weight calculation value 6084.9, the sequencing result of matter language test molecule amount 6086.3 and SEQ ID NO:161 is consistent;
II -124:Molecular weight calculation value 6659.5, the sequencing result of matter Venezuelan test molecule amount 6660.7 and SEQ ID NO:162 is consistent.Compound
The modification of compound based on IGF-1
The acylation of polypeptide
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, it is cooled with an ice bath.Gradually add 2- methyl-2-propanols（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) mixtures are 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-NMRiCDC) 5:2.35 (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), it is added 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. In order to seal HPLC grades of Yue alcohol of addition in active triphenylmethyl, resin（2.4 mL), mix 15 minutes.Resin filter, after being cleaned with DCM (mL of 3 X 30), DMF (mL of 2 X 30), DCM (mL of 3 X 30), Yue alcohol (mL of 3 X 30), is 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, lO 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-Glu-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 (l g, 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 HCI and saturated common salt water washing, after stone gram acid magnesium is dried, reduction vaporization obtains tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu.Nuclear magnetic resonance data is： ^-NMRiCDC ) 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).
III-7 synthesis.
Synthesized with above-mentioned A, B chain connection method.Calculate molecular weight 5746.7, mass spectrometric measurement molecular weight 5747.8.At room temperature by polypeptide（55 mg) it is dissolved in lOOmM Na2C03(5 mL, pH 10).Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (6.6 mg) is dissolved in acetonitrile（5mL), polypeptide solution is added.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), vacuum evaporating solvent after 30 minutes, is dissolved in Slow fliud flushings A by crude product and freezes.Purified using RP-HPLC C5 posts.Molecular weight calculation value 6144.2, mass spectrometric measurement molecular weight 6146.0.After B chain warp trypsin hydrolysises, the calculating molecular weight 1269.5 of the terminal fragment containing aliphatic acid, mass spectrometric measurement molecular weight 1270.4.
111-28 synthesis：
Method 1:
B chains（0.1 mmol) Boc chemical syntheses are used, lysine side chain amino groups are protected with Fmoc.After the synthesis of B chains is finished, the Boc of B chain N- ends should not be removed.20% piperidines/DMF (6 mL) adds resin, and mixture vibrates 15 minutes, drains reagent.Reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Fmoc-L-Glu-OtBu (425 mg, 1 mmol), DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, l mmol) and DIPEA (0.2 mL, 1.15mmol) be dissolved in DMF, add resin.After vibration 2 hours, solution is drained, and resin is washed with DMF and DCM.20% piperidines/DMF adds resin, and mixture vibrates 15 minutes, drains reagent.Reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (550 mg) and DIPEA (0.2 mL) are dissolved in DMF, add resin.After vibration 3 hours, solution is taken out thousand, and resin is washed with DMF and DCM.After vacuum drying, the polypeptide on resin is cut with HF, is added to Yue phenol reagents, mixture is stirred 1 hour under condition of ice bath, after HF vacuum takes out thousand, and polypeptide Bing Yi Mi are precipitated, and precipitation is collected by centrifugation.Molecular weight calculation value 3601.2, mass spectrometric measurement molecular weight 3602.8.
B chains crude product is crossed chromatographic column desalination, isolated and purified, freeze-drying obtains final product IGF-1 B through sulfydryl sulfonating reaction Chain S sulphonic acid esters.IGF-1 A (Q5, S12, N18) are synthesized by universal method, molecular weight calculation value 2436.9, mass spectrometric measurement molecular weight 2437.6.IGF-1 A chain S sulphonic acid esters and B chain S sulphonic acid esters are synthesized using above-mentioned general A chains with the connection method of B chains.Molecular weight calculation value 6032.0, mass spectrometric measurement molecular weight 6033.4.
Method 2:
B chains Boc chemical syntheses, lysine Side chain amino is protected with Fmoc, and B18Cys (corresponds in formula or X44!) sulfydryl protected with Acm.After synthesis is finished, the Boc of B chain N- ends should not be removed.20% piperazine DMF (6 mL) adds resin（0.1 mmol), mixture vibrates 15 minutes, drains reagent.Reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Fmoc-L-Glu-OtBu (425 mg), DIC (126 mg, 0.1 ml), HOBT (135 mg) and DIPEA (0.2 mL) add resin.After vibration 2 hours, solution is taken out thousand, after resin is washed with DMF and DCM, adds 20% piperidines/DMF, and mixture vibrates 15 minutes, drains reagent.Pyridine is sent to be reacted by two-wheeled, resin is washed 3 times with DMF and DCM.Tert-butyl hexadecandioyl diacyl-L-Glu-OtBu (715 mg), DIC (126 mg; 0.1 ml), HOBT (135 mg) and DIPEA (0.2 mL) be dissolved in DMF; add resin; after vibration 2 hours; solution is drained, and resin is washed with DMF and DCM.After vacuum thousand is dry, the polypeptide on resin is cut with HF, adds paracresol reagent, mixture is stirred 1 hour under condition of ice bath, after HF vacuum is drained, and polypeptide ice ether is precipitated, and precipitation is collected by centrifugation.Calculate molecular weight 3672.3, mass spectrometric measurement molecular weight 3673.5.
Β chains are dissolved in DMF or DMSO, add equimolar 2, and the sulphur of 2'- bis- is double (5- nitropyridines).Reaction is detected and purified with HPLC, obtains Β-(S-Npys)6 ( S-Acm ) 18 [H9,F15,P27,K28-NeB28-(Na-(HOOC (CH2)14CO)-y- G1U-N- (Y-G1U))] calculate molecular weight 3826.4, mass spectrometric measurement molecular weight 3827.0.
A chains synthesize IGF-1 A- (Q5, S12, N18) (SH)7 ( S-Acm ) 6' 20Calculate molecular weight 2650.1, mass spectrometric measurement molecular weight 2651.5.The connection method of A chains and B chains is according to above-mentioned A chains and B chain connection methods（ 2 ).Molecular weight calculation value 6032.0, mass spectrometric measurement molecular weight 6034.2.
111-29 synthesis.
After B chains (O.lmmol) are finished with Boc chemical syntheses, the Boc of B chain N- ends is removed with TFA.Fmoc-L-Glu-OtBu (425 mg, 1 mmol), DIC (126 mg, 0.1 ml, 1 mmol), HOBT (135 mg, lmmol) and DIPEA (0.2 ml, DMF 1.15mmol) is dissolved in, resin is added.After vibration 2 hours, solution is drained, and resin is washed with DMF and DCM.20% piperidines/DMF adds resin, and mixture vibrates 15 minutes.Reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Palmitic acid (256 mg, 1 mmol)>DIC (126 mg, 0.1ml, lmmol), HOBT (135 mg, lmmol) and DIPEA (0.2 ml, 1.15mmol) are dissolved in DMF, add resin.After 2 hours, solution is drained, and resin is washed with DMF and DCM.After vacuum drying, the polypeptide on resin is cut with HF, is added to Yue distiller's yeast reagents, mixture is stirred 1 hour under condition of ice bath, after HF vacuum is drained, and polypeptide ice ether is precipitated, and precipitation is collected by centrifugation.Molecular weight calculation value 3512.1, mass spectrometric measurement molecular weight 3513.5.B chains crude product reacts (Sulfitolysis) through fearful base horizontalization, crosses color Pan Zhu desalinations, isolates and purifies, freeze-drying obtains final product B chain S sulphonic acid esters.A chain S sulphonic acid esters and B chain S sulphonic acid esters are synthesized using above-mentioned general A chains with the connection method of B chains.Molecular weight calculation value 5943.0, mass spectrometric measurement molecular weight 5943.6.
III- 12 is synthesized：
A is synthesized in aforementioned manners.Molecular weight calculation value 6432.3, mass spectrometric measurement molecular weight 6434.1.At room temperature by polypeptide（643 mg) it is dissolved in lOOmM Na2C03(5 mL, pH 10).Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (68 mg) is dissolved in acetonitrile（5mL), polypeptide solution is added.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), vacuum evaporating solvent after 30 minutes, Slow is dissolved in by crude product Fliud flushing A is simultaneously freezed.Purified using RP-HPLC C5 posts.Molecular weight calculation value 6829.8, mass spectrometric measurement molecular weight 6831.6.A small amount of product is reduced and trypsin degradation through DTT, and liquid chromatogram-mass spectrometry observes GPETLCGAHLVDALFFVCGDR (calculating molecular weight 2220.6, test molecule amount 2221.4), GFYFNPTGK- [N8-(Na-(HOOC(CH2)14CO)-Y-Glu)]-GSSSAAPQT GIVDQCCFR (calculating molecular weight 3236.7, test molecule amount 3237.5).It is thus identified that aliphatic acid is combined in C peptide lysine side-chains.Through analysis, gained compound is 111-12.
(CH300C(CH2)! iNHCO(CH2)3CO)-Glu(OSu)-OCH3Synthesis
Methanol（40 ml) ice bath cooling, S0C1 is added dropwise2(4 ml).Add 12-aminolauric acid（3g, 13.9 mmol), it is stirred overnight.Mixture and drying are filtered, 3g 12-aminolauric acid Yue ester hydrochlorides are obtained.
1H-NMR ( DMSO-d6) δ:7.97 (bs, 3H), 3.58 (s, 3H), 2.73 (m, 2H), 2.28 (t, 2H), 1.52 (m, 4H), 1.25 (m, 14H).
12-aminolauric acid Yue ester hydrochlorides（Lg, 3.8 mmol) THF (15 ml) is suspended in, glutaric acid liver (1.29g, 3.8 mmol) and TEA (0.52 ml, 3.8 mmol) are added, is stirred overnight at room temperature.Add water（75 ml) stirring 1 hour after filter, be washed with water, dry after obtain lg l2- (4- carboxyls amide-based small) lauric acid/dodecanoic acid Yue esters.
1H-NMR ( DMSO-d6) δ:12 (bs, 1H), 7.73 (t, 1H), 3.57 (s, 3H), 3.00 (q, 2H), 2.28 (t, 2H), 2.18 (t, 2H), 2.06 (t, 2H), 1.69 (p, 2H), 1.50 (p, 2H), 1.36 (p, 2H), 1.23 (m, 14H).
12- (4- carboxyls amide-based small) lauric acid/dodecanoic acid Yue esters（0.33g, 0.95 mmol) it is dissolved in dry DMF/DCM (0.5 ml:0.5 ml), add DCC (0.2g, 1 mmol) and N- hydroxysuccinimides（0.115 g, 1 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 the U.S. of sulfuric acid 4 is dried, 12- (the 4- carboxyls amide-based small) methyl dodecanoate for obtaining OSu activation is evaporated under reduced pressure.The compound is dissolved in DMF (1.5 ml), adds DIEA (0.19g, 1.5 mmol) and H-Glu-OMe (0.168g, 1.04 mmol), is stirred overnight at room temperature.Ethyl acetate is added after solvent is evaporated under reduced pressure, with 0.1 N HC1 and saturated common salt water washing, after magnesium sulfate is dried, reduction vaporization obtains (CH3OOC(CH2)1 !NHCOCCH^jCOi-Glu-OCHj。
1H-NMR ( DMSO-d6) δ:12 (bs, 1 Η), 8.22 (d, 1H), 7.73 (t, 1H), 4.24 (m, 1H), 3.61 (s, 3H), 3.57 (s, 3H), 3.00 (q, 2H), 2.27 (m, 4H), 2.10 (t, 2H), 2.04 (t, 2H), 1.9 (m, 1H), 1.8 (m, 1H), 1.68 (t, 2H), 1.50 (m, 2H), 1.36 (m, 2H), 1.23 (m, 14H).
(CH3OOC(CH2)nNHCO(CH2)3CO)-Glu-OCH3(0.36g, 0.36 mmol) is dissolved in dry DMF/DCM (0.5 ml:0.5 ml), add DCC (0.08 g, 0.4 mmol) and N- hydroxysuccinimides（0.046 g, 0.4 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 (CHsO CH nNHCC CH^CC-Gh^OSuHDCH
1H-NMR ( DMSO-d6) δ:8.27 (d, 1H), 7.72 (t, 1H), 4.31 (m, 1H), 3.63 (s, 3H), 3.57 (s, 3H),
3.00 (q, 2H), 2.81 (s, 4H), 2.28 (t, 2H), 2.12 (t, 2H), 2.05 (t, 2H), 1.70 (m, 2H), 1.50 (m, 2H), 1.35 (m, 2H), 1.23 (m, 14H).
III- 10 is synthesized
With above-mentioned A, B chain connection method synthesis polypeptide.Calculate molecular weight 5746.7, mass spectrometric measurement molecular weight 5748.0.At room temperature by polypeptide（58 mg) it is dissolved in lOOmM Na2C03(5 mL, pH 10).（CH3OOC(CH2)uNHCO(CH2)3CO)- Glu(OSu)-OCH3(7 mg) is dissolved in acetonitrile（5mL), polypeptide solution is added.After stirring 30 minutes, it is cooled with an ice bath, adds 0.1N NaOH, acidifying with acetic acid, upper RP-HPLC C5 posts purifying are used in stirring after 1 hour.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 6187.2, mass spectrometric measurement molecular weight 6188.7.Through analysis, gained compound is 111-10.
OSu-CO-(CH2CH20)5-(CH2)2-NH-[Na-(HOOC (CH2)I6CO)-Y-G1U] synthesis Octadecane diacid（The mmol of 2.5g 8.0) 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 (mmol of 0.097g 0.8).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（1.12g, 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 sulfuric acid town is dried, reduction vaporization obtains the benzyl octadecane diacid ester of succinimido one.
Ή-NMR ( CDC13 ) 5: 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 HC1, saturated common salt water washing, solvent is evaporated under reduced pressure after magnesium sulfate thousand is dry, BzlO- octadecandioyls-L-Glu-OBzl is obtained.
'H-NMR( CDC13 )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.
1H-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 ol) and H2N-(CH2)2-(OCH2CH2)5COOH (31mg, 0.1mmol) is dissolved in DMF/DCM (0.5 ml:1.5ml), 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 the U.S. of sulfuric acid 4, 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] propanoic acid).Calculate molecular weight 915.2, the ο of test molecule amount 916.5
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 (91mg; O.lmmol DMF (1 ml) and DCM (1 ml)) are dissolved in, is cooled down with water-bath.Add DCC (24.7mg, 0.12 mmol) and N- hydroxysuccinimides（13.8 mg 0.12 mmol).Mixture is 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 the U.S. of sulfuric acid 4 is dried, reduction vaporization obtains benzene Yue bases 18- [[1- benzyloxycarbonyl groups -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -3- oxos-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino] -4- oxo-butyls] amino] -18- oxos-stearate（benzyl 18-[[l-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. Benzyl 18- [[1- benzyloxycarbonyl groups -4- [2- [2- [2- [2- [2- [3- (2, 5- dioxo pyrrolidins -1-yl) oxygen -3- oxos-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino] -4- oxo-butyls] amino] -18- oxos-stearate (51mg, 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- [[1- carboxyls -4- [2- [2- [2- [2- [2- [3- (2, 5- dioxo pyrrolidins -1-yl) oxygen -3- oxos-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethylamino] _4- oxo-butyl] amino]-I8- oxo-stearic acid（ 18-[[l-carboxy-4-[2-[2-[2-[2- [2-[3-(2,5- dioxopyrrolidin- 1 -yl)oxy-3-oxo-propoxy]ethoxy] ethoxy] ethoxy] ethoxy] ethylamino] -4-oxo-butyl] amino]- 18-oxo-octadecanoic acid ).Calculate molecular weight 832.0, test molecule amount 833.4.
III-36 is synthesized
With above-mentioned Α, Β chain connection method synthesis polypeptide.Calculate molecular weight 5531.4, mass spectrometric measurement molecular weight 5533.7.At room temperature by polypeptide（56 mg, 10 μ η ι ο) it is dissolved in lOOmM Na2C03(1 ml, pH 10).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]-4- oxo-butyl] amino] -18- oxos-stearic acid (9.2 mg, 11 μ π ι ο) are 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 6428.3, mass spectrometric measurement molecular weight 6430.2.Through analysis, gained compound is 111-36.The PEGylation processes of polypeptide（PEGylation):
A) reduction pit foundation (reductive alkylation)
Polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH3CN 1) is pressed:2:45 ratios are dissolved in the acetums of pH 4.3（0.1M 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.
The synthetic methods of III- 1：
A (Q5, S12, N18):B (H9, F15, P27) synthetic method is ibid.B1 reductive alkylations obtain product.Molecular weight calculation value 25576.4, matter falsely charges test and obtains a broad peak, intermediate molecular weight 25580.2, a small amount of product is reduced through DTT, liquid chromatograph mass spectrography observes A (Q5, S12, N18) (molecular weight calculation value 2436.9, mass spectrometric measurement molecular weight 2438.0) and G (Na- PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT (molecular weight calculation value 23144.6, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 23140.5), without molecular weight 22436.9.Through analysis, gained compound is 111-1.
B) NHS esters (n-hydroxysuccinimide) are acylated
Polypeptide and mPEG20K-NHS in molar ratio 1：1 is dissolved in 0.1N Ν, Ν-bis- (2- ethoxys) glycine solution（PH 8), peptide concentration 0.5mg/mL.Reaction is carried out 2 hours in room temperature, is purified with HPLC.Yield about 90%.
III-2 synthetic methods：
A(Q5,S12,N18):B (H9, F15, P27, K28, desT) is synthesized with above-mentioned A, B chain connection method.Calculate molecular weight 5505.4, matter language test molecule amount 5506.8.Polypeptide after mPEG20K-NHS reactions with obtaining 111-2.Molecular weight calculation value 25505.4, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 25501.7.A small amount of product is through DTT reduction and trypsin degradation B chains, liquid chromatograph mass spectrography observes GPETLCGAH LVDALFFVCGDR (molecular weight calculation values 2220.6, mass spectrometric measurement molecular weight 2221.7), GFYFNPK (molecular weight calculation value 872.0) is not observed, but mass spectrum observes a broad peak, intermediate molecular weight 20871.9.Through analysis, gained compound is 111-2.
III-4 synthetic method：
Essentially identical with above-mentioned Ι Π -2, simply C2Lys side-chain amino group reacts with mPEG20K-NHS, obtains 111-4.Molecule Calculated value 26600.5 is measured, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 26604.5.A small amount of product observes GPETLCGAHLVDALFFVCGDR (molecular weight calculation value 2220.6, test molecule amount 2220.8) and GFYFNPPT-GK (N through DTT reduction and trypsin degradation B chains, liquid chromatogram-matter Pan combination£- PEG 20K)-GSSSAAAPQTGIVDQCCFR (molecular weight calculation value 23007.4, test obtains a broad peak, intermediate molecular weight 23010.0).Through analysis, gained compound is 111-4.
Synthesize Al, B27-diBoc-IGF-l analogs
A(Gl-Na- Boc, Q5, S12, N18): B(H9, F15, K27-NE-Boc)
1-15 A(Q5,S12,N18):B (H9, F15) is synthesized with above universal method.Calculate molecular weight 5606.5, mass spectrometric measurement molecular weight 5607.3.Polypeptide（100 mg, 17.8 μ π ι ο 1) it is dissolved in water（1 mL )、 IN NaHC03(0.3 mL) and DMF (3 mL).Add t-Boc- azide（5.5mg, 37.5 μ π ι ο 1).Mixture is 40.C is stirred 3 hours, adds 50% acetic acid（0.35 mL) terminating reaction.Unreacted t-Boc-azide Yong Yi Mi extractions（2 X 15 mL ).Water layer vacuum freeze drying.It is monosubstituted that crude product contains Boc, disubstituted and three substitution polypeptides.Mixture SP-Sephadex C-25 ion exchange column purifications.Ion exchange column is first balanced with the 1.5 M acetic acid containing 6M urea, and polypeptide elution flow velocity is 48 mL/h, 1.5 M acetic acid of the mL 6M urea of linear gradient 0.04-0.4 M sodium chloride/1000.Di-t-Boc polypeptides are further purified with DEAE- Sephadex A-25 posts.Color knows post well in advance with the 0.01 M Tris Slow fliud flushings containing 7M urea（PH 8.5) balance.Elute the mL/h of flow velocity 35, the mL Tris Slow fliud flushings of gradient 0.14-0.28 M sodium chloride/100.Molecular weight calculation value 5806.7, matter knows test molecule amount 5808.3 well.A small amount of polypeptide is dissolved in 0.05M NH4HCO3/ 20%ACN, mass spectral analysis after being reduced 10 minutes with DTT. A(Gl-Na- Boc, Q5, S12, N18) molecular weight calculation value 2537.0, mass spectrometric measurement molecular weight 2537.9.B (H9, F15, K27-Ns- Boc) molecular weight calculation value 3275.8, shield spectrum test molecule amount 3276.0.After tryptose enzymolysis, the molecular weight calculation value 1073.2 of the fragment containing Boc, matter Spectrum test molecules amount 1074.5.The B1 amino of Al, B27-di-Boc polypeptide can combine to form long-acting polypeptides with polyethylene glycol, albumin, aliphatic acid etc..
111-30 synthesis.
A(Gl-Na- Boc, Q5, S12, N18): B(H9, F15 , K27-Ns- Boc) synthetic method is as above.Polypeptide (58 mg) is dissolved in DMF (3 mL), adds Mal-dPEG12-NHS (8.7mg) (Quanta Biodesign) and triethylamine (30 L).Reaction is stirred at room temperature 2 hours.Depressurize after solvent flashing, add TFA (2 mL) and TIS (Ι Ο Ο μ Ι).After stirring 10 minutes, volatilize TFA.Crude product is dissolved in H20/ACN (3:1) purified with RP- HPLC.Molecular weight calculation value 6556.5, mass spectrometric measurement molecular weight 6558.0.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% 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, eluent is 0.05M ammonium bicarbonate aqueous solutions.Sterling is obtained after vacuum freeze drying.Compound molecular weight calculated value 73028.7, mass spectrometric measurement molecular weight 73030.2 is compound 111-30 through analysis.
Composite result:
The compound of the respectively modification based on IGF-1, including single chain compound and double chain compound are respectively synthesized according to the method described above, and the structure of each compound is detected by matter language, it is as a result as follows：
III-3:Molecular weight calculation value 26673.6, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 26680.5, and through analysis, gained compound is Π Ι -3;
III-5:Molecular weight calculation value 26618.5, mass spectrometric measurement obtains a broad peak, intermediate molecular weight 26624.9, and through analysis, gained compound is Π Ι -5;
III-6:Molecular weight calculation value 5845.8, shield spectrum test molecule amount 5847.2, through analysis, gained compound is Π Ι -6;
III-8:Molecular weight calculation value 6172.2, mass spectrometric measurement molecular weight 6173.4, through analysis, gained compound is Π Ι -8;
III-9:Molecular weight calculation value 6116.1, mass spectrometric measurement molecular weight 6118.2, through analysis, gained compound is Π Ι -9;
111-11 :Molecular weight calculation value 6273.3, mass spectrometric measurement molecular weight 6274.5, through analysis, gained compound is Π Ι -11; 111-13:The mass spectrometric measurement molecular weight 6830.4 of molecular weight calculation value 6829.8 is through analysis, and gained compound is the Π Ι -14 of III- 13:The mass spectrometric measurement molecular weight 6831.1 of molecular weight calculation value 6829.8 is through analysis, and gained compound is Ι Π -14 111-15:The Zhi Wrong test molecules amount 6846.9 of molecular weight calculation value 6845.8 is through analysis, and gained compound is Ι Π -15 111-16:The mass spectrometric measurement molecular weight 6973.3 of molecular weight calculation value 6972.0 is through analysis, and gained compound is Π Ι -16 111-17:The matter language test molecule amount 6458.0 of molecular weight calculation value 6457.4 is through analysis, and gained compound is Ι Π -17 111-18:The mass spectrometric measurement molecular weight 6732.2 of molecular weight calculation value 6730.7 is through analysis, and gained compound is Π Ι -18 111-19:The mass spectrometric measurement molecular weight 5903.4 of molecular weight calculation value 5902.9 is through analysis, and gained compound is Ι Π -19 111-20:The mass spectrometric measurement molecular weight 6662.7 of molecular weight calculation value 6661.6 is through analysis, and gained compound is Π Ι -20 111-21:The mass spectrometric measurement molecular weight 6187.8 of molecular weight calculation value 6186.2 is through analysis, and gained compound is 111-21 111-22:The mass spectrometric measurement molecular weight 5946.0 of molecular weight calculation value 5944.9 is through analysis, and gained compound is Π Ι -22 Ι Ι Ι -23:The mass spectrometric measurement molecular weight 6101.6 of molecular weight calculation value 6101.1 is through analysis, and gained compound is Π Ι -23 111-24:The mass spectrometric measurement molecular weight 6258.9 of molecular weight calculation value 6257.3 is through analysis, and gained compound is Π Ι -24 111-25:The mass spectrometric measurement molecular weight 6344.1 of molecular weight calculation value 6342.4 is through analysis, and gained compound is Ι Π -25 111-26:The mass spectrometric measurement molecular weight 6472.3 of molecular weight calculation value 6471.5 is through analysis, and gained compound is Π Ι -26 111-27:The mass spectrometric measurement molecular weight 6529.1 of molecular weight calculation value 6528.6 is through analysis, and gained compound is Ι Π -27 111-31:The matter of molecular weight calculation value 6241.3 borrows test molecule amount 6242.4 through analysis, and gained compound is Π Ι -31 111-32:The mass spectrometric measurement molecular weight 6101.8 of molecular weight calculation value 6101.1 is through analysis, and gained compound is Ι Π -32 111-33:The mass spectrometric measurement molecular weight 6231.5 of molecular weight calculation value 6230.2 is through analysis, and gained compound is Ι Π -33 111-34:The matter language test molecule amount 6288.2 of molecular weight calculation value 6287.3 is through analysis, and gained compound is Ι Π -34: ΙΠ -35:The mass spectrometric measurement of molecular weight calculation value 26462.3 obtains a broad peak, intermediate molecular weight 26470.1, and through analysis, gained compound is Π Ι -35;
III -37:Molecular weight calculation value 7246.3, mass spectrometric measurement molecular weight 7248.0, through analysis, gained compound is 111-37.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 (1): 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、 lll.l lnM. 37.04nM、 12.35nM、 4.12nM、 1.37nM、 0.46nM ).For its IGF-1 in the present invention The activity of acceptor 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, H 8.0,100 mM NaCl, 10 mM MgCl2, 0.5 % (w/v) BSA, 0.025 % (w/v) Triton X- 100], (^L is cultivated 48 hours cumulative volume 20 at 4 °C.Acceptor and the polypeptide and part that are combined with acceptor are with 0.2 % gamma globulins and 50 (L 25 % (w/v) PEG 8000 is precipitated, 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 combining Slow fliud flushings (DMEM, containing 0.50% BSA, 20 mM Hepes, pH 7.8), adds 40 (L125I-IGF-1 (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.Cultivated 3 hours at 16 °C, 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 be used for tyrosine iodination reaction（ iodination ).125I-IGF-1 (50-80 Ci/g, 50 fmol), Human plactnta film（0.2 mg) and the polypeptide of 3 times of dilutions of series add 0.2 milliliter of 0.1 M Hepes Slow fliud flushing, pH 8, comprising 120mM sodium chloride, 5mM potassium chloride, the sour magnesium of 0.12mM stones gram and 0.1% bovine albumin, is cultivated 1 hour at 20 °C.Sample is filtered with Whatman GF/F filters to be combined and uncombined polypeptide compound with separating.Filter is handled with 0.1% polyethyleneimine 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 Γ 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 μM) measure 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, Μ Α).Polypeptide is relative to actrapid monotard or activity=IC of IGF-1 standards5.Insulin or IGF-1 standards/IC5QPolypeptide.
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：
1st, based on IGF-1 compound of the invention and the binding ability of insulin receptor and IGF-1 acceptors is tested
In this part, the bioactivity to the compound with duplex structure is detected, examines these compounds and insulin receptor and IGF-1 receptor binding capacities.Using natural insulin and IGF-1 respectively as the benchmark with combination rate of insulin receptor and with IGF-1 acceptor binding forces（ 100% ).It the results are shown in Table two.
Although IGF-1 A chains and B chains and the A chains and B chains of insulin have high homology, and the result of NMR and X-ray crystal diffraction displays that IGF-1 and the A chains of insulin and the three-dimensional conformation of B chains are also almost completely superposed, double-strand IGF-1 A:The adhesion of B and insulin receptor is very low, but compared with insulin and too high to the activity of IGF-1 acceptors.Therefore must be that the amino acid residue of IGF-1 A chains and the specific site in B chains causes above-mentioned difference.After B15Q is replaced by phenylalanine or tryptophan, IGF-1 A:B analogue is suitable with insulin in the adhesion of insulin receptor, it was demonstrated that the amino acid residue of B15 be and insulin receptor combination key.IGF-1 has obvious selectivity to IR-A, and double chain compound 11 ~ 16 also show that degree is relatively low, but same receptor-selective.The substitution of A8 can improve adhesion of the polypeptide in 3 acceptors simultaneously, but without the selectivity changed to acceptor.Preferable trypsin class medicine should have high and IR-A and IR-B acceptor binding forces, and alap IGF-1 acceptor binding forces IGF-1 in a balanced way.The side chain negative electrical charge for replacing A5 and A12 with neutral side chain has recovered balance of the polypeptide on IR-A and IR-B, and significantly reduces the activity in IGF-1 acceptors.
Research shows that B9E is the important amino acid residue of IGF-1 growth promoting functions, IGF-1 is had the danger for causing cancer.Replace glutamic acid with histidine, arginine, glutamine or phenylalanine, be maintained to suitable insulin receptor activity.
Lysine residue is added at A22, A23, insulin receptor is combined and had no adverse effect, but lysine Side chains amino can provide the site of an acylation reaction.
Table two：The bioactivity result of the IGF-1 analogs of duplex structure
The 1-17 195 112 3.2 of compound IR-A (%) IR-B (%) IGF-1R (%) 1 compound IR-A (%) IR-B (%) IGF-1R (%) insulin 100 100 0.5
IGF-1 3.66 0.7 100 1-18 333 197 0.9
IGF-1A:
14 4.7 6.3 1-19 149 80 0.9 IGF-1B
1-1 158 63 5.7 1-20 96 73 0.8
1-2 102 52 4.2 1-21 105 102 0.7
1-3 87 49 2.8 1-22 98 71 2.3
1-4 150 61 3.9 1-23 163 155 0.8
1-5 139 73 3.5 1-24 97 101 0.5
1-6 164 99 3.6 1-25 89 92 0.4
1-7 330 173 10.6 1-26 83 86 0.5
1-8 305 162 8.2 1-27 81 82 0.2
1-9 101 119 0.9 1-28 83 80 0.5
1-10 93 89 0.7 1-29 145 134 0.4
1-11 80 85 1.1 1-30 133 127 0.7
1-12 72 69 1.0 1-31 129 120 0.1
1-13 84 76 1.2 1-32 118 115 0.1
Note：IR-A and IR-B are insulin receptor isoform A and B;IGF-1 R insulin-like growth factor-1 receptors； IGF-1A:IGF-1B represents IGF- 1A chains and IGF-1B chain duplex structures.
2nd, the experimental result of the unmodified single chain compound based on IGF-1
In this part, the bioactivity to the unmodified single chain compound based on IGF-1 is detected, examines these single chain compounds and insulin receptor and IGF-1 receptor binding capacities.Using natural insulin and IGF-1 respectively as the benchmark with combination rate of insulin receptor and with IGF-1 acceptor binding forces（ 100% ).It the results are shown in Table three.
Natural IGF-1 is single chain polypeptide, and the adhesion with insulin receptor is less than the 5% of insulin.Since the substitution of B15 amino acids can make the bioactivity of the double chain compound based on IGF-1 reach the level of insulin, same substitution can also improve the activity of the single chain compound based on IGF-1.II -1 displays only change B15 amino acid residues, remove the D domains it is generally acknowledged that being had no significant effect to bioactivity, just improve the insulin receptor activity of compound up to more than 10 times.C2Tyr is a very prominent amino acid residue in IGF-1 crystal structures, display be probably and GF-1 acceptors combination key.After tyrosine is replaced by serine or alanine, insulin receptor activity retains substantially, but considerably reduces IGF-1 receptor actives, exactly expected effect.In addition, result is shown, the length of C peptides is not necessarily required to 12 amino acid residues.Former IGF-1 C more amino acid all can be lacked 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 residues of IGF-1 B chain C-terminals, i.e. X^XK XKHX^X^ are that 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 three：The bioactivity result of unmodified single chain compound based on IGF-1
Chemical combination IR IGF-1R chemical combination IR IGF-1 chemical combination IR IGF-1 chemical combination IR IGF-1 things (%) (%) thing (%) R (%) thing (%) R (%) thing (%) R (%)
II -1 107 5.9 II -32 83 0.4 II -63 86 0.2 II -94 65 0.4
II -2 95 1.6 II -33 85 0.3 II -64 75 0.4 II -95 87 0.2
II -3 87 0.9 II -34 78 0.3 11 -65 58 0.5 11 -96 76 0.3
II -4 90 0.8 11 -35 80 0.4 II -66 89 0.1 II -97 72 0.5
II -5 89 0.7 II -36 84 0.5 II -67 72 0.2 II -98 68 <0.1
II -6 79 1.3 II -37 93 0.7 II -68 76 0.5 II -99 71 0.2
II -7 102 1.1 II -38 79 0.6 11 -69 50 0.4 11 -100 83 <0.1
II -8 99 1.0 II -39 57 0.4 11 -70 57 0.6 11 -101 78 <0.1
II -9 92 1.4 11 -40 62 0.4 II -71 51 0.6 11 -102 80 <0.1
11 -10 78 0.9 11 -41 53 0.7 II -72 63 0.4 11 -103 70 <0.1
11 -11 80 0.7 II -42 66 0.6 II -73 49 0.3 11 -104 76 <0.1
11 -12 77 0.6 II -43 50 0.6 II -74 87 <0.1 11 -105 57 <0.1 II -13 75 0.6 II -44 49 0.5 11 -75 71 0.3 11 -106 90 <0.1
II -14 63 0.5 11 -45 51 0.6 II -76 67 <0.1 11 -107 73 <0.1
II -15 82 0.7 II -46 43 0.4 II -77 59 <0.1 11 -108 62 <0.1
II -16 65 0.6 II -47 47 0.3 II -78 40 0.4 11 -109 44 <0.1
II -17 92 1.0 II -48 45 0.6 II -79 58 0.5 11 -110 39 <0.1
II -18 95 0.4 II -49 87 0.3 11 -80 46 0.3 11 -111 43 <0.1
11 -19 97 0.2 II -50 62 0.2 II -81 53 0.4 11 -112 81 <0.1
II -20 88 0.6 II -51 44 88 <0.1 11 -113 56 <0.1
II -21 87 0.5 II -52 51 0.5 1 11 -83 75 <0.1 11 -114 87 <0.1
II -22 62 0.6 II -53 47 0.5 11 -84 62 0.2 11 -115 79 <0.1
II -23 73 0.8 II -54 63 0.4 11 -85 55 <0.1 11 -116 68 <0.1
II -24 102 0.9 II -55 85 0 o.1 11 -86 82 <0.1 11 -117 43 <0.1
It is very little
II -25 98 0.9 II -56 78 0.3 11 -87 91 0.2 11 -118 65 <0.1
II -26 93 0.4 II -57 90 0.5 11 -88 79 <0.1 11 -119 82 0.8
II -27 91 0.3 II -58 69 0.4 11 -89 86 0.1 11 -120 91 0.7
II -28 106 0.5 II -59 93 0.1 11 -90 56 0.2 11 -121 89 0.7
II -29 85 0.4 II -60 48 0.4 11 -91 78 0.2 11 -122 73 0.6
11 -30 82 0.5 II -61 44 0.3 II -92 49 0.3 11 -123 85 1.0
II -31 91 0.5 II -62 53 0.3 II -93 53 0.5 11 -124 89 0.9
3rd, the experimental result of the compound based on IGF-1 of PEG or fatty acid modifying
In this part, the bioactivity to the compound based on IGF-1 of PEG, albumin or fatty acid modifying is detected, examines these compounds and insulin receptor and IGF-1 receptor binding capacities.Using natural insulin and IGF-1 respectively as the benchmark with combination rate of insulin receptor and with IGF-1 acceptor binding forces（ 100% ).It the results are shown in Table four.
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 new lysine etc. that can introduce has the acylation sites of amino, and there is acylate enough bioactivity to have blunt important meaning for exploitation long-acting polypeptides.
It is demonstrated experimentally that extending to multiple sites of B31, B32, single-stranded IGF-1 analogs C peptides corresponding to the C-terminal of IGF-1 Bl, B28, A14, A15, A22, B chain, suitable insulin receptor activity is all remained after 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 the valid density of polypeptide in an experiment is reduced, therefore the Receptor-Binding Data of acyl polypeptide seems relatively low.But a large amount of zooperies of Past 30 Years show, external activity and the activity in vivo of insulin analog do not have clear and definite corresponding relation.External activity is substantially less than the activity of the analog of actrapid monotard in vivo with actrapid monotard substantially quite (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 zoopery of inventor also indicates that the internal substantial activity of Pegylation and fatty-acylation polypeptide is better than insulin control.
Table four：The bioactivity result of the compound based on IGF-1 of PEG and fatty acid modifying
Compound IR (%) IGF-1R of compound IR (%) IGF-1R 1 compound IR (%) IGF-1R 1
The another compound for providing the present invention has the data of prolongation of effect in the application.
Fig. 1 shows the average value that blood glucose is changed over time after the III-1 of mouse subcutaneous injection physiological saline, actrapid monotard and 3 kinds of various doses.Under Isodose, polyethyleneglycol modified polypeptide action time is considerably longer than actrapid monotard, can reach 24 hours or even the longer time.And the polyethyleneglycol modified flat Slow of polypeptide hypoglycemic effect, even if being increased to 3 multiple doses, do not cause blood glucose low ebb yet.Therefore, Π Ι -1 are better than actrapid monotard in terms of control blood glucose and security.
Fig. 2 show mouse subcutaneous injection physiological saline, actrapid monotard and Π Ι -12 (be 80 nanomoles/kilogram）The average value that blood glucose is changed over time afterwards.Under Isodose, Π Ι -12 onset time is suitable with actrapid monotard, but upon administration 8 hours still have it is obvious suppress hypoglycemic effect, at this moment insulin lost suppression hypoglycemic effect.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.And Ι Π -12 show the hypoglycemic curve of L-type, glycemic control is steady and lasting, and effect is better than actrapid monotard
Fig. 3 be mouse subcutaneous injection physiological saline, insulin detemir and the present invention Π Ι -7 compounds after blood glucose change over time value.Insulin detemir is one of only two kinds of Recent Development of Long-acting Insulin Analogs medicines on current international market, is 14 hours in people's Half-life in vivo, is usually one day pin when using.III-7 only uses the dosage of insulin detemir 1/3, it becomes possible to reach longer action time and more stably blood sugar decreasing effect.
Three above is it is demonstrated experimentally that with the IGF-1 analogs in polyethylene glycol or the fatty acid modifying present invention, can improve the therapeutic effect and pharmacokinetic characteristic of IGF-1 analogs.

Claims (1)

  1. Claims
    1st, a kind of compound with hypoglycemic effect, it is characterised in that the compound includes A chains and B chains, wherein, the amino ^ of A chains is classified as：X^ GIVDXsCCTCwXsX^ioCwX LRRLEX YCwX X, B chain amino is classified as：
    X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFX47X48X49X5oX5iX52X53, wherein,
    It is lysine, arginine or missing；It is lysine, arginine or missing； Χ5It is glutamic acid, asparagine, glutamine or serine； Χ8It is histidine, arginine, phenylalanine or threonine； Χ9It is arginine or serine；10It is serine or isoleucine；Xl2 is aspartic acid, serine, glutamine or asparagine； Χ18It is asparagine, Yue methyllanthionines or threonine； Χ21It is asparagine, alanine or glycine； Χ22It is lysine, arginine-lysine dipeptides or missing； χ23.26It is phenylalanine-valine-asparagine-glutamin tetrapeptide, or GPE, valine-asparagine-glutamin tripeptides, or proline-glutamicacid, asparagine-glutamin dipeptides, or glutamic acid, glutamine, lysine or arginine, or with the sequence after any one amino acid residue in lysine or arginine substitution above-mentioned two, three, tetrapeptide array, or missing； χ27It is histidine or threonine； χ32It is histidine, glutamic acid, glutamine, arginine or phenylalanine； Χ38It is phenylalanine or tryptophan； Χ39It is phenylalanine or tryptophan；4It is arginine, glutamic acid, aspartic acid or alanine；7It is phenylalanine, tyrosine or histidine；8It is-NH2、 dA-NH2, phenylalanine or tyrosine or missing；9It is asparagine, aspartic acid, glutamic acid, threonine or missing； X5()It is lysine, proline, arginine, aspartic acid, glutamic acid or missing； X51It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X52It is threonine or missing； X53It is glutamic acid, aspartic acid, Glu-Glu, Asp-Asp dipeptides or missing；In the compound, [1]-[6] represent the numbering of cysteine；Key is dredged by 63 pair two of cysteine formation in the compound, wherein A chains and B chains is connected by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, and the particular location of three pairs of disulfide bond is： Ctl]And C[4]Form two and dredge key, C[2]With C [6]Form two stone gram keys, Ct3] and C [5] form disulfide bond.
    2nd, compound according to claim 1, it is characterised in that the compound includes A chains and B chains, wherein, the amino acid sequence of A chains is： GIVDX5C[3]C[4]X8RSC[5]X12LRRLEX18YC[6]X21X22,
    B chain amino acid sequences are：
    X23-26X27LC[1]GAX32LVDALX38X39VC[2]GDX44GFYX48X49 X50 X51 X52>Wherein,
    X5It is glutamic acid, asparagine, glutamine or serine； X8It is histidine, arginine or phenylalanine；12It is aspartic acid, serine, glutamine or asparagine； X18It is asparagine, Yue methyllanthionines or threonine； Χ2ι is asparagine, alanine or glycine； X22It is lysine, arginine-lysine dipeptides or missing； X23.26 it is Gly-Pro-glutamic acid tripeptides or phenylalanine-valine-asparagine-glutamin tetrapeptide； X27It is histidine or threonine； X32It is histidine, glutamic acid, glutamine, arginine or phenylalanine； X38It is phenylalanine or tryptophan； X39It is phenylalanine or tryptophan；4 be arginine, glutamic acid, aspartic acid or alanine；8It is-NH2、 dA-NH2Or phenylalanine；9It is asparagine or missing； Χ5.It is lysine, proline or missing； X51It is proline, lysine or missing； X52It is threonine or missing.
    3rd, the compound according to claim 2, it is characterised in that the sequence of the B chains is GPEX27LCGAX32LVDALX3gX39VCGDX44GFY-NH2; GPEX27LCGAX32LVDALX38X39VCGDX44
    GFYFNKPT; GPEX27LCGAX32LVDALX3gX39VCGDX44GFYdA-NH2;Or FVNQX27LCGAX32L VDALX38X39VCGDX44GFYFNKPT, wherein X27、 X32 X38 . X39As defined in claim 2.
    4th, the compound according to claim 2, it is characterised in that the A chain-orderings are GIVDX5CCX8RSCX12LRRLEX18YCA or GrVDX5CCX8RSCX12LRRLEX18YCN, wherein X5、 X8、 X12、 X18As defined in claim 2. 5th, the compound according to claim any one of 1-4, it is characterised in that the compound is selected from following compound： 1-1 ：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 1；The sequence of B chains is
    GPETLCGAELVDALFFVCGDRGFY-NH2;
    1-2：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 1；The sequence of B chains is
    GPETLCGAELVDALWFVCGDRGFY-NH2;
    1-3：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 1；The sequence of B chains is
    GPETLCGAELVDALWWVCGD GFY-NH2;
    1-4:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 7；The sequence of B chains is SEQ ID NO:Sequence shown in 3； 1-5:Wherein A chain-orderings are SEQIDNO:Sequence shown in 8；The sequence of B chains is SEQIDNO:Sequence shown in 3； 1-6:Wherein A chain-orderings are SEQIDNO:Sequence shown in 9；The sequence of B chains is SEQ ID NO:Sequence shown in 3； 1-7:Wherein A chain-orderings are SEQIDNO:Sequence shown in 10;The sequence of B chains is SEQ ID NO:Sequence shown in 3； 1-8:Wherein A chain-orderings are SEQIDNO:Sequence shown in 11；The sequence of B chains is SEQIDNO:Sequence shown in 3； 1-9:Wherein A chain-orderings are SEQIDNO:Sequence shown in 12；The sequence of B chains is SEQ IDNO:Sequence shown in 3； 1-10:Wherein A chain-orderings are SEQIDNO:Sequence shown in 13；The sequence of B chains is SEQ ID NO:Sequence shown in 3; 1-11:Wherein A chain-orderings are SEQIDNO:Sequence shown in 14；The sequence of B chains is SEQ ID NO:Sequence shown in 3 is bad ' J； 1-12:Wherein A chain-orderings are SEQIDNO:Sequence shown in 15；The sequence of B chains is SEQ ID NO:Sequence shown in 3； 1-13:Wherein A chain-orderings are SEQIDNO:Sequence shown in 16;The sequence of B chains is SEQ ID NO:Sequence shown in 3; 1-14:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is SEQ ID NO:Sequence shown in 3； 1-15:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17；The sequence of B chains is SEQIDNO:Sequence shown in 18； 1-16:Wherein A chain-orderings are SEQIDNO:Sequence shown in 19；The sequence of B chains is SEQ ID NO:Sequence shown in 18; 1-17:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 20；The sequence of B chains is SEQ ID NO:Sequence shown in 18； 1-18：Wherein A chain-orderings are SEQ ID NO:Sequence shown in 17；The sequence of B chains is
    GPETLCG AHLVD ALFFVCGDRGFYdA-NH2；
    1-19:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17;The sequence of B chains is SEQ ID NO:Sequence shown in 22; 1-20:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17；The sequence of B chains is SEQ ID NO:Sequence shown in 23； 1-21:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17；The sequence of B chains is SEQ ID NO:Sequence shown in 24； 1-22:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 8；The sequence of B chains is SEQ ID NO:Sequence shown in 25； 1-23:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 26；The sequence of B chains is SEQ ID NO:Sequence shown in 25； 1-24:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17；The sequence of B chains is SEQ ID NO:Sequence shown in 25； 1-25:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17;The sequence of B chains is SEQIDNO:Sequence shown in 27; 1-26:Wherein A chain-orderings are SEQIDNO:Sequence shown in 17；The sequence of B chains is SEQ ID NO:Sequence shown in 28； 1-27:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 151；The sequence of B chains is SEQ ID NO:Sequence shown in 152 is bad ' J； 1-28:Wherein A chain-orderings are SEQID NO:Sequence shown in 153；The sequence of B chains is SEQ ID NO:Sequence shown in 152； 1-29:Wherein A chain-orderings are SEQ ID NO:Sequence shown in 154；The sequence of B chains is SEQ ID NO:Sequence shown in 155 is bad ' J； 1-30:Wherein A chain-orderings are SEQIDNO:Sequence shown in 156；The sequence of B chains is SEQ ID NO:Sequence shown in 155 is bad ' J； 1-31:Wherein A chain-orderings are SEQ IDNO:Sequence shown in 157；The sequence of B chains is SEQ ID NO:Sequence shown in 155； 1-32:Wherein A chain-orderings are SEQ IDNO:Sequence shown in 158；The sequence of B chains is SEQ ID NO:Sequence shown in 155； 1-33:Wherein A chain-orderings are SEQ IDNO:Sequence shown in 159；The sequence of B chains is SEQ ID NO:Sequence shown in 160； 1-34:Wherein A chain-orderings are SEQ IDNO:Sequence shown in 159；The sequence of B chains is SEQ ID NO:Sequence shown in 155.
    6th, a kind of single chain compound with hypoglycemic effect, the compound structure is： Xi01ALC [！] GAX1oibLVDALXLOLCX101dVC[2]GDRGFX1oie i02Xio3Xi04Xio5 i06-CL-GIVDQC[3]C[4]X1 07RSC[5]SLRRLENYC[6]X108 X109, wherein,
    X10Ia is GPE-threonine, GPE-histidine tetrapeptide or phenylalanine-valine-asparagine-glutamin-histidine pentapeptide, or with the sequence after any of the GPE or phenylalanine-Valerian propylhomoserins-asparagine-glutamin in lysine or the above-mentioned tetrapeptide of arginine substitution or pentapeptide amino acid residue；XlOlb is histidine, glutamic acid, glutamine, arginine or phenylalanine；XiOlc is phenylalanine or tryptophan；XlOld is phenylalanine or tryptophan； X10ieIt is phenylalanine, tyrosine or histidine； X1()2It is phenylalanine or missing； X1()3It is asparagine or missing； X104It is lysine, proline or missing； X1G5It is proline, lysine or missing； XIQ6It is threonine or missing； x1()7It is phenylalanine, arginine or histidine； x1()8It is alanine, glycine or asparagine； x109It is lysine, arginine-lysine dipeptides or missing；CL is junction fragment.
    7th, compound according to claim 6, it is characterised in that the structure for being confused compound is：
    X1o1aLC[1]GAHLVDALFFVC[2]GDRGFYX1o2Xio3Xio4Xio5Xi06-CL-GIVDQC[3]C[4]FRSC[5]SL
    RRLENYC[6]A X109, wherein,
    XlOia is GPE-threonine tetrapeptide or phenylalanine-valine-asparagine-glutamin-histidine pentapeptide； X,.2It is phenylalanine or missing； X1()3It is asparagine or missing； X,.4 be lysine, proline or missing； X105It is proline, lysine or missing； XiD6It is threonine or missing； X1Q9It is lysine, arginine-lysine dipeptides or missing；CL is junction fragment.
    8th, compound according to claim 7, it is characterised in that the structure of the compound is：
    GPETLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;
    GPETLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA;
    FVNQHLCGAHLVDALFFVCGDRGFY-CL-GIVDQCCFRSCSLRRLENYCA;
    FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCA;Or FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-CL-GIVDQCCFRSCSLRRLENYCARK。
    9th, the compound according to claim any one of 6-8, it is characterised in that the junction fragment CLIt is the peptide sequence of 6-60 amino acid, each of which amino acid residue is all independently selected from glycine, alanine, serine, threonine or proline.
    10th, compound according to claim 9, it is characterised in that the junction fragment includes 1 or more than 1 aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine.
    11st, compound according to claim 10, it is characterised in that the junction fragment CLIt 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；
    GASPGTSSTSGR ; GSGSSSAAAPQT； GSGSSSAAPQT ; GSGSSSAPQT ; GSGSSSRRA ;
    GSPAGSPTSTSR; GSGPSSATPASR; GSGSSSRGR; GSGPSTRSAPQR; GPETPSGPSSAPQT;
    GAGSSSRAPPPSAPSPSRAPGPSAPQR; GSGSSAGR; GASSPSTSRPGR; GSSSGSSGSPSGR; GSSPSASTGTGR; GAGSSSAPSAPSPSRAPGPSAPQR; GSGSGSGR; GSPSSPTRGSAPQT; GASTSSRGAPSR ； GPSGTSTSAPGR ； GAGSSSAPQT; SSSSAPSAPSPSRPQR ； GSGASSPTSPQR ； GSSPATSATPQT ； GAGSSSAPPPSAPSPSRAPGPSAPQR ； GASTSPSRPSGR; GSTAGSRTSTGR; GSTAGSRTSPQR; GSGTATSGSPQT; GASSSATSASGR; GAGSATRGSASR; GSSSRSPSGSGR; SSSSAPPPSAPSPSRAPGPSAPQR; GSSPSGRSSSPGR; GSPAGSPSSSAGSSASASPASPGR； GSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGR； RREAEDGGGPGAGSSQRK; GGGSGGGR; RRGGGPGAGSSQRK; RGGGPGAGSSQRK; RGGGPGAGSSQRK ； SSSAPPPSAPSPSRAPGPSPQR ； SAASSSASSSSASSASAGR ； GAGGPSSGAPPPSPQT; GSGSSGGR; GAGSPAAPASPAPAPSAGR; SSSAPSPSRSPGPSPQR; SSSAPSAPSPSPQR; GSGSSSRRAPQT; SSSSAASAASASSSASGR; SSSRAPPSAPSPQR; GGPSSGAPPPSR; SSSSGAPPPGR; GPSSGAPSR; GPSSGAPQT ; GGPSSGAPPPSPQT ; SSSAPPPSAPSPSRAPQT； GAGPSSGAPPPSPQT； GGGGAPQT; GAGGPSSGAPPPQT； GGPSSGAPPPSPSPSRPGPSPQR ； SSASSASSSSAGR ; SSASSSAASSSASSSASGR ； SSSGAPPPSPSRAPGPSPQR； GSGSASRGR; SSSSAASSASGR ; SASASASASSASSGR； SASSPSPSAPSSPSPAS ； GPSSPSPSAPSSPSPASPSSGR； SSSAPPPASPSPSRAPGPQR； SASASASASASSAGR; GSGASSRGR; GSGAAPASPAAPAPSAGR; 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.
    12nd, the compound according to any one of claim 6 or 7, the compound is selected from SEQ ID NO:29~ SEQ ID NO: 150, SEQ ID NO: 161、 SEQ ID NO:Compound shown in 162.
    13rd, a kind of compound with hypoglycemic effect, the compound includes A chains and B chains, wherein,
    The amino acid sequence of A chains is：
    X3 9X 00GIVDX405C[3]C[4]X408X409X410C[5]X412LX414¾ 15L 17 8 YC【6]X421 22,
    B chain amino acid sequences are：
    X423-426X427LC[i]GAHLVDALX438X439 C[2]GDRGFX447 448X44 450X45lX452 453 54 55, in the compound, [1]-[6] represent the numbering of cysteine；The compound is connected by 6 cysteine 3 pairs of disulfide bond of formation, wherein A chains and B chains by two pairs of ^ ■ keys of interchain two, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is： Cn]And C[4]Form two stones and fill key, C[2]With C [6]Form disulfide bond, C[3]And C[5]Form two stones and fill key, wherein,
    X399 be arginine, lysine or missing；00 is arginine, lysine or missing； 05It is glutamic acid, asparagine, glutamine or serine；.8It is histidine, arginine, phenylalanine, threonine or formula（I) structure；09It is arginine, serine or formula（I) structure；1()It is histidine, arginine, phenylalanine or formula（I) structure； 12It is serine, isoleucine or formula（I) structure；14It is arginine or formula（I) structure；15It is arginine or formula（I) structure；17It is glutamic acid or formula（I) structure；18It is asparagine or formula（I) structure；21 is alanine, glycine or Tian Dong zhen amine；22It is lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；When 22 be dipeptides, one of amino acid is formula（I) structure；X423 6 be GPE tripeptides,
    UL- GPE, phenylalanine-valine-two propylhomoserins of asparagine-glutamin tetrapeptide or benzene-valine-asparagine-glutamin；27 be histidine or threonine；38 be phenylalanine or tryptophan；39Be phenylalanine or Tryptophan；Xw is phenylalanine, histidine or tyrosine；48It is-NH2, phenylalanine, tyrosine or missing；49 be asparagine, threonine, glutamic acid, aspartic acid or missing；X45Q is lysine, arginine, glutamic acid, aspartic acid, proline or missing；51It is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or is formula
    (I) structure；52It is threonine, lysine or missing, or is formula（I) structure；53It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；54It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；55It is lysine or missing, or is formula（I) structure,
    The formula（I) ：
    ULIt is-W-X-Y-Z structures, aliphatic acid, polyethylene glycol, albumin, L-MLStructure, hydrogen atom or Na-( a-(HOOC(CH2)„CO)-Y-Glu)- , Na-(Na-(CH3(CH2)nCO)-y-Glu)-, wherein η is 8-20 integer, such as 8,10,12,14,16,20, ΝαThe 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 ,-ML structures or hydrogen atom.
    14th, compound according to claim 13, it is characterised in that the compound includes A chains and B chains, wherein, the amino acid sequence of A chains is：
    GIVDQC[3]C[4]FRSC[5]X4i2LX414X415LX4i7X418YC[6]AX22)
    B chain amino acid sequences are：
    In compound described in X423^26 27LC [i] GAHLVDALFFVC [2] GDRGFYX448X449X45oX45lX452X453X454 55', [1]-[6] represent the numbering of cysteine；The compound is connected by 6 cysteine 3 pairs of disulfide bond of formation, wherein A chains and B chains by two pairs of interchain disulfide bonds, there are a pair of intrachain disulfide bonds in A chains, the particular location of three pairs of disulfide bond is： Ctl]And C[4]Form disulfide bond, C[2]And C[6]Form disulfide bond, C[3] and C[5]Form disulfide bond；Wherein, 12For serine or formula（I) structure；14For arginine or formula（I) structure；15It is arginine or formula（I) structure；17For glutamic acid or formula（I) structure；18For asparagine or formula（I) structure；22For lysine, arginine-lysine dipeptides or missing, or it is formula（I) structure；When22During for dipeptides, one of amino acid is formula（I) structure；23_ 426 be GPE tripeptides, UL- GPEs, phenylalanine-valine-asparagine-glutamin tetrapeptide or UL- phenylalanine-valine-asparagine-glutamin；27It is histidine or threonine；48 be-NH2, phenylalanine or missing；49It is asparagine or missing；50 is lysine, arginine, glutamic acid, aspartic acid, proline or missing；51It is proline, lysine or missing, or is formula（I) structure；52It is threonine, lysine or missing, or is formula（I) structure；53Glutamic acid, it is sweet as acid, lysine or missing, or be logical formula (I) structure；54It is glutamic acid, glycine, lysine or missing, or is formula（I) structure；55It is lysine or scarce Lose, or be formula（I) structure, ULAnd formula（I) as defined in claim 13.
    15th, the compound according to claim any one of 13-14, it is characterised in that the compound is selected from：
    III- 1：Duplex structure, including A chains and B chains, wherein, A chain-orderings are SEQ ID NO:Sequence shown in 17；B chain-orderings are G (Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT;
    III-2:Duplex structure, including A chains and B chains, wherein, A chain-orderings are SEQ ID NO:Sequence shown in 17；B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPK (Ne-PEG 20K);
    ΙΠ-6:Duplex structure, including A chains and B chains, wherein, A chain-orderings are： SEQ ID NO:Sequence shown in 17；B chain-orderings are： G(Na-CO(CH2)14COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT;
    ΠΙ-7:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)14CO)-y-Glu)]；
    III-8:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N (Na- (HOOC(CH2)16CO)-y-Glu)];
    III-9:Duplex structure, including Α chains and Β chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FV QHLCGAHLVDALFFVCGDRGFYFNPK[Ne-(Na- (HOOC(CH2)12CO)-y-Glu)]；
    111-10:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK{NE-[Na- (HOOC(C¾)„NHCO(CH2)3CO)
    -γ-Glu]} ;
    III-11 :Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are： FVNQHLCGAHLVDALFFVCGDRGFYFNPK[NE-(Na- (HOOC(CH2)i4CO)-y-Glu -N-(Y-GIU)] ;
    111-19:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPK [NS- (Na-(HOOC (CH2)14 CO)-y-Glu)];
    III -21 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are： GIVDQCCFRSCSLK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]RLENYCA ；B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 274 );
    III -22 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are： GIVDQCCFRSCSLRK[Ne-(Na-(HOOC(CH2),4CO)-y-Glu)]LENYCA ；B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO:275 );
    III -23 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCAK [NE-( a-(HOOC(CH2)14CO)-y-Glu)] ;B chain-orderings are SEQ ID NO:Sequence shown in 275；
    III -24 ：Duplex structure, including A chains and B chains, wherein A chain-orderings are GIVDQCCFRSCSLRRLENYCARK [Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)], B chain-orderings are SEQ ID NO:Sequence shown in 275；
    111-25:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK [NE- (Na-(HOOC(CH2)14CO)-y-Glu)]；
    ΠΙ-26:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [NE-(Na-(HOOC(CH2)i4CO)-y-Glu)]； 111-27:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK [NE- (Na-(HOOC(CH2)i4CO)-Y-Glu)]; ΠΙ-28:Duplex structure, including Α chains and Β chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPK [NE- (Να- (HOOC(CH2)14CO)-y-Glu-N-
    (γ-Giu))];
    111-29:Duplex structure, including Α chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are G [Na-(Na-(CH3(CH2)14CO)-y-L-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT;
    111-30:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are G (Na-dPEG12- maleimide-albumin） PETLCGAHLVDALFFVCGDRGFYFNKPT;
    111-31 :Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are FVNQHLCGAHLVDALFFVCGDRGFYFNPPK [NS- (Na-(HOOC(CH2),4CO)-Y-Glu)];
    III-32:Duplex structure, including Α chains and Β chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTK [N£- (Na-(HOOC(CH2)14CO)-Y-Glu)]；
    111-33:Duplex structure, including A chains and B chains, wherein A chain-orderings are sequence shown in SEQ ID NO 17, and B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTEK [N (Na-(HOOC(CH2)14CO)-Y-Glu)]；
    111-34:Duplex structure, including A chains and B chains, wherein A chain-orderings are SEQ ID NO:Sequence shown in 17, B chain-orderings are GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK [NE- (Na-(HOOC(CH2)14CO)-Y-Glu)]；
    111-36:Duplex structure, including A chains and B chains, wherein A chain-orderings are GIVDQCCHRSCSLRRLENYCA, and B chain-orderings are GPEHLCGAHLVDALFFVCGDRGFYFNPK [Ne-CO-(CH2CH20)5-(CH2)2- NH- (Na-(HOOC
    ΝαRepresent the a- amino of amino acid or amino acid residue； ΝεRepresent the epsilon-amino of amino acid or amino acid residue, the epsilon-amino of such as lysine side-chain.
    16th, a kind of compound with hypoglycemic effect, the structure of the compound is：
    X20iaLC [！] GAX201bLVDALX201cX201d C[2]GDRGFX201eX202 203 204X205X206GX207X207a 208X 209X210 21l 212X213X214X215 216GIVDQC[3]C[4]X2i7RSC[5]X2i8LX219X220LX221X222YC[6]X223X224, wherein,
    X2oiaIt is GPE-threonine, GPE-histidine, phenylalanine-valine-asparagine-glutamin-histidine, UL- GPE-threonine, UL_- GPEs-histidine or UL- phenylalanines-valine-asparagine-glutamin-histidine； X2。lbIt is histidine, glutamic acid, glutamine, arginine or phenylalanine；201(；It is phenylalanine or tryptophan； X2()ldIt is phenylalanine or tryptophan； X201eIt is phenylalanine, tyrosine or histidine； X2Q2It is phenylalanine, tyrosine or missing； X2。3It is asparagine, threonine, aspartic acid, glutamic acid or missing； X2G4It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X2Q5It is proline, lysine, arginine, aspartic acid, glutamic acid or missing or formula（I) structure； X2。6It is threonine, relies ammonia Acid or missing or formula（I) structure； X2Q7It is serine, alanine, glycine, formula（I) structure or missing； x207aIt is serine, alanine, glycine, formula（I) structure or missing； X2G8It is serine, formula（I) structure or missing； X209It is serine, formula（I) structure or missing； X21QIt is serine, formula（I) structure or missing； x211It is arginine, alanine, glycine, formula（I) structure or missing； X212It is arginine, alanine, glycine, formula（I) structure or missing； X213It is alanine, proline, arginine, glycine, formula（I) structure or missing； X214It is proline, glutamine, glycine, formula（I) structure or missing； X215It is glutamine, threonine, glycine, formula（I) structure or missing； X216It is threonine, arginine, lysine, formula（I) structure or missing； x217It is phenylalanine, arginine, histidine or formula（I) structure； X218It is aspartic acid, serine, paddy ammonia barefoot amine, asparagine or formula（I) structure；
    X219 be arginine or formula（I) structure；X22Q is arginine or formula（I) structure；221It is glutamic acid or formula（I) structure； X222It is asparagine or formula（I) structure； X223It is alanine, glycine or asparagine； X224 be lysine, arginine-lysine dipeptides or missing, or is formula（I) structure；Work as X224During for dipeptides, one of amino acid is formula
    (I) structure, wherein ULAnd formula（I) as defined in claim 13.
    17th, the compound according to claim 16, it is characterised in that the structure of the compound is：
    X201aLC[1]GAHLVDALFFVC[2]GDRGFYX2o2X2o3X204X2o5X206GX207GX2o8X209X2ioX2iiX2i2X2i3X 2i4 2i5 2i6GIVDQC[3]C[4]FRSC[5]X218LX219X220LX221X222YC[6] A, wherein,
    X20La is GPE-threonine, phenylalanine-valine-asparagine-glutamin-histidine, UL- GPEs-threonine or the western propylhomoserin-valine-asparagine-glutamin-histidine of UL- benzene； X202 is phenylalanine or missing； X2Q3It is asparagine or missing； X2Q4It is proline, lysine, arginine, aspartic acid or missing；X205 is proline, lysine or formula（I) structure；X206 is threonine, lysine or formula（I) structure； X207It is serine, alanine or formula（I) structure； X2G8It is serine or formula（I) structure； X2o9It is serine or formula（I) structure； X21QIt is serine or formula（I) structure； X211It is arginine, alanine, formula（I) structure or missing； X212It is arginine, alanine, glycine, formula（I) structure or missing； X213It is alanine, proline, arginine, logical formula (I) structure or missing； X2I4 is proline, glutamine, formula（I) structure or missing； X215It is glutamine, threonine, formula（I) structure or missing； X216It is threonine, arginine, lysine, formula（I) structure or missing； X218It is serine or formula（I) structure；219It is arginine or formula（I) structure； X22QIt is arginine or formula（I) structure； X22I is glutamic acid or formula（I) structure； X222It is asparagine or formula（I) structure, wherein ULAnd formula（I) as defined in claim 13.
    18th, the compound according to claim any one of 16-17, it is characterised in that the compound is selected from：
    III-3:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGK( E-PEG20K)GSSSRRAPQTGIVDQCCFR SCSLRRLENYCA;
    III-4:
    GPETLCGAHLVDALFFVCGDRGFYFNPPTG (N£-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
    ΙΠ-5:
    GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(Ne-PEG20K)GSSSAAAPQTGIVDQCCF RSCSLRRLENYCA;
    III- 12:
    GPETLCGAHLVDALFFVCGD GFYFNPTGK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]GSSSA APQTGIVDQCCFRSCSLRRLENYCA; ΠΙ-13:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N£-(Na-(HOOC(CH2)14CO)- Y-Glu)]SSAAPQTGIVDQCCFRSCSLRRLENYCA;
    111-14:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)]A APQTGIVDQCCFRSCSLRRLENYCA;
    111-15:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)]A PQTGIVDQCCFRSCSLRRLENYCA;
    III- 16:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N£-(Na-(HOOC(CH2)14
    CO)-y-Glu)]AAPQT GIVDQCCFRSCSLRRLENYCA;
    111-17:
    GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[NE-(Na-(HOOC (CH2)14CO) -γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA;
    111-18:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[NE-(Na-(HOOC(CH2)14CO)-Y-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA;
    111-20:
    GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[Ne-(Na-(HOOC(CH2)14CO)-Y-Glu)] GRGIVDQCCFRSCSLRRLENYCA;
    111-35: G(Na-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAA PQTGIVDQCCFRSCSLRRLENYCA;
    111-37: GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[Ne-CO-(CH2CH20)5- (CH2)2-NH-(Na-(HOOC (CH2)16 CO)-y-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA;
    NaRepresent the a- amino of amino acid or amino acid residue； ΝεRepresent the epsilon-amino of amino acid or amino acid residue, the epsilon-amino of such as lysine side-chain.
    19th, acceptable carrier in a kind of pharmaceutical composition, including compound and pharmaceutics described in claim any one of 1-18.
    20th, pharmaceutical composition according to claim 19, further comprises Semilente Insulin or insulin analog and/or additive.
    21st, application of the compound according to claim any one of 1-18 in the medicine for the treatment of diabetes or hyperglycemia is prepared.
    22nd, a kind of method for treating diabetes or hyperglycemia, including the compound according to claim any one of 1-18 or the composition described in claim any one of 19-20 are applied to the sufferer of needs.
CN201280061410.6A 2011-12-15 2012-12-14 Compound And Composition Having Hypoglycemic Effect And Use Thereof Pending CN104011070A (en)

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