CN109180802A - Compound, composition with hypoglycemic effect and application thereof - Google Patents

Abstract

The present invention provides a kind of application of the compound with hypoglycemic effect, composition and the compound or composition in treatment diabetes, hyperglycemia.The present invention also provides the methods for the treatment of diabetes, hyperglycemia etc., apply the compound of the present invention or composition including the sufferer to needs.Compared with existing insulin and the like, the compound of the present invention good water solubility, cycle period in vivo is long, the high activity with bound insulin receptor, and is substantially reduced to the toxic effect of individual, and prepare and be easy.

Description

Compound, composition with hypoglycemic effect and application thereof

The application be December 14 2012 applying date, application number 201280061410.6, it is entitled " have drop blood The divisional application of the application for a patent for invention of the sugared compound acted on, composition and application thereof ".

Technical field

The invention belongs to field of biological pharmacy, and in particular to the compound of hypoglycemic effect, composition and they Purposes.

Background technique

Special efficacy and choice drug of the insulin as treatment diabetes, the history for up to the present having been subjected to three generations are drilled Become: first generation product is extracted from the animal pancreas such as pig or ox.Due to heterologous allergic reaction, such product curative effect compared with Difference.Second generation product be rh-insulin, be that insulin gene is obtained from human cell, be then inserted into saccharomycete or It is cultivated in Escherichia coli, is produced by complicated modernization biological gene technology.Third on behalf of insulin analog, It is to be obtained and insulin to people carries out structural modification, including Semilente Insulin and protamine zine insulin.

The large-scale clinical researches such as the NHANS research in the beginning of this century U.S., the CODE research in Europe show to diabetes and its The control that complication is increasingly stringenter, so that the hypertension of diabetic, hyperlipidemia, total cholesterol compliance rate are higher and higher, And the most fundamental blood glucose fluctuation rate does not rise anti-drop, main problem is that existing insulin medicament is still not able to mould well Quasi- physiological insulin secretion mode.Therefore, exploitation curative effect, anti-immunity source, treatment it is up to standard, simulation in terms of all The novel insulin analogs being improved largely will be one of the main solution for improving treating diabetes effect.

American diabetes association (ADA) suggests with European diabetology meeting (EASD) guide, lifestyle modification with After oral hypoglycemic drug therapy, if the glycemic control of diabetic is still undesirable, insulin therapy should be started as early as possible, and And preferred basal insulin is shared with oral hypoglycemic agents.If this therapy not can control blood glucose still, it is proposed that having dinner on this basis Shi Jiayong Semilente Insulin.

Basal insulin is used to maintain euglycemia on an empty stomach to secrete.Many metabolism researchs are found between dinner and night The late time keeps basic insulin level that can reduce the decomposition of three phosphide of glycerol, inhibits liver to export glucose, make fasting blood-glucose It keeps stablizing, to reduce whole blood glucose level.Ideal basal insulin, such as Recent Development of Long-acting Insulin Analogs, it should Neng Goumo Quasi- physiological insulin secretion mode avoids that hypoglycemia especially Nocturnal hypoglycemia occurs, does not put on weight.

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

Second is insulin detemir (detemir).It is that tetradecanoic acid is connected to the insulin type of B29 lysine seemingly Object.Insulin detemir absorbs slowly after injection.Extinction time T at injection_50%About 10 hours.It with it is white in blood Albumen is combined by B29 fatty acid, is then slowly dissociated from complex.Double six is multimerizing (Dihexamerization), six aggressiveness and dimer all extend retention of the insulin detemir at injection in conjunction with albumin Time.After insulin detemir enters blood circulation, in conjunction with albumin, the internal residence time is further extended.

The third is insulin glargine.Drug dissolves in the formulation in pH 3.0, and when pH rises to about after injecting It is crystallized when 7.4.The slow decomposition of injection site brings the effect of delay.But absorbent properties and pharmacokinetics are in crowd It is all very big with a variation in vivo.

Insulin glargine and insulin detemir are that only two kinds of Recent Development of Long-acting Insulin Analogs, longest act on currently on the market Time is no more than 24 hours.Insulin glargine activity on insulin-like growth factor-1 receptor (IGF-1R) is much higher than natural human Insulin.It is disputable always long-term because the generation of insulin-like growth factor-1 receptor and kinds cancer is closely related Whether will increase the cancered risk of patient using insulin glargine.Insulin detemir is about day in the intracorporal bioactivity of people The 20% of right actrapid monotard, therefore its dosage is 5 times of insulin regular dosage, this dramatically increases production and is used Cost.

Insulin requirements when rh-insulin is difficult to meet meal.Human insulin molecule is usually formed six dimeric structures, skin It is gradually depolymerized to dimer after lower injection, circulation could be entered through capillary by being further dissociated into monomer, played hypoglycemic and made With.Since there are depolymerization, absorption process, rh-insulin just works for about 30 minutes after subcutaneous injection, and peak time is long, make With continuing about 6~7 hours.And because of individual difference, injects and eventually enter into the amount of circulation after same dose actrapid monotard and also can There is notable difference.

The limitation of actrapid monotard brings two negative consequences.On the one hand, to guarantee to reduce postprandial blood sugar, diabetes patient must Poor blood glucose control must be easily led in advance, is delayed, is easily caused low in 30 minutes before the meal subcutaneous injection actrapid monotards, meal time Blood glucose.On the other hand, due to, there are depolymerization and individual absorption difference, eventually entering into the pancreas islet of circulation after actrapid monotard's subcutaneous injection Element amount can not be estimated accurately, and insulin excess or deficiency is be easy to cause.

The research and development of Insulin Asp (such as insulin aspart, insulin lispro) are precisely in order to make up actrapid monotard Deficiency.Insulin Asp absorbs fastly, and peak time is short, and peak value is higher, and Cmax continues 1~3 hour, effect lasts Time is 3~5 hours, hence it is evident that is better than actrapid monotard.But the onset time of insulin aspart and insulin lispro is about 20 points Clock, still not convenient enough for diabetes patient, be still improved leeway.

Therefore, exploitation novel insulin analogs, raising bioactivity and bioavilability, extension action time are (long-acting Insulin) or it is individual difference when shortening onset time (Semilente Insulin), improving water solubility, reduce medication, more effective Ground prevents risk of hypoglycemia, increases the important directions that stability is trypsin class medicine exploitation.

Summary of the invention

The object of the present invention is to provide with insulin active, can in conjunction with insulin receptor height, have it is hypoglycemic The compound of effect, pharmaceutically acceptable composition and its application in hypoglycemic.

The first purpose of the invention is to provide a kind of compound with hypoglycemic effect, the compound includes A chain With B chain, wherein

The amino acid sequence of A chain are as follows: X_-1X₀GIVDX₅C_[3]C_[4]X₈X₉X₁₀C_[5]X₁₂LRRLEX₁₈YC_[6]X₂₁X₂₂,

B chain amino acid sequence are as follows:

X_23-26X₂₇LC_[1]GAX₃₂LVDALX₃₈X₃₉VC_[2]GDX₄₄GFX₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃, wherein

X_-1It is lysine, arginine or missing；X₀It is lysine, arginine or missing；X₅Be glutamic acid, asparagine, Glutamine or serine；X₈It is histidine, arginine, phenylalanine or threonine；X₉It is arginine or serine；X₁₀It is silk Propylhomoserin or isoleucine；X₁₂It is aspartic acid, serine, glutamine or asparagine；X₁₈It is asparagine, methionine Or threonine；X₂₁It is asparagine, alanine or glycine；X₂₂It is lysine, arginine-lysine dipeptides or missing；X_23-26 It is phenylalanine-valine-asparagine-glutamin tetrapeptide or glycine-proline-glutamic acid, valine-asparagus fern acyl Amine-glutamine tripeptides or proline-glutamicacid, asparagine-glutamin dipeptides or glutamic acid, rely ammonia at glutamine Acid or arginine, or with lysine or arginine replace above-mentioned two, three, in tetrapeptide array after any one amino acid residue Sequence, 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；X₄₄It is arginine, glutamic acid, aspartic acid or the third ammonia Acid；X₄₇It is phenylalanine, tyrosine or histidine；X₄₈It is-NH₂、dA-NH₂, phenylalanine or tyrosine or missing；X₄₉It is day Winter amide, aspartic acid, glutamic acid, threonine or missing；X₅₀It 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] indicate the number of cysteine；Pass through 6 cysteine shapes in the compound At 3 pairs of disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, a pair of of intrachain disulfide bond of presence in A chain, and three pair two The specific location of sulfide linkage is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond.

In the second aspect, the present invention provide it is a kind of it is single-stranded, can with insulin receptor ining conjunction with, with hypoglycemic effect Compound, the amino acid sequence structure of the compound are as follows:

X_101aLC_[1]GAX_101bLVDALX_101cX_101dVC_[2]GDRGFX_101eX₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆-C_L-GIVDQC_[3]C_[4] X₁₀₇RSC_[5]SLRRLENYC_[6]X₁₀₈X₁₀₉, wherein

X_101aBe glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine tetrapeptide or Phenylalanine-valine-asparagine-glutamin-histidine pentapeptide, or above-mentioned tetrapeptide is replaced with lysine or arginine Or any of glycine-proline-glutamic acid in pentapeptide or phenylalanine-valine-asparagine-glutamin amino Sequence after sour residue；X_101bIt is histidine, glutamic acid, glutamine, arginine or phenylalanine；X_101cBe phenylalanine or Tryptophan；X_101dIt is phenylalanine or tryptophan；X_101eIt is phenylalanine, tyrosine or histidine；X₁₀₂Be phenylalanine or lack It loses；X₁₀₃It is asparagine or missing；X₁₀₄It is lysine, proline or missing；X₁₀₅It is proline, lysine or missing；X₁₀₆ It is threonine or missing；X₁₀₇It is phenylalanine, arginine or histidine；X₁₀₈It is alanine, glycine or asparagine；X₁₀₉ It is lysine, arginine-lysine dipeptides or missing；C_LIt is the junction fragment being defined herein.

In the third aspect, the present invention further provides it is a kind of it is with hypoglycemic effect, repaired on the basis of polypeptide The compound of decorations, with the solubility, stability, body-internal-circulation action time etc. for further increasing the compound.The modification It is the alpha-amido or single-stranded chemical combination that will modify the -terminal amino acid residue for the B chain that side chain is connected to double chain compound of the invention The alpha-amido of the -terminal amino acid residue of object, or it is connected to lysine present in double-strand or single chain compound of the invention Epsilon-amino.

In one embodiment, the compound includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows:

X₃₉₉X₄₀₀GIVDX₄₀₅C_[3]C_[4]X₄₀₈X₄₀₉X₄₁₀C_[5]X₄₁₂LX₄₁₄X₄₁₅LX₄₁₇X₄₁₈YC_[6]X₄₂₁X₄₂₂,

The amino acid sequence of B chain are as follows:

X_423-426X₄₂₇LC_[1]GAHLVDALX₄₃₈X₄₃₉VC_[2]GDRGFX₄₄₇X₄₄₈X₄₄₉X₄₅₀X₄₅₁X₄₅₂X₄₅₃X₄₅₄X₄₅₅；

In the compound, [1]-[6] indicate the number of cysteine；The compound forms 3 by 6 cysteines To disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond, three pair of two sulphur in A chain The specific location of key is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond.Wherein,

X₃₉₉It is lysine, arginine or missing；X₄₀₀It is lysine, arginine or missing；X₄₀₅It is glutamic acid, asparagus fern acyl Amine, glutamine or serine；X₄₀₈It is histidine, arginine, phenylalanine, threonine or logical formula (I) structure；X₄₀₉It is smart ammonia Acid, serine or logical formula (I) structure；X₄₁₀It is serine, isoleucine or logical formula (I) structure；X₄₁₂It is aspartic acid, silk ammonia Acid, glutamine, asparagine or logical formula (I) structure；X₄₁₄It is arginine or logical formula (I) structure；X₄₁₅It is arginine or general formula (I) structure；X₄₁₇It is glutamic acid or logical formula (I) structure；X₄₁₈It is asparagine or logical formula (I) structure；X₄₂₁It is alanine, sweet ammonia Acid or asparagine；X₄₂₂It is lysine, arginine-lysine dipeptides or missing, or is logical formula (I) structure；Work as X₄₂₂For dipeptides When, one of amino acid is logical formula (I) structure；X_423-426It is glycine-proline-glutamic acid tripeptides, U_LGlycine-dried meat ammonia Acid-glutamic acid, phenylalanine-valine-asparagine-glutamin tetrapeptide or U_LPhenylalanine-valine-asparagine- Glutamine；X₄₂₇It is histidine or threonine；X₄₃₈It is phenylalanine or tryptophan；X₄₃₉It is phenylalanine or tryptophan；X₄₄₇ It is phenylalanine, histidine or tyrosine；X₄₄₈It is-NH₂, phenylalanine, tyrosine or missing；X₄₄₉It is asparagine, Soviet Union's ammonia Acid, glutamic acid, aspartic acid or missing；X₄₅₀It is lysine, arginine, glutamic acid, aspartic acid, proline or missing；X₄₅₁ It is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or is logical formula (I) structure；X₄₅₂It is threonine, relies ammonia Acid or missing, or be logical formula (I) structure；X₄₅₃It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₄ It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₅It is lysine or missing, or is logical formula (I) knot Structure；U_LSuch as present invention is as defined below with logical formula (I) structure.

In another embodiment, the compound is single-stranded structure, amino acid sequence structure are as follows:

X_201aLC_[1]GAX_201bLVDALX_201cX_201dVC_[2]GDRGFX_201eX₂₀₂X₂₀₃X₂₀₄X₂₀₅X₂₀₆GX₂₀₇X_207aX₂₀₈X_{20 9}X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆GIVDQC_[3]C_[4]X₂₁₇RSC_[5]X₂₁₈LX₂₁₉X₂₂₀LX₂₂₁X₂₂₂YC_[6]X₂₂₃X₂₂₄, wherein

X_201aIt is glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine, phenylpropyl alcohol ammonia Acid-valine-asparagine-glutamin-histidine, U_LGlycine-proline-glutamic acid-threonine, U_LGlycine-dried meat Propylhomoserin-glutamic acid-histidine or U_LPhenylalanine-valine-asparagine-glutamin-histidine；X_201bBe histidine, Glutamic acid, glutamine, arginine or phenylalanine；X_201cIt is phenylalanine or tryptophan；X_201dIt is phenylalanine or color ammonia Acid；X_201eIt is phenylalanine, tyrosine or histidine；X₂₀₂It is phenylalanine, tyrosine or missing；X₂₀₃It is asparagine, Soviet Union Propylhomoserin, aspartic acid, glutamic acid or missing；X₂₀₄It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X₂₀₅It is proline, lysine, arginine, aspartic acid, glutamic acid or missing or logical formula (I) structure；X₂₀₆It is threonine, relies ammonia Acid or missing or logical formula (I) structure；X₂₀₇It is serine, alanine, glycine, logical formula (I) structure or missing；X_207aIt is ammonia Acid, alanine, glycine, logical formula (I) structure or missing；X₂₀₈It is serine, logical formula (I) structure or missing；X₂₀₉Be serine, Logical formula (I) structure or missing；X₂₁₀It is serine, logical formula (I) structure or missing；X₂₁₁It is arginine, alanine, glycine, leads to Formula (I) structure or missing；X₂₁₂It is arginine, alanine, glycine, logical formula (I) structure or missing；X₂₁₃It is alanine, dried meat ammonia Acid, arginine, glycine, logical formula (I) structure or missing；X₂₁₄Be proline, glutamine, glycine, logical formula (I) structure or Missing；X₂₁₅It is glutamine, threonine, glycine, logical formula (I) structure or missing；X₂₁₆Be threonine, arginine, lysine, Logical formula (I) structure or missing；X₂₁₇It is phenylalanine, arginine, histidine or logical formula (I) structure；X₂₁₈It is aspartic acid, silk ammonia Acid, glutamine, asparagine or logical formula (I) structure；X₂₁₉It is arginine or logical formula (I) structure；X₂₂₀It is arginine or general formula (I) structure；X₂₂₁It is glutamic acid or logical formula (I) structure；X₂₂₂It is asparagine or logical formula (I) structure；X₂₂₃It is alanine, sweet ammonia Acid or asparagine；X₂₂₄It is lysine, arginine-lysine dipeptides or missing, or is logical formula (I) structure；Work as X₄₂₂For dipeptides When, one of amino acid is logical formula (I) structure；U_LSuch as present invention is defined hereinafter with logical formula (I) structure.

The fourth aspect of the invention is to provide a kind of pharmaceutical composition, by the compound with blood sugar reducing function of the invention It is mixed with pharmaceutically acceptable carrier, 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 with It is Asp^B28Actrapid monotard, Lys^B28Pro^B29Actrapid monotard or Lys^B3Glu^B29Actrapid monotard.

The fifth aspect of the invention is to provide the compound of the present invention and treats the medicines such as diabetes or hyperglycemia in preparation Application in object.

The sixth aspect of the invention is to provide a kind of method for treating diabetes or hyperglycemia etc., including to needs Sufferer applies the compound of the present invention or composition.

Compared with existing insulin and the like, the compound of the present invention good water solubility, have bound insulin by The high activity of body, low to the toxic effect of individual, preparation is easy.The circulation time of compound in vivo after modification obviously prolongs It is long.

Detailed description of the invention

Fig. 1 is III -1 compound of the invention of mouse subcutaneous injection physiological saline, actrapid monotard and three kinds of various doses Blood glucose changes over time value afterwards；

Fig. 2 is that blood glucose becomes at any time after mouse subcutaneous injection physiological saline, actrapid monotard and III -12 compound of the invention Change value；

Fig. 3 is that blood glucose becomes at any time after mouse subcutaneous injection physiological saline, actrapid monotard and III -7 compound of the invention Change value.

Specific embodiment

Definition and term

Unless otherwise stated, following definitions are suitable for present invention full text.Undefined term can be according to arranging in industry Custom at definition understand.

" amino acid " refers to that molecule that is any while including amino and carboxyl functional group, the amino of a-amino acid are connected with carboxyl On the same carbon atom (α carbon).α carbon can have 1-2 organic substituent.Amino acid includes L and dexiroisomer and racemization Mixture.Unless otherwise instructed, the amino acid residue in the present invention in polypeptide sequence is all L isomer i.e. l-amino acid, D- amino acid is before amino acid name or abbreviation plus lowercase " d " is indicated, such as dK.

Expression way " codified amino acid " or " codified amino acid residue " can be by nucleotide triplets for expression The amino acid or amino acid residue of coding.

HGlu is high glutamic acid；

α-hGlu is-HNCH (CO-) CH₂CH₂CH₂The L isomer of COOH；

δ-hGlu is-HNCH (COOH) CH₂CH₂CH₂The L isomer of CO-；

α-Asp is-HNCH (CO-) CH₂The L isomer of COOH；

β-Asp is-HNCH (COOH) CH₂The L isomer of CO-；

α-Glu is-HNCH (CO-) CH₂CH₂The L isomer of COOH；

γ-Glu is-HNCH (COOH) CH₂CH₂The L isomer of CO-；

β-Ala is-HN-CH₂-CH₂-COOH；

Sar is sarcosine.

Amino acid residue can be indicated with three letter amino acid code or one letter amino code；Amino acid table is as follows It is shown:

Table one: it amino acid name and writes a Chinese character in simplified form

Chinese

English name

Trigram

Single-letter

Chinese

English name

Trigram

Single-letter

Glycine

Glycine

Gly

G

Threonine

Threonine

Thr

T

Alanine

Alanine

Ala

A

Cysteine

Cysteine

Cys

C

Valine

Valine

Val

V

Methionine

Methionine

Met

M

Leucine

Leucine

Leu

L

Asparagine

Asparagine

Asn

N

Isoleucine

Isoleucine

Ile

I

Glutamine

Glutamine

Gln

Q

Proline

Proline

Pro

P

Tryptophan

Tryptophan

Trp

W

Phenylalanine

Phenylalanine

Phe

F

Serine

Serine

Ser

S

Tyrosine

Tyrosine

Tyr

Y

Lysine

Lysine

Lys

K

Aspartic acid

Aspartic acid

Asp

D

Arginine

Arginine

Arg

R

Glutamic acid

Glutamic acid

Glu

E

Histidine

Histidine

His

H

" natural insulin " refers to plain (such as people's pancreas of mammalian islet from natural, chemical synthesis, genetic engineering production Island element, bovine insulin, pork insulin etc.).Actrapid monotard includes the A chain of 21 amino acid compositions and the B of 30 amino acid composition Chain.Two chains are connected by 3 disulfide bond: A7 and B7, A20 and B19, A6 and A11.B7, A7 refer to natural insulin B chain position Set the amino acid residue of 7 (from N-terminal numbers) 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 modified, including having homologous sequence with natural insulin The duplex molecule and single-chain insulin analogues being made of A chain and B chain." insulin analog " has natural insulin Partially, whole or enhancing activity, or it can be converted into part, whole or enhancing with natural insulin in vivo or in vitro Active polypeptide, such as than natural insulin increase, reduction or the polypeptide for replacing one or more amino acid residues.People, animal Or even proinsulin, preproinsulin, insulin precurosor, single-chain insulin precursor and the analog of nonmammalian are referred to as " Insulin analog ".Many insulin analogs are seen in document.Except non-specifically other explanation, " insulin analog " broad sense Including natural insulin and insulin analog.

IGF refers to insulin-like growth factor (insulin-like growth factor), including insulin-like growth factor - 1 (IGF-1) of son and insulin-like growth factor-2 (IGF-2).

The sequence of the A chain of people IGF-1 is sequence shown in SEQ ID NO:1, and the B chain-ordering of people IGF-1 is SEQ ID NO:2 Shown sequence.IGF-1 analog relative to natural human IGF-1 molecule have one or more amino acid mutation, substitution, missing or Addition.IGF-1 analog includes double-strand and single-stranded IGF-1 analog.

IGF-2 analog relative to natural human IGF-2 molecule have the mutation of one or more more amino acids, replace, Missing or addition.IGF-2 analog includes double-strand and single-stranded IGF-2 analog.

Unless otherwise specified, insulin involved in the application refers to that actrapid monotard, IGF-1 refer 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 chain of double chain compound and B chain Number defers to following principle:

The A chain of the compound is from 1 (X of number₁Or X₄₀₁) start to 22 (X of number₂₂Or X₄₂₂), except the position changed in the application Point is outer, and preceding 21 amino acids of compound A chain are corresponding with the amino acid of A chain of people IGF-1, especially compound A chain the 6th (X₆Or X₄₀₆)、7(X₇Or X₄₀₇)、11(X₁₁Or X₄₁₁) and 20 (X₂₀Or X₄₂₀) position is cysteine.If in X₁N-terminal add amino Sour residue, then the number of amino acid residue is X_-1And X₀；If in X₄₀₁N-terminal add amino acid residue, then amino acid The number of residue is X₃₉₉And X₄₀₀。

The B chain of the compound is from 23 (X of number₂₃Or X₄₂₃) start, in addition to the site changed in the application, compound B chain 28 (X of number₂₈Or X₄₂₈) extremely 47 (X of number₄₇Or X₄₄₇) amino acid and 5 to 24 of the B chain of people IGF-1 amino acid phases It is corresponding, the especially 29 (X of number of compound B chain₂₉Or X₄₂₉)、41(X₄₁Or X₄₄₁) position is cysteine.

It when individually referring to some amino acid, can be indicated with such as A1G, B1G or B9H, respectively refer to first in A chain Amino acid, the first of B chain and the 9th amino acid residue are G, G, H respectively.

Subject to explanation of the number of single chain compound according to each compound.

Single chain compound is referred to general structure B chain-C_LThe polypeptide sequence of-A chain or the polypeptide sequence of modification, wherein B chain is B chain of IGF-1 or the like, and A chain is A chain of IGF-1 or the like, C_LIt is connection B chain C-terminal amino acid residue and A The peptide chain of chain N-terminal.

Unless otherwise specified, with A chain or the amino acid of B chain position description in the application, such as A14, B28 are indicated and IGF- 1 A chain or the amino acid of B chain opposite position or its variation, wherein the A chain of IGF-1 or the number of B chain are since 1.

The number of cysteine in compound:

For convenience of description, the cysteine in each compound in the present invention is numbered, respectively C_[1]~C_[6], It is corresponding to be:

When the compound is double-strand, C_[1]And C_[2]It respectively corresponds in the B chain of double chain compound from N-terminal to the two of C-terminal A cysteine；C_[3]~C_[6]Be corresponding in turn in A chain from N-terminal to the 4 of C-terminal cysteines, that is, respectively correspond A chain number be 6, 7,11 and 20 cysteines；

When the compound is single-stranded, C_[1]~C_[6]It is corresponding in turn to 6 half of the single chain compound from N-terminal to C-terminal Cystine.

The compound of the present invention is the structure based on IGF-1, and IGF-1 and IGF-2 and insulin shape in an identical manner At disulfide bond.All include disulfide bond in the tertiary structure of any one compound in the present invention, all single-stranded IGF-1 analogs and Double-strand IGF-1 analog has three pairs of disulfide bond that mode is equally constituted with insulin.Therefore, those skilled in the art are based on upper State the disulfide bond position that explanation and common knowledge are understood that completely and know any one compound in the present invention.

" modification group "

IGF-1 analog may include one or more modification groups.Modification group is capable of providing IGF-1 analog needs Feature.For example, modification group can reduce the degradation rate of IGF-1 analog (such as alimentary canal, blood) under circumstances. Preferred modification group is that those allow IGF-1 analog to retain suitable insulin receptor in conjunction with active group.Preferably repair Decorations group includes amphiprotic group, water soluble group, or makes IGF-1 analog lipophilicity lower than non-modified analog, more The group of highly-water-soluble.Modification group may include degradable linker, such as PAG；It may include linker susceptible to hydrolysis, Such as lactide, glycolide, carbonic acid, ester, carbamate.This method can make polymer be degraded into small-molecular-weight segment.

Modification group may include one or more hydrophilic radicals, lipophilic group, amphiprotic group, salt forming group, interval base The combination of group, linking group, end-capping group or these groups.Various groups can be with covalent bond, or with hydrolyzable or can not water The key connection of solution is together.Representative hydrophilic radical and lipophilic group are described below.

Hydrophilic radical

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

Polyalkylene glycol (Polyalkylene Glycol, PAG) group is made of multiple aklylene glycol monomers.? In one embodiment, all monomers are identical (such as polyethylene glycol (PEG) or polypropylene glycols (PPG)).In another implementation In example, aklylene glycol is different.Condensate can be random copolymer (such as the copolymerization of ethylene oxide and propylene oxide Object) or branch or graft copolymer.

" PEG " or polyethylene glycol used herein refer to any water-soluble polyethylene glycol or polyethylene glycol oxide.Polyethylene glycol Chemical structural formula is-(CH₂CH₂O)_n, wherein n can be the integer from 2 to 2000.One end of PEG is usually to be relatively free of work The functional group of property, such as alkyl or alkoxy.Alkyl includes the linear chain or branched chain alkyl of saturation.The example of alkoxy stands is Methoxyl group, ethyoxyl, propoxyl group (such as 1- propoxyl group and 2- propoxyl group), butoxy (such as 1- butoxy, 2- butoxy and 2- Methyl -2- propoxyl group), amoxy, hexyloxy etc..MPEG, structural formula CH are named as using the PEG of methoxy group₃O (CH₂CH₂O)_n, but general still referred to as PEG.PEG20K refers to that molecular weight is 20,000 peg molecule.

The PEG other end is usually activating functional group or the functional group for being easily formed covalent bond, such as amino, carboxyl, hydroxyl Base, sulfydryl, aldehyde etc..PEG- maleimide, PEG- vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan) and PEG- iodoacteyl (CO-CH₂- I) etc. can be with half Guang Sulfydryl-the SH of propylhomoserin side chain reacts to form stable covalent bond；PEG-NHS (succinimide) can be with polypeptide N-terminal α-ammonia Base or lysine side chain amino groups are engaged by nucleophilic substitution (acylation)；The amino of PEG- aldehyde and polypeptide is in reducing agent (such as cyanogen Base sodium borohydride) effect is lower can pass through standard reductive alkylation reaction engagement.

PEG molecule in the present invention can be straight chain, branch, bifurcated or dumbbell shaped PEG.In one embodiment In, branch PEG can use general formula R (- PEG-_nOH)_mIt indicates, wherein R (usually polyhydroxy) is core group, such as season penta Tetrol, sugar, lysine or glycerol；M represents branch number, can be from 2 and plays core group attachment site maximum number；N is represented The quantity of the quantity of PEG segment, the PEG segment on each branch can not wait.Under normal circumstances, n is the integer of 2-1800.? In another embodiment, branch PEG can use general formula (CH₃O-PEG-_n)_pR-Z indicates that p is equal to 2 or 3, and R is lysine or sweet Oil, Z represent the activating functional group that can be reacted.In one embodiment, bifurcated PEG general formula PEG (- L-X)_nIt indicates, L It is linker, X is terminal activating functional group.

PEG is usually polydispersion, and polydispersity index is less than 1.05.PEG group is also possible to monodispersed.Monodisperse refers to PEG has single length (molecular weight), rather than the mixture of various length (molecular weight).

Glycosyl group

Representative glycosyl group includes, but are not limited to, glycerol, monosaccharide, disaccharides, trisaccharide, oligosaccharides and polysaccharide such as starch, sugar Former, cellulose and/or polysaccharide gum.Special monosaccharide includes C6 or more (especially C6 and C8) sugar such as glucose, fructose, sweet Reveal sugar, galactolipin, ribose or sedoheptose；Disaccharides and trisaccharide include containing two or three monosaccharide unit (especially 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, such as poly- bad The amino acid polymer with multiple positive charges of propylhomoserin 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 methylamino acrylic acid Ester, quaternary polyamines etc..Biocompatible polyanion group includes the group on skeleton or side chain with multiple carboxyls, such as poly- Aspartic acid, polyglutamic acid etc..Other hydrophilic radicals include natural or synthetic polysaccharide, such as chitosan, glucan.

Polyanion bioadhesive polymer

Certain hydrophilic radicals have potential bio-adhesive properties.Such example is found in United States Patent (USP) US 6,197, 346.These polymer with multiple carboxyls show bio-adhesive properties.The quick bio drop of multiple carboxyls is manifested when degradation Depolymerize object, as lactic-co-glycolic acid (poly (lactide-co-glycolide)), polyanhydride, polyorthoester are also all Bioadhesive polymer.These polymer can be launched IGF-1 analog to gastrointestinal tract.The carboxyl that polymer is exposed when degrading It can be firmly attached to gastrointestinal tract, assist to launch IGF-1 analog.

Lipophilic group

In one embodiment, modification group includes one or more lipophilic groups.Lipophilic group can be people from this field Member it is well known that including, but are not limited to: alkyl, alkenyl, alkynyl, aryl, aryl alkyl, alkylaryl, fatty acid, Cholesterine and lipophilicity polymer and oligomer.

Alkyl can be saturation, unsaturation, straight chain, branch or cyclic hydrocarbon, have one or more carbon atoms.At one In 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 group, this A little substituent groups will not preferably make conjugate lose bioactivity.

Lipophilic group is also possible to fatty acid, as it is natural, synthesis, saturation, it is unsaturated, straight chain or branch Fatty acid.In one embodiment, fatty acid have 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 atom.

In conjunction with (Conjugation) strategy

The selection of the combination degree, binding site of modification group and polypeptide, the selection of modification group will be varied as desired, Such as to make conjugate not degradable in vivo, to extend plasma half-life.Such as IGF-1 analog modification after have one, Two, three, four or more modification group.Binding site may include an amino acid residue, such as lysine residue.? In one embodiment, IGF-1 conjugate is single conjugate.In another embodiment, IGF-1 conjugate is more conjugates.? In another embodiment, IGF-1 conjugate is the mixture of single conjugate, double combination object, three conjugates, four conjugates etc..It repairs Decorations group may be the same or different.When IGF-1 conjugate has multiple modification groups, one or more modification groups Preferably by hydrolyzable bond be connected with IGF-1 and other one or more modification groups preferably by non-hydrolysable key and IGF-1 Conjugate is connected.It is connected with IGF-1 alternatively, all modification groups all pass through hydrolyzable bond, but each modification group is in vivo Hydrolysis rate faster or slower.

Preferably combining strategy is that conjugate is made to have the part or all of bioactivity of original IGF-1 analog.Preferred knot Closing position includes the end B1-N, B chain lysine side chain amino groups or single-stranded N-terminal, lysine side chain amino groups of double-chain polypeptides etc.. The mono- conjugate of B1 and B chain double combination object are most common.It in addition can be more by the junction fragment or double-strand in single chain compound Peptide A chain, B chain introduce the natural or non-natural amino acids with amino or sulfydryl to create other binding sites.

Modification group and IGF-1 analog can be tied by hydrolyzable bond (such as ester, carbonic acid, hydrolyzable carbamate) It closes.Hydrolyzable bond makes IGF-1 conjugate have the effect of prodrug.If it is desired to modification group and the no activity of IGF-1 conjugate, Such as the binding site of modification group in IGF-1 analog to insulin receptor combined area, prodrug strategies are exactly preferred method.When One or more modification groups are detached from from IGF-1 conjugate whithin a period of time, to release active IGF-1 analog, are made The effect of delay release or sustained release is capable of providing with hydrolyzable bond.

In one embodiment, IGF-1 analog is connected by non-hydrolytic key (such as amido bond, ehter bond) with modification group. When necessary, non-hydrolytic key helps to extend circulation time of the IGF-1 conjugate in blood plasma.

IGF-1 homologue can be connected by various nucleophilic functional groups with modification group, including but not limited to nucleophilic hydroxyl Base or amino.Such as serine, threonine, tyrosine have nucleophilic hydroxy, histidine, lysine or IGF-1 analog A chain, B The end chain N- all has nucleophilic amino.IGF-1 homologue can also be connected by free sulfhydryl group-SH with modification group, such as shape At thioesters, thioether, sulfanilamide (SN) key.

Circulation time of the lesser polypeptide compound of molecular weight in a blood plasma short key factor is exactly that kidney is removed.Increase Peptide compounds molecular weight is added until being more than that renal clearance can be significantly reduced in kidney removing critical point, extends polypeptide and acts in vivo Time.Common method is that polypeptide and natural or synthetic macromolecular is made to form hydrolyzable or non-hydrolysable key.Large biological molecule packet Include albumin, polysaccharide, antibody (such as IgG).70% albumin is mercaptalbumin (mercaptalbumin) in blood vessel, The side chain thiol of cysteine -34 is the strongest sulfydryl of activity in blood plasma.IGF-1 analog can have horse by an one end The linker for carrying out the activating functional groups such as acid imide, which reacts with it, generates IGF-1- albumin conjugates.Linker itself can be length Chain fatty acid or PEG molecule.Specific example is referred to Bioconjugate Chem.2005,16,1000-1008.Synthesis is big Molecule includes polyethylene glycol and glucan.Another way is fatty-acylation, will be discussed in acylated IGF-1 analog part.

Occur being catalyzed the method for combining two molecules by amido bond with protease sortase in recent years.Sortase It is a kind of mediation gram-positive bacteria cell wall and the covalently bound transpeptidase of surface protein, is primarily present in gram-positive bacteria In.The sequential analysis of protein of GeneBank CDS and NCBI data bank is shown, sortase family includes 150 multiple protein sequences It arranges, wherein Staphylococcus aureus sortase A (SrtA or SrtA_Staph) be current most study isotype (isoform).There are more documents to disclose the molecular mechanism of the transpeptidation reaction of SrtA catalysis.SrtA identification includes LPXTG (Leu- Pro-X-Thr-Gly) the substrate of motif, 184 cysteines are as the peptide bond in nucleophilic group attack LPXTG motif Thus Thr-Gly generates an acyl-enzyme intermediate.The thioesters intermediate of threonine carboxyl passes through the oligomeric sweet ammonia with substrate Acid (is the pentaglycine (Gly of the precursor commissure bridge of branched lipids II in S.aureus₅)) amino group occur nucleophilic it is anti- It answers, generates new connection product.

Another relevant Streptococcus pyogenes sortase can receive the parent being made of two alanine Core group, but S.aureus enzyme cannot.This sortase (SrtA_strep) cutting LPXTA motif in peptide bond Thr-Ala, allow Nucleophilic group based on alanine.SrtA_strepIt can also identify LPXTG motif, but activity is lower.LPXTA motif will not By SrtA_StaphCutting.For the sake of simplicity, following methods are discussed with SrtA_StaphFor, but SrtA_strepEqual isotypes can also be with With the same or similar method.

Glycine of the SrtA to LPXTG motif and N-terminal with free amino repeats (a-Gly_n) highly single-minded.X can be with It is all natural amino acids (not yet tested) in addition to cysteine and tryptophan.Show N-terminal with one in spite of experiment The polypeptide of glycine can participate in the transpeptidation reaction of sortase catalysis, and N-terminal has the substrate of two or more glycine It can achieve maximum reaction efficiency.The main application of one of Sortase auxiliary connection (Sortase mediated ligation) It is exactly that non-natural functional group is introduced into albumen or polypeptide.Non-natural functional group can be small molecule, synthesis polypeptide or albumen, polymerization Object etc..These functional groups and LPXTG or a-Gly_nMolecule made of fusion can become the substrate of SrtA.Specific method and anti- Condition is answered to be referred to pertinent literature (such as Tsukiji, " Sortase-Mediated Ligation:A Gift from Gram-Positive Bacteria to Protein Engineering",ChemBioChem,2009,10,787-798； Popp etc., " Sortase-catalyzed transformations that improve the properties of Cytokines ", PNAS, 2011,108,3169-3174).

SrtA can introduce non-natural functional group on IGF-1 analog, and specific strategy will be according to the knot of IGF-1 analog Depending on structure.For double-strand IGF-1 analog, the binding site of general little effect bioactivity is the N-terminal or A chain, B chain of B chain C-terminal.If binding site is the N-terminal of B chain, the N-terminal of IGF-1B chain is preferably introduced multiple glycine, such as GGGGG-IGF- 1B chain；And the C-terminal of the modification group to be introduced such as PEG, long chain fatty acids or albumin etc. will have LPXTG motif, such as PEG- LPATGGGG, albumin-LPETGGG or fatty acid LPGTGGGGG etc..If binding site is the C-terminal of A or B chain, A or B The c terminal amino acid sequence of chain will include LPXTG motif, for example can become IGF-1A-LPATGGGGG or IGF-1B- LPGTGGGG etc., and the N-terminal of the modification group to be introduced such as PEG, long chain fatty acids or albumin etc. will have one or more sweet Propylhomoserin, such as GGG-PEG, GGGG- long chain fatty acids, GGGGG- albumin.For single-stranded IGF-1 analog, the knot being easiest to Coincidence point is that the N-terminal of B chain or the C-terminal of A chain, 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 of 70 amino acid, There are four domain (claiming " chain " in some documents) for tool.Wherein the A chain and B chain structure of the domain A and B and insulin are homologous；And the domain C connects A It is corresponding with the C peptide of proinsulin with the domain B；The domain D is the C-terminal extension in the domain A.The three-dimensional structure of IGF-1 has had NMR With the report of X-ray method for crystallising.The crystalline texture of the structure and insulin in its domain B and the domain A and the NMR structure of proinsulin It is closely similar.

Insulin Receptor Family includes insulin receptor (IR), insulin-like growth factor-1 receptor (IGF-1R) and pancreas islet Plain receptor associated receptor, they are all receptor tyrosine kinases, transmit letter and increasing phosphoric acid molecules on specific tyrosine Number.The member of Insulin Receptor Family is made of two acceptor portions, each includes the β subunit of extracellular α subunit and cross-film, It is connected by disulfide bond.When not combining aglucon, this receptor exists with dimeric forms, is formed through two between two α subunits Combined 2 beta 2 receptor of α of sulfide linkage.Depending on the region being compared, insulin receptor is similar with the sequence of IGF-1 receptor Degree is 41-84%.

The ability of IGF-1 bound insulin receptor is 100 times lower than insulin, with IGF-1 induced insulin receptor in vivo Phosphorylation and the practical capacity of hypoglycemia are not inconsistent (it is about the 10% of insulin that IGF-1, which activates the ability of insulin receptor). Therefore part signal conduction may be by IGF-1 receptor/insulin receptor heterodimer.

Almost each of human body cell is all influenced by IGF-1, especially muscle, cartilage, bone, liver, kidney, mind Through cell in, skin and lung.In addition to insulin-like effects, the equally adjustable cell growth and development of IGF-1 are especially neural The DNA of cell and cell synthesis.

About 98% IGF-1 is always combined with one of six kinds of IGF-1 binding proteins (IGF-1-BP).Wherein IGF-1BP3 Quantity is maximum, combines 80% IGF-1.The combination molar ratio of IGF-1 and IGF-1BP-3 is 1:1.

IGF-1 is similar with the metabolic response of insulin: two kinds of hormones increase in a very similar way glucose absorption and Oxidation inhibits glucose production, free fatty acids horizontal and fat oxidation rate.

Insulin resistance is made of many common and several uncommon clinical symptoms.Insulin receptor gene or with There is the patient of mutation that there is different phenotypes, including lipodystrophy, partial fat in the relevant gene of signal transduction path It is dysbolism, the A pattern synthesis disease of insulin resistance with insulin receptor gene mutation, false acromegaloidism, short Evil spirit's looks syndrome and Rabson-Mendenhall syndrome.In many such sufferers, hyperglycemia is quite difficult to control It treats, because insulin is invalid.

Insulin resistance is equally common in the sufferer of adult-onset diabetes.Human body is by increasing insulin Secretion overcomes this problem, but causes the Expression of Insulin Receptor on target tissue to reduce, is degrading insulin resistance.High glycerine Three ester mass formed by blood stasis are cometabolism exception in these sufferers.Therefore, can obviously change specific to the treatment of insulin resistance Kind treating diabetes effect.

IGF-1 has been proposed for the treatment of severe insulin Antagonism.Because of its biological action and insulin It is similar, it is possible to avoid the physical function defect for hindering insulin to play a role.It has been found that intravenous injection recombination IGF-1 subtracts Lacked two A pattern synthesis diseases with insulin resistance sufferer and one with Rabson-Mendenhall syndrome The blood glucose and serum insulin concentration of child.In the research of the sufferer of several severe insulin Antagonisms with different phenotypes In, reduce fasting and 24 hourly average serum insulin concentrations using IGF-1, improve glucose tolerance, increases insulin Susceptibility and reduce fasting serum triglyceride concentrations.

About 60% diabetic can lead to hand or foot stolidity or have tingling by neurotrosis in life Sense.Similar neurotrosis may weaken control of the human body to blood pressure, cause incontinence or impotence, or cause diarrhea or constipation.Sugar It urinates sick rat studies and shows that neurotrosis caused by this paradiabetes can treat recovery by IGF-1.According to conjecture, diabetes Nerve cell is prevented to be unable to normal development, and IGF-1 is allowed to restore normal.

But the beneficial effect of IGF-1 has cost.IGF-1 may cause cardiovascular vigorous reaction, including the rhythm of the heart Stopping and low blood pressure.Certain reactions may be because IGF-1 causes blood phosphate sharply very few.Long-term, high-dose subcutaneous injection IGF-1 can generate uncomfortable temporomandibular joint, face and hand edema, weight gain, expiratory dyspnea, sinus tachycardia.But most recently Studies have shown that can be resistant to very well and effective dosage can be accomplished.But the best way is exactly to modify molecular structure, IGF-1 analog is improved in the binding force of insulin receptor, while reducing its activity on IGF-1 receptor.

The further advantage of IGF-1 analog includes, but are not limited to, water solubility more better than actrapid monotard, higher production Rate develops the potentiality etc. of the double agonists of insulin receptor and IGF-1 receptor.

Inventor is studied for a long period of time discovery, is connected in a manner of the A chain of IGF-1 is connected with B chain by insulin, and by B chain B15 amino acids residue W or F are replaced with by Q after, such IGF-1 analog and insulin receptor binding ability and people's pancreas Island element is quite.

Further, the IGF-1 analog of the invention in conjunction with insulin receptor height be the B chain of IGF-1 in B15 and Amino acid residue on two positions B16 is substituted.After the QF on the former position B15 and B16 is replaced by FF, WF or WW, new To IGF-1 analog also show and the comparable performance in conjunction with insulin receptor of natural insulin.It is taken by FF, WF or WW The IGF-1 analog in generation also maintains ability of the part in conjunction with IGF-1 binding protein.IGF-1 analog combination IGF-1 is combined This ability of albumen, which is believed to be helpful in, extends the time that IGF-1 analog is recycled and acted in serum.

Two chain analogs including people IGF-1A chain and actrapid monotard's B chain are tested in insulin-like activity (as fat generates) In show and be equivalent to about the 40% of insulin ability, but in growth factor experiment (such as thymidine incorporation), activity is obvious It is higher than insulin, it is equivalent to about 730%.But the compound is the growth factor more inefficient than IGF-1 itself, is equivalent to About the 26.5% of IGF-1.Although it have been demonstrated that the domain C of IGF-1 is the principal element of IGF-1 acceptor binding force, but including people The two chain analogs of IGF-1A chain and actrapid monotard's B chain show decrease but still apparent IGF-1 acceptor binding force, display The structural property for including in the domain A of IGF-1 causes raised growth promotion ability.And multiple studies have shown that, excessively active IGF-1 Receptor may cause cancer.

There are two hypotypes for insulin receptor: IR-A and IR-B.IR-A has the growth promoting function similar to IGF-1 receptor, and The major function of IR-B is Metabolism regulation (such as glycometabolism).The basic phase of binding ability of natural human insulin and two receptor subtypes When.And IGF-1 and IGF-2 is significantly higher than the binding ability to IR-B to the binding ability of IR-A.IGF-1 analog still part Maintain this selectivity.And ideal IGF-1 analog should be able in conjunction with insulin receptor height, but with IGF-1 by The ability that body combines is lower.In addition, IGF-1 analog should have equilibrium on insulin receptor two hypotypes IR-A and IR-B Bioactivity.

IGF-1A chain and INSULIN A chain have the sequence homology of height.In the sequence of INSULIN A chain and IGF-1A chain Two significant differences are A5Gln-A5Glu and A12Ser-A12Asp.These residues are conservative in IGF-1 and IGF-2.Sequence These variations in column are related to the variation between the acidic residues in the tral residue and IGF-1 in insulin.Inventors have found that A5 glutamic acid of A chain and A12 Asp side chain carboxyls are the key that determine receptor-selective.A5 glutamic acid are converted For glutamine, asparagine or serine, A12 aspartic acids are converted into serine, glutamine or asparagine etc. There are approximate size and polarity, but the amino acid residue of the side chain without negatively charged functional group, thus obtained analog has more Good receptor-selective.A5 and A10 replaces the single substitution effect than alternative one to become apparent from simultaneously.

Based on the above inventive concept, the present invention provides a kind of compound with hypoglycemic effect, and the compound includes A Chain and B chain, wherein

The amino acid sequence of A chain are as follows: X_-1X₀GIVDX₅C_[3]C_[4]X₈X₉X₁₀C_[5]X₁₂LRRLEX₁₈YC_[6]X₂₁X₂₂,

B chain amino acid sequence are as follows:

X_23-26X₂₇LC_[1]GAX₃₂LVDALX₃₈X₃₉VC_[2]GDX₄₄GFX₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃, wherein

X_-1It is lysine, arginine or missing；X₀It is lysine, arginine or missing；X₅Be glutamic acid, asparagine, Glutamine or serine；X₈It is histidine, arginine, phenylalanine or threonine；X₉It is arginine or serine；X₁₀It is silk Propylhomoserin or isoleucine；X₁₂It is aspartic acid, serine, glutamine or asparagine；X₁₈It is asparagine, methionine Or threonine；X₂₁It is asparagine, alanine or glycine；X₂₂It is lysine, arginine-lysine dipeptides or missing；X_23-26 It is phenylalanine-valine-asparagine-glutamin tetrapeptide or glycine-proline-glutamic acid, valine-asparagus fern acyl Amine-glutamine tripeptides or proline-glutamicacid, asparagine-glutamin dipeptides or glutamic acid, rely ammonia at glutamine Acid or arginine, or with lysine or arginine replace above-mentioned two, three, in tetrapeptide array after any one amino acid residue Sequence, 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；X₄₄It is arginine, glutamic acid, aspartic acid or the third ammonia Acid；X₄₇It is phenylalanine, tyrosine or histidine；X₄₈It is-NH₂、dA-NH₂, phenylalanine or tyrosine or missing；X₄₉It is day Winter amide, aspartic acid, glutamic acid, threonine or missing；X₅₀It 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] indicate the number of cysteine；Pass through 6 cysteine shapes in the compound At 3 pairs of disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, a pair of of intrachain disulfide bond of presence in A chain, and three pair two The specific location of sulfide linkage is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond.

It is important to note that X₃₂、X₄₄、X₄₉、X₅₀、X₅₁Amino acid residue whether be related to compound as people's pancreas islet Element is equally produced from connection (self association).Actrapid monotard, which generally passes through, to be formed six aggressiveness from connection to be stored in pancreas islet β thin In born of the same parents.It gradually is depolymerized to dimer by six aggressiveness after the subcutaneous injection of rh-insulin's molecule, is further dissociated into monomer ability Enter circulation through capillary, plays blood sugar reducing function.Since there are depolymerization, absorption process, rh-insulin is subcutaneously being infused It is long (referring to " Monomeric insulins and their experimental and such as Brange to penetrate rear onset time clinical implications"Diabetes Care,Vol 13 No.9,923-54,1990).If X₃₂It is histidine, So be conducive to compound and form six dimeric structures under the assistance of zinc ion.If X₃₂It is aspartic acid, glutamic acid, phenylpropyl alcohol ammonia The amino acid residues such as acid, glutamine, arginine can not then form six stable dimeric structures.If X₄₄、X₅₀、X₅₁Equal sites Amino acid residue be aspartic acid or glutamic acid, be also not easy to be formed stable from joining.Therefore, if X₃₂It is non-histidine amino Sour residue or X₄₄、X₅₀、X₅₁One or more sites of the amino acid residue in equal sites are aspartic acid or glutamic acid, then correspondingization It closes object to be easier to exist with dimer or monomeric form, rapidly enters blood after subcutaneous injection, can achieve and drop in a short time The effect of hypoglycemia.

In a preferred embodiment of present aspect, the compound with hypoglycemic effect includes A chain and B chain, Wherein,

The amino acid sequence of A chain are as follows: GIVDX₅C_[3]C_[4]X₈RSC_[5]X₁₂LRRLEX₁₈YC_[6]X₂₁X₂₂,

B chain amino acid sequence are as follows:

X_23-26X₂₇LC_[1]GAX₃₂LVDALX₃₈X₃₉VC_[2]GDX₄₄GFYX₄₈X₄₉X₅₀X₅₁X₅₂, wherein

X₅It is glutamic acid, asparagine, glutamine or serine；X₈It is histidine, arginine or phenylalanine；X₁₂It is Aspartic acid, serine, glutamine or asparagine；X₁₈It is asparagine, methionine or threonine；X₂₁It is asparagus fern acyl Amine, alanine or glycine；X₂₂It is lysine, arginine-lysine dipeptides or missing；X_23-26It is Gly-Pro-paddy Propylhomoserin tripeptides or phenylalanine-valine-asparagine-glutamin tetrapeptide；X₂₇It is histidine or threonine；X₃₂It is a group ammonia Acid, glutamic acid, glutamine, arginine or phenylalanine；X₃₈It is phenylalanine or tryptophan；X₃₉It is phenylalanine or color ammonia Acid；X₄₄It is arginine, glutamic acid, aspartic acid or alanine；X₄₈It is-NH₂、dA-NH₂Or phenylalanine；X₄₉It is asparagine Or missing；X₅₀It is lysine, proline or missing；X₅₁It is proline, lysine or missing；X₅₂It is threonine or missing；

In the compound, [1]-[6] indicate the number of cysteine；Pass through 6 cysteine shapes in the compound At 3 pairs of disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, a pair of of intrachain disulfide bond of presence in A chain, and three pair two The specific location of sulfide linkage is: C_[1]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 chain is:

GPEX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFY-NH₂；

In further preferred embodiment, the sequence of the B chain is:

GPEX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFYFNKPT；

In further preferred embodiment, the sequence of the B chain is:

GPEX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFYdA-NH₂；

In further preferred embodiment, the sequence of the B chain is:

FVNQX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFYFNKPT；

In further preferred embodiment, the sequence of the A chain is:

GIVDX₅CCX₈RSCX₁₂LRRLEX₁₈YCA；

In further preferred embodiment, the sequence of the A chain is:

GIVDX₅CCX₈RSCX₁₂LRRLEX₁₈YCN；

It further applies in mode at these, the X in A chain₅It is glutamic acid, asparagine, glutamine or serine；X₈It is Histidine, arginine or phenylalanine；X₁₂It is aspartic acid, serine, glutamine or asparagine；X₁₈Be asparagine, Methionine or threonine；X in B chain₂₇It is histidine or threonine；X₃₂It is histidine, glutamic acid, glutamine, arginine Or phenylalanine；X₃₈It is phenylalanine or tryptophan；X₃₉It is phenylalanine or tryptophan；X₄₄It is arginine, glutamic acid, asparagus fern Propylhomoserin or alanine.

In this regard, having the compound of high binding capacity with insulin receptor includes A chain and B chain, A chain and B chain It is connected by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond in A chain, specifically: C_[1]And C_[4]Form disulfide bond, C_[2] And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond；The compound is selected from following double-chain polypeptides:

I-1: wherein A chain-ordering is GIVDECCFRSCDLRRLEMYCA (SEQ ID NO:1)；The sequence of B chain is GPETLCGAELVDALFFVCGDRGFY-NH₂(SEQ ID NO:4)；

I-2: wherein A chain-ordering is sequence shown in SEQ ID NO:1；The sequence of B chain is GPETLCGAELVDALWFVCGDRGFY-NH₂(SEQ ID NO:5)；

I-3: wherein A chain-ordering is sequence shown in SEQ ID NO:1；The sequence of B chain is GPETLCGAELVDALWWVCGDRGFY-NH₂(SEQ ID NO:6)；

I-4: wherein A chain-ordering is GIVDECCFRSCDLRRLEMYCN (SEQ ID NO:7)；The sequence of B chain is GPETLCGAELVDALFFVCGDRGFYFNKPT(SEQ ID NO:3)；

I-5: wherein A chain-ordering is GIVDECCFRSCDLRRLENYCA (SEQ ID NO:8)；The sequence of B chain is SEQ ID Sequence shown in NO:3；

I-6: wherein A chain-ordering is GIVDECCFRSCDLRRLETYCA (SEQ ID NO:9)；The sequence of B chain is SEQ ID Sequence shown in NO:3；

I-7: wherein A chain-ordering is GIVDECCRRSCDLRRLENYCN (SEQ ID NO:10)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-8: wherein A chain-ordering is GIVDECCHRSCDLRRLENYCN (SEQ ID NO:11)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-9: wherein A chain-ordering is GIVDQCCFRSCDLRRLENYCA (SEQ ID NO:12)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-10: wherein A chain-ordering is GIVDNCCFRSCDLRRLENYCA (SEQ ID NO:13)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-11: wherein A chain-ordering is GIVDECCFRSCSLRRLENYCA (SEQ ID NO:14)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-12: wherein A chain-ordering is GIVDECCFRSCNLRRLENYCA (SEQ ID NO:15)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-13: wherein A chain-ordering is GIVDECCFRSCQLRRLENYCA (SEQ ID NO:16)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-14: wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCA (SEQ ID NO:17)；The sequence of B chain is SEQ Sequence shown in ID NO:3；

I-15: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPETLCGAHLVDALFFVCGDRGFYFNKPT(SEQ ID NO:18)；

I-16: wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCAK (SEQ ID NO:19)；The sequence of B chain is SEQ Sequence shown in ID NO:18；

I-17: wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCARK (SEQ ID NO:20)；The sequence of B chain is Sequence shown in SEQ ID NO:18；

I-18: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPETLCGAHLVDALFFVCGDRGFYdA-NH₂(SEQ ID NO:21)；

I-19: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPEHLCGARLVDALFFVCGDRGFYFNKPT(SEQ ID NO:22)；

I-20: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPEHLCGAFLVDALFFVCGDRGFYFNKPT(SEQ ID NO:23)；

I-21: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPEHLCGAHLVDALFFVCGDEGFYFNKPT(SEQ ID NO:24)；

I-22: wherein A chain-ordering is sequence shown in SEQ ID NO:8；The sequence of B chain is FVNQHLCGAHLVDALFFVCGDRGFYFNKPT(SEQ ID NO:25)；

I-23: wherein A chain-ordering is GIVDQCCHRSCSLRRLENYCA (SEQ ID NO:26)；The sequence of B chain is SEQ Sequence shown in ID NO:25；

I-24: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:25 Column；

I-25: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPEHLCGAHLVDALFFVCGDAGFYFNKPT(SEQ ID NO:27)；

I-26: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPEHLCGAQLVDALFFVCGDRGFYFNKPT(SEQ ID NO:28)；

I-27: wherein A chain-ordering is KGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:151)；The sequence of B chain is GPEHLCGAHLVDALFFVCGDRGFYFNKPT(SEQ ID NO:152)；

I-28: wherein A chain-ordering is RGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:153)；The sequence of B chain is Sequence shown in SEQ ID NO:152；

I-29: wherein A chain-ordering is KGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:154)；The sequence of B chain is GPEHLCGAHLVDALFFVCGDRGFYFNPKT(SEQ ID NO:155)；

I-30: wherein A chain-ordering is RGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:156)；The sequence of B chain is Sequence shown in SEQ ID NO:155；

I-31: wherein A chain-ordering is RRGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:157)；The sequence of B chain is Sequence shown in SEQ ID NO:155；

I-32: wherein A chain-ordering is KKGIVDQCCHRSCSLRRLENYCN (SEQ ID NO:158)；The sequence of B chain is Sequence shown in SEQ ID NO:155；

I-33: wherein A chain-ordering is GIVDQCCHRSCSLRRLENYCN (SEQ ID NO:159)；The sequence of B chain is GPEHLCGAHLVDALFFVCGDRGFYFNPKTE(SEQ ID NO:160)；

I-34: wherein A chain-ordering is sequence shown in SEQ ID NO:159；The sequence of B chain is shown in SEQ ID NO:155 Sequence.

Single chain compound

In mammals, it is synthesized in the β cell of pancreas islet of the insulin in pancreas.Proinsulin is containing 86 amino The single chain precursor of acid, construction are as follows: B chain-ArgArg-C peptide-LysArg-A chain." the connection that C peptide is made of 31 amino acid Peptide ".Arg-Arg and Lys-Arg is the split point that followed by action of proteolytic enzymes divides C peptide from A and B chain, it is known that proteolysis Enzyme is prohormone convertase (PC1 and PC2) and external form protease Carboxypeptidase E.These changes of proinsulin remove C peptide. Remaining B chain is together with A chain is by disulfide-bonded.

The duplex structure of insulin makes insulin have a variety of conformations.Insulin has the latent of sizable conformation change Can, insulin receptor is significantly reduced to the affinity of ligand to the limitation of these variations.The aminoterminal for closing Gly A1 is same Weaken receptor binding capacity.Proinsulin and insulin receptor affinity only have the 1-2% of insulin.

Effect of the current not clear C peptide in proinsulin folding.The length of C peptide is in 26- in different animals type Change between 38 amino acid.Binary amino acid residue in B chain-C peptide (B-C) and the junction C peptide-A chain (C-A) is conservative , and think that insulin conservative is needed to be the smallest.The three-dimensional structure of insulin shows that A chain and B chain can pass through The link peptide more much smaller than the C peptide of 31 amino acid combines.

In insulin molecule physics and chemical stability be diabetes insulinization prerequisite and pancreas Island element conformation, the basis of the pot-life of applicable insulin administration methods and pharmaceutical preparation and preservation condition.In pancreas islet Element administration when use solution that insulin molecule is made to be exposed to many factors, such as raised temperature, it is gas-liquid-solid it is alternate variation and Shearing force may cause the expendable conformation change of insulin molecule, as fibrillation acts on.This and the insulin in syringe pump Solution is in close relations, because either external application is still implanted into, insulin molecule is all exposed to these factors and from pump length In generation shearing force in phase moving process.Therefore, when using syringe pump as insulin delivery system, fibrillation effect It is a very big problem.In addition, the solubility of insulin is affected by many factors, and obvious in the range of pH4.2-6.6 It reduces.Limitation usually is brought to formula in the decanting zone pH.

Therefore, an important factor for stability and solubility of insulin are current insulin therapies.This invention address that this A little problems provide stable single chain compound by introducing C peptide between B and A chain to reduce molecular flexibility and reduce original simultaneously Tendency for fibrillation, limitation or the modification decanting zone pH.In addition, the main method of the insulin of genetic engineering production at present is to produce first By insulin B chain and A chain by the single-chain insulin precursor of small peptide head and the tail connection, generated after then insulin precurosor is digested double Chain insulin.If directly producing single-chain insulin analogues, production process is enormously simplified, is reduced costs.

As the member of insulin family, insulin-like growth factor-i (IGF-1) is that have 70 amino acid residues Single-stranded peptide includes the domain A, B, C and D.The domain A of IGF-1 and the basic structure in the domain B are similar to the A chain of insulin and B chain height, point There is not 52% and 45% homology.Their three-dimensional structure is also closely similar.

The domain C of IGF-1 acts on very little in insulin receptor combination.Remove whole domains IGF-1 C, with 4 glycine The bridge of composition replaces, and combination rate of insulin receptor is caused to increase by twice compared with wild type, and the domain IGF-1 C is added to insulin The C-terminal of B chain causes insulin receptor affinity to reduce 3.5 times compared with wild type.It is made of insulin and the domain IGF-1 C Single-chain insulin/IGF-1 mixture insulin binding force and natural human insulin are not significantly different.Ironically, IGF-1 CII mixture all has increased affinity to IR-A and IR-B, and IGF-2CI has weaker affinity, shows C Domain determines IR binding specificity.

Tyr31 is most important by high affinity for keeping IGF-1's in IGF-1, but it seems obstruction and pancreas islet Plain receptor combines, because when tyrosine is replaced by alanine, results in very little but apparent Human plactnta insulin receptor combines Double increase.

In the present invention, inventor further design, synthesize and characterize can in conjunction with insulin receptor, have it is hypoglycemic The single-stranded IGF-1 analog of effect.6-12 or the junction fragment (C of 8-12 amino acid are used in the IGF-1 analog Chain) position A1 of the C-terminal of the B chain analog of IGF-1 and IGF-1A chain analog is connected, the single-stranded IGF-1 of gained is similar Object is expressed as B chain-C chain-A chain.Ideal single-stranded IGF-1 analog should have height binding force to insulin receptor, realize quiet Electric equilibrium；There are excellent thermodynamic stability, no self assembly.

Further, in these IGF-1 analogs, the A chain and B chain be IGF-1 A chain and B chain or they Analog.Based on first part of the invention, the B chain is that B15 amino acid Q (glutamine) are replaced by F (phenylpropyl alcohol ammonia Acid) or W (tryptophan) IGF-1 B chain variant.Combination rate of insulin receptor can be enhanced in double-strand IGF-1 analog, change Kind receptor-selective increases the various amino acid variation of polypeptide water solubility and stability, such as A5, A12, A18, A22, B9 equipotential The variation of point, is also applied for single-stranded IGF-1 analog.It is allowed to for changing compound from ability is joined in double-strand IGF-1 analog Mainly with the selection and variation of amino acid existing for dimer or monomer, it is also applied for single-stranded IGF-1 analog.

Junction fragment

Inventor has found under study for action, and the double-strand of IGF-1 molecule is connected the single chain compound to be formed by junction fragment, Equally there is insulin active, and there is easily preparation, the advantages such as more peptide modified sites are provided.

Junction fragment C_LIt is the peptide sequence of 6-60 amino acid, wherein each amino acid residue is independently selected from sweet ammonia Acid, alanine, serine, threonine, proline.Applicable junction fragment C_LWith three point features: first, junction fragment needs Length appropriate.When B chain is 30 amino acid overall lengths, junction fragment length is preferably equivalent at least 6 amino acid；When B chain is 25 When a amino acid, junction fragment length is preferably equivalent at least 10 amino acid.Junction fragment be shorter in length than above-mentioned amino acid number or When person is longer than 60 amino acid, the insulin receptor binding ability of single chain analogs has decreasing trend；Second, junction fragment is best There is no secondary structure, space conformation can flexibly change；Third, junction fragment itself can provide more without bioactivity Peptide decorating site, such as acylated, glycosylation.

The junction fragment C designed with above method_LCan by amino acid residue replace and be inserted into comprising 1 or 1 with Upper aspartic acid, glutamic acid, arginine, lysine, cysteine or asparagine.C_LIt may include 1,2,3,4 asparagus fern ammonia Acid, glutamic acid, arginine or lysine improve solubility to adjust the charge balance of polypeptide sequence.The sequence may include 1, 2, the serine or threonine of 3,4,5 asparagines and identical quantity constitutes the required N-X-S/T of N glycosylation to form Consensus sequence (natural amino acid that X is codified).Further, which can also include 1,2,3 or 4 lysine or half Guang Propylhomoserin, side-chain amino group or sulfydryl can be natural or synthetic with fatty acid, polyethylene glycol, albumin etc. modification group pass through water It solves key or non-hydrolytic key is connected, to make the IGF-1 molecule after modification that there is different physics, chemistry and biological nature.

According to a kind of embodiment, C_LC-terminal amino acid can be selected from by glycine-lysine, glycine-essence ammonia Acid, Arg-Arg, lysine-lysine, arginine-lysine, Lys-Arg, proline-glutamine-Soviet Union Propylhomoserin, proline-glutamine-lysine or proline-glutamine-arginine composition group.According to a kind of embodiment party Formula, C_LC-terminal amino acid be selected from lysine or arginine.

In a particular embodiment, C_LCan be all or part of sequence of following polypeptide fragment, or with it is following more Peptide fragment has the difference of 1,2 or 3 amino acid residue, perhaps have with following polypeptide fragment 70%, 80%, 90% it is similar or It is 1,2,3,4 or 5 repetitive sequence of all or part of sequence of following polypeptide fragment:

(GASPGGSSGS)_nGR, wherein n is 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。

Based on the studies above as a result, the present invention further provides a kind of single-stranded compound with blood sugar decreasing effect, institute It states compound to be transformed based on the structure of people IGF-1, the structure of the compound are as follows:

X_101aLC_[1]GAX_101bLVDALX_101cX_101dVC_[2]GDRGFX_101eX₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆-C_L-GIVDQC_[3]C_[4] X₁₀₇RSC_[5]SLRRLENYC_[6]X₁₀₈X₁₀₉, wherein

X_101aBe glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine tetrapeptide or Phenylalanine-valine-asparagine-glutamin-histidine pentapeptide, or above-mentioned tetrapeptide is replaced with lysine or arginine Or any of glycine-proline-glutamic acid in pentapeptide or phenylalanine-valine-asparagine-glutamin amino Sequence after sour residue；X_101bIt is histidine, glutamic acid, glutamine, arginine or phenylalanine；X_101cBe phenylalanine or Tryptophan；X_101dIt is phenylalanine or tryptophan；X_101eIt is phenylalanine, tyrosine or histidine；X₁₀₂Be phenylalanine or lack It loses；X₁₀₃It is asparagine or missing；X₁₀₄It is lysine, proline or missing；X₁₀₅It is proline, lysine or missing；X₁₀₆ It is threonine or missing；X₁₀₇It is phenylalanine, arginine or histidine；X₁₀₈It is alanine, glycine or asparagine；X₁₀₉ It is lysine, arginine-lysine dipeptides or missing；C_LIt is selected from above-mentioned junction fragment.

In above compound structure, [1]-[6] indicate the number of cysteine.Single chain compound of the invention is in three-level knot In structure, the disulfide bond in chain is formed with the frame mode of insulin, specifically: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Shape At disulfide bond, C_[3]And C_[5]Form disulfide bond.

In a preferred embodiment of present aspect, the structure of the single chain compound are as follows:

X_101aLC_[1]GAHLVDALFFVC_[2]GDRGFYX₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆-C_L-GIVDQC_[3]C_[4]FRSC_[5] SLRRLENYC_[6]A X₁₀₉, wherein

X_101aIt is glycine-proline-glutamic acid-threonine tetrapeptide or phenylalanine-valine-asparagine-paddy Glutamine-histidine pentapeptide；X₁₀₂It is phenylalanine or missing；X₁₀₃It is asparagine or missing；X₁₀₄It is lysine, proline Or missing；X₁₀₅It is proline, lysine or missing；X₁₀₆It is threonine or missing；X₁₀₉It is lysine, arginine-lysine two Peptide or missing；C_LIt is selected from above-mentioned junction fragment.

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

GPETLCGAHLVDALFFVCGDRGFY-C_L-GIVDQCCFRSCSLRRLENYCA；

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

GPETLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCA；

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

FVNQHLCGAHLVDALFFVCGDRGFY-C_L-GIVDQCCFRSCSLRRLENYCA；

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

FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCA；

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

FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCARK；

The C_LStructure as defined herein.

In this regard, of the invention being capable of single chain compound in conjunction with insulin receptor, with hypoglycemic effect It is selected from:

II -1:

GPETLCGAELVDALFFVCGDRGFYFNKPTGSGSSSRRAPQTGIVDECCFRSCDLRRLEMYCA(SEQ ID NO:29)；

II -2:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSRRAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:30)；

II -3:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:31)；

II -4:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSAAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:32)；

II -5:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:33)；

II -6:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSRRAGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:34)；

II -7:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSRGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:35)；

II -8:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGASSRGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:36)；

II -9:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSASRGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:37)；

II -10:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSAGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 38)；

II -11:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSGGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 39)；

II -12:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 40)；

II -13:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 41)；

II -14:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGGGSGGGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 42)；

II -15:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSGSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 43)；

II -16:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGGSGGSGGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:44)；

II -17:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGSSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:45)；

II -18:

GPETLCGAHLVDALFFVCGDRGFYGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 46)；

II -19:

GPETLCGAHLVDALFFVCGDRGFYFGSGSSSAAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 47)；

II -20:

GPETLCGAHLVDALFFVCGDRGFYFNGSGSSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 48)；

II -21:

GPETLCGAHLVDALFFVCGDRGFYFNKGSGSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 49)；

II -22:

GPETLCGAHLVDALFFVCGDRGFYFNKGGGGAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 50)；

II -23:

FVNQHLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSAARGIVDQCCFRSCSLRRLENYCARK(SEQ ID NO:51)；

II -24:

FVNQHLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:52)；

II -25:

FVNQHLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:53)。

II -26:

GPETLCGAHLVDALFFVCGDRGFYGSGSSAAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 54)；

Ⅱ-27:

GPETLCGAHLVDALFFVCGDRGFYSSSSRRAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 55)；

Ⅱ-28:

GPETLCGAHLVDALFFVCGDRGFYSSSGSGSSAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 56)；

Ⅱ-29:

GPETLCGAHLVDALFFVCGDRGFYSSGSGSSSAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 57)；

Ⅱ-30:

GPETLCGAHLVDALFFVCGDRGFYFNGSGSSAAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 58)；

II -31:

GPETLCGAHLVDALFFVCGDRGFYFGSGSSSRSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 59)；

II -32:

GPETLCGAHLVDALFFVCGDRGFYFNGSGSSSRGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 60)；

II -33:

GPETLCGAHLVDALFFVCGDRGFYFNDSSSSRAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 61)；

II -34:

GPETLCGAHLVDALFFVCGDRGFYSSSSAPSAPSPSRAPGPSPAPQRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:62)；

II -35:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASPGGSSGSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:63)；

II -36:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSGSSGPGSSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:64)；

II -37:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSGSGSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:65)；

II -38:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGGAPSRSGSSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:66)；

II -39:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSPAGSPTSTGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:67)；

II -40:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSPATSGSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:68)；

II -41:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASSSATPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:69)；

II -42:

GPETLCGAHLVDALFFVCGDRGFYGSGSSSRAPPSAPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:70)；

II -43:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSSESPSGAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:71)；

II -44:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGTPASGSAPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:72)；

II -45:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSPSGGSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:73)；

II -46:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSTSSTARSPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:74)；

II -47:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGPSGTASPSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:75)；

II -48:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSTPSGAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:76)；

II -49:

GPETLCGAHLVDALFFVCGDRGFYSSSSAPPPSAPSPSRAPQRGIVEQCCTSICSLYQLEN YCN(SEQ ID NO:77)；

II -50:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASPGTSSTSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:78)；

II -51:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSPAGSPTSTSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:79)；

II -52:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGPSSATPASRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:80)；

II -53:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGPSTRSAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:81)；

II -54:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPETPSGPSSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:82)；

II -55:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGSSSRAPPPSAPSPSRAPGPSAPQRGIVDQCCFRSCS LRRLENYCA(SEQ ID NO:83)；

II -56:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASSPSTSRPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:84)；

II -57:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSSGSSGSPSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:85)；

II -58:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSPSASTGTGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:86)；

II -59:

GPETLCGAHLVDALFFVCGDRGFYGAGSSSAPSAPSPSRAPGPSAPQRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:87)；

II -60:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSPSSPTRGSAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:88)；

II -61:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASTSSRGAPSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:89)；

II -62:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPSGTSTSAPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:90)；

II -63:

GPETLCGAHLVDALFFVCGDRGFYSSSSAPSAPSPSRPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:91)；

II -64:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGASSPTSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:92)；

II -65:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSPATSATPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:93)；

II -66:

GPETLCGAHLVDALFFVCGDRGFYGAGSSSAPPPSAPSPSRAPGPSAPQRGIVDQCCFRSCSLRRLEN YCA(SEQ ID NO:94)；

II -67:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASTSPSRPSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:95)；

II -68:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSTAGSRTSTGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:96)；

II -69:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSTAGSRTSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:97)；

II -70:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGTATSGSPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:98)；

II -71:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASSSATSASGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:99)；

II -72:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGSATRGSASRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:100)；

II -73:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSSRSPSGSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:101)；

II -74:

GPETLCGAHLVDALFFVCGDRGFYSSSSAPPPSAPSPSRAPGPSAPQRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:102)；

II -75:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSSPSGRSSSPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:103)；

II -76:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSPAGSPSSSAGSSASASPASPGRGIVDQCCFRSCSLRR LENYCA(SEQ ID NO:104)；

II -77:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSPAGSPSSSAGSSASASPASGPGSSSAPSAGSPGRGIV DQCCFRSCSLRRLENYCA(SEQ ID NO:105)；

II -78:

GPETLCGAHLVDALFFVCGDRGFYFNKPTRREAEDGGGPGAGSSQRKGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:106)；

II -79:

GPETLCGAHLVDALFFVCGDRGFYFNKPTRRGGGPGAGSSQRKGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:107)；

II -80:

GPETLCGAHLVDALFFVCGDRGFYGGGPGAGSSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 108)；

II -81:

GPETLCGAHLVDALFFVCGDRGFYFNPRGGGPGAGSSQRKGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:109)；

II -82:

GPETLCGAHLVDALFFVCGDRGFYSSSAPPPSAPSPSRAPGPSPQRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:110)；

II -83:

GPETLCGAHLVDALFFVCGDRGFYFNKPTSAASSSASSSSASSASAGRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:111)；

II -84:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGGPSSGAPPPSPQTGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:112)；

II -85:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGSPAAPASPAPAPSAGRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:113)；

II -86:

GPETLCGAHLVDALFFVCGDRGFYSSSAPSPSRSPGPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:114)；

II -87:

GPETLCGAHLVDALFFVCGDRGFYSSSAPSAPSPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:115)；

II -88:

GPETLCGAHLVDALFFVCGDRGFYSSSSAASAASASSSASGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:116)；

II -89:

GPETLCGAHLVDALFFVCGDRGFYSSSRAPPSAPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:117)；

II -90:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGGPSSGAPPPSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:118)；

II -91:

GPETLCGAHLVDALFFVCGDRGFYSSSSGAPPPGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 119)；

II -92:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPSSGAPSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:120)；

II -93:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPSSGAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:121)；

II -94:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGGPSSGAPPPSPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:122)；

II -95:

GPETLCGAHLVDALFFVCGDRGFYSSSAPPPSAPSPSRAPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:123)；

II -96:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGPSSGAPPPSPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:124)；

II -97:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGGPSSGAPPPQTGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:125)；

II -98:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGGPSSGAPPPSPSPSRPGPSPQRGIVDQCCFRSCSLRRL ENYCA(SEQ ID NO:126)；

II -99:

GPETLCGAHLVDALFFVCGDRGFYSSASSASSSSAGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 127)；

II -100:

GPETLCGAHLVDALFFVCGDRGFYSSASSSAASSSASSSASGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:128)；

II -101:

GPETLCGAHLVDALFFVCGDRGFYSSSGAPPPSPSRAPGPSPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:129)；

II -102:

GPETLCGAHLVDALFFVCGDRGFYSSSSAASSASGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 130)；

II -103:

GPETLCGAHLVDALFFVCGDRGFYFNKPTSASASASASSASSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:131)；

II -104:

GPETLCGAHLVDALFFVCGDRGFYFNKPTSASSPSPSAPSSPSPASGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:132)；

II -105:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPSSPSPSAPSSPSPASPSSGRGIVDQCCFRSCSLRRLE NYCA(SEQ ID NO:133)；

II -106:

GPETLCGAHLVDALFFVCGDRGFYSSSAPPPASPSPSRAPGPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:134)；

II -107:

GPETLCGAHLVDALFFVCGDRGFYFNKPTSASASASASASSAGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:135)；

II -108:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGAAPASPAAPAPSAGRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:136)；

II -109:

GPETLCGAHLVDALFFVCGDRGFYSSPSASPSSPASPSSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:137)；

II -110:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAPASPAPSAPAPAAPSGRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:138)；

II -111:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGPSSPSPSAPSSPSPASPSSAPQTGIVDQCCFRSCSLRR LENYCA(SEQ ID NO:139)；

II -112:

GPETLCGAHLVDALFFVCGDRGFYSSASSASSSSSASAGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:140)；

II -113:

GPETLCGAHLVDALFFVCGDRGFYFNKPTSAPSSPSPSAPSSPSASPSGRGIVDQCCFRSCSLRRLEN YCA(SEQ ID NO:141)；

II -114:

GPETLCGAHLVDALFFVCGDRGFYSSSAPPPSAPSPSAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:142)；

II -115:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGASSPSPSAPSSPSPASGRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:143)；

II -116:

GPETLCGAHLVDALFFVCGDRGFYSSPSAPSPSSPASPSSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO:144)；

II -117:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGAGPAAPSAPPAASPAAPSAGRGIVDQCCFRSCSLRRLE NYCA(SEQ ID NO:145)；

II -118:

GPETLCGAHLVDALFFVCGDRGFYSSSSPSAPSPSSPASPSPSSAPQRGIVDQCCFRSCSLRRLENYCA (SEQ ID NO:146)；

II -119:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 147)；

II -120:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSARGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 148)；

II -121:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGSSSGRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 149)；

II -122:

GPETLCGAHLVDALFFVCGDRGFYFNKPTGSGAPQRGIVDQCCFRSCSLRRLENYCA(SEQ ID NO: 150)；

II -123:

GPEHLCGAHLVDALFFVCGDRGFYFNKGGGGAPQTGIVDQCCHRSCSLRRLENYCN(SEQ ID NO: 161)；

II -124:

GPEHLCGAHLVDALFFVCGDRGFYFNPKTGSGSSSAAAPQTGIVDQCCHRSCSLRRLENYCN(SEQ ID NO:162)。

Modified compound

Chemiluminescent polypeptide man is less than the difficulty that the drug molecule of 67kDa is quickly removed by kidney to solve blood plasma middle-molecular-weihydroxyethyl Topic, is used for several method.1, " storehouse " (depot) is built in injection site；2, with the carrier protein in blood plasma with non-covalent bond Combine to prevent glomerular filtration；3 and carrier protein to be covalently keyed；4, in conjunction with macromolecule modification group, such as greatly Molecular weight PEG, polysaccharide etc. (as disclosed in the chapters and sections before present specification)." hydrophobic to store sth. in a cellar " (hydrophobic Depoting it increases considerably the hydrophobicity of peptide) to reduce solubility, and it is made to form " storehouse " in injection portion.Peptide storehouse slowly solves From rear, polypeptide is bound to the carrier protein (such as albumin) of cell membrane and/or whole body.Carrier protein molecule amount is greater than glomerulus Maximum molecular weight is filtered, therefore is not easy to be removed by kidney, can be recycled in blood plasma more days.Therefore, in conjunction with carrier protein Polypeptide is not easy by glomerular filtration or by the proteasome degradation on inner membrance.

Fatty acid generally extends polypeptide action time in vivo by three kinds of modes.First, fatty acid can be infused in drug Penetrate position and albumin with non-covalent bond in conjunction with, polypeptide-fatty acid-albumin macromolecule conjugate release of formation is slow；The Two, polypeptide-fatty acid-albumin conjugates macromolecule reduces renal clearance；Third, albumin provide guarantor for polypeptide Shield, is not easy to be easily degraded by proteases.4th, fatty acid reduces the immunogenicity of polypeptide.The effect of three features and long-chain PEG modification afterwards Fruit is seemingly.More mechanism and experiment are supported to be 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。

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

In addition, insulin is modified with macromoleculars such as polyethylene glycol (PEG, molecular weight are not less than 20K) and human albumins, it can also To achieve the effect that action time in the extension body similar with above-mentioned fatty acid modifying.Therefore, fatty acid acyl can be used all The site of change can use the modification of the macromoleculars such as polyethylene glycol or human albumin.

The present invention is based on such a understanding: the bulk hydrophobicity of the compound with hypoglycemic effect of the invention is at this It plays an important role in terms of the in vivo efficacy of compound.The present invention further provides it is a kind of it is with hypoglycemic effect, in polypeptide base The compound modified on plinth, to further increase the compound body-internal-circulation action time.The modification is will to modify Side chain is connected to the alpha-amido of the -terminal amino acid residue of the B chain of double chain compound of the invention or is connected to of the invention The alpha-amido of the -terminal amino acid residue of single chain compound is connected to present in double-strand or single chain compound of the invention The epsilon-amino of lysine.

In one embodiment, the compound is transformed based on IGF-1 analog, which includes A chain and B Chain, wherein

The amino acid sequence of A chain are as follows:

X₃₉₉X₄₀₀GIVDX₄₀₅C_[3]C_[4]X₄₀₈X₄₀₉X₄₁₀C_[5]X₄₁₂LX₄₁₄X₄₁₅LX₄₁₇X₄₁₈YC_[6]X₄₂₁X₄₂₂,

B chain amino acid sequence are as follows:

X_423-426X₄₂₇LC_[1]GAHLVDALX₄₃₈X₄₃₉VC_[2]GDRGFX₄₄₇X₄₄₈X₄₄₉X₄₅₀X₄₅₁X₄₅₂X₄₅₃X₄₅₄X₄₅₅,

In the compound, [1]-[6] indicate the number of cysteine；The compound forms 3 by 6 cysteines To disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond, three pair of two sulphur in A chain The specific location of key is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond, wherein

X₃₉₉It is arginine, lysine or missing；X₄₀₀It is arginine, lysine or missing；X₄₀₅It is glutamic acid, asparagus fern acyl Amine, glutamine or serine；X₄₀₈It is histidine, arginine, phenylalanine, threonine or logical formula (I) structure, the general formula (I) structure are as follows:

X₄₀₉It is arginine, serine or logical formula (I) structure；X₄₁₀It is histidine, arginine, phenylalanine or logical formula (I) Structure；X₄₁₂It is serine, isoleucine or logical formula (I) structure；X₄₁₄It is arginine or logical formula (I) structure；X₄₁₅Be arginine or Logical formula (I) structure；X₄₁₇It is glutamic acid or logical formula (I) structure；X₄₁₈It is asparagine or logical formula (I) structure；X₄₂₁Be alanine, Glycine or asparagine；X₄₂₂It is lysine, arginine-lysine dipeptides or missing, or is logical formula (I) structure；Work as X₄₂₂For When dipeptides, one of amino acid is logical formula (I) structure；X_423-426It is glycine-proline-glutamic acid tripeptides, U_LGlycine- Proline-glutamicacid, phenylalanine-valine-asparagine-glutamin tetrapeptide or U_LPhenylalanine-valine-asparagus fern Amide-glutamine；X₄₂₇It is histidine or threonine；X₄₃₈It is phenylalanine or tryptophan；X₄₃₉It is phenylalanine or color ammonia Acid；X₄₄₇It is phenylalanine, histidine or tyrosine；X₄₄₈It is-NH₂, phenylalanine, tyrosine or missing；X₄₄₉It is asparagus fern acyl Amine, threonine, glutamic acid, aspartic acid or missing；X₄₅₀It is lysine, arginine, glutamic acid, aspartic acid, proline or scarce It loses；X₄₅₁It is proline, lysine, arginine, glutamic acid, aspartic acid or missing, or is logical formula (I) structure；X₄₅₂It is Soviet Union's ammonia Acid, lysine or missing, or be logical formula (I) structure；X₄₅₃It is glutamic acid, glycine, lysine or missing, or is logical formula (I) knot Structure；X₄₅₄It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₅It is lysine or missing, or is general formula (I) structure；

U_LIt is-W-X-Y-Z structure, fatty acid, polyethylene glycol, albumin, L_n-M_LStructure, hydrogen atom or N^α-(N^α-(HOOC (CH₂)_nCO)-γ-Glu)-、N^α-(N^α-(CH₃(CH₂)_nCO)-γ-Glu)-, wherein n is the integer of 8-20, such as 8,10,12, 14,16,20, N^αIt indicates the alpha-amido of amino acid or amino acid residue, or is logical formula (II) structure, the logical formula (II) structure It is:

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

Wherein L_n-M_LIn structure, M_LIt is modification group, it is including but not limited to-W-X-Y-Z, fatty acid, polyethylene glycol, white Albumen, IgG Fc, glycosyl group etc..

L_nIt is optional linker, covalent bond or is not present.Optional linker includes but is not limited to, polyethylene glycol, Long chain fatty acids, or one or more peg molecules and long-chain fat acid molecule pass through the long-chain for being covalently keyed formation. L_nIt can be-NH- (CH₂)_n-CO-、-NH-(CH₂CH₂O)_n-CH₂-CO-、-NH-(CH₂CH₂O)_n-(CH₂)_r-CO-, n are 1-20 Integer, r are the integers of 1-10.In one embodiment, L_nIt is-NH- (CH₂CH₂O)₂-CH₂-CONH-(CH₂CH₂O)₂-CH₂-CO-.In one embodiment, L_nIt is-NH- (CH₂)_n1-O-(CH₂CH₂O)_n2-(CH₂)_n3-CO-, n1, n2, n3 are 1-16 respectively Integer.In one embodiment, L_nIt is-NH- (CH₂)_n1-(OCH₂CH₂)_n2 CO-, n1, n2 are the integer of 1-16 respectively.? In embodiment of above, L_nAmido bond is formed by the amino of key and polypeptide compound from the carbonyl carbon underlined.Separately One end and M_LForm covalent bond.In one embodiment, L_nPass through key and and peptide from the carbonyl carbon underlined The amino of object forms amido bond, and the other end and-W-X-Y-Z form amido bond.

In the present invention ,-W-X-Y-Z structure is:

W is the a-amino acid residue that side chain has carboxyl, and the residue is with the B of a carboxyl and double center chain compound of the present invention The alpha-amido of the -terminal amino acid residue of chain or with the alpha-amido of the -terminal amino acid residue of single chain compound or with it is single-stranded Or the epsilon-amino of the lysine residue in double-strand is formed together amide groups；

Or W is the chain connected by 2,3 or 4 a-amino acid residues by amido bond, which is connected by amido bond It is connected to the alpha-amido of the -terminal amino acid residue of the B chain of double chain compound or is connected to the -terminal amino acid of single chain compound The amino acid residue of the epsilon-amino of the alpha-amido of residue or the lysine residue being connected on single-stranded or double-stranded compound, W is selected from Amino acid residue and side chain with neutral side chain have the amino acid residue of carboxyl, have so that W contains at least one in side chain There is the amino acid residue of carboxyl；

Or W is the alpha-amido of the -terminal amino acid of the B chain from X to double chain compound or the end N- to single chain compound The alpha-amido of terminal amino acid residue or to double-strand or the covalent bond of the epsilon-amino of the lysine residue of single chain compound；

X is-CO-、-CH(COOH)CO-、-N(CH₂COOH)CH₂ CO-、-N(CH₂COOH)CH₂CON(CH₂COOH) CH₂ CO-、-N(CH₂CH₂COOH)CH₂CH₂ CO-、-N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂ CO-、-NHCH (COOH)(CH₂)₄NHCO-、-N(CH₂CH₂COOH)CH₂ CO- or-N (CH₂COOH)CH₂CH₂ CO-, wherein

A) when W is amino acid residue or amino acid residue chain, key and W that above-mentioned X passes through the carbonyl carbon by underlining In amino formed amido bond；Or

B) when W is covalent bond, above-mentioned X passes through the B chain of key and double chain compound from the carbonyl carbon underlined N- terminal aamino relies with the alpha-amido of the -terminal amino acid residue of single chain compound or with double-strand or single chain compound The epsilon-amino of histidine residue forms amido bond；

Y is-(CH₂)_m, wherein m is the integer of 6-32；

Or include 1,2 or 3-CH=CH- group and multiple-CH₂The bivalent hydrocarbon chain of group, the multiple-CH₂Group Number to meet the total number of carbon atoms range in hydrocarbon chain be 10-32；

Or general formula-(CH₂)_VC₆H₄(CH_2)wBivalent hydrocarbon chain, wherein v and w be integer or they first is that zero so that The range of v and w summation is 6-30；And

Z is-COOH ,-CO-Asp ,-CO-Glu ,-CO-Gly ,-CO-Sar ,-CH (COOH)₂、-N(CH₂COOH)₂、- SO₃H、-PO₃H is not present；Condition is when W is covalent bond and X is-CO-, and Z is not-COOH.

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

In another embodiment, the chain that W is made of two a-amino acid residues, one of a-amino acid residue With 4-10 carbon atom and side chain has a carboxyl, and another is with 2-11 carbon atom but no free carboxyl group.Described There is no the a-amino acid residue of free carboxyl group to can be the a-amino acid residue of neutral codified.According to this embodiment The example of W is: α-Asp-Gly, Gly- α-Asp, β-Asp-Gly, Gly- β-Asp, α-Glu-Gly, Gly- α-Glu, γ-Glu- Gly, Gly- γ-Glu, α-hGlu-Gly, Gly- α-hGlu, δ-hGlu-Gly and Gly- δ-hGlu.

In another embodiment, the chain that W is made of two a-amino acid residues, two a-amino acid residue difference With 4-10 carbon atom, carboxyl is all had on side chain.One or two of these a-amino acid residues can be the α-of codified Amino acid residue.Example according to the W of this embodiment is: α-Asp- α-Asp, α-Asp- α-Glu, α-Asp- α-hGlu, α-Asp-β-Asp、α-Asp-γ-Glu、α-Asp-δ-hGlu、β-Asp-α-Asp、β-Asp-α-Glu、β-Asp-α-hGlu、β- Asp-β-Asp、β-Asp-γ-Glu、β-Asp-δ-hGlu、α-Glu-α-Asp、α-Glu-α-Glu、α-Glu-α-hGlu、α- Glu-β-Asp、α-Glu-γ-Glu、α-Glu-δ-hGlu、γ-Glu-α-Asp、γ-Glu-α-Glu、γ-Glu-α-hGlu、 γ-Glu-β-Asp、γ-Glu-γ-Glu、γ-Glu-δ-hGlu、α-hGlu-α-Asp、α-hGlu-α-Glu、α-hGlu-α- hGlu、α-hGlu-β-Asp、α-hGlu-γ-Glu、α-hGlu-δ-hGlu、δ-hGlu-α-Asp、δ-hGlu-α-Glu、δ- HGlu- α-hGlu, δ-hGlu- β-Asp, δ-hGlu- γ-Glu and δ-hGlu- δ-hGlu.

In another embodiment, what the a-amino acid residue that W is respectively provided with 4-10 carbon atom by three formed Chain, the amino acid residue of the chain is selected from the residue of residue and side chain with carboxyl with neutral side chain so that the chain contain to A few side chain has the residue of carboxyl.In one embodiment, the amino acid residue is the residue of codified.

In another embodiment, W is to be respectively provided with 4-10 carbon atom by four, a-amino acid residue composition Chain, the amino acid residue of the chain are selected from the residue of residue and side chain with carboxyl with neutral side chain so that the chain contain to A few side chain has the residue of 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 ε-ammonia of lysine residue by urea derivative Base.

X in side chain-W-X-Y-Z can be general formula-CThe group of O- passes through key and W from the carbonyl carbon underlined In amino formed amido bond；Or when W is covalent bond, X passes through key and double chain compound from the carbonyl carbon underlined B chain the end N- alpha-amido or with the alpha-amido of the end N- of single chain compound or in single-stranded or double-stranded compound rely The epsilon-amino of histidine residue forms amido bond.

In further embodiment, the X in the side chain-W-X-Y-Z can be general formula-CH (COOH)CThe base of O- Group forms amido bond by the amino in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X is by coming From the alpha-amido of the end N- of the B chain of the key and double chain compound of the carbonyl carbon underlined or with the end N- of single chain compound Alpha-amido or form amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in side chain-W-X-Y-Z can be general formula-N (CH₂COOH)CH₂ CThe base of O- Group forms amido bond by the amino in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X is by coming From the alpha-amido of the end N- of the B chain of the key and double chain compound of the carbonyl carbon underlined or with the end N- of single chain compound Alpha-amido or form amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in side chain-W-X-Y-Z can be general formula-N (CH₂CH₂COOH)CH₂ CO-'s Group forms amido bond by the amino in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X passes through The alpha-amido of the end N- of the B chain of key and double chain compound from the carbonyl carbon underlined is last with the N- of single chain compound The alpha-amido at end forms amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in-W-X-Y-Z can be general formula-N (CH₂COOH)CH₂CH₂ CThe base of O- Group forms amido bond by the amino in key and W from the carbonyl carbon underlined；Or when W is covalent bond, X is by coming From the alpha-amido of the end N- of the B chain of the key and double chain compound of the carbonyl carbon underlined or with the end N- of single chain compound Alpha-amido or form amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in-W-X-Y-Z can be general formula-N (CH₂COOH)CH₂CON(CH₂COOH) CH₂ CThe group of O- forms amido bond by the amino in key and W from the carbonyl carbon underlined；Or when W is covalent bond When, X by the alpha-amido of the end N- of the B chain of key and double chain compound from the carbonyl carbon underlined or with single-stranded chemical combination The alpha-amido of the end N- of object forms amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in-W-X-Y-Z can be general formula-N (CH₂CH₂COOH)CH₂CH₂ CO-'s Group forms amido bond by the amino in the key and W for underlining carbonyl carbon；Or when W is covalent bond, X is by coming From the alpha-amido of the end N- of the B chain of the key and double chain compound of the carbonyl carbon underlined or with the end N- of single chain compound Alpha-amido or form amido bond with the epsilon-amino of the lysine residue in double-strand or single chain compound.

In further embodiment, the X in-W-X-Y-Z can be general formula-N (CH₂CH₂COOH)CH₂CH₂CON (CH₂CH₂COOH)CH₂CH₂ CThe group of O- forms amido bond by the amino in key and W from the carbonyl carbon underlined； Or when W is covalent bond, α-ammonia that X passes through the end N- of the B chain of key and double chain compound from the carbonyl carbon underlined Base or epsilon-amino shape with the alpha-amido of the end N- of single chain compound or with the lysine residue in double-strand or single chain compound At amido bond.

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

In another embodiment, the Y in-W-X-Y-Z is comprising 1,2 or 3-CH=CH- group and multiple-CH₂- The bivalent hydrocarbon chain of group, the multiple-CH₂The number of group meet the total number of carbon atoms range in hydrocarbon chain be 6-32,10-32, 12-20 or 12-16.

In another embodiment, the Y in-W-X-Y-Z is general formula-(CH₂)_VC₆H₄(CH_2)wBivalent hydrocarbon chain, wherein V and w is integer, or one of them is zero, so that the range of v and w summation is 6-30,10-20 or 12-16.

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

In another embodiment, the Z in-W-X-Y-Z is-CO-Asp ,-CO-Glu ,-CO-Gly ,-CO-Sar ,-CH (COOH)₂、-N(CH₂COOH)₂、-SO₃H or-PO₃H。

In further embodiment, the W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu；X is-CO- Or-CH (COOH) CO-；Y is-(CH₂)_m, wherein m is the integer of 12-18；Z is-COOH- ,-CH (COOH)₂Or it is not present.

In another embodiment, the W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu；- X-Y-Z is-CO(CH₂)_n, amido bond is formed by the amino in key and W from the carbonyl carbon underlined, wherein n is whole in 10-18 Number.

The W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu in a more specific embodiment,；-X-Y-Z It is-CO (CH₂)₁₄。

The W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu in a more specific embodiment,；-X-Y-Z It is-CO (CH₂)₁₆。

The W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu in a more specific embodiment,；-X-Y-Z It is-CO (CH₂)₁₈。

The W in-W-X-Y-Z is α-Asp, β-Asp, α-Glu or γ-Glu in a more specific embodiment,；-X-Y-Z It is cholesterol, bile acid (such as cholic acid, chenodesoxycholic acid, liver and gallbladder acid, taurocholate, deoxycholic acid, lithocholic acid)；

In a preferred embodiment, which includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows:

GIVDQC_[3]C_[4]FRSC_[5]X₄₁₂LX₄₁₄X₄₁₅LX₄₁₇X₄₁₈YC_[6]AX₄₂₂,

B chain amino acid sequence are as follows:

X_423-426X₄₂₇LC_[1]GAHLVDALFFVC_[2]GDRGFYX₄₄₈X₄₄₉X₄₅₀X₄₅₁X₄₅₂X₄₅₃X₄₅₄X₄₅₅,

In the compound, [1]-[6] indicate the number of cysteine；The compound forms 3 by 6 cysteines To disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond, three pair of two sulphur in A chain The specific location of key is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond；

Wherein X₄₁₂For serine or logical formula (I) structure；X₄₁₄For arginine or logical formula (I) structure；X₄₁₅It is arginine or logical Formula (I) structure；X₄₁₇For glutamic acid or logical formula (I) structure；X₄₁₈For asparagine or logical formula (I) structure；X₄₂₂For lysine, essence Propylhomoserin-lysine dipeptides or missing, or be logical formula (I) structure；Work as X₄₂₂When for dipeptides, one of amino acid is logical formula (I) knot Structure；X_423-426It is glycine-proline-glutamic acid tripeptides, U_LGlycine-proline-glutamic acid, phenylalanine-valine-day Winter amide-glutamine tetrapeptide or U_LPhenylalanine-valine-asparagine-glutamin；X₄₂₇It is histidine or Soviet Union's ammonia Acid；X₄₄₈It is-NH₂, phenylalanine or missing；X₄₄₉It is asparagine or missing；X₄₅₀It is lysine, arginine, glutamic acid, day Aspartic acid, proline or missing；X₄₅₁It is proline, lysine or missing, or is logical formula (I) structure；X₄₅₂It is threonine, relies ammonia Acid or missing, or be logical formula (I) structure；X₄₅₃It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₄ It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₅It is lysine or missing, or is logical formula (I) knot Structure；U_LIt is as defined in the present invention with logical formula (I) structure.

In another embodiment, the compound is the single-stranded structure based on IGF-1 transformation, the structure of the compound Are as follows:

X_201aLC_[1]GAX_201bLVDALX_201cX_201dVC_[2]GDRGFX_201eX₂₀₂X₂₀₃X₂₀₄X₂₀₅X₂₀₆GX₂₀₇X_207aX₂₀₈X_{20 9}X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆GIVDQC_[3]C_[4]X₂₁₇RSC_[5]X₂₁₈LX₂₁₉X₂₂₀LX₂₂₁X₂₂₂YC_[6]X₂₂₃X₂₂₄, wherein

X_201aIt is glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine, phenylpropyl alcohol ammonia Acid-valine-asparagine-glutamin-histidine, U_LGlycine-proline-glutamic acid-threonine, U_LGlycine-dried meat Propylhomoserin-glutamic acid-histidine or U_LPhenylalanine-valine-asparagine-glutamin-histidine；X_201bBe histidine, Glutamic acid, glutamine, arginine or phenylalanine；X_201cIt is phenylalanine or tryptophan；X_201dIt is phenylalanine or color ammonia Acid；X_201eIt is phenylalanine, tyrosine or histidine；X₂₀₂It is phenylalanine, tyrosine or missing；X₂₀₃It is asparagine, Soviet Union Propylhomoserin, aspartic acid, glutamic acid or missing；X₂₀₄It is proline, lysine, arginine, aspartic acid, glutamic acid or missing； X₂₀₅It is proline, lysine, arginine, aspartic acid, glutamic acid or missing or logical formula (I) structure；X₂₀₆It is threonine, relies ammonia Acid or missing or logical formula (I) structure；X₂₀₇It is serine, alanine, glycine, logical formula (I) structure or missing；X_207aIt is ammonia Acid, alanine, glycine, logical formula (I) structure or missing；X₂₀₈It is serine, logical formula (I) structure or missing；X₂₀₉Be serine, Logical formula (I) structure or missing；X₂₁₀It is serine, logical formula (I) structure or missing；X₂₁₁It is arginine, alanine, glycine, leads to Formula (I) structure or missing；X₂₁₂It is arginine, alanine, glycine, logical formula (I) structure or missing；X₂₁₃It is alanine, dried meat ammonia Acid, arginine, glycine, logical formula (I) structure or missing；X₂₁₄Be proline, glutamine, glycine, logical formula (I) structure or Missing；X₂₁₅It is glutamine, threonine, glycine, logical formula (I) structure or missing；X₂₁₆Be threonine, arginine, lysine, Logical formula (I) structure or missing；X₂₁₇It is phenylalanine, arginine, histidine or logical formula (I) structure；X₂₁₈It is aspartic acid, silk ammonia Acid, glutamine, asparagine or logical formula (I) structure；X₂₁₉It is arginine or logical formula (I) structure；X₂₂₀It is arginine or general formula (I) structure；X₂₂₁It is glutamic acid or logical formula (I) structure；X₂₂₂It is asparagine or logical formula (I) structure；X₂₂₃It is alanine, sweet ammonia Acid or asparagine；X₂₂₄It is lysine, arginine-lysine dipeptides or missing, or is logical formula (I) structure, works as X₂₂₄For dipeptides When, one of amino acid is logical formula (I) structure；U_LIt is as defined in the present invention with logical formula (I) structure；

In above compound structure, [1]-[6] indicate the number of cysteine.Single chain compound of the invention is in three-level knot In structure, the disulfide bond in chain is formed with the frame mode of insulin, specifically: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Shape At disulfide bond, C_[3]And C_[5]Form disulfide bond.

In a preferred embodiment, the structure of the single chain compound are as follows:

X_201aLC_[1]GAHLVDALFFVC_[2]GDRGFYX₂₀₂X₂₀₃X₂₀₄X₂₀₅X₂₀₆GX₂₀₇GX₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X_{21 4}X₂₁₅X₂₁₆GIVDQC_[3]C_[4]FRSC_[5]X₂₁₈LX₂₁₉X₂₂₀LX₂₂₁X₂₂₂YC_[6]A, wherein

X_201aIt is glycine-proline-glutamic acid-threonine, phenylalanine-valine-asparagine-glutamin- Histidine, U_LGlycine-proline-glutamic acid-threonine or U_LPhenylalanine-valine-asparagine-glutamin-group Propylhomoserin；X₂₀₂It is phenylalanine or missing；X₂₀₃It is asparagine or missing；X₂₀₄It is proline, lysine, arginine, asparagus fern ammonia Acid or missing；X₂₀₅It is proline, lysine or logical formula (I) structure；X₂₀₆It is threonine, lysine or logical formula (I) structure；X₂₀₇ It is serine, alanine or logical formula (I) structure；X₂₀₈It is serine or logical formula (I) structure；X₂₀₉It is serine or logical formula (I) knot Structure；X₂₁₀It is serine or logical formula (I) structure；X₂₁₁It is arginine, alanine, logical formula (I) structure or missing；X₂₁₂Be arginine, Alanine, glycine, logical formula (I) structure or missing；X₂₁₃It is alanine, proline, arginine, logical formula (I) structure or missing； X₂₁₄It is proline, glutamine, logical formula (I) structure or missing；X₂₁₅It is glutamine, threonine, logical formula (I) structure or scarce It loses；X₂₁₆It is threonine, arginine, lysine, logical formula (I) structure or missing；X₂₁₈It is serine or logical formula (I) structure；X₂₁₉It is Arginine or logical formula (I) structure；X₂₂₀It is arginine or logical formula (I) structure；X₂₂₁It is glutamic acid or logical formula (I) structure；X₂₂₂It is day Winter amide or logical formula (I) structure；U_LAs defined herein with logical formula (I) structure.

In terms of the compound for the modification being transformed based on IGF-1, the compound of modification of the invention is selected from:

III -1: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17；B chain-ordering For G (N^α-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT；

III -2: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17；B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPK (N^ε-PEG 20K)；

III -3: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGK(N^ε-PEG20K)GSSSRRAPQTGIVDQCCFRSCSLRRLEN YCA；

III -4: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPPTGK(N^ε-PEG20K)GSSSAAAPQTGIVDQCCFRSCSLRRLE NYCA；

III -5: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N^ε-PEG20K)GSSSAAAPQTGIVDQCCFRSCSLRRLE NYCA；

III -6: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: sequence shown in SEQ ID NO:17；B chain sequence It is classified as: G (N^α-CO(CH₂)₁₄COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT；

III -7: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering Are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -8: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering Are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₆CO)-γ-Glu)]；

III -9: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering Are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₂CO)-γ-Glu)]；

III -10: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering Are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK { N^ε-[N^α-(HOOC(CH₂)₁₁NHCO(CH₂)₃CO)-γ-Glu]}；

III -11: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering Are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu-N-(γ-Glu)]；

III -12: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] GSSSAAPQTGIVDQCCFRSCSLRRLENYCA；

III -13: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] SSAAPQTGIVDQCCFRSCSLRRLENYCA；

III -14: single-stranded structure, sequence are as follows: GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK [N^ε-(N^α- (HOOC(CH₂)₁₄CO)-γ-Glu)]AAPQTGIVDQCCFRSCSLRRLENYCA；

III -15: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] APQTGIVDQCCFRSCSLRRLENYCA；

III -16: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA；

III -17: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA；

III -18: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA；

III -19: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -20: single-stranded structure, sequence are as follows:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] GRGIVDQCCFRSCSLRRLENYCA；

III -21: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: GIVDQCCFRSCSLK [N^ε-(N^α-(HOOC (CH₂)₁₄CO)-γ-Glu)]RLENYCA；B chain-ordering be FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 163)；

III -22: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: GIVDQCCFRSCSLRK [N^ε-(N^α-(HOOC (CH₂)₁₄CO)-γ-Glu)]LENYCA；B chain-ordering be GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 164)；

III -23: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCAK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；B chain-ordering is sequence shown in SEQ ID NO:164；

III -24: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCARK [N^ε- (N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)], B chain-ordering is sequence shown in SEQ ID NO:164；

III -25: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -26: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For FVNQHLCGAHLVDALFFVCGDRGFYFNPPTEK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -27: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For FVNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -28: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu-N-(γ-Glu))]；

III -29: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For G [N^α-(N^α-(CH₃(CH₂)₁₄CO)-γ-L-Glu)]PETLCGAHLV DALFFVCGDRGFYFNPPT；

III -30: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For G (N^α-dPEG₁₂Maleimide-albumin) PETLCGAHLVDALFFVCGDRGFYFNKPT；

III -31: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For FVNQHLCGAHLVDALFFVCGDRGFYFNPPK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -32: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPPTK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -33: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO 17, B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPPTEK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -34: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering For GPETLCGAHLVDALFFVCGDRGFYFNPPTGEK [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III-35: single-stranded structure G (N^α-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAAPQTGI VDQCCFRSCSLRRLENYCA；

III-36: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCHRSCSLRRLENYCA, B chain sequence It is classified as GPEHLCGAHLVDALFFVCGDRGFYFNPK [N^ε-CO-(CH₂CH₂O)₅-(CH₂)₂-NH-(N^α-(HOOC(CH₂)₁₆CO)- γ-Glu)], structure is as follows；

III-37: single-stranded structure GPEHLCGAHLVDALFFVCGDRGFYFNPTGK [N^ε-CO-(CH₂CH₂O)₅-(CH₂)₂- NH-(N^α-(HOOC(CH₂)₁₆CO)-γ-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA；

N^αIndicate the alpha-amido of amino acid or amino acid residue；N^εIndicate the epsilon-amino of amino acid or amino acid residue, such as The epsilon-amino of lysine side-chain.

Two sulphur in chain are formed in the tertiary structure of above-mentioned double-strand or single chain compound with the frame mode of insulin Key, specifically: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond.Cysteine Number is as defined herein.

Can be provided with the composite form of the compound form or zinc that are substantially free of zinc of the invention has hypoglycemic work Compound.When providing the zinc complexes of the 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 invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition includes that treatment is effective Acceptable carrier on the compound according to the present invention and pharmaceutics of amount, for treating type 1 diabetes, diabetes B and drawing Play other situations of hyperglycemia.Compound according to the present invention can be used for preparing treatment type 1 diabetes, diabetes B and Cause the pharmaceutical composition of other situations of hyperglycemia.

In another aspect of the invention, it provides and a kind of treat type 1 diabetes, diabetes B and cause hyperglycemia Other situations pharmaceutical composition, described pharmaceutical composition includes the compound according to the present invention of therapeutically effective amount, mixing There are acceptable carrier and additive in insulin or insulin analog and pharmaceutics with snap action effect.

The routine techniques that pharmaceuticals industry can be used prepares the Injectable composition of IGF-1 analog of the present invention, including molten It solves and mixes appropriate component and obtain required finished product.Therefore, according to a set of operating procedure, by IGF-1 analog of the invention It is dissolved in a certain amount of water, volume is slightly less than the final volume of composition to be prepared.If desired, preservative, isotonic is added Agent and buffer.If it is necessary, adjusting the pH of solution using sour (such as hydrochloric acid) or alkali (such as sodium hydroxide).Finally use water The volume of solution is adjusted to required concentration.

In another embodiment of the present invention, it is sweet to be selected from sodium acetate, sodium carbonate, citrate, glycyl for buffer Propylhomoserin, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and three (methylol)-ammonia Methylmethane, N- bis- (ethoxy) glycine, N- (methylol) methylglycine, malic acid, succinate, maleic acid, fumaric acid, Or mixtures thereof tartaric acid, aspartic acid.Each in these specific buffers constitutes alternative embodiment of the invention.

In another embodiment of the present invention, the preparation includes pharmaceutically acceptable preservative, it is selected from benzene Phenol, o-cresol, m-cresol, p-Cresol, methyl p-hydroxybenzoate, ethyl-para-hydroxybenzoate, P-hydroxybenzoic acid third It is ester, butyl p-hydroxybenzoate, 2- phenoxetol, benzylalcohol, methaform, thimerosal, bronopol, benzoic acid, miaow urea, double Or mixtures thereof chlorhexidine, sodium dehydroacetate, chloreresol, benzyl rope chloramines, Chlorphenesin.In another reality of the invention It applies in scheme, the concentration of preservative is 0.1mg/mL-20mg/mL.In another embodiment of the present invention, preservative is dense Degree is 0.1mg/mL-5mg/mL.In another embodiment of the present invention, the concentration of preservative is 5mg/mL-10mg/mL. Each in these specific preservatives constitutes alternative embodiment of the invention.It is this that preservative is applied in pharmaceutical composition Field technical staff is well-known.Reference Remington:The Science and Practice of Pharmacy, the 19 editions, 1995.

In another embodiment of the present invention, the preparation further comprises isotonic agent, selected from salt (such as chlorination Sodium), sugar or sugar alcohol, amino acid, alditol (such as glycerol, propylene glycol, 1,3- propylene glycol, 1,3 butylene glycol), polyethylene glycol (example Or mixtures thereof such as PEG400).Any sugar, such as monosaccharide, disaccharides, polysaccharide or water-soluble dextran, including such as fructose, grape It is sugar, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, general Shandong indigo plant, dextrin, cyclodextrin, solvable Property starch, hydroxyethyl starch and carboxymethyl cellulose-Na.In one embodiment, sugar additives are sucrose.Sugar alcohol is defined For the C4-C8 hydrocarbon at least one-OH group, including for example mannitol, sorbierite, inositol, galactitol, dulcitol, Xylitol and arabite.In one embodiment, which is mannitol.Above-mentioned carbohydrate or glycitols can To be used singly or in combination.To the unfixed limitation of dosage, as long as the sugar or sugar alcohol are dissolved in liquid preparation and will not Adverse effect is generated to the static stabilization for using the method for the present invention to obtain.In one embodiment, sugared or sugar alcohol Concentration is 1mg/mL-150mg/mL.In another embodiment, the concentration of isotonic agent is 1mg/mL-50mg/mL.Another In a embodiment, the concentration of isotonic agent is 1mg/mL-7mg/mL.In another embodiment, the concentration of isotonic agent is 8mg/mL-24mg/mL.In another embodiment, the concentration of isotonic agent is 25mg/mL-50mg/mL.These are specific isotonic Each in agent constitutes alternative embodiment of the invention.It is those skilled in the art crowd that isotonic agent is applied in pharmaceutical composition Well known.Referring to Remington:The Science and Practice of Pharmacy, the 19th edition, 1995.

Typical isotonic agent is sodium chloride, mannitol, dimethyl sulfoxide and glycerol, and typical preservative is phenol, m- first Phenol, methyl p-hydroxybenzoate and benzylalcohol.

The example of surfactant includes sodium acetate, glycylglycine, hydroxyethyl piperazineethanesulfonic acid (HEPES) and phosphoric acid Sodium.

Embodiment

Protecting group:

Acm acetamidomethyl: acetamide methyl；Alloc or AOC allyloxycarbonyl: allyl oxygen carbonyl Base；Bom, benzyloxymethyl: benzyloxymethyl；2-Br-Z, 2-bromobenzyloxycarbonyl:2- bromo-benzyloxycarbonyl； TBu, t-butyl: tert-butyl；Bz, benzoyl: benzoyl；Bzl, benzyl: benzyl；Boc: tertbutyloxycarbonyl；CHO Formyl: formoxyl；CHx, cyclohexyl: cyclohexyl；Cbz or Z benzyloxycarbonyl: benzyloxycarbonyl group；Cl-Z,2- Chlorobenzyloxycarbonyl:2- benzyloxycarbonylchloride base；Fm, 9-fluorenylmethyl:9- fluorenyl methyl；Fmoc,9- Fluorenylmethoxycarbonyl:9- fluorenylmethyloxycarbonyl；Mtt, 4-methyltrityl:4- methyltrityl；Npys, 3-nitro-2-pyridinesulfenyl:3- nitro -2- pyridine sulfenyl；Pmc,(2,2,5,7,8- Pentametylchroman-6-sulphonyl:2,2,5,7,8- pentamethyl -6- hydroxychroman；Tos,4- Toluenesulphonyl: tolysulfonyl；Trt, tripheylmethyl: trityl；Xan, xanthyl: ton base, oxygen (miscellaneous) anthryl.

Reagent and solvent:

ACN, acetonitrile: acetonitrile；BOP,benzotriazol-1-yloxytris(dimethylamino) (the Ka Te condensation of phosphoniumhexafluorophosphate: benzotriazole -1- three (dimethylamino)-hexafluorophosphoric acid ester Agent)；DCC, N, N'-Dicyclohexylcarbodiimide: dicyclohexyl carbodiimide；DCM: methylene chloride；DEPBT, 3- (Diethoxyphosphoryloxy) -1,2,3-benzotriazin-4 (3H)-one:3- (diethoxy neighbour acyloxy) - 1,2,3- phentriazine -4- ketone；DIC, N, N'-Diisopropylcarbodiimide:N, N'- diisopropylcarbodiimide； DIPEA (or DIEA), diisopropylethylamine: diisopropylethylamine；DMAP,4-N,N- Dimethylaminopyridine:4-N, N dimethylamino naphthyridine；DMF:N, dinethylformamide；DMSO: dimethyl sulfoxide； DTT, dithiothreitol: dithiothreitol (DTT)；EDC or EDCI, 1-ethyl-3- (3-dimethylaminopropyl) Carbodiimide:1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate；EtOAc: ethyl acetate；HBTUO- (1H-benzotriazole-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate benzo Triazole-N, N, N', N'- tetramethylurea hexafluorophosphate；HOBT 1-hydroxybenzotriazole:1- hydroxyl-benzo- Triazole；NMM, N-Methylmorpholine:N- methyl morpholine；NMP, N-methylpyrrolidinone:N- methylpyrrole Alkanone；Piperidine: piperidines；Su succinimide: succinimide；

TEA, triethylamine: triethylamine；TFA, trifluoroacetic acid trifluoroacetic acid；TFE 2,2,2- Trifluoroethanol trifluoroethanol；THF tetrahydrofuran tetrahydrofuran；TIS triisopropylsilane Tri isopropyl silane.

Chemiluminescent polypeptide synthetic method

Linear polypeptide uses Boc or Fmoc solid phase polypeptide synthesis.If the use of the end Fmoc chemical synthesis C- being carboxyl Polypeptide, it is general to select Wang resin；The end C- is that the polypeptide of amide usually selects Rink amide resin.If using Boc The end chemical synthesis C- is the polypeptide of carboxyl, general to select Pam resin；The end C- is that the polypeptide of amide usually selects MBHA tree Rouge.Condensing agent and activator are DIC and HOBT, other optional peptide bond condensing agents include BOP, HBTU, DEPBT etc..5 times of amino acid It is excessive.Being condensed the time is 1 hour.Fmoc protecting group is removed with 50% piperidines/DMF.Boc protecting group is removed with TFA.Peptide bond condensation Reaction is monitored with ninhydrin (Ninhydrin, 2,2-Dihydroxyindane-1,3-dione) reagent.

When using Fmoc solid phase polypeptide synthesis, universal amino acid and protecting 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, allyloxycarbonyl is can be used in the side-chain amino group of lysine (aloc) it protects.Peptide chain synthesis finishes, and removing allyloxycarbonyl can use tetrakis triphenylphosphine palladium (0) and 37:2:1 ratio DCM, glacial acetic acid and NMM (15mL/g resin) are stirred 2 hours in ar gas environment, under room temperature.Resin needs to use after reaction 0.5%DIPEA/DMF (10mL), 0.5% 3 hydration diethyl-dithio sodium carbonate/DMF (3X 10mL), 1:1 DCM:DMF (5X10mL) cleaning.4- methyltrityl (Mtt) protection also can be used in the side-chain amino group of lysine.Resin is suspended in DCM, It is added TFA/TIS/DCM (1:2:97), shakes 10 minutes.After repeating 2 times, resin is washed with DCM, DMF and isopropanol.

After solid phase Fmoc chemistry synthesis polypeptide, common cutting reagent is TFA.Dried resin is placed in a shaking flask, is added Enter appropriate amount TFA/TIS/H₂O (95:2.5:2.5,10-25mL/g resin), closes the lid, carries out intermittent rotation at room temperature Concussion.Resin is filtered after 2 hours, is cleaned resin 2-3 times with new TFA, merging filtrate, the ice ether of 8-10 times of volume is added dropwise. Finally, the polypeptide crude product being precipitated out is collected by centrifugation.

When using Boc solid phase polypeptide synthesis, universal amino acid and protecting 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, allyloxycarbonyl is can be used in the side-chain amino group of lysine (aloc) protection or Fmoc protection.If the side chain carboxyl group of aspartic acid or glutamic acid is used for acylation reaction, carboxyl should be converted It is protected for allyl ester (allyl ester) or 9- fluorenyl methyl.

After solid phase Boc chemically synthesized polypeptide, for PAM, MBHA resin, generally cut using HF, every 0.1 mM of resin Add 5 milliliters of HF, while the reagents such as p-cresol, p-mercaptophenol or methyl phenyl ethers anisole are added, it is small that mixture stirs 1 under condition of ice bath When.After HF vacuum is drained, polypeptide ice ether is precipitated, and precipitating is collected by centrifugation, isolates and purifies by HPLC, and freeze-drying obtains most Product afterwards.

The synthesis of double chain compound

Method 1:

Sulfydryl sulfonating reaction (Sulfitolysis)

Reaction dissolvent ingredient is as follows:

0.5mM IGF-1A chain or B chain are dissolved in above-mentioned reaction dissolvent, and pH value is adjusted to 8.5-8.7.Mixture is in room Temperature is vigorously stirred 1-1.5 hours, then uses the desalination of G10 or G25 column.Buffer solution A is 0.05M ammonium bicarbonate aqueous solution, buffering Liquid B is 0.05M ammonium hydrogen carbonate/50% acetonitrile solution.Sterling low-pressure refrigeration after desalination is dry (lyophilization).

The connection of A chain and B chain

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).By sulfydryl sulfonating reaction Obtained A chain S sulphonic acid ester and B chain S sulphonic acid ester is mixed by 2:1 weight ratio, is dissolved in (pH in 0.1M glycine solution 10.5), peptide concentration is 5-10 mg/ml.DTT is added, making SH:S sulphonic acid ester ratio is 1.2.Reaction stirs 12 at 4 DEG C Hour.Reaction gained mixture is purified using RP-HPLC.

The synthesis of I-15:

A chain and the Fmoc chemical synthesis of B chain.A chain calculates molecular weight 2436.9, mass spectrometric measurement molecular weight 2437.6；B chain meter 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 5607.3。

Method 2:

The connection of A chain and B chain

Literature method (Han etc., " Insulin chemical synthesis using a two-step orthogonal formation of the three disulfides”,21^st American Peptide Society Symposium,2009).A chain and B chain are synthesized with Fmoc or Boc chemical synthesis process, and A7, B6 (correspond to X in general formula₂₉Or X₄₂₉) the general protecting group of cysteine, but A6, A11, A20 and B18 (correspond to X in general formula₄₁Or X₄₄₁) cysteine side chain Sulfydryl is protected with Acm.Synthetic A chain and B chain becomes A- (SH) after cutting down from resin⁷(S-Acm)^6,11,20With B- (SH)⁶ (S-Acm)¹⁸.B chain is dissolved in DMF or DMSO, and equimolar 2 is added, and bis- sulphur of 2'- is bis- (5- nitropyridine).Reaction with HPLC detection and Purifying, obtains B- (S-Npys)⁶(S-Acm)¹⁸.Equimolar A- (SH)⁷(S-Acm)^6,11,20With B- (S-Npys)⁶(S-Acm)¹⁸It is molten In DMSO, peptide concentration 15mg/mL.After A7-B6 disulfide bond formation, 80% aqueous acetic acid is added, peptide concentration is diluted to 1mg/mL.Add 40 times of I₂.After reaction is stirred at room temperature 1 hour, aqueous ascorbic acid is added and terminates reaction.Mixture Purified with HPLC, final product is confirmed with mass spectrum.

The synthesis of I-24

A chain and the Fmoc chemical synthesis of B chain.A-(SH)⁷(S-Acm)^6,11,20Calculate molecular weight 2650.1, mass spectrometric measurement point Son amount 2651.3；B-(SH)⁷(S-Acm)¹⁹Calculate molecular weight 3246.7, mass spectrometric measurement molecular weight 3247.5.The meter of final product Calculate molecular weight 5847.8, mass spectrometric measurement molecular weight 5849.0.

Other double chain compounds are synthesized with same method.

The connection type for further confirming that pair disulfide bond of the structure of compound, especially three, using in Chance document HPLC " fingerprint " 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.In brief, 2mg polypeptide Sample is dissolved in 0.2ml 0.01N hydrochloric acid, and the 0.05M NH that 0.8ml contains 100 μ g S.aureus V8 protease is added₄HCO₃, make PH reaches 7.9.It is cultivated 24 hours at 37 DEG C.Sample fraction is added DTT and cultivates 30 minutes.Use LC-MS contrast sample and mark The retention time (retention time) and molecular weight of each segment of quasi- product can disulfide bond and compound structure.This A 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:

It is respectively synthesized double chain compound using the above-mentioned synthetic method of the present invention, utilizes the method pair of sequencing and mass spectrometric measurement These compounds are tested analysis, and sequencing result shows that the amino acid sequence of compound is correct, and mass spectral results show chemical combination The structure of object is consistent with former design structure, that is, the compound synthesized is desired compound.Data result is as follows:

I-1: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5055.9, mass spectrometric measurement molecular weight 5057.3；

I-2: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5094.9, mass spectrometric measurement molecular weight 5095.7；

I-3: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5133.9, mass spectrometric measurement molecular weight 5135.1；

I-4: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5687.6, mass spectrometric measurement molecular weight 5689.3；

I-5: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5627.4, mass spectrometric measurement molecular weight 5628.5；

I-6: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5614.4, mass spectrometric measurement molecular weight 5615.9；

I-7: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5636.5, mass spectrometric measurement molecular weight 5636.7；

I-8: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5660.4, mass spectrometric measurement molecular weight 5661.2；

I-9: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5626.5, mass spectrometric measurement molecular weight 5627.6；

I-10: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5612.4, mass spectrometric measurement molecular weight 5614.1；

I-11: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5599.4, mass spectrometric measurement molecular weight 5601.7；

I-12: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5626.5, mass spectrometric measurement molecular weight 5627.3；

I-13: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5640.5, mass spectrometric measurement molecular weight 5641.2；

I-14: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5598.5, mass spectrometric measurement molecular weight 5599.1；

I-16: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5734.7, mass spectrometric measurement molecular weight 5735.4；

I-17: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5890.8, mass spectrometric measurement molecular weight 5992.2；

I-18: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5088.9, mass spectrometric measurement molecular weight 5090.5；

I-19: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5661.6, mass spectrometric measurement molecular weight 5673.2；

I-20: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5652.6, mass spectrometric measurement molecular weight 5652.9；

I-21: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5642.5, mass spectrometric measurement molecular weight 5643.3；

I-22: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5876.8, mass spectrometric measurement molecular weight 5877.6；

I-23: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5837.7, mass spectrometric measurement molecular weight 5839.0；

I-25: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5557.4, mass spectrometric measurement molecular weight 5558.7；

I-26: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5633.5, mass spectrometric measurement molecular weight 5634.9；

I-27: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5770.7, mass spectrometric measurement molecular weight 5772.1；

I-28: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5798.7, mass spectrometric measurement molecular weight 5799.6；

I-29: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5803.7, mass spectrometric measurement molecular weight 5805.3；

I-30: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5831.7, mass spectrometric measurement molecular weight 5832.8；

I-31: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5987.9, mass spectrometric measurement molecular weight 5990.4；

I-32: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5931.9, mass spectrometric measurement molecular weight 5932.5；

I-33: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated value 5804.6, mass spectrometric measurement molecular weight 5805.9；

I-34: Mass Spectrometer Method shows that A chain and B chain pass through two pairs of intermolecular disulfide bonds and a pair of of intramolecular disulfide key connection: C_[1]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 Calculated 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., " Comparative Properties of Insulin-like Growth Factor 1(IGF-1)and[Gly7D-Ala]IGF-1 Prepared by Total Chemical Synthesis.” Angew.Chem.Int.Ed.2008,47,1102–1106).It is partly improved according to amino acid sequence.

IGF-1 analog is divided into two segment synthesis.One section is with Boc chemical synthesis IGF-1B chain [1-17]-COS- (CH₂)₂CO-(Arg)₄A.With S- trityl -3- mercaptopropionic acid when thioesters residue synthesizes.Second segment includes (corresponding from B18Cys The X in general formula₄₁Or X₄₄₁) play B chain C-terminal whole amino acid, C peptide and A chain amino acid.Two segment polypeptides universal method solid phase Synthesis is cut, purifying.

Naturally reaction is connected chemically to carry out in buffer.Buffer guanidine hydrochloride containing 6M, 200mM phosphate, 200mM4- Carboxymethyl benzenethiol (MPAA), 20mM tri- (2- carboxyethyl) phosphine (TCEP), pH 6.9, polypeptide are dissolved by 1:1 molar ratio, concentration 2mM.Reaction HPLC detection, purifying.

The IGF-1 (SH) of purifying₆It is dissolved in 0.5M guanidine hydrochloride, 20mM Tris, 8mM cysteine, 1mM cystine salt Phthalate buffer, pH 7.8, peptide concentration 0.5mg/mL.After the completion of HPLC display folds, buffer is acidified with 0.1N hydrochloric acid To pH3.Polypeptide preparation RP-HPLC is purified.

II -3 preparation.

First segment GPETLCGAHLVDALFFV- (COS-CH₂CH₂- CO)-RRRRA Boc chemical synthesis, thick peptide RP- HPLC purifying.Molecular weight calculated value 2573.1, mass spectrometric measurement molecular weight 2573.8.

Second segment CGDRGFYFNKPTGSGSSSAAAPQTGIVDQCCFRSCSLRRLENYCA is synthesized by universal method, Thick peptide is purified with RP-HPLC.Molecular weight calculated value 4825.4, mass spectrometric measurement molecular weight 4827.0.

First segment 25.7mg (10 μm of ol) and the second segment 48.3mg (10 μm of ol) are dissolved in buffer (5mL).Buffering Liquid includes 6M guanidine hydrochloride, 200mM phosphate, 200mM 4- carboxymethyl benzenethiol, 20mM tri- (2- carboxyethyl) phosphine, pH 6.9.It reacts and completes after 10 hours.Molecular weight calculated value 6596.5, mass spectrometric measurement molecular weight 6597.1.Mixture is transferred to ruler Very little exclusion chromatography column, elution buffer are 0.5M guanidine hydrochloride, 20mM Tris, pH 7.8.It collects comprising correct polypeptide molecular weight Part, after merging be added 8mM cysteine, 1mM cystine hydrochloride buffer, after 2 hours polypeptide folding finish.It will buffering Liquid is acidified to pH 3 with 0.1N hydrochloric acid, is then purified with RP-HPLC.Molecular weight calculated value 6590.5, mass spectrometric measurement molecular weight 6591.6 sequencing result is consistent with SEQ ID NO:31.

The single chain compound based on IGF-1 is in the above way prepared, using the molecular weight of Mass Spectrometer Method molecule, passes through sequencing The structure of the single chain polypeptide of preparation is detected to verify synthesized compound, as a result:

II -1: molecular weight calculated value 6798.8, mass spectrometric measurement molecular weight 6799.3, sequencing result and SEQ ID NO:29 mono- It causes；

II -2: molecular weight calculated value 6760.7, mass spectrometric measurement molecular weight 6763.4, sequencing result and SEQ ID NO:30 mono- It causes；

II -4: molecular weight calculated value 6519.4, mass spectrometric measurement molecular weight 6521.5, sequencing result and SEQ ID NO:32 mono- It causes；

II -5: molecular weight calculated value 6448.3, mass spectrometric measurement molecular weight 6450.1, sequencing result and SEQ ID NO:33 mono- It causes；

II -6: molecular weight calculated value 6434.3, mass spectrometric measurement molecular weight 6436.0, sequencing result and SEQ ID NO:34 mono- It causes；

II -7: molecular weight calculated value 6420.3, mass spectrometric measurement molecular weight 6421.9, sequencing result and SEQ ID NO:35 mono- It causes；

II -8: molecular weight calculated value 6404.3, mass spectrometric measurement molecular weight 6406.2, sequencing result and SEQ ID NO:36 mono- It causes；

II -9: molecular weight calculated value 6404.3, mass spectrometric measurement molecular weight 6405.6, sequencing result and SEQ ID NO:37 mono- It causes；

II -10: molecular weight calculated value 6248.1, mass spectrometric measurement molecular weight 6250.8, sequencing result and SEQ ID NO:38 Unanimously；

II -11: molecular weight calculated value 6234.1, mass spectrometric measurement molecular weight 6235.7, sequencing result and SEQ ID NO:39 Unanimously；

II -12: molecular weight calculated value 6120.0, mass spectrometric measurement molecular weight 6121.4, sequencing result and SEQ ID NO:40 Unanimously；

II -13: molecular weight calculated value 6207.1, mass spectrometric measurement molecular weight 6208.0, sequencing result and SEQ ID NO:41 Unanimously；

II -14: molecular weight calculated value 6174.0, mass spectrometric measurement molecular weight 6175.5, sequencing result and SEQ ID NO:42 Unanimously；

II -15: molecular weight calculated value 6234.1, mass spectrometric measurement molecular weight 6235.4, sequencing result and SEQ ID NO:43 Unanimously；

II -16: molecular weight calculated value 6261.1, mass spectrometric measurement molecular weight 6261.6, sequencing result and SEQ ID NO:44 Unanimously；

II -17: molecular weight calculated value 6432.3, mass spectrometric measurement molecular weight 6433.5, sequencing result and SEQ ID NO:45 Unanimously；

II -18: molecular weight calculated value 6002.8, mass spectrometric measurement molecular weight 6004.1, sequencing result and SEQ ID NO:46 Unanimously；

II -19: molecular weight calculated value 6078.9, mass spectrometric measurement molecular weight 6080.2, sequencing result and SEQ ID NO:47 Unanimously；

II -20: molecular weight calculated value 6121.9, mass spectrometric measurement molecular weight 6122.7, sequencing result and SEQ ID NO:48 Unanimously；

II -21: molecular weight calculated value 6163.0, mass spectrometric measurement molecular weight 6164.4, sequencing result and SEQ ID NO:49 Unanimously；

II -22: molecular weight calculated value 6071.0, mass spectrometric measurement molecular weight 6072.8, sequencing result and SEQ ID NO:50 Unanimously；

II -23: molecular weight calculated value 6590.5, mass spectrometric measurement molecular weight 6592.3, sequencing result and SEQ ID NO:51 Unanimously；

II -24: molecular weight calculated value 6505.4, mass spectrometric measurement molecular weight 6506.0, sequencing result and SEQ ID NO:52 Unanimously；

II -25: molecular weight calculated value 6831.8, mass spectrometric measurement molecular weight 6833.7, sequencing result and SEQ ID NO:53 Unanimously；

II -26: molecular weight calculated value 5899.7, mass spectrometric measurement molecular weight 5900.6, sequencing result and SEQ ID NO:54 Unanimously；

II -27: molecular weight calculated value 6114.0, mass spectrometric measurement molecular weight 6115.9, sequencing result and SEQ ID NO:55 Unanimously；

II -28: molecular weight calculated value 6089.9, mass spectrometric measurement molecular weight 6091.5, sequencing result and SEQ ID NO:56 Unanimously；

II -29: molecular weight calculated value 6089.9, mass spectrometric measurement molecular weight 6090.7, sequencing result and SEQ ID NO:57 Unanimously；

II -30: molecular weight calculated value 6161.0, mass spectrometric measurement molecular weight 6162.4, sequencing result and SEQ ID NO:58 Unanimously；

II -31: molecular weight calculated value 6066.9, mass spectrometric measurement molecular weight 6068.1, sequencing result and SEQ ID NO:59 Unanimously；

II -32: molecular weight calculated value 6093.9, mass spectrometric measurement molecular weight 6095.6, sequencing result and SEQ ID NO:60 Unanimously；

II -33: molecular weight calculated value 6334.2, mass spectrometric measurement molecular weight 6336.0, sequencing result and SEQ ID NO:61 Unanimously；

II -34: molecular weight calculated value 7159.1, mass spectrometric measurement molecular weight 7161.2, sequencing result and SEQ ID NO:62 Unanimously；

II -35: molecular weight calculated value 6546.4, mass spectrometric measurement molecular weight 6546.0, sequencing result and SEQ ID NO:63 Unanimously；

II -36: molecular weight calculated value 6592.5, mass spectrometric measurement molecular weight 6593.8, sequencing result and SEQ ID NO:64 Unanimously；

II -37: molecular weight calculated value 6592.4, mass spectrometric measurement molecular weight 6594.1, sequencing result and SEQ ID NO:65 Unanimously；

II -38: molecular weight calculated value 6732.6, mass spectrometric measurement molecular weight 6733.4, sequencing result and SEQ ID NO:66 Unanimously；

II -39: molecular weight calculated value 6644.6, mass spectrometric measurement molecular weight 6646.7, sequencing result and SEQ ID NO:67 Unanimously；

II -40: molecular weight calculated value 6701.6, mass spectrometric measurement molecular weight 6702.9, sequencing result and SEQ ID NO:68 Unanimously；

II -41: molecular weight calculated value 6715.7, mass spectrometric measurement molecular weight 6716.6, sequencing result and SEQ ID NO:69 Unanimously；

II -42: molecular weight calculated value 6608.5, mass spectrometric measurement molecular weight 6609.7, sequencing result and SEQ ID NO:70 Unanimously；

II -43: molecular weight calculated value 6761.6, mass spectrometric measurement molecular weight 6762.5, sequencing result and SEQ ID NO:71 Unanimously；

II -44: molecular weight calculated value 6659.6, mass spectrometric measurement molecular weight 6660.4, sequencing result and SEQ ID NO:72 Unanimously；

II -45: molecular weight calculated value 6689.6, mass spectrometric measurement molecular weight 6691.2, sequencing result and SEQ ID NO:73 Unanimously；

II -46: molecular weight calculated value 6733.7, mass spectrometric measurement molecular weight 6734.1, sequencing result and SEQ ID NO:74 Unanimously；

II -47: molecular weight calculated value 6614.5, mass spectrometric measurement molecular weight 6615.8, sequencing result and SEQ ID NO:75 Unanimously；

II -48: molecular weight calculated value 6559.5, mass spectrometric measurement molecular weight 6560.7, sequencing result and SEQ ID NO:76 Unanimously；

II -49: molecular weight calculated value 6846.8, mass spectrometric measurement molecular weight 6848.1, sequencing result and SEQ ID NO:77 Unanimously；

II -50: molecular weight calculated value 6634.5, mass spectrometric measurement molecular weight 6636.0, sequencing result and SEQ ID NO:78 Unanimously；

II -51: molecular weight calculated value 6774.6, mass spectrometric measurement molecular weight 6675.9, sequencing result and SEQ ID NO:79 Unanimously；

II -52: molecular weight calculated value 6644.6, mass spectrometric measurement molecular weight 6645.3, sequencing result and SEQ ID NO:80 Unanimously；

II -53: molecular weight calculated value 6770.7, mass spectrometric measurement molecular weight 6772.2, sequencing result and SEQ ID NO:81 Unanimously；

II -54: molecular weight calculated value 6882.8, mass spectrometric measurement molecular weight 6884.1, sequencing result and SEQ ID NO:82 Unanimously；

II -55: molecular weight calculated value 8098.2, mass spectrometric measurement molecular weight 8099.6, sequencing result and SEQ ID NO:83 Unanimously；

II -56: molecular weight calculated value 6729.7, mass spectrometric measurement molecular weight 6731.5, sequencing result and SEQ ID NO:84 Unanimously；

II -57: molecular weight calculated value 6679.5, mass spectrometric measurement molecular weight 6680.2, sequencing result and SEQ ID NO:85 Unanimously；

II -58: molecular weight calculated value 6960.9, mass spectrometric measurement molecular weight 6961.6, sequencing result and SEQ ID NO:86 Unanimously；

II -59: molecular weight calculated value 7160.1, mass spectrometric measurement molecular weight 7161.5, sequencing result and SEQ ID NO:87 Unanimously；

II -60: molecular weight calculated value 6899.8, mass spectrometric measurement molecular weight 6901.6, sequencing result and SEQ ID NO:88 Unanimously；

II -61: molecular weight calculated value 6703.6, mass spectrometric measurement molecular weight 6704.3, sequencing result and SEQ ID NO:89 Unanimously；

II -62: molecular weight calculated value 6644.6, mass spectrometric measurement molecular weight 6645.8, sequencing result and SEQ ID NO:90 Unanimously；

II -63: molecular weight calculated value 6581.5, mass spectrometric measurement molecular weight 6583.3, sequencing result and SEQ ID NO:91 Unanimously；

II -64: molecular weight calculated value 6701.6, mass spectrometric measurement molecular weight 6702.4, sequencing result and SEQ ID NO:92 Unanimously；

II -65: molecular weight calculated value 6674.6, mass spectrometric measurement molecular weight 6675.7, sequencing result and SEQ ID NO:93 Unanimously；

II -66: molecular weight calculated value 7354.3, mass spectrometric measurement molecular weight 7356.2, sequencing result and SEQ ID NO:94 Unanimously；

II -67: molecular weight calculated value 6729.7, mass spectrometric measurement molecular weight 6730.2, sequencing result and SEQ ID NO:95 Unanimously；

II -68: molecular weight calculated value 6707.6, mass spectrometric measurement molecular weight 6709.0, sequencing result and SEQ ID NO:96 Unanimously；

II -69: molecular weight calculated value 6774.7, mass spectrometric measurement molecular weight 6775.4, sequencing result and SEQ ID NO:97 Unanimously；

II -70: molecular weight calculated value 6620.5, mass spectrometric measurement molecular weight 6621.1, sequencing result and SEQ ID NO:98 Unanimously；

II -71: molecular weight calculated value 6608.5, mass spectrometric measurement molecular weight 6609.7, sequencing result and SEQ ID NO:99 Unanimously；

II -72: molecular weight calculated value 6647.6, mass spectrometric measurement molecular weight 6648.6, sequencing result and SEQ ID NO:100 Unanimously；

II -73: molecular weight calculated value 6691.6, mass spectrometric measurement molecular weight 6693.0, sequencing result and SEQ ID NO:101 Unanimously；

II -74: molecular weight calculated value 7256.2, mass spectrometric measurement molecular weight 7257.9, sequencing result and SEQ ID NO:102 Unanimously；

II -75: molecular weight calculated value 6788.7, mass spectrometric measurement molecular weight 6790.9, sequencing result and SEQ ID NO:103 Unanimously；

II -76: molecular weight calculated value 7587.5, mass spectrometric measurement molecular weight 7588.3, sequencing result and SEQ ID NO:104 Unanimously；

II -77: molecular weight calculated value 8530.5, mass spectrometric measurement molecular weight 8532.8, sequencing result and SEQ ID NO:105 Unanimously；

II -78: molecular weight calculated value 7385.4, mass spectrometric measurement molecular weight 7386.0, sequencing result and SEQ ID NO:106 Unanimously；

II -79: molecular weight calculated value 6941.0, mass spectrometric measurement molecular weight 6941.5, sequencing result and SEQ ID NO:107 Unanimously；

II -80: molecular weight calculated value 6009.8, mass spectrometric measurement molecular weight 6011.4, sequencing result and SEQ ID NO:108 Unanimously；

II -81: molecular weight calculated value 6555.5, mass spectrometric measurement molecular weight 6556.6, sequencing result and SEQ ID NO:109 Unanimously；

II -82: molecular weight calculated value 7098.1, mass spectrometric measurement molecular weight 7100.2, sequencing result and SEQ ID NO:110 Unanimously；

II -83: molecular weight calculated value 7186.0, mass spectrometric measurement molecular weight 7187.1, sequencing result and SEQ ID NO:111 Unanimously；

II -84: molecular weight calculated value 6934.9, mass spectrometric measurement molecular weight 6935.6, sequencing result and SEQ ID NO:112 Unanimously；

II -85: molecular weight calculated value 7160.2, mass spectrometric measurement molecular weight 7161.9, sequencing result and SEQ ID NO:113 Unanimously；

II -86: molecular weight calculated value 6664.6, mass spectrometric measurement molecular weight 6665.1, sequencing result and SEQ ID NO:114 Unanimously；

II -87: molecular weight calculated value 6338.2, mass spectrometric measurement molecular weight 6339.8, sequencing result and SEQ ID NO:115 Unanimously；

II -88: molecular weight calculated value 6511.3, mass spectrometric measurement molecular weight 6512.4, sequencing result and SEQ ID NO:116 Unanimously；

II -89: molecular weight calculated value 6407.3, mass spectrometric measurement molecular weight 6408.2, sequencing result and SEQ ID NO:117 Unanimously；

II -90: molecular weight calculated value 6636.6, mass spectrometric measurement molecular weight 6637.5, sequencing result and SEQ ID NO:118 Unanimously；

II -91: molecular weight calculated value 6569.5, mass spectrometric measurement molecular weight 6570.9, sequencing result and SEQ ID NO:119 Unanimously；

II -92: molecular weight calculated value 6385.3, mass spectrometric measurement molecular weight 6387.0, sequencing result and SEQ ID NO:120 Unanimously；

II -93: molecular weight calculated value 6371.3, mass spectrometric measurement molecular weight 6372.3, sequencing result and SEQ ID NO:121 Unanimously；

II -94: molecular weight calculated value 6806.8, mass spectrometric measurement molecular weight 6808.1, sequencing result and SEQ ID NO:122 Unanimously；

II -95: molecular weight calculated value 6704.6, mass spectrometric measurement molecular weight 6705.9, sequencing result and SEQ ID NO:123 Unanimously；

II -96: molecular weight calculated value 6877.8, mass spectrometric measurement molecular weight 6878.7, sequencing result and SEQ ID NO:124 Unanimously；

II -97: molecular weight calculated value 6750.7, mass spectrometric measurement molecular weight 6771.9, sequencing result and SEQ ID NO:125 Unanimously；

II -98: molecular weight calculated value 7724.8, mass spectrometric measurement molecular weight 7726.2, sequencing result and SEQ ID NO:126 Unanimously；

II -99: molecular weight calculated value 6123.9, mass spectrometric measurement molecular weight 6125.8, sequencing result and SEQ ID NO:127 Unanimously；

II -100: molecular weight calculated value 6614.4, mass spectrometric measurement molecular weight 6615.3, sequencing result and SEQ ID NO: 128 is consistent；

II -101: molecular weight calculated value 6899.8, mass spectrometric measurement molecular weight 6901.4, sequencing result and SEQ ID NO: 129 is consistent；

II -102: molecular weight calculated value 6036.8, mass spectrometric measurement molecular weight 6038.0, sequencing result and SEQ ID NO: 130 is consistent；

II -103: molecular weight calculated value 6853.7, mass spectrometric measurement molecular weight 6854.5, sequencing result and SEQ ID NO: 131 is consistent；

II -104: molecular weight calculated value 7284.2, mass spectrometric measurement molecular weight 7285.6, sequencing result and SEQ ID NO: 132 is consistent；

II -105: molecular weight calculated value 7551.5, mass spectrometric measurement molecular weight 7552.9, sequencing result and SEQ ID NO: 133 is consistent；

II -106: molecular weight calculated value 6913.9, mass spectrometric measurement molecular weight 6915.3, sequencing result and SEQ ID NO: 134 is consistent；

II -107: molecular weight calculated value 6837.7, mass spectrometric measurement molecular weight 6839.3, sequencing result and SEQ ID NO: 135 is consistent；

II -108: molecular weight calculated value 7063.1, mass spectrometric measurement molecular weight 7064.7, sequencing result and SEQ ID NO: 136 is consistent；

II -109: molecular weight calculated value 6528.4, mass spectrometric measurement molecular weight 6529.8, sequencing result and SEQ ID NO: 137 is consistent；

II -110: molecular weight calculated value 7200.3, mass spectrometric measurement molecular weight 7201.4, sequencing result and SEQ ID NO: 138 is consistent；

II -111: molecular weight calculated value 7735.7, mass spectrometric measurement molecular weight 7737.0, sequencing result and SEQ ID NO: 139 is consistent；

II -112: molecular weight calculated value 6369.1, mass spectrometric measurement molecular weight 6370.6, sequencing result and SEQ ID NO: 140 is consistent；

II -113: molecular weight calculated value 7468.4, mass spectrometric measurement molecular weight 7470.2, sequencing result and SEQ ID NO: 141 is consistent；

II -114: molecular weight calculated value 6603.5, mass spectrometric measurement molecular weight 6604.7, sequencing result and SEQ ID NO: 142 is consistent；

II -115: molecular weight calculated value 7254.2, mass spectrometric measurement molecular weight 7255.5, sequencing result and SEQ ID NO: 143 is consistent；

II -116: molecular weight calculated value 6625.5, mass spectrometric measurement molecular weight 6627.1, sequencing result and SEQ ID NO: 144 is consistent；

II -117: molecular weight calculated value 7399.5, mass spectrometric measurement molecular weight 7401.3, sequencing result and SEQ ID NO: 145 is consistent；

II -118: molecular weight calculated value 7223.1, mass spectrometric measurement molecular weight 7224.4, sequencing result and SEQ ID NO: 146 is consistent；

II -119: molecular weight calculated value 6120.0, mass spectrometric measurement molecular weight 6121.6, sequencing result and SEQ ID NO: 147 is consistent；

II -120: molecular weight calculated value 6278.2, mass spectrometric measurement molecular weight 6277.9, sequencing result and SEQ ID NO: 148 is consistent；

II -121: molecular weight calculated value 6264.1, mass spectrometric measurement molecular weight 6265.8, sequencing result and SEQ ID NO: 149 is consistent；

II -122: molecular weight calculated value 6242.2, mass spectrometric measurement molecular weight 6243.4, sequencing result and SEQ ID NO: 150 is consistent；

II -123: molecular weight calculated value 6084.9, mass spectrometric measurement molecular weight 6086.3, sequencing result and SEQ ID NO: 161 is consistent；

II -124: molecular weight calculated value 6659.5, mass spectrometric measurement molecular weight 6660.7, sequencing result and SEQ ID NO: 162 is consistent.

The modification of compound

The modification of compound based on IGF-1

The acylation of polypeptide

Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu preparation

Hexadecandioic acid (hexadecane diacid) (5.72g, 20mmol) is dissolved in anhydrous DMF (240mL), is cooled with an ice bath.Gradually add 2- first Base -2- propyl alcohol (1.48g, 20mmol), DIC (2.7g, 2.25mL, 21.4mmol), HOBT (2.88g, 21.4mmol), NMM (2.16g, 2.34mL, 21.4mmol), DMAP (244mg, 2mmol).Mixture is stirred at room temperature overnight.80mL water is added, It is acidified to pH 3, is extracted with ethyl acetate, organic layer is washed with 0.1N HCl and saturated common salt, and after magnesium sulfate is dry, solvent subtracts Pressure evaporation obtains one tert-butyl ester of hexadecandioic acid (hexadecane diacid) (3.32g, yield 47%).Nuclear magnetic resonance data is¹H-NMR(CDCl₃) δ: 2.35 (t,2H),1.56-1.66(m,4H),1.44(s,9H),1.21-1.35(m,20H)。

Fmoc-Glu-OtBu (4.25g, 10mmol) is dissolved in DCM (30mL), is added to 3 grams of 2-CTC resin (2- Chlorotrityl chloride resin, sub.1mmol/g), continuously add DIPEA (1.29g, 10mmol, 1.74mL). Mixture vibrates after five minutes in shaking machine, adds DIPEA (1.93g, 15mmol, 2.6mL).Mixture high vibration 1 is small When.

In order to seal active triphenylmethyl, HPLC grades of methanol (2.4mL) are added in resin, mix 15 minutes.Resin mistake Filter, after being cleaned with DCM (3X 30mL), DMF (2X 30mL), DCM (3X 30mL), methanol (3X 30mL), is dried in a vacuum.

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

Prepare the cutting liquid (20mL/g resin) of AcOH/TFE/DCM (1:1:8).Resin is suspended in the cutting liquid of half, room Temperature is lower to place 30 minutes.Resin is filtered, washs resin three times with the other half cutting liquid.Mixed filtrate be added 15 times of volumes just oneself Alkane, revolving remove extra acetic acid, obtain tert-butyl hexadecandioyl diacyl-L-Glu-OtBu.Nuclear magnetic resonance data is¹H-NMR (CDCl₃) δ: 6.25 (d, 1H), 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 (1g, 1.9mmol) is dissolved in anhydrous DMF/DCM (1mL:4mL) and adds Enter DCC (0.412g, 2mmol) and N- hydroxysuccinimide (0.23g, 2mmol).Mixture is stirred at room temperature overnight.Filtering Mixture, filtrate are diluted with ethyl acetate, are evaporated under reduced pressure after magnesium sulfate is dry with 0.1N HCl and saturated common salt water washing To tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu.Nuclear magnetic resonance data are as follows:¹H-NMR(CDCl₃) δ: 6.17 (d, 1H), 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.

It is synthesized with above-mentioned A, B chain link method.Calculate molecular weight 5746.7, mass spectrometric measurement molecular weight 5747.8.At room temperature Polypeptide (55mg) is dissolved in 100mM Na₂CO₃(5mL,pH 10).Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (6.6mg) is dissolved in acetonitrile (5mL), and polypeptide solution is added.After stirring 30 minutes, with 50% acidifying with acetic acid, upper RP-HPLC C5 column Purifying.Buffer solution A: 0.1%TFA aqueous solution, 10% acetonitrile buffer B:0.1%TFA aqueous solution, 80% acetonitrile.Preliminary purification TFA/TIS/H is added in polypeptide after freeze-drying₂O (95:2.5:2.5,10mL), vacuum evaporating solvent after 30 minutes are molten by crude product In buffer solution A and it is lyophilized.Use RP-HPLC C5 column purification.Molecular weight calculated value 6144.2, mass spectrometric measurement molecular weight 6146.0. After B chain warp trypsin hydrolysis, the calculating molecular weight 1269.5 of the terminal fragment containing fatty acid, mass spectrometric measurement molecular weight 1270.4。

III -28 synthesis:

Method 1:

B chain (0.1mmol) Boc chemical synthesis, lysine side chain amino groups are protected with Fmoc.After B chain synthesizes, B chain The Boc of the end N- not removed.Resin is added in 20% piperidines/DMF (6mL), and mixture vibrates 15 minutes, drains reagent.By The reaction of two-wheeled piperidines, resin are washed 3 times with DMF and DCM.Fmoc-L-Glu-OtBu(425mg,1mmol),DIC(126mg, 0.1ml, 1mmol), HOBT (135mg, 1mmol) and DIPEA (0.2mL, 1.15mmol) be dissolved in DMF, resin is added.It is small to vibrate 2 Shi Hou, solution are drained, and resin is washed with DMF and DCM.Resin is added in 20% piperidines/DMF, and mixture is vibrated 15 minutes, drained Reagent.It is reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)- OtBu (550mg) and DIPEA (0.2mL) is dissolved in DMF, and resin is added.After vibration 3 hours, solution is drained, resin with DMF and DCM washing.After vacuum drying, the polypeptide on resin is cut with HF, and paracresol reagent is added, and mixture stirs under condition of ice bath 1 hour, after HF vacuum is drained, polypeptide ice ether was precipitated, and precipitating is collected by centrifugation.Molecular weight calculated value 3601.2, mass spectrometric measurement Molecular weight 3602.8.

B chain crude product is crossed chromatographic column desalination, is isolated and purified, freeze-drying obtains final product IGF- through sulfydryl sulfonating reaction 1B chain S sulphonic acid ester.IGF-1A (Q5, S12, N18) is synthesized by universal method, molecular weight calculated value 2436.9, mass spectrometric measurement molecule Amount 2437.6.IGF-1A chain S sulphonic acid ester and B chain S sulphonic acid ester are synthesized using above-mentioned general A chain with the connection method of B chain.Molecular weight Calculated value 6032.0, mass spectrometric measurement molecular weight 6033.4.

Method 2:

The Boc chemical synthesis of B chain, lysine side chain amino groups are protected with Fmoc, and B18Cys (corresponds to X in general formula₄₁Or X₄₄₁) Sulfydryl protected with Acm.After synthesis, the Boc of the end B chain N- not removed.Resin is added in 20% piperidines/DMF (6mL) (0.1mmol), mixture vibrate 15 minutes, drain reagent.It is reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM. Resin is added in Fmoc-L-Glu-OtBu (425mg), DIC (126mg, 0.1ml), HOBT (135mg) and DIPEA (0.2mL).Vibration After 2 hours dynamic, solution is drained, and after resin is washed with DMF and DCM, 20% piperidines/DMF is added, and mixture vibrates 15 minutes, Drain reagent.It is reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Tert-butyl hexadecandioyl diacyl-L-Glu-OtBu (715mg), DIC (126mg, 0.1ml), HOBT (135mg) and DIPEA (0.2mL) are dissolved in DMF, and resin is added, and vibrate 2 hours Afterwards, solution is drained, and resin is washed with DMF and DCM.After vacuum drying, the polypeptide on resin is cut with HF, and paracresol examination is added Agent, mixture stirs 1 hour under condition of ice bath, and after HF vacuum is drained, polypeptide ice ether is precipitated, and precipitating is collected by centrifugation.Meter Calculate molecular weight 3672.3, mass spectrometric measurement molecular weight 3673.5.

B chain is dissolved in DMF or DMSO, and equimolar 2 is added, and bis- sulphur of 2'- is bis- (5- nitropyridine).Reaction with HPLC detection and it is pure Change, obtains B- (S-Npys)⁶(S-Acm)¹⁸[H9,F15,P27,K28-N^εB28-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu-N- (γ-Glu))].Calculate molecular weight 3826.4, mass spectrometric measurement molecular weight 3827.0.

A chain synthesizes IGF-1A- (Q5, S12, N18) (SH)⁷(S-Acm)^6,11,20Calculate molecular weight 2650.1, mass spectrometric measurement Molecular weight 2651.5.The connection method of A chain and B chain is according to above-mentioned A chain and B chain link method (2).Molecular weight calculated value 6032.0 mass spectrometric measurement molecular weight 6034.2.

III -29 synthesis.

After B chain (0.1mmol) uses Boc chemical synthesis, the Boc of the end B chain N- is removed with TFA.Fmoc-L-Glu- OtBu (425mg, 1mmol), DIC (126mg, 0.1ml, 1mmol), HOBT (135mg, 1mmol) and DIPEA (0.2ml, It 1.15mmol) is dissolved in DMF, resin is added.After vibration 2 hours, solution is drained, and resin is washed with DMF and DCM.20% piperidines/ Resin is added in DMF, and mixture vibrates 15 minutes.It is reacted by two-wheeled piperidines, resin is washed 3 times with DMF and DCM.Palmitinic acid (256mg, 1mmol), DIC (126mg, 0.1ml, 1mmol), HOBT (135mg, 1mmol) and DIPEA (0.2ml, 1.15mmol) It is dissolved in DMF, resin is added.After 2 hours, solution is drained, and resin is washed with DMF and DCM.It is more on resin after vacuum drying Peptide is cut with HF, and paracresol reagent is added, and mixture stirs 1 hour under condition of ice bath, after HF vacuum is drained, polypeptide ice second Ether precipitating, is collected by centrifugation precipitating.Molecular weight calculated value 3512.1, mass spectrometric measurement molecular weight 3513.5.B chain crude product is through sulfydryl sulfonation It reacts (Sulfitolysis), crosses chromatographic column desalination, isolates and purifies, freeze-drying obtains final product B chain S sulphonic acid ester.A chain S Sulphonic acid ester and B chain S sulphonic acid ester are synthesized using above-mentioned general A chain with the connection method of B chain.Molecular weight calculated value 5943.0, mass spectrum Test molecule amount 5943.6.

III -12 synthesis:

GPETLCGAHLVDALFFVCGDRGFYFNPTGKGSSSAAPQTGIVDQCCFRSCSLRRLE NYCA is with above-mentioned side Method synthesis.Molecular weight calculated value 6432.3, mass spectrometric measurement molecular weight 6434.1.Polypeptide (643mg) is dissolved in 100mM at room temperature Na₂CO₃(5mL,pH 10).Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (68mg) is dissolved in acetonitrile (5mL), is added more Peptide solution.After stirring 30 minutes, with 50% acidifying with acetic acid, upper RP-HPLC C5 column purification.Buffer solution A: 0.1%TFA aqueous solution, 10% acetonitrile buffer B:0.1%TFA aqueous solution, 80% acetonitrile.TFA/TIS/H is added in polypeptide after preliminary purification freeze-drying₂O Crude product is dissolved in buffer solution A and is lyophilized by (95:2.5:2.5,10mL), vacuum evaporating solvent after 30 minutes.Use RP-HPLC C5 column purification.Molecular weight calculated value 6829.8, mass spectrometric measurement molecular weight 6831.6.A small amount of product is restored through DTT and trypsase Degradation, liquid chromatograph mass spectrography observe that GPETLCGAHLVDALFFVCGDR (calculates molecular weight 2220.6, test molecule It measures 2221.4), GFYFNPTGK- [N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]-GSSSAAPQT GIVDQCCFR (calculating point Son amount 3236.7, test molecule amount 3237.5).It is thus identified that fatty acid is incorporated in C peptide lysine side-chain.Through analyzing, gained Close object i.e. III -12.

(CH₃OOC(CH₂)₁₁NHCO(CH₂)₃CO)-Glu(OSu)-OCH₃Synthesis

Methanol (40ml) ice bath is cooling, and SOCl is added dropwise₂(4ml).It is added 12-aminolauric acid (3g, 13.9mmol), stirring Overnight.Mixture and drying are filtered, 3g 12-aminolauric acid methyl ester hydrochloride is 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 methyl ester hydrochloride (1g, 3.8mmol) is suspended in THF (15ml), and glutaric anhydride is added (1.29g, 3.8mmol) and TEA (0.52ml, 3.8mmol), is stirred overnight at room temperature.It is filtered after adding water (75ml) to stir 1 hour, It is washed with water, obtains 1g 12- (4- carboxyl amide-based small) methyl dodecanoate after dry.

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- carboxyl amide-based small) methyl dodecanoate (0.33g, 0.95mmol) be dissolved in anhydrous DMF/DCM (0.5ml: 0.5ml), DCC (0.2g, 1mmol) and N- hydroxysuccinimide (0.115g, 1mmol) is added.Mixture was stirred at room temperature Night.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1N HCl and saturated common salt water washing, after magnesium sulfate is dry, decompression Evaporation obtains 12- (4- carboxyl amide-based small) methyl dodecanoate of OSu activation.The compound is dissolved in DMF (1.5ml), is added DIEA (0.19g, 1.5mmol) and H-Glu-OMe (0.168g, 1.04mmol), is stirred overnight at room temperature.Add after solvent is evaporated under reduced pressure Enter ethyl acetate, with 0.1N HCl and saturated common salt water washing, after magnesium sulfate is dry, reduction vaporization obtains (CH₃OOC(CH₂)₁₁NHCO(CH₂)₃CO)-Glu-OCH₃。

¹H-NMR(DMSO-d₆)δ:12(bs,1H),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)₁₁NHCO(CH₂)₃CO)-Glu-OCH₃(0.36g, 0.36mmol) is dissolved in anhydrous DMF/DCM DCC (0.08g, 0.4mmol) and N- hydroxysuccinimide (0.046g, 0.4mmol) is added in (0.5ml:0.5ml).Mixture It is stirred at room temperature 24 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1N HCl and saturated common salt water washing, sulphur After sour magnesium is dry, reduction vaporization obtains (CH₃OOC(CH₂)₁₁NHCO(CH₂)₃CO)-Glu(OSu)-OCH₃。

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 synthesis

With above-mentioned A, B chain link method synthesis polypeptide.Calculate molecular weight 5746.7, mass spectrometric measurement molecular weight 5748.0.Room Polypeptide (58mg) is dissolved in 100mM Na under temperature₂CO₃(5mL,pH 10)。(CH₃OOC(CH₂)₁₁NHCO(CH₂)₃CO)-Glu (OSu)-OCH₃(7mg) is dissolved in acetonitrile (5mL), and polypeptide solution is added.It after stirring 30 minutes, is cooled with an ice bath, 0.1N is added NaOH uses acidifying with acetic acid, upper RP-HPLC C5 column purification after stirring 1 hour.Buffer solution A: 0.1%TFA aqueous solution, 10% acetonitrile Buffer solution B: 0.1%TFA aqueous solution, 80% acetonitrile.Molecular weight calculated value 6187.2, mass spectrometric measurement molecular weight 6188.7.Through dividing Analysis, gained compound i.e. III -10.

OSu-CO-(CH₂CH₂O)₅-(CH₂)₂-NH-[N^α-(HOOC(CH₂)₁₆CO)-γ-Glu] synthesis

Octadecane diacid (2.5g, 8.0mmol) is suspended in DCM (60ml), and triethylamine (1.16ml, 8.3mmol) is added simultaneously It is cooled with an ice bath.Benzyl chloroformate (1.14ml) is added dropwise in nitrogen environment, adds DMAP (0.097g, 0.8mmol).It stirs After mixing 30 minutes, solvent is evaporated under reduced pressure, by crude product with silica gel column purification (ethyl acetate: heptane 1:7-1:1), evaporates after solvent To one benzyl ester of octadecane diacid (1.12g, 35%).

¹H-NMR(CDCl₃)δ:7.35(m,5H),5.11(s,2H),2.35(t,4H),1.63(t,4H),1.30-1.22 (m,24)。

One benzyl ester of octadecane diacid (0.8g, 2mmol) is dissolved in DMF (3ml) and DCM (3ml), is cooled with an ice bath.DCC is added (0.408g, 2mmol) and N- hydroxysuccinimide (0.23g, 2mmol).Mixture is stirred at room temperature 24 hours.Filtering mixing Object, filtrate are diluted with ethyl acetate, and with 0.1N HCl and saturated common salt water washing, after magnesium sulfate is dry, reduction vaporization obtains amber One benzyl octadecane diacid ester of amber imide.

¹H-NMR(CDCl₃)δ:7.35(m,5H),5.11(s,2H),2.83(s,4H),2.60(t,2H),2.35(t,2H), 1.80-1.60(m,4H),1.40-1.20(m,24H)。

One benzyl octadecane diacid ester (95mg, 0.19mmol) of succinimido is dissolved in DMF (1.5ml), and L- is added Glu-OBzl (49mg, 0.21mmol) and DIEA (52 μ l, 0.3mmol) is stirred 16 hours.Solvent is evaporated under reduced pressure, acetic acid is added Ethyl ester is evaporated under reduced pressure solvent after magnesium sulfate is dry, is obtained two acyl of BzlO- octadecane with 0.1N HCl, saturated common salt water washing Base-L-Glu-OBzl.

¹H-NMR(CDCl₃)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- octadecandioyl-L-Glu-OBzl (110mg, 0.18mmol) is dissolved in DMF (1ml) and DCM (1ml), uses Ice bath is cooling.DCC (41mg, 0.2mmol) and N- hydroxysuccinimide (23mg, 0.2mmol) is added.Mixture is stirred in room temperature It mixes 12 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, and with 0.1N HCl and saturated common salt water washing, magnesium sulfate is dry Afterwards, it is evaporated under reduced pressure and obtains BzlO- octadecandioyl-L-Glu (OSu)-OBzl.

¹H-NMR(CDCl₃)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- octadecandioyl-L-Glu (OSu)-OBzl (72mg, 0.1mmol) and H₂N-(CH₂)₂-(OCH₂CH₂)₅COOH (31mg, 0.1mmol) is dissolved in DMF/DCM (0.5ml:1.5ml), is added DIEA (26 μ L, 0.15mmol), and stirring 16 is small When.Solvent is evaporated under reduced pressure, ethyl acetate is added, with 0.1N HCl, saturated common salt water washing, is evaporated under reduced pressure after magnesium sulfate is dry molten Agent obtains 3- [2- [2- [2- [2- [2- [[5- benzyloxy -4- [(18- benzyloxy -18- oxo-octadecanoyl) amino] -5- Oxo-pentanoyl] 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, test Molecular weight 916.5.

3- [2- [2- [2- [2- [2- [[5- benzyloxy -4- [(18- benzyloxy -18- oxo-octadecanoyl) amino] -5- Oxo-pentanoyl] amino] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] propionic acid (91mg, 0.1mmol) is dissolved in DMF (1ml) and DCM (1ml), is cooled with an ice bath.Be added DCC (24.7mg, 0.12mmol) and N- hydroxysuccinimide (13.8mg, 0.12mmol).Mixture is stirred at room temperature 12 hours.Mixture is filtered, filtrate is diluted with ethyl acetate, with 0.1N HCl and is satisfied And brine It, after magnesium sulfate is dry, reduction vaporization obtains benzyl 18- [[1- benzyloxycarbonyl group -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -3- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] second ammonia Base] -4- oxo-butyl] amino] -18- oxo-stearate (benzyl 18- [[1-benzyloxycarbonyl-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-octadecanoate).Calculate molecular weight 1012.2 test molecule amount 1013.1.

Benzyl 18- [[1- benzyloxycarbonyl group -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- oxo - Stearate (51mg, 0.05mmol) is dissolved in methanol/acetone/0.1%TFA, and Pd/C is added, 5 are stirred at room temperature in a nitrogen environment Hour, it is filtered by diatomite, precipitating and evaporation residue solvent, obtain 18- [[1- carboxyl -4- [2- [2- [2- [2- from heptane [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -3- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] second Amino] -4- oxo-butyl] amino] -18- oxo-stearic acid (18- [[1-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 Molecular weight 833.4.

III-36 synthesis

With above-mentioned A, B chain link method synthesis polypeptide.Calculate molecular weight 5531.4, mass spectrometric measurement molecular weight 5533.7.Room Polypeptide (56mg, 10 μm of ol) is dissolved in 100mM Na under temperature₂CO₃(1ml,pH 10).18- [[1- carboxyl -4- [2- [2- [2- [2- [2- [3- (2,5- dioxo pyrrolidin -1-yl) oxygen -3- oxo-propoxyl group] ethyoxyl] ethyoxyl] ethyoxyl] ethyoxyl] second Amino] -4- oxo-butyl] amino] -18- oxo-stearic acid (9.2mg, 11 μm of ol) is dissolved in acetonitrile (0.5ml), polypeptide is added Solution.Acidifying with acetic acid, upper RP-HPLC C5 column purification are used after stirring 30 minutes.Buffer solution A: 0.1%TFA aqueous solution, 10% second Nitrile buffer solution B: 0.1%TFA aqueous solution, 80% acetonitrile.Molecular weight calculated value 6428.3, mass spectrometric measurement molecular weight 6430.2.Through Analysis, gained compound i.e. III -36.

The pegylation method (PEGylation) of polypeptide:

A) reductive alkylation (reductive alkylation)

Polypeptide, mPEG20K-CHO, sodium cyanoborohydride (NaBH₃CN) 4.3 acetum of pH is dissolved in 1:2:45 ratio (0.1M NaCl,0.2M CH₃COOH, 0.1M Na₂CO₃).Peptide concentration is 0.5-1mg/mL.Reaction with HPLC detection and it is pure Change.Yield about 55%.Polyethylene glycol can be selectively combined in B1 by reductive alkylation reaction.

III -1 synthetic method:

A (Q5, S12, N18): B (H9, F15, P27) synthetic method is same as above.B1 reductive alkylation obtains product.Molecular weights Calculation value 25576.4, mass spectrometric measurement obtain a broad peak, intermediate molecular weight 25580.2, and a small amount of product is restored through DTT, liquid chromatogram- Mass spectrometry observes A (Q5, S12, N18) (molecular weight calculated value 2436.9, mass spectrometric measurement molecular weight 2438.0) and G (N^α- PEG20K) PETLCGAHLVDALFFVCGDRGFYFNPPT (molecular weight calculated value 23144.6, mass spectrometric measurement obtain a broad peak, in Between molecular weight 23140.5), without molecular weight 22436.9.Through analyzing, gained compound i.e. III -1.

B) NHS ester (n-hydroxysuccinimide) is acylated

1:1 is dissolved in 0.1N N, bis- (2- ethoxy) glycine solution (pH of N- in molar ratio by polypeptide and mPEG20K-NHS 8), peptide concentration 0.5mg/mL.Reaction carries out 2 hours in room temperature, is purified with HPLC.Yield about 90%.

III -2 synthetic method:

A (Q5, S12, N18): B (H9, F15, P27, K28, desT) is synthesized with above-mentioned A, B chain link method.Calculate molecule Amount 5505.4, mass spectrometric measurement molecular weight 5506.8.Polypeptide obtains III -2 after reacting with mPEG20K-NHS.Molecular weight calculated 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 Chain, liquid chromatograph mass spectrography observe GPETLCGAH LVDALFFVCGDR (molecular weight calculated value 2220.6, mass spectrometric measurement Molecular weight 2221.7), it does not observe GFYFNPK (molecular weight calculated value 872.0), but mass spectrum observes a broad peak, centre point Son amount 20871.9.Through analyzing, gained compound i.e. III -2.

III -4 synthetic method:

Essentially identical with above-mentioned III -2, only the side-chain amino group of C2Lys is reacted with mPEG20K-NHS, obtains III -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 is through DTT reduction and pancreas egg White enzyme is degraded B chain, liquid chromatograph mass spectrography observe GPETLCGAHLVDALFFVCGDR (molecular weight calculated value 2220.6, Test molecule amount 2220.8) and GFYFNPPT-GK (N^ε- PEG20K)-GSSSAAAPQTGIVDQCCFR (molecular weight calculated value 23007.4, test obtains a broad peak, intermediate molecular weight 23010.0).Through analyzing, gained compound i.e. III -4.

Synthesize A1, B27-diBoc-IGF-1 analog

A(G1-N^α- Boc, Q5, S12, N18): B (H9, F15, K27-N^ε-Boc)

I-15A (Q5, S12, N18): B (H9, F15) is synthesized with the above universal method.Molecular weight 5606.5 is calculated, mass spectrum is surveyed Try molecular weight 5607.3.Polypeptide (100mg, 17.8 μm of ol) is dissolved in water (1mL), 1N NaHCO₃(0.3mL) and DMF (3mL).Add Enter t-Boc- azide (5.5mg, 37.5 μm of ol).Mixture stirs 3 hours at 40 DEG C, and 50% acetic acid (0.35mL) is added eventually Only react.Unreacted t-Boc-azide extracts (2X 15mL) with ether.Water layer vacuum freeze drying.It is mono- that crude product contains Boc Replace, disubstituted and three substitution polypeptides.Mixture SP-Sephadex C-25 ion exchange column purification.Ion exchange column is first used 1.5M acetic acid balance containing 6M urea, it is 48mL/h, linear gradient 0.04-0.4M sodium chloride/1000mL that polypeptide, which elutes flow velocity, The 1.5M acetic acid of 6M urea.Di-t-Boc polypeptide further uses DEAE-Sephadex A-25 column purification.Chromatographic column is in advance with containing There is the 0.01M Tris buffer (pH 8.5) of 7M urea to balance.Elution flow velocity 35mL/h, gradient 0.14-0.28M sodium chloride/ 100mL Tris buffer.Molecular weight calculated value 5806.7, mass spectrometric measurement molecular weight 5808.3.A small amount of polypeptide is dissolved in 0.05M NH₄HCO₃/ 20%ACN restores mass spectral analysis after ten minutes with DTT.A(G1-N^α- Boc, Q5, S12, N18) molecular weight calculated value 2537.0 mass spectrometric measurement molecular weight 2537.9.B (H9, F15, K27-N^ε- Boc) molecular weight calculated value 3275.8, mass spectrometric measurement point Son amount 3276.0.After tryptose enzymatic hydrolysis, the molecular weight calculated value 1073.2 of the segment containing Boc, mass spectrometric measurement molecular weight 1074.5. The B1 amino of A1, B27-di-Boc polypeptide can form long-acting polypeptides with combinations such as polyethylene glycol, albumin, fatty acid.

III -30 synthesis.

A(G1-N^α- Boc, Q5, S12, N18): B (H9, F15, K27-N^ε- Boc) synthetic method is as above.Polypeptide (58mg) is molten In DMF (3mL), Mal-dPEG12-NHS (8.7mg) (Quanta Biodesign) and triethylamine (30 μ L) is added.Reaction is in room Temperature lower stirring 2 hours.After depressurizing solvent flashing, TFA (2mL) and TIS (100 μ L) is added.After ten minutes, volatilize TFA for stirring.Slightly Product are dissolved in H₂O/ACN (3:1) is purified with RP-HPLC.Molecular weight calculated value 6556.5, mass spectrometric measurement molecular weight 6558.0.Malaysia Acid imide polypeptide is dissolved in pure water, peptide concentration 10mM.It is added human albumin (665mg), is cultivated 30 minutes at 37 DEG C.Then it uses 20mM sodium radio-phosphate,P-32 solution containing 5mM Sodium Caprylate and 750mM ammonium sulfate is diluted to 5%.It is removed with gel-filtration chromatography not anti- The reagent answered, eluent are 0.05M ammonium bicarbonate aqueous solution.Sterling is obtained after vacuum freeze drying.Compound molecular weight calculates Value 73028.7, mass spectrometric measurement molecular weight 73030.2 are analyzed as compound III -30.

Composite result:

It is respectively synthesized the compound of the respectively modification based on IGF-1 according to the method described above, including single chain compound and duplexed Object is closed, as a result as follows by the structure of each compound of Mass Spectrometer Method:

III -3: molecular weight calculated value 26673.6, mass spectrometric measurement obtain a broad peak, intermediate molecular weight 26680.5, through analyzing, institute Obtain compound i.e. III -3；

III -5: molecular weight calculated value 26618.5, mass spectrometric measurement obtain a broad peak, intermediate molecular weight 26624.9, through analyzing, institute Obtain compound i.e. III -5；

III -6: molecular weight calculated value 5845.8, mass spectrometric measurement molecular weight 5847.2, through analyzing, gained compound i.e. III -6；

III -8: molecular weight calculated value 6172.2, mass spectrometric measurement molecular weight 6173.4, through analyzing, gained compound i.e. III -8；

III -9: molecular weight calculated value 6116.1, mass spectrometric measurement molecular weight 6118.2, through analyzing, gained compound i.e. III -9；

III -11: molecular weight calculated value 6273.3, mass spectrometric measurement molecular weight 6274.5, through analyzing, gained compound i.e. III - 11；

III -13: molecular weight calculated value 6829.8, mass spectrometric measurement molecular weight 6830.4, through analyzing, gained compound i.e. III - 13；

III -14: molecular weight calculated value 6829.8, mass spectrometric measurement molecular weight 6831.1, through analyzing, gained compound i.e. III - 14；

III -15: molecular weight calculated value 6845.8, mass spectrometric measurement molecular weight 6846.9, through analyzing, gained compound i.e. III - 15；

III -16: molecular weight calculated value 6972.0, mass spectrometric measurement molecular weight 6973.3, through analyzing, gained compound i.e. III - 16；

III -17: molecular weight calculated value 6457.4, mass spectrometric measurement molecular weight 6458.0, through analyzing, gained compound i.e. III - 17；

III -18: molecular weight calculated value 6730.7, mass spectrometric measurement molecular weight 6732.2, through analyzing, gained compound i.e. III - 18；

III -19: molecular weight calculated value 5902.9, mass spectrometric measurement molecular weight 5903.4, through analyzing, gained compound i.e. III - 19；

III -20: molecular weight calculated value 6661.6, mass spectrometric measurement molecular weight 6662.7, through analyzing, gained compound i.e. III - 20；

III -21: molecular weight calculated value 6186.2, mass spectrometric measurement molecular weight 6187.8, through analyzing, gained compound i.e. III - 21；

III -22: molecular weight calculated value 5944.9, mass spectrometric measurement molecular weight 5946.0, through analyzing, gained compound i.e. III - 22；

III -23: molecular weight calculated value 6101.1, mass spectrometric measurement molecular weight 6101.6, through analyzing, gained compound i.e. III - 23；

III -24: molecular weight calculated value 6257.3, mass spectrometric measurement molecular weight 6258.9, through analyzing, gained compound i.e. III - 24；

III -25: molecular weight calculated value 6342.4, mass spectrometric measurement molecular weight 6344.1, through analyzing, gained compound i.e. III - 25；

III -26: molecular weight calculated value 6471.5, mass spectrometric measurement molecular weight 6472.3, through analyzing, gained compound i.e. III - 26；

III -27: molecular weight calculated value 6528.6, mass spectrometric measurement molecular weight 6529.1, through analyzing, gained compound i.e. III - 27；

III -31: molecular weight calculated value 6241.3, mass spectrometric measurement molecular weight 6242.4, through analyzing, gained compound i.e. III - 31；

III -32: molecular weight calculated value 6101.1, mass spectrometric measurement molecular weight 6101.8, through analyzing, gained compound i.e. III - 32；

III -33: molecular weight calculated value 6230.2, mass spectrometric measurement molecular weight 6231.5, through analyzing, gained compound i.e. III - 33；

III -34: molecular weight calculated value 6287.3, mass spectrometric measurement molecular weight 6288.2, through analyzing, gained compound i.e. III - 34；

III -35: molecular weight calculated value 26462.3, mass spectrometric measurement obtain a broad peak, intermediate molecular weight 26470.1, through analyzing, Gained compound i.e. III -35；

III -37: molecular weight calculated value 7246.3, mass spectrometric measurement molecular weight 7248.0, through analyzing, gained compound i.e. III - 37。

Receptor binding assay

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

2, 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 following methods it One.Unless otherwise instructed, by preparation also such as literature method, user's placental membrane.Under normal circumstances, insulin receptor knot It closes experiment and uses 0.025 milligram of placental membrane；IGF-1 Receptor Binding Assay uses 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 100nM respectively obtains the control and compound of 7 various concentrations then by the 3 times of dilutions of insulin and the compounds of this invention series Solution (100nM, 33.33nM, 11.11nM, 3.70nM, 1.23nM, 0.41nM, 0.13nM, 0.04nM).For its in the present invention The activity of insulin receptor is lower than the compound of actrapid monotard's standard 10%, and compound initial concentration is 500nM.IGF-1 by In the experiment of body binding analysis, the initial concentration of IGF-1 standard is 100nM, and the compounds of this invention initial concentration is 1000nM, then By 3 times of dilutions of IGF-1 and the compounds of this invention series, the control and compound solution of 7 various concentrations are respectively obtained (1000nM,333.33nM,111.11nM,37.04nM,12.35nM,4.12nM,1.37nM,0.46nM).For in the present invention The activity of its IGF-1 receptor is lower than the compound of IGF-1 standard 1%, and compound initial concentration is 5000nM.

Receptor binding assay (1)

The water-soluble receptor of truncation

IGF-1 or insulin receptor,¹²⁵I-IGF-1 (3-10pM) or¹²⁵3 times of I- insulin (3pM) and series are diluted more Buffer [100mM Hepes, pH 8.0,100mM NaCl, 10mM MgCl is added in peptide₂, 0.5% (w/v) BSA, 0.025% (w/v) Triton X-100], 200 μ L of total volume is cultivated 48 hours at 4 DEG C.Receptor and polypeptide and ligand use in conjunction with receptor 0.2% gamma globulin and 500 μ L 25% (w/v) PEG 8000 precipitating, measure the radioactivity in precipitating.The concentration of receptor will be adjusted Save the binding of receptor and ligand for having 15-20% when being not added with polypeptide.

Membrane-bound receptor

BHK of the membrane-bound receptor that receptor binding assay uses from height expression overall length insulin or IGF-1 receptor is thin Born of the same parents.The transfection bhk cell (2000-5000) of equivalent is evenly distributed on each hole of 96 orifice plates, is including 10% (v/v) tire ox blood Receptor binding assay is carried out again after culture 24 hours in the Eagle's culture medium (DMEM) of clear Dulbecco's improvement.Cell It is first washed one time with combination buffer (DMEM contains 0.50%BSA, 20mM Hepes, pH 7.8), 400 μ L is added¹²⁵I-IGF-1 (6.5pM) or¹²⁵I- insulin (6.5pM) and the 3 times of diluted polypeptides of series for being dissolved in combination buffer.It is small in 16 DEG C of cultures 3 When, unbonded polypeptide is sucked out with aspirator, is washed one time with 1.2ml combination buffer.Cell dissolution is in 500 μ L 1% (w/v) SDS, 100mM NaCl, 25mM Hepes (pH 7.8), then measure.Cell quantity will be adjusted to have 16- when not adding peptide 28% binding of receptor and ligand.

Receptor binding assay (2)

IGF-1 receptor: [Thr⁵⁹] IGF-1 be used for tyrosine iodination reaction (iodination).¹²⁵I-IGF-1(50- 80Ci/g, 50fmol), 0.2 milliliter of 0.1M Hepes buffer is added in Human plactnta film (0.2mg) and the diluted polypeptide of 3 times of series, PH 8 includes 120mM sodium chloride, 5mM potassium chloride, 0.12mM magnesium sulfate and 0.1% bovine albumin, cultivates 1 hour at 20 DEG C. Sample is filtered with Whatman GF/F filter to separate and combine and unbonded polypeptide compound.Filter uses 0.1% in advance Polyethyleneimine processing.Culture tube and filter are washed 4 times with 2.5 milliliters of cold buffer liquid without bovine albumin.There is no placental membrane In the case where, the polypeptide compound attachment less than 5% is on the filter.Under conditions of the competition of no polypeptide, placental membrane is combined About 38% ligand.Non- iodate [the Thr of excessive addition can be passed through to placental membrane non-specific binding⁵⁹] IGF-1 (0.3 μM) arrives Culture mix measures.Non-specific binding usually account for ligand in conjunction with placental membrane total amount 5%.

Insulin receptor:¹²⁵I- insulin (30nCi), serial 3 times of diluted polypeptides and placental membrane (0.025mg) are 0.05 In the above-mentioned buffer of milliliter, 20 DEG C are cultivated 1 hour.Sample is filtered with EHWP filter, culture tube and filter with 2.5 milliliters not Cold buffer liquid containing bovine albumin is washed 4 times.In the case where not having placental membrane, the polypeptide compound less than 5% is attached to filter On.Placental membrane non-specific binding can be measured by the non-Iodinated Insulin-125I (1 μM) of excessive addition to culture mix.It is non- Specific bond usually account for ligand in conjunction with placental membrane total amount 1% or less.

Specific bond percentage=(in conjunction with exit dose-non-specific binding exit dose/all in conjunction with the non-specific knot of exit dose- Close exit dose) x 100.It is all the radiation total amount measured when being not added with polypeptide in conjunction with exit dose.It is that addition is more in conjunction with exit dose The exit dose measured after peptide.The IC of polypeptide compound₅₀It is calculated using Origin software (OriginLab, Northampton, MA). Polypeptide is relative to actrapid monotard or activity=IC of IGF-1 standard₅₀Insulin or IGF-1 standard/IC₅₀Polypeptide.

3, zoopery

7-9 week old C57BL/6 male mice, average weight 20-25g, 6 are divided into one group, prohibit within 4 hours before experiment starts Food.Experiment measures blood glucose, and each specified time point sampling and measuring blood glucose afterwards before starting.Control group is physiological saline, more Peptide is dissolved in physiological saline, subcutaneous injection.Mouse is observed in the reaction of experiment overall process, records any abnormal behaviour.

Experimental result:

1, the compound of the present invention based on IGF-1 and the binding ability of insulin receptor and IGF-1 receptor are tested

In this part, the bioactivity of the compound with duplex structure is detected, examines these compounds With insulin receptor and IGF-1 receptor binding capacity.Using natural insulin and IGF-1 respectively as in conjunction with insulin receptor Power and benchmark (100%) with IGF-1 acceptor binding force.It the results are shown in Table two.

Although the A chain and B chain of IGF-1 and the A chain and B chain of insulin have high homology, and NMR and X-ray crystal The three-dimensional conformation of A chain and B chain that the result of diffraction also shows IGF-1 and insulin is also almost overlapped, but double-strand IGF-1A: The binding force of B and insulin receptor is very low, but again excessively high compared with insulin to the activity of IGF-1 receptor.Therefore must be The amino acid residue of IGF-1A chain and the specific site in B chain leads to above-mentioned difference.When B15Q is taken by phenylalanine or tryptophan Dai Hou, IGF-1A:B analog are suitable with insulin in the binding force of insulin receptor, it was demonstrated that B15 amino acid residues be with The key of the combination of insulin receptor.IGF-1 has apparent selectivity to IR-A, and double chain compound I1~I6 also shows degree It is lower, but same receptor-selective.A8 substitutions can improve polypeptide simultaneously and change in the binding force of 3 receptors, but not Become the selectivity to receptor.Ideal trypsin class medicine should have high and balanced IR-A and IR-B acceptor binding force, With alap IGF-1 acceptor binding force IGF-1.The side chain negative electrical charge of A5 and A12 is replaced to restore polypeptide with neutral side chain Balance on IR-A and IR-B, and significantly reduce the activity in IGF-1 receptor.

Studies have shown that B9E is the important amino acid residue of IGF-1 growth promoting function, IGF-1 is also made to have the danger for leading to cancer Danger.Replace glutamic acid with histidine, arginine, glutamine or phenylalanine, comparable insulin receptor can still be maintained Activity.

In A22, A23 addition lysine residues, insulin receptor is combined and is had no adverse effect, but lysine side-chain ammonia Base can provide the site of an acylation reaction.

Table two: the bioactivity result of the IGF-1 analog of duplex structure

Note: IR-A and IR-B is insulin receptor isoform A and B；IGF-1R insulin-like growth factor-1 receptor；IGF- 1A:IGF-1B indicates IGF-1A chain and IGF-1B chain duplex structure.

2, the experimental result of the unmodified single chain compound based on IGF-1

In this part, the bioactivity of the unmodified single chain compound based on IGF-1 is detected, is examined These single chain compounds and insulin receptor and IGF-1 receptor binding capacity.Using natural insulin and IGF-1 respectively as with Combination rate of insulin receptor and benchmark (100%) with IGF-1 acceptor binding force.It the results are shown in Table three.

Natural IGF-1 is single chain polypeptide, with the binding force of insulin receptor less than insulin 5%.Since B15 bit amino Acid replaces the level that the bioactivity of the double chain compound based on IGF-1 can be made to reach insulin, and same substitution also can Improve the activity of the single chain compound based on IGF-1.II -1 display only change B15 amino acid residue, remove it is generally acknowledged that pair The insulin receptor activity of compound is just improved up to 10 times or more in the domain D that bioactivity has no significant effect.C2Tyr is in IGF-1 It is an amino acid residue very outstanding in crystal structure, display may be and the key of the combination of GF-1 receptor.Tyrosine After being replaced by serine or alanine, insulin receptor activity retains substantially, but considerably reduces IGF-1 receptor active, just It is expected effect.In addition, the results show that the length of C peptide is not necessarily required to 12 amino acid residues.Multiple ammonia of former IGF-1C Base acid residue all can be lacked or be replaced.Especially IGF-1 C-terminal removes 3 amino acid residue PQT and 4 residues APQT not only has no adverse effect the activity of insulin receptor, and its IGF-1 receptor active is made to be reduced to the water of insulin It is flat.Further investigation revealed that in the case where retaining the length of C peptide is 12 amino acid residues, the 5 of IGF-1B chain C-terminal A amino acid residue, i.e. X₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆It is can have 1,2,3,4 or all missing, without reducing pancreas Island element acceptor binding force.Further study showed that IGF-1 C peptide is only a selection in a variety of junction fragments.These Data explanation, the amino acid substitution of C chain length appropriate, flexible C chain space conformation and specific site be improve insulin by An important factor for body activity, reduction IGF-1 acceptor binding force.

Table three: the bioactivity result of the unmodified single chain compound based on IGF-1

3, the experimental result of the compound based on IGF-1 of PEG or fatty acid modifying

In this part, to the bioactivity of the compound based on IGF-1 of PEG, albumin or fatty acid modifying into Row detection, examines these compounds and insulin receptor and IGF-1 receptor binding capacity.Use natural insulin and IGF-1 points Not as with combination rate of insulin receptor and with the benchmark (100%) of IGF-1 acceptor binding force.It the results are shown in Table four.

Pegylation and fatty-acylation be extend polypeptide body in action time common method, but Pegylation and Bioactivity is generally greatly lowered in fatty-acylation.Therefore the new acylation position that can be introduced lysine etc. and there is amino is found Point, and there is acylate enough bioactivity to have very important significance for exploitation long-acting polypeptides.

It is demonstrated experimentally that the C-terminal of B1, B28, A14, A15, A22, B chain corresponding to IGF-1 extends to B31, B32, single-stranded Multiple sites of IGF-1 analog C peptide, all remain comparable insulin receptor activity after modification.It is real due to being combined in receptor Albumen non-specific binding is reduced in testing using bovine albumin, but the fatty acid on acyl polypeptide can be in conjunction with albumin (this is also the mechanism of fatty acid prolonging action time), to reduce the effective concentration of polypeptide in an experiment, therefore acylation is more The Receptor-Binding Data of peptide seems lower.But Past 30 Years a large amount of animal experiments show that, the external activity of insulin analog There is no specific corresponding relationship with activity in vivo.External activity be substantially less than the analog of actrapid monotard in vivo with actrapid monotard Activity substantially quite (referring to " the In vitro and in vivo potency of insulin analogs such as Volund designed for clinical use",Diabetic Medicine,1991,8:839-47；" the Equivalent such as Ribel 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 table The internal substantial activity of bright Pegylation and fatty-acylation polypeptide is better than insulin control.

The bioactivity result of the compound based on IGF-1 of table four: PEG and fatty acid modifying

The data that the compound of the present invention has prolongation of effect are separately provided in the application.

Fig. 1 show mouse subcutaneous injection physiological saline, actrapid monotard and 3 kinds of various doses III -1 after blood glucose at any time The average value of variation.Under Isodose, polyethyleneglycol modified polypeptide action time is considerably longer than actrapid monotard, can achieve 24 hours or even longer time.And polyethyleneglycol modified polypeptide hypoglycemic effect is gentle, even if being increased to 3 multiple doses, Blood glucose low ebb is not caused.Therefore, III -1 actrapid monotard is better than in terms of control blood glucose and safety.

Fig. 2 shows mouse subcutaneous injection physiological saline, actrapid monotard and III -12 (be 80 nanomoles/kilogram) blood afterwards The average value that sugar changes over time.Under Isodose, III -12 onset time is suitable with actrapid monotard, but upon administration 8 Hour still has apparent inhibition hypoglycemic effect, at this moment insulin has lost inhibition hypoglycemic effect.Actrapid monotard is testing In show the hypoglycemic curve of typical V-shape.The shortcomings that this hypoglycemic curve, is that the decline of initial stage blood glucose is too fast, is easy to make At hypoglycemia, and the later period is unable to control blood glucose.And III -12 shows the hypoglycemic curve of L-type, glycemic control is steady and holds Long, effect is better than actrapid monotard.

Fig. 3 is that blood glucose is at any time after mouse subcutaneous injection physiological saline, insulin detemir and III-7 compound of the invention Changing value.Insulin detemir is one of only two kinds of Recent Development of Long-acting Insulin Analogs drugs on current international market, in human body Half-life period is 14 hours, usually one day needle when use.III-7 only uses the dosage of insulin detemir 1/3, it will be able to reach Longer action time and more stably blood sugar decreasing effect.

Three above can improve it is demonstrated experimentally that with the IGF-1 analog in polyethylene glycol or the fatty acid modifying present invention The therapeutic effect and pharmacokinetic characteristic of IGF-1 analog.

Sequence table

<110>like moral Dean (Beijing) Bioisystech Co., Ltd

<120>compound with hypoglycemic effect, composition and application thereof

<130>DP1F181973ZX- division

<150> CN201110421619.6

<151> 2011-12-15

<160> 164

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> PRT

<213>people

<400> 1

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Met Tyr Cys Ala

20

<210> 2

<211> 29

<212> PRT

<213>people

<400> 2

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 3

<211> 29

<212> PRT

<213>artificial sequence

<400> 3

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 4

<211> 24

<212> PRT

<213>artificial sequence

<400> 4

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr

20

<210> 5

<211> 24

<212> PRT

<213>artificial sequence

<400> 5

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Trp Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr

20

<210> 6

<211> 24

<212> PRT

<213>artificial sequence

<400> 6

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Trp Trp

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr

20

<210> 7

<211> 21

<212> PRT

<213>artificial sequence

<400> 7

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Met Tyr Cys Asn

20

<210> 8

<211> 21

<212> PRT

<213>artificial sequence

<400> 8

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 9

<211> 21

<212> PRT

<213>artificial sequence

<400> 9

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Thr Tyr Cys Ala

20

<210> 10

<211> 21

<212> PRT

<213>artificial sequence

<400> 10

Gly Ile Val Asp Glu Cys Cys Arg Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 11

<211> 21

<212> PRT

<213>artificial sequence

<400> 11

Gly Ile Val Asp Glu Cys Cys His Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 12

<211> 21

<212> PRT

<213>artificial sequence

<400> 12

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 13

<211> 21

<212> PRT

<213>artificial sequence

<400> 13

Gly Ile Val Asp Asn Cys Cys Phe Arg Ser Cys Asp Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 14

<211> 21

<212> PRT

<213>artificial sequence

<400> 14

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 15

<211> 21

<212> PRT

<213>artificial sequence

<400> 15

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asn Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 16

<211> 21

<212> PRT

<213>artificial sequence

<400> 16

Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Gln Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 17

<211> 21

<212> PRT

<213>artificial sequence

<400> 17

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 18

<211> 29

<212> PRT

<213>artificial sequence

<400> 18

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 19

<211> 22

<212> PRT

<213>artificial sequence

<400> 19

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala Lys

20

<210> 20

<211> 23

<212> PRT

<213>artificial sequence

<400> 20

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala Arg Lys

20

<210> 21

<211> 25

<212> PRT

<213>artificial sequence

<400> 21

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ala

20 25

<210> 22

<211> 29

<212> PRT

<213>artificial sequence

<400> 22

Gly Pro Glu His Leu Cys Gly Ala Arg Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 23

<211> 29

<212> PRT

<213>artificial sequence

<400> 23

Gly Pro Glu His Leu Cys Gly Ala Phe Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 24

<211> 29

<212> PRT

<213>artificial sequence

<400> 24

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Glu Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 25

<211> 30

<212> PRT

<213>artificial sequence

<400> 25

Phe Val Asn Gln His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe

1 5 10 15

Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25 30

<210> 26

<211> 21

<212> PRT

<213>artificial sequence

<400> 26

Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Ala

20

<210> 27

<211> 29

<212> PRT

<213>artificial sequence

<400> 27

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Ala Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 28

<211> 29

<212> PRT

<213>artificial sequence

<400> 28

Gly Pro Glu His Leu Cys Gly Ala Gln Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 29

<211> 62

<212> PRT

<213>artificial sequence

<400> 29

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

35 40 45

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala

50 55 60

<210> 30

<211> 62

<212> PRT

<213>artificial sequence

<400> 30

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 31

<211> 62

<212> PRT

<213>artificial sequence

<400> 31

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Ala Ala Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 32

<211> 61

<212> PRT

<213>artificial sequence

<400> 32

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Ala Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe

35 40 45

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 33

<211> 60

<212> PRT

<213>artificial sequence

<400> 33

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg

35 40 45

Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 34

<211> 59

<212> PRT

<213>artificial sequence

<400> 34

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Arg Arg Ala Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 35

<211> 59

<212> PRT

<213>artificial sequence

<400> 35

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Arg Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 36

<211> 59

<212> PRT

<213>artificial sequence

<400> 36

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ala Ser Ser Arg Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 37

<211> 59

<212> PRT

<213>artificial sequence

<400> 37

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ala Ser Arg Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 38

<211> 58

<212> PRT

<213>artificial sequence

<400> 38

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ala Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 39

<211> 58

<212> PRT

<213>artificial sequence

<400> 39

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Gly Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 40

<211> 56

<212> PRT

<213>artificial sequence

<400> 40

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 41

<211> 57

<212> PRT

<213>artificial sequence

<400> 41

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 42

<211> 58

<212> PRT

<213>artificial sequence

<400> 42

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Gly Gly

20 25 30

Ser Gly Gly Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 43

<211> 58

<212> PRT

<213>artificial sequence

<400> 43

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Gly Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 44

<211> 59

<212> PRT

<213>artificial sequence

<400> 44

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Gly Ser

20 25 30

Gly Gly Ser Gly Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 45

<211> 60

<212> PRT

<213>artificial sequence

<400> 45

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Ser Ser Ser Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg

35 40 45

Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 46

<211> 57

<212> PRT

<213>artificial sequence

<400> 46

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Ser Gly Ser Ser Ser Ala Ala

20 25 30

Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 47

<211> 57

<212> PRT

<213>artificial sequence

<400> 47

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Gly Ser Gly Ser Ser Ser Ala

20 25 30

Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 48

<211> 57

<212> PRT

<213>artificial sequence

<400> 48

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Gly Ser Gly Ser Ser Ser

20 25 30

Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 49

<211> 57

<212> PRT

<213>artificial sequence

<400> 49

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Gly Ser Gly Ser Ser

20 25 30

Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 50

<211> 56

<212> PRT

<213>artificial sequence

<400> 50

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Gly Gly Gly Gly Ala

20 25 30

Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 51

<211> 62

<212> PRT

<213>artificial sequence

<400> 51

Phe Val Asn Gln His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe

1 5 10 15

Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser

20 25 30

Gly Ser Ser Ser Ala Ala Arg Gly Ile Val Asp Gln Cys Cys Phe Arg

35 40 45

Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala Arg Lys

50 55 60

<210> 52

<211> 59

<212> PRT

<213>artificial sequence

<400> 52

Phe Val Asn Gln His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe

1 5 10 15

Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser

20 25 30

Gly Ser Ser Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 53

<211> 63

<212> PRT

<213>artificial sequence

<400> 53

Phe Val Asn Gln His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe

1 5 10 15

Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser

20 25 30

Gly Ser Ser Ser Ala Ala Ala Pro Gln Thr Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 54

<211> 55

<212> PRT

<213>artificial sequence

<400> 54

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Ser Gly Ser Ser Ala Ala Pro

20 25 30

Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg

35 40 45

Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 55

<211> 55

<212> PRT

<213>artificial sequence

<400> 55

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Arg Arg Ala Pro

20 25 30

Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg

35 40 45

Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 56

<211> 57

<212> PRT

<213>artificial sequence

<400> 56

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Gly Ser Gly Ser Ser

20 25 30

Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 57

<211> 57

<212> PRT

<213>artificial sequence

<400> 57

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Gly Ser Gly Ser Ser Ser

20 25 30

Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 58

<211> 57

<212> PRT

<213>artificial sequence

<400> 58

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Gly Ser Gly Ser Ser Ala

20 25 30

Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 59

<211> 56

<212> PRT

<213>artificial sequence

<400> 59

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Gly Ser Gly Ser Ser Ser Arg

20 25 30

Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 60

<211> 56

<212> PRT

<213>artificial sequence

<400> 60

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Gly Ser Gly Ser Ser Ser

20 25 30

Arg Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 61

<211> 57

<212> PRT

<213>artificial sequence

<400> 61

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Asp Ser Ser Ser Ser Arg

20 25 30

Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 62

<211> 68

<212> PRT

<213>artificial sequence

<400> 62

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Pro Ser Ala

20 25 30

Pro Ser Pro Ser Arg Ala Pro Gly Pro Ser Pro Ala Pro Gln Arg Gly

35 40 45

Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu

50 55 60

Asn Tyr Cys Ala

65

<210> 63

<211> 62

<212> PRT

<213>artificial sequence

<400> 63

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Pro Gly Gly Ser Ser Gly Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 64

<211> 62

<212> PRT

<213>artificial sequence

<400> 64

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Gly Ser Ser Gly Pro Gly Ser Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 65

<211> 62

<212> PRT

<213>artificial sequence

<400> 65

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Gly Ser Gly Ser Ser Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 66

<211> 63

<212> PRT

<213>artificial sequence

<400> 66

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Gly Ala Pro Ser Arg Ser Gly Ser Ser Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 67

<211> 62

<212> PRT

<213>artificial sequence

<400> 67

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Pro

20 25 30

Ala Gly Ser Pro Thr Ser Thr Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 68

<211> 62

<212> PRT

<213>artificial sequence

<400> 68

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Pro Ala Thr Ser Gly Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 69

<211> 62

<212> PRT

<213>artificial sequence

<400> 69

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Ser Ser Ala Thr Pro Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 70

<211> 62

<212> PRT

<213>artificial sequence

<400> 70

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Ser Gly Ser Ser Ser Arg Ala

20 25 30

Pro Pro Ser Ala Pro Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 71

<211> 63

<212> PRT

<213>artificial sequence

<400> 71

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Ser Glu Ser Pro Ser Gly Ala Pro Gln Thr Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 72

<211> 63

<212> PRT

<213>artificial sequence

<400> 72

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Thr Pro Ala Ser Gly Ser Ala Pro Gly Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 73

<211> 63

<212> PRT

<213>artificial sequence

<400> 73

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Pro Ser Gly Gly Ser Ser Ala Pro Gln Thr Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 74

<211> 62

<212> PRT

<213>artificial sequence

<400> 74

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Thr

20 25 30

Ser Ser Thr Ala Arg Ser Pro Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 75

<211> 62

<212> PRT

<213>artificial sequence

<400> 75

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Pro Ser Gly Thr Ala Ser Pro Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 76

<211> 61

<212> PRT

<213>artificial sequence

<400> 76

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Thr Pro Ser Gly Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe

35 40 45

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 77

<211> 64

<212> PRT

<213>artificial sequence

<400> 77

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Pro Pro Pro

20 25 30

Ser Ala Pro Ser Pro Ser Arg Ala Pro Gln Arg Gly Ile Val Glu Gln

35 40 45

Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

50 55 60

<210> 78

<211> 62

<212> PRT

<213>artificial sequence

<400> 78

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Pro Gly Thr Ser Ser Thr Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 79

<211> 62

<212> PRT

<213>artificial sequence

<400> 79

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Pro

20 25 30

Ala Gly Ser Pro Thr Ser Thr Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 80

<211> 62

<212> PRT

<213>artificial sequence

<400> 80

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Pro Ser Ser Ala Thr Pro Ala Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 81

<211> 62

<212> PRT

<213>artificial sequence

<400> 81

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Pro Ser Thr Arg Ser Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 82

<211> 64

<212> PRT

<213>artificial sequence

<400> 82

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Glu

20 25 30

Thr Pro Ser Gly Pro Ser Ser Ala Pro Gln Thr Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 83

<211> 77

<212> PRT

<213>artificial sequence

<400> 83

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Ser Ser Ser Arg Ala Pro Pro Pro Ser Ala Pro Ser Pro Ser Arg Ala

35 40 45

Pro Gly Pro Ser Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe

50 55 60

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

65 70 75

<210> 84

<211> 62

<212> PRT

<213>artificial sequence

<400> 84

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Ser Pro Ser Thr Ser Arg Pro Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 85

<211> 63

<212> PRT

<213>artificial sequence

<400> 85

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Ser Gly Ser Ser Gly Ser Pro Ser Gly Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 86

<211> 62

<212> PRT

<213>artificial sequence

<400> 86

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Pro Ser Ala Ser Thr Gly Thr Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 87

<211> 69

<212> PRT

<213>artificial sequence

<400> 87

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Ala Gly Ser Ser Ser Ala Pro

20 25 30

Ser Ala Pro Ser Pro Ser Arg Ala Pro Gly Pro Ser Ala Pro Gln Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 88

<211> 64

<212> PRT

<213>artificial sequence

<400> 88

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Pro

20 25 30

Ser Ser Pro Thr Arg Gly Ser Ala Pro Gln Thr Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 89

<211> 62

<212> PRT

<213>artificial sequence

<400> 89

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Thr Ser Ser Arg Gly Ala Pro Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 90

<211> 62

<212> PRT

<213>artificial sequence

<400> 90

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Ser

20 25 30

Gly Thr Ser Thr Ser Ala Pro Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 91

<211> 61

<212> PRT

<213>artificial sequence

<400> 91

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Pro Ser Ala

20 25 30

Pro Ser Pro Ser Arg Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe

35 40 45

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 92

<211> 62

<212> PRT

<213>artificial sequence

<400> 92

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ala Ser Ser Pro Thr Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 93

<211> 62

<212> PRT

<213>artificial sequence

<400> 93

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Pro Ala Thr Ser Ala Thr Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 94

<211> 71

<212> PRT

<213>artificial sequence

<400> 94

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Ala Gly Ser Ser Ser Ala Pro

20 25 30

Pro Pro Ser Ala Pro Ser Pro Ser Arg Ala Pro Gly Pro Ser Ala Pro

35 40 45

Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg

50 55 60

Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 95

<211> 62

<212> PRT

<213>artificial sequence

<400> 95

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Thr Ser Pro Ser Arg Pro Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 96

<211> 62

<212> PRT

<213>artificial sequence

<400> 96

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Thr

20 25 30

Ala Gly Ser Arg Thr Ser Thr Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 97

<211> 62

<212> PRT

<213>artificial sequence

<400> 97

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Thr

20 25 30

Ala Gly Ser Arg Thr Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 98

<211> 62

<212> PRT

<213>artificial sequence

<400> 98

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Thr Ala Thr Ser Gly Ser Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 99

<211> 62

<212> PRT

<213>artificial sequence

<400> 99

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Ser Ser Ala Thr Ser Ala Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 100

<211> 62

<212> PRT

<213>artificial sequence

<400> 100

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Ser Ala Thr Arg Gly Ser Ala Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 101

<211> 62

<212> PRT

<213>artificial sequence

<400> 101

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Ser Arg Ser Pro Ser Gly Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 102

<211> 69

<212> PRT

<213>artificial sequence

<400> 102

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Pro Pro Pro

20 25 30

Ser Ala Pro Ser Pro Ser Arg Ala Pro Gly Pro Ser Ala Pro Gln Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 103

<211> 63

<212> PRT

<213>artificial sequence

<400> 103

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Ser

20 25 30

Pro Ser Gly Arg Ser Ser Ser Pro Gly Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 104

<211> 74

<212> PRT

<213>artificial sequence

<400> 104

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Pro

20 25 30

Ala Gly Ser Pro Ser Ser Ser Ala Gly Ser Ser Ala Ser Ala Ser Pro

35 40 45

Ala Ser Pro Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

50 55 60

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 105

<211> 86

<212> PRT

<213>artificial sequence

<400> 105

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Pro

20 25 30

Ala Gly Ser Pro Ser Ser Ser Ala Gly Ser Ser Ala Ser Ala Ser Pro

35 40 45

Ala Ser Gly Pro Gly Ser Ser Ser Ala Pro Ser Ala Gly Ser Pro Gly

50 55 60

Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg

65 70 75 80

Leu Glu Asn Tyr Cys Ala

85

<210> 106

<211> 68

<212> PRT

<213>artificial sequence

<400> 106

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Arg Arg Glu

20 25 30

Ala Glu Asp Gly Gly Gly Pro Gly Ala Gly Ser Ser Gln Arg Lys Gly

35 40 45

Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu

50 55 60

Asn Tyr Cys Ala

65

<210> 107

<211> 64

<212> PRT

<213>artificial sequence

<400> 107

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Arg Arg Gly

20 25 30

Gly Gly Pro Gly Ala Gly Ser Ser Gln Arg Lys Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 108

<211> 57

<212> PRT

<213>artificial sequence

<400> 108

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Gly Gly Gly Pro Gly Ala Gly Ser

20 25 30

Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 109

<211> 61

<212> PRT

<213>artificial sequence

<400> 109

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Arg Gly Gly Gly Pro

20 25 30

Gly Ala Gly Ser Ser Gln Arg Lys Gly Ile Val Asp Gln Cys Cys Phe

35 40 45

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 110

<211> 67

<212> PRT

<213>artificial sequence

<400> 110

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Pro Pro Ser

20 25 30

Ala Pro Ser Pro Ser Arg Ala Pro Gly Pro Ser Pro Gln Arg Gly Ile

35 40 45

Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn

50 55 60

Tyr Cys Ala

65

<210> 111

<211> 69

<212> PRT

<213>artificial sequence

<400> 111

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Ser Ala Ala

20 25 30

Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ala Ser Ala Gly Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 112

<211> 66

<212> PRT

<213>artificial sequence

<400> 112

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser Pro Gln Thr Gly Ile Val

35 40 45

Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr

50 55 60

Cys Ala

65

<210> 113

<211> 69

<212> PRT

<213>artificial sequence

<400> 113

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Ser Pro Ala Ala Pro Ala Ser Pro Ala Pro Ala Pro Ser Ala Gly Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 114

<211> 62

<212> PRT

<213>artificial sequence

<400> 114

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Ser Pro Ser

20 25 30

Arg Ser Pro Gly Pro Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 115

<211> 59

<212> PRT

<213>artificial sequence

<400> 115

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Ser Ala Pro

20 25 30

Ser Pro Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 116

<211> 63

<212> PRT

<213>artificial sequence

<400> 116

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Ala Ser Ala

20 25 30

Ala Ser Ala Ser Ser Ser Ala Ser Gly Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 117

<211> 59

<212> PRT

<213>artificial sequence

<400> 117

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Arg Ala Pro Pro Ser

20 25 30

Ala Pro Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 118

<211> 62

<212> PRT

<213>artificial sequence

<400> 118

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 119

<211> 56

<212> PRT

<213>artificial sequence

<400> 119

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Gly Ala Pro Pro

20 25 30

Pro Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 120

<211> 59

<212> PRT

<213>artificial sequence

<400> 120

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Ser Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 121

<211> 59

<212> PRT

<213>artificial sequence

<400> 121

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser

35 40 45

Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 122

<211> 64

<212> PRT

<213>artificial sequence

<400> 122

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Pro Gln Thr Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 123

<211> 63

<212> PRT

<213>artificial sequence

<400> 123

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Pro Pro Ser

20 25 30

Ala Pro Ser Pro Ser Arg Ala Pro Gln Thr Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 124

<211> 65

<212> PRT

<213>artificial sequence

<400> 124

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Pro Ser Ser Gly Ala Pro Pro Pro Ser Pro Gln Thr Gly Ile Val Asp

35 40 45

Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys

50 55 60

Ala

65

<210> 125

<211> 64

<212> PRT

<213>artificial sequence

<400> 125

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Gly Pro Ser Ser Gly Ala Pro Pro Pro Gln Thr Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 126

<211> 73

<212> PRT

<213>artificial sequence

<400> 126

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Pro Ser Pro Ser Arg Pro Gly Pro

35 40 45

Ser Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

50 55 60

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 127

<211> 58

<212> PRT

<213>artificial sequence

<400> 127

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ala Ser Ser Ala Ser Ser

20 25 30

Ser Ser Ala Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 128

<211> 64

<212> PRT

<213>artificial sequence

<400> 128

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ala Ser Ser Ser Ala Ala

20 25 30

Ser Ser Ser Ala Ser Ser Ser Ala Ser Gly Arg Gly Ile Val Asp Gln

35 40 45

Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 129

<211> 65

<212> PRT

<213>artificial sequence

<400> 129

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Gly Ala Pro Pro Pro

20 25 30

Ser Pro Ser Arg Ala Pro Gly Pro Ser Pro Gln Arg Gly Ile Val Asp

35 40 45

Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys

50 55 60

Ala

65

<210> 130

<211> 57

<212> PRT

<213>artificial sequence

<400> 130

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Ala Ala Ser Ser

20 25 30

Ala Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 131

<211> 65

<212> PRT

<213>artificial sequence

<400> 131

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Ser Ala Ser

20 25 30

Ala Ser Ala Ser Ala Ser Ser Ala Ser Ser Gly Arg Gly Ile Val Asp

35 40 45

Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys

50 55 60

Ala

65

<210> 132

<211> 67

<212> PRT

<213>artificial sequence

<400> 132

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Ser Ala Ser

20 25 30

Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala Ser Gly Ile

35 40 45

Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn

50 55 60

Tyr Cys Ala

65

<210> 133

<211> 72

<212> PRT

<213>artificial sequence

<400> 133

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Ser

20 25 30

Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala Ser Pro Ser

35 40 45

Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

50 55 60

Arg Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 134

<211> 65

<212> PRT

<213>artificial sequence

<400> 134

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Pro Pro Ala

20 25 30

Ser Pro Ser Pro Ser Arg Ala Pro Gly Pro Gln Arg Gly Ile Val Asp

35 40 45

Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys

50 55 60

Ala

65

<210> 135

<211> 65

<212> PRT

<213>artificial sequence

<400> 135

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Ser Ala Ser

20 25 30

Ala Ser Ala Ser Ala Ser Ala Ser Ser Ala Gly Arg Gly Ile Val Asp

35 40 45

Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys

50 55 60

Ala

65

<210> 136

<211> 68

<212> PRT

<213>artificial sequence

<400> 136

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ala Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro Ser Ala Gly Arg Gly

35 40 45

Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu

50 55 60

Asn Tyr Cys Ala

65

<210> 137

<211> 62

<212> PRT

<213>artificial sequence

<400> 137

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Pro Ser Ala Ser Pro Ser

20 25 30

Ser Pro Ala Ser Pro Ser Ser Gly Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 138

<211> 69

<212> PRT

<213>artificial sequence

<400> 138

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Pro

20 25 30

Ala Ser Pro Ala Pro Ser Ala Pro Ala Pro Ala Ala Pro Ser Gly Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 139

<211> 74

<212> PRT

<213>artificial sequence

<400> 139

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Pro Ser

20 25 30

Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala Ser Pro Ser

35 40 45

Ser Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

50 55 60

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 140

<211> 61

<212> PRT

<213>artificial sequence

<400> 140

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ala Ser Ser Ala Ser Ser

20 25 30

Ser Ser Ser Ala Ser Ala Gly Arg Gly Ile Val Asp Gln Cys Cys Phe

35 40 45

Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 141

<211> 71

<212> PRT

<213>artificial sequence

<400> 141

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Ser Ala Pro

20 25 30

Ser Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Ala Ser Pro Ser

35 40 45

Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg

50 55 60

Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 142

<211> 62

<212> PRT

<213>artificial sequence

<400> 142

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ala Pro Pro Pro Ser

20 25 30

Ala Pro Ser Pro Ser Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys

35 40 45

Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 143

<211> 69

<212> PRT

<213>artificial sequence

<400> 143

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Ser

20 25 30

Ser Pro Ser Pro Ser Ala Pro Ser Ser Pro Ser Pro Ala Ser Gly Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 144

<211> 63

<212> PRT

<213>artificial sequence

<400> 144

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Pro Ser Ala Pro Ser Pro

20 25 30

Ser Ser Pro Ala Ser Pro Ser Ser Gly Arg Gly Ile Val Asp Gln Cys

35 40 45

Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55 60

<210> 145

<211> 72

<212> PRT

<213>artificial sequence

<400> 145

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ala Gly

20 25 30

Pro Ala Ala Pro Ser Ala Pro Pro Ala Ala Ser Pro Ala Ala Pro Ser

35 40 45

Ala Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

50 55 60

Arg Arg Leu Glu Asn Tyr Cys Ala

65 70

<210> 146

<211> 69

<212> PRT

<213>artificial sequence

<400> 146

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Ser Ser Ser Ser Pro Ser Ala Pro

20 25 30

Ser Pro Ser Ser Pro Ala Ser Pro Ser Pro Ser Ser Ala Pro Gln Arg

35 40 45

Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg Leu

50 55 60

Glu Asn Tyr Cys Ala

65

<210> 147

<211> 56

<212> PRT

<213>artificial sequence

<400> 147

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 148

<211> 58

<212> PRT

<213>artificial sequence

<400> 148

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Ala Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 149

<211> 58

<212> PRT

<213>artificial sequence

<400> 149

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ser Ser Ser Gly Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys

35 40 45

Ser Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 150

<211> 57

<212> PRT

<213>artificial sequence

<400> 150

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Ser Gly

20 25 30

Ala Pro Gln Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser

35 40 45

Leu Arg Arg Leu Glu Asn Tyr Cys Ala

50 55

<210> 151

<211> 22

<212> PRT

<213>artificial sequence

<400> 151

Lys Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg

1 5 10 15

Leu Glu Asn Tyr Cys Ala

20

<210> 152

<211> 29

<212> PRT

<213>artificial sequence

<400> 152

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr

20 25

<210> 153

<211> 22

<212> PRT

<213>artificial sequence

<400> 153

Arg Gly Ile Val Asp Gln Cys Cys Phe Arg Ser Cys Ser Leu Arg Arg

1 5 10 15

Leu Glu Asn Tyr Cys Ala

20

<210> 154

<211> 22

<212> PRT

<213>artificial sequence

<400> 154

Lys Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg Arg

1 5 10 15

Leu Glu Asn Tyr Cys Asn

20

<210> 155

<211> 29

<212> PRT

<213>artificial sequence

<400> 155

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Lys Thr

20 25

<210> 156

<211> 22

<212> PRT

<213>artificial sequence

<400> 156

Arg Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg Arg

1 5 10 15

Leu Glu Asn Tyr Cys Asn

20

<210> 157

<211> 23

<212> PRT

<213>artificial sequence

<400> 157

Arg Arg Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg

1 5 10 15

Arg Leu Glu Asn Tyr Cys Asn

20

<210> 158

<211> 23

<212> PRT

<213>artificial sequence

<400> 158

Lys Lys Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg

1 5 10 15

Arg Leu Glu Asn Tyr Cys Asn

20

<210> 159

<211> 21

<212> PRT

<213>artificial sequence

<400> 159

Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu Arg Arg Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210> 160

<211> 30

<212> PRT

<213>artificial sequence

<400> 160

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Lys Thr Glu

20 25 30

<210> 161

<211> 56

<212> PRT

<213>artificial sequence

<400> 161

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Gly Gly Gly Gly Ala

20 25 30

Pro Gln Thr Gly Ile Val Asp Gln Cys Cys His Arg Ser Cys Ser Leu

35 40 45

Arg Arg Leu Glu Asn Tyr Cys Asn

50 55

<210> 162

<211> 62

<212> PRT

<213>artificial sequence

<400> 162

Gly Pro Glu His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Lys Thr Gly Ser Gly

20 25 30

Ser Ser Ser Ala Ala Ala Pro Gln Thr Gly Ile Val Asp Gln Cys Cys

35 40 45

His Arg Ser Cys Ser Leu Arg Arg Leu Glu Asn Tyr Cys Asn

50 55 60

<210> 163

<211> 30

<212> PRT

<213>artificial sequence

<400> 163

Phe Val Asn Gln His Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe

1 5 10 15

Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Pro Thr

20 25 30

<210> 164

<211> 29

<212> PRT

<213>artificial sequence

<400> 164

Gly Pro Glu Thr Leu Cys Gly Ala His Leu Val Asp Ala Leu Phe Phe

1 5 10 15

Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Pro Pro Thr

20 25

Claims (22)

1. a kind of compound with hypoglycemic effect, which is characterized in that the compound includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows: X_-1X₀GIVDX₅C_[3]C_[4]X₈X₉X₁₀C_[5]X₁₂LRRLEX₁₈YC_[6]X₂₁X₂₂,

B chain amino acid sequence are as follows:

X_23-26X₂₇LC_[1]GAX₃₂LVDALX₃₈X₃₉VC_[2]GDX₄₄GFX₄₇X₄₈X₄₉X₅₀X₅₁X₅₂X₅₃, wherein

X_-1It is lysine, arginine or missing；X₀It is lysine, arginine or missing；X₅It is asparagine, glutamine or silk Propylhomoserin；X₈It is histidine, arginine, phenylalanine or threonine；X₉It is arginine or serine；X₁₀It is serine or different bright ammonia Acid；X₁₂It is serine, glutamine or asparagine；X₁₈It is asparagine, methionine or threonine；X₂₁Be asparagine, Alanine or glycine；X₂₂It is lysine, arginine-lysine dipeptides or missing；X_23-26It is phenylalanine-valine-asparagus fern Amide-glutamine tetrapeptide or glycine-proline-glutamic acid, valine-asparagine-glutamin tripeptides or dried meat ammonia Acid-glutamic acid, asparagine-glutamin dipeptides or glutamic acid, glutamine, lysine or arginine, or with lysine Or arginine replaces above-mentioned two, three, the sequence in tetrapeptide array after any one amino acid residue, or missing；X₂₇It is histidine Or threonine；X₃₂It is histidine, glutamic acid, glutamine, arginine or phenylalanine；X₃₈It is phenylalanine or tryptophan；X₃₉ It is phenylalanine or tryptophan；X₄₄It is arginine, glutamic acid, aspartic acid or alanine；X₄₇Be phenylalanine, tyrosine or Histidine；X₄₈It is-NH₂、dA-NH₂, phenylalanine or tyrosine or missing；X₄₉It is asparagine, aspartic acid, glutamic acid, Soviet Union Propylhomoserin or missing；X₅₀It is lysine, proline, arginine, aspartic acid, glutamic acid or missing；X₅₁Be proline, lysine, Arginine, aspartic acid, glutamic acid or missing；X₅₂It is threonine or missing；X₅₃It is glutamic acid, aspartic acid, glutamic acid-paddy Propylhomoserin, Asp-Asp dipeptides or missing；

In the compound, [1]-[6] indicate the number of cysteine；3 pairs are formed by 6 cysteines in the compound Disulfide bond, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there are a pair of of intrachain disulfide bond, three pairs of disulfide bond in A chain Specific location be: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond.

2. compound according to claim 1, which is characterized in that the compound includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows: GIVDX₅C_[3]C_[4]X₈RSC_[5]X₁₂LRRLEX₁₈YC_[6]X₂₁X₂₂,

B chain amino acid sequence are as follows:

X_23-26X₂₇LC_[1]GAX₃₂LVDALX₃₈X₃₉VC_[2]GDX₄₄GFYX₄₈X₄₉X₅₀X₅₁X₅₂, wherein

X₅It is asparagine, glutamine or serine；X₈It is histidine, arginine or phenylalanine；X₁₂It is serine, paddy ammonia Amide or asparagine；X₁₈It is asparagine, methionine or threonine；X₂₁It is asparagine, alanine or glycine；X₂₂ It is lysine, arginine-lysine dipeptides or missing；X_23-26It is glycine-proline-glutamic acid tripeptides or phenylalanine-figured silk fabrics Propylhomoserin-asparagine-glutamin tetrapeptide；X₂₇It is histidine or threonine；X₃₂It is histidine, glutamic acid, glutamine, essence Propylhomoserin or phenylalanine；X₃₈It is phenylalanine or tryptophan；X₃₉It is phenylalanine or tryptophan；X₄₄Be arginine, glutamic acid, Aspartic acid or alanine；X₄₈It is-NH₂、dA-NH₂Or phenylalanine；X₄₉It is asparagine or missing；X₅₀It is lysine, dried meat ammonia Acid or missing；X₅₁It is proline, lysine or missing；X₅₂It is threonine or missing.

3. compound according to claim 2, which is characterized in that the sequence of the B chain is GPEX₂₇LCGAX₃₂LVDALX_{3 8}X₃₉VCGDX₄₄GFY-NH₂；GPEX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFYFNKPT；GPEX₂₇LCGAX₃₂LVDALX₃₈X₃₉V CGDX₄₄GFYdA-NH₂；Or FVNQX₂₇LCGAX₃₂LVDALX₃₈X₃₉VCGDX₄₄GFYFNKPT, wherein X₂₇、X₃₂、X₃₈、X₃₉、X₄₄Such as Defined in claim 2.

4. compound according to claim 2, which is characterized in that the A chain-ordering is GIVDX₅CCX₈RSCX₁₂LRRLEX₁₈YCA or GIVDX₅CCX₈RSCX₁₂LRRLEX₁₈YCN, wherein X₅、X₈、X₁₂、X₁₈As right is wanted It asks defined in 2.

5. compound according to claim 1-4, which is characterized in that the compound is selected from following compound:

I-9: wherein A chain-ordering is sequence shown in SEQ ID NO:12；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-10: wherein A chain-ordering is sequence shown in SEQ ID NO:13；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-11: wherein A chain-ordering is sequence shown in SEQ ID NO:14；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-12: wherein A chain-ordering is sequence shown in SEQ ID NO:15；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-13: wherein A chain-ordering is sequence shown in SEQ ID NO:16；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-14: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:3；

I-15: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:18；

I-16: wherein A chain-ordering is sequence shown in SEQ ID NO:19；The sequence of B chain is sequence shown in SEQ ID NO:18；

I-17: wherein A chain-ordering is sequence shown in SEQ ID NO:20；The sequence of B chain is sequence shown in SEQ ID NO:18；

I-18: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is GPETLCGAHLVDALFFVCGDRGFYdA-NH₂；

I-19: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:22；

I-20: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:23；

I-21: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:24；

I-23: wherein A chain-ordering is sequence shown in SEQ ID NO:26；The sequence of B chain is sequence shown in SEQ ID NO:25；

I-24: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:25；

I-25: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:27；

I-26: wherein A chain-ordering is sequence shown in SEQ ID NO:17；The sequence of B chain is sequence shown in SEQ ID NO:28；

I-27: wherein A chain-ordering is sequence shown in SEQ ID NO:151；The sequence of B chain is sequence shown in SEQ ID NO:152；

I-28: wherein A chain-ordering is sequence shown in SEQ ID NO:153；The sequence of B chain is sequence shown in SEQ ID NO:152；

I-29: wherein A chain-ordering is sequence shown in SEQ ID NO:154；The sequence of B chain is sequence shown in SEQ ID NO:155；

I-30: wherein A chain-ordering is sequence shown in SEQ ID NO:156；The sequence of B chain is sequence shown in SEQ ID NO:155；

I-31: wherein A chain-ordering is sequence shown in SEQ ID NO:157；The sequence of B chain is sequence shown in SEQ ID NO:155；

I-32: wherein A chain-ordering is sequence shown in SEQ ID NO:158；The sequence of B chain is sequence shown in SEQ ID NO:155；

I-33: wherein A chain-ordering is sequence shown in SEQ ID NO:159；The sequence of B chain is sequence shown in SEQ ID NO:160；

I-34: wherein A chain-ordering is sequence shown in SEQ ID NO:159；The sequence of B chain is sequence shown in SEQ ID NO:155.

6. a kind of single chain compound with hypoglycemic effect, the compound structure are as follows: X_101aLC_[1] GAX_101bLVDALX_101cX_101dVC_[2]GDRGFX_101eX₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆-C_L-GIVDQC_[3]C_[4]X₁₀₇RSC_[5] SLRRLENYC_[6]X₁₀₈X₁₀₉, wherein

X_101aIt is glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine tetrapeptide or phenylpropyl alcohol Propylhomoserin-valine-asparagine-glutamin-histidine pentapeptide, or above-mentioned tetrapeptide or five are replaced with lysine or arginine Any of glycine-proline-glutamic acid or phenylalanine-valine-asparagine-glutamin in peptide amino acid is residual Sequence after base；X_101bIt is histidine, glutamic acid, glutamine, arginine or phenylalanine；X_101cIt is phenylalanine or color ammonia Acid；X_101dIt is phenylalanine or tryptophan；X_101eIt is phenylalanine, tyrosine or histidine；X₁₀₂It is phenylalanine or missing； X₁₀₃It is asparagine or missing；X₁₀₄It is lysine, proline or missing；X₁₀₅It is proline, lysine or missing；X₁₀₆It is Soviet Union Propylhomoserin or missing；X₁₀₇It is phenylalanine, arginine or histidine；X₁₀₈It is alanine, glycine or asparagine；X₁₀₉It is bad Propylhomoserin, arginine-lysine dipeptides or missing；C_LIt is junction fragment.

7. compound according to claim 6, which is characterized in that the structure of the compound are as follows:

X_101aLC_[1]GAHLVDALFFVC_[2]GDRGFYX₁₀₂X₁₀₃X₁₀₄X₁₀₅X₁₀₆-C_L-GIVDQC_[3]C_[4]FRSC_[5] SLRRLENYC_[6]A X₁₀₉, wherein

X_101aIt is glycine-proline-glutamic acid-threonine tetrapeptide or phenylalanine-valine-asparagine-glutamy Amine-histidine pentapeptide；X₁₀₂It is phenylalanine or missing；X₁₀₃It is asparagine or missing；X₁₀₄It is lysine, proline or scarce It loses；X₁₀₅It is proline, lysine or missing；X₁₀₆It is threonine or missing；X₁₀₉Be lysine, arginine-lysine dipeptides or Missing；C_LIt is junction fragment.

8. compound according to claim 7, which is characterized in that the structure of the compound is:

GPETLCGAHLVDALFFVCGDRGFY-C_L-GIVDQCCFRSCSLRRLENYCA；

GPETLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCA；

FVNQHLCGAHLVDALFFVCGDRGFY-C_L-GIVDQCCFRSCSLRRLENYCA；

FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCA；Or

FVNQHLCGAHLVDALFFVCGDRGFYFNKPT-C_L-GIVDQCCFRSCSLRRLENYCARK。

9. according to the described in any item compounds of claim 6-8, which is characterized in that the junction fragment C_LIt is 6-60 amino The peptide sequence of acid, wherein each amino acid residue is independently selected from glycine, alanine, serine, threonine or proline.

10. compound according to claim 9, which is characterized in that the junction fragment C_LInclude 1 or 1 or more asparagus fern Propylhomoserin, glutamic acid, arginine, lysine, cysteine or asparagine.

11. compound according to claim 10, which is characterized in that the junction fragment C_LIt is the complete of following polypeptide fragment Portion or partial sequence, perhaps have with following polypeptide fragment 1,2 or 3 amino acid residue difference or with following polypeptide fragment There is 1,2,3,4 or 5 repetition sequence of all or part of sequence of 70%, 80%, 90% similar or following polypeptide fragment Column:

(GASPGGSSGS)_nGR, wherein n is 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；GSPAGSPSSSAG SSASASPASGPGSSSAPSAGSPGR；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。

12. the described in any item compounds of according to claim 6 or 7, the compound is selected from SEQ ID NO:30~SEQ ID NO:150, SEQ ID NO:161, SEQ ID NO:162 compound represented.

13. a kind of compound with hypoglycemic effect, the compound includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows:

X₃₉₉X₄₀₀GIVDX₄₀₅C_[3]C_[4]X₄₀₈X₄₀₉X₄₁₀C_[5]X₄₁₂LX₄₁₄X₄₁₅LX₄₁₇X₄₁₈YC_[6]X₄₂₁X₄₂₂,

B chain amino acid sequence are as follows:

X_423-426X₄₂₇LC_[1]GAHLVDALX₄₃₈X₄₃₉VC_[2]GDRGFX₄₄₇X₄₄₈X₄₄₉X₄₅₀X₄₅₁X₄₅₂X₄₅₃X₄₅₄X₄₅₅,

In the compound, [1]-[6] indicate the number of cysteine；The compound forms 3 pair two by 6 cysteines Sulfide linkage, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond in A chain, three pairs of disulfide bond Specific location is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond, wherein

X₃₉₉It is arginine, lysine or missing；X₄₀₀It is arginine, lysine or missing；X₄₀₅It is asparagine, glutamine Or serine；X₄₀₈It is histidine, arginine, phenylalanine, threonine or logical formula (I) structure；X₄₀₉Be arginine, serine or Logical formula (I) structure；X₄₁₀It is histidine, arginine, phenylalanine or logical formula (I) structure；X₄₁₂It is serine, isoleucine or logical Formula (I) structure；X₄₁₄It is arginine or logical formula (I) structure；X₄₁₅It is arginine or logical formula (I) structure；X₄₁₇It is glutamic acid or general formula (I) structure；X₄₁₈It is asparagine or logical formula (I) structure；X₄₂₁It is alanine, glycine or asparagine；X₄₂₂Be lysine, Arginine-lysine dipeptides or missing, or be logical formula (I) structure；Work as X₄₂₂When for dipeptides, one of amino acid is logical formula (I) Structure；X_423-426It is glycine-proline-glutamic acid tripeptides, U_LGlycine-proline-glutamic acid, phenylalanine-valine- Asparagine-glutamin tetrapeptide or U_LPhenylalanine-valine-asparagine-glutamin；X₄₂₇It is histidine or Soviet Union's ammonia Acid；X₄₃₈It is phenylalanine or tryptophan；X₄₃₉It is phenylalanine or tryptophan；X₄₄₇It is phenylalanine, histidine or tyrosine； X₄₄₈It is-NH₂, phenylalanine, tyrosine or missing；X₄₄₉It is asparagine, threonine, glutamic acid, aspartic acid or missing； X₄₅₀It is lysine, arginine, glutamic acid, aspartic acid, proline or missing；X₄₅₁It is proline, lysine, arginine, paddy Propylhomoserin, aspartic acid or missing, or be logical formula (I) structure；X₄₅₂It is threonine, lysine or missing, or is logical formula (I) structure； X₄₅₃It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₄It is glutamic acid, glycine, lysine or scarce It loses, or is logical formula (I) structure；X₄₅₅It is lysine or missing, or is logical formula (I) structure,

The logical formula (I) structure are as follows:

U_LIt is-W-X-Y-Z structure, fatty acid, polyethylene glycol, albumin, L_n-M_LStructure, hydrogen atom or N^α-(N^α-(HOOC(CH₂)_nCO)-γ-Glu)-、N^α-(N^α-(CH₃(CH₂)_nCO)-γ-Glu)-, wherein n is the integer of 8-20, such as 8,10,12,14,16, 20, N^αIt indicates the alpha-amido of amino acid or amino acid residue, or is logical formula (II) structure, the logical formula (II) structure is:

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

14. compound according to claim 13, which is characterized in that the compound includes A chain and B chain, wherein

The amino acid sequence of A chain are as follows:

GIVDQC_[3]C_[4]FRSC_[5]X₄₁₂LX₄₁₄X₄₁₅LX₄₁₇X₄₁₈YC_[6]AX₄₂₂,

B chain amino acid sequence are as follows:

X_423-426X₄₂₇LC_[1]GAHLVDALFFVC_[2]GDRGFYX₄₄₈X₄₄₉X₄₅₀X₄₅₁X₄₅₂X₄₅₃X₄₅₄X₄₅₅,

In the compound, [1]-[6] indicate the number of cysteine；The compound forms 3 pair two by 6 cysteines Sulfide linkage, wherein A chain is connected with B chain by two pairs of interchain disulfide bonds, there is a pair of of intrachain disulfide bond in A chain, three pairs of disulfide bond Specific location is: C_[1]And C_[4]Form disulfide bond, C_[2]And C_[6]Form disulfide bond, C_[3]And C_[5]Form disulfide bond；Wherein,

X₄₁₂For serine or logical formula (I) structure；X₄₁₄For arginine or logical formula (I) structure；X₄₁₅It is arginine or logical formula (I) knot Structure；X₄₁₇For glutamic acid or logical formula (I) structure；X₄₁₈For asparagine or logical formula (I) structure；X₄₂₂Rely for lysine, arginine- Propylhomoserin dipeptides or missing, or be logical formula (I) structure；Work as X₄₂₂When for dipeptides, one of amino acid is logical formula (I) structure； X_423-426It is glycine-proline-glutamic acid tripeptides, U_LGlycine-proline-glutamic acid, phenylalanine-valine-asparagus fern Amide-glutamine tetrapeptide or U_LPhenylalanine-valine-asparagine-glutamin；X₄₂₇It is histidine or threonine； X₄₄₈It is-NH₂, phenylalanine or missing；X₄₄₉It is asparagine or missing；X₄₅₀It is lysine, arginine, glutamic acid, asparagus fern ammonia Acid, proline or missing；X₄₅₁It is proline, lysine or missing, or is logical formula (I) structure；X₄₅₂Be threonine, lysine or Missing, or be logical formula (I) structure；X₄₅₃It is glutamic acid, glycine, lysine or missing, or is logical formula (I) structure；X₄₅₄It is paddy Propylhomoserin, glycine, lysine or missing, or be logical formula (I) structure；X₄₅₅It is lysine or missing, or is logical formula (I) structure, U_L It is as defined in claim 13 with logical formula (I).

15. the described in any item compounds of 3-14 according to claim 1, which is characterized in that the compound is selected from:

III -1: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17；B chain-ordering is G (N^α-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPPT；

III -2: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17；B chain-ordering is GPETLCGAHLVDALFFVCGDRGFYFNPK(N^ε-PEG 20K)；

III -6: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: sequence shown in SEQ ID NO:17；B chain-ordering are as follows: G(N^α-CO(CH₂)₁₄COOH)PETLCGAHLVDALFFVCGDRGFYFNPPT；

III -7: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -8: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₆CO)-γ-Glu)]；

III -9: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₂CO)-γ-Glu)]；

III -10: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK{N^ε-[N^α-(HOOC(CH₂)₁₁NHCO(CH₂)₃CO)-γ-Glu]}；

III -11: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, B chain-ordering are as follows: FVNQHLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu-N-(γ-Glu)]；

III -19: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is GPETLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -21: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: GIVDQCCFRSCSLK [N^ε-(N^α-(HOOC (CH₂)₁₄CO)-γ-Glu)]RLENYCA；B chain-ordering be FVNQHLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 274)；

III -22: duplex structure, including A chain and B chain, wherein A chain-ordering are as follows: GIVDQCCFRSCSLRK [N^ε-(N^α-(HOOC (CH₂)₁₄CO)-γ-Glu)]LENYCA；B chain-ordering be GPETLCGAHLVDALFFVCGDRGFYFNPPT (SEQ ID NO: 275)；

III -23: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCAK [N^ε-(N^α- (HOOC(CH₂)₁₄CO)-γ-Glu)]；B chain-ordering is sequence shown in SEQ ID NO:275；

III -24: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCFRSCSLRRLENYCARK [N^ε-(N^α- (HOOC(CH₂)₁₄CO)-γ-Glu)], B chain-ordering is sequence shown in SEQ ID NO:275；

III -25: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is FVNQHLCGAHLVDALFFVCGDRGFYFNPPTK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -26: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is F VNQHLCGAHLVDALFFVCGDRGFYFNPPTEK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -27: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is F VNQHLCGAHLVDALFFVCGDRGFYFNPPTGEK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -28: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is GPETLCGAHLVDALFFVCGDRGFYFNPK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu-N-(γ-Glu))]；

III -29: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is G [N^α-(N^α-(CH₃(CH₂)₁₄CO)-γ-L-Glu)]PETLCGAHLVDALFFVCGDRGFYFNPPT；

III -30: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is G (N^α-dPEG₁₂Maleimide-albumin) PETLCGAHLVDALFFVCGDRGFYFNKPT；

III -31: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is FVNQHLCGAHLVDALFFVCGDRGFYFNPPK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -32: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is GPETLCGAHLVDALFFVCGDRGFYFNPPTK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -33: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is GPETLCGAHLVDALFFVCGDRGFYFNPPTEK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III -34: duplex structure, including A chain and B chain, wherein A chain-ordering is sequence shown in SEQ ID NO:17, and B chain-ordering is G PETLCGAHLVDALFFVCGDRGFYFNPPTGEK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)]；

III-36: duplex structure, including A chain and B chain, wherein A chain-ordering is GIVDQCCHRSCSLRRLENYCA, and B chain-ordering is GPEHLCGAHLVDALFFVCGDRGFYFNPK[N^ε-CO-(CH₂CH₂O)₅-(CH₂)₂-NH-(N^α-(HOOC(CH₂)₁₆CO)-γ- Glu)], structure is as follows；

N^αIndicate the alpha-amido of amino acid or amino acid residue；N^εIt indicates the epsilon-amino of amino acid or amino acid residue, such as relies ammonia The epsilon-amino of sour side chain.

16. a kind of compound with hypoglycemic effect, the structure of the compound are as follows:

X_201aLC_[1]GAX_201bLVDALX_201cX_201dVC_[2]GDRGFX_201eX₂₀₂X₂₀₃X₂₀₄X₂₀₅X₂₀₆GX₂₀₇X_207aX₂₀₈X₂₀₉X₂₁₀X_{21 1}X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆GIVDQC_[3]C_[4]X₂₁₇RSC_[5]X₂₁₈LX₂₁₉X₂₂₀LX₂₂₁X₂₂₂YC_[6]X₂₂₃X₂₂₄, wherein

X_201aIt is glycine-proline-glutamic acid-threonine, glycine-proline-glutamic acid-histidine, phenylalanine-figured silk fabrics Propylhomoserin-asparagine-glutamin-histidine, U_LGlycine-proline-glutamic acid-threonine, U_LGly-Pro- Glutamic acid-histidine or U_LPhenylalanine-valine-asparagine-glutamin-histidine；X_201bIt is histidine, paddy ammonia Acid, glutamine, arginine or phenylalanine；X_201cIt is phenylalanine or tryptophan；X_201dIt is phenylalanine or tryptophan； X_201eIt is phenylalanine, tyrosine or histidine；X₂₀₂It is phenylalanine, tyrosine or missing；X₂₀₃It is asparagine, Soviet Union's ammonia Acid, aspartic acid, glutamic acid or missing；X₂₀₄It is proline, lysine, arginine, aspartic acid, glutamic acid or missing；X₂₀₅ It is proline, lysine, arginine, aspartic acid, glutamic acid or missing or logical formula (I) structure；X₂₀₆It is threonine, lysine Missing or logical formula (I) structure；X₂₀₇It is serine, alanine, glycine, logical formula (I) structure or missing；X_207aBe serine, Alanine, glycine, logical formula (I) structure or missing；X₂₀₈It is serine, logical formula (I) structure or missing；X₂₀₉It is serine, logical Formula (I) structure or missing；X₂₁₀It is serine, logical formula (I) structure or missing；X₂₁₁It is arginine, alanine, glycine, general formula (I) structure or missing；X₂₁₂It is arginine, alanine, glycine, logical formula (I) structure or missing；X₂₁₃Be alanine, proline, Arginine, glycine, logical formula (I) structure or missing；X₂₁₄It is proline, glutamine, glycine, logical formula (I) structure or scarce It loses；X₂₁₅It is glutamine, threonine, glycine, logical formula (I) structure or missing；X₂₁₆It is threonine, arginine, lysine, leads to Formula (I) structure or missing；X₂₁₇It is phenylalanine, arginine, histidine or logical formula (I) structure；X₂₁₈It is serine, glutamy Amine, asparagine or logical formula (I) structure；X₂₁₉It is arginine or logical formula (I) structure；X₂₂₀It is arginine or logical formula (I) structure； X₂₂₁It is glutamic acid or logical formula (I) structure；X₂₂₂It is asparagine or logical formula (I) structure；X₂₂₃It is alanine, glycine or asparagus fern Amide；X₂₂₄It is lysine, arginine-lysine dipeptides or missing, or is logical formula (I) structure；Work as X₂₂₄When for dipeptides, wherein one A amino acid is logical formula (I) structure, wherein U_LIt is as defined in claim 13 with logical formula (I).

17. compound according to claim 16, which is characterized in that the structure of the compound is: X_201aLC_[1] GAHLVDALFFVC_[2]GDRGFYX₂₀₂X₂₀₃X₂₀₄X₂₀₅X₂₀₆GX₂₀₇GX₂₀₈X₂₀₉X₂₁₀X₂₁₁X₂₁₂X₂₁₃X₂₁₄X₂₁₅X₂₁₆GIVDQC_[3] C_[4]FRSC_[5]X₂₁₈LX₂₁₉X₂₂₀LX₂₂₁X₂₂₂YC_[6]A, wherein

X_201aIt is glycine-proline-glutamic acid-threonine, phenylalanine-valine-asparagine-glutamin-group ammonia Acid, U_LGlycine-proline-glutamic acid-threonine or U_LPhenylalanine-valine-asparagine-glutamin-group ammonia Acid；X₂₀₂It is phenylalanine or missing；X₂₀₃It is asparagine or missing；X₂₀₄It is proline, lysine, arginine, aspartic acid Or missing；X₂₀₅It is proline, lysine or logical formula (I) structure；X₂₀₆It is threonine, lysine or logical formula (I) structure；X₂₀₇It is Serine, alanine or logical formula (I) structure；X₂₀₈It is serine or logical formula (I) structure；X₂₀₉It is serine or logical formula (I) structure； X₂₁₀It is serine or logical formula (I) structure；X₂₁₁It is arginine, alanine, logical formula (I) structure or missing；X₂₁₂It is arginine, third Propylhomoserin, glycine, logical formula (I) structure or missing；X₂₁₃It is alanine, proline, arginine, logical formula (I) structure or missing；X₂₁₄ It is proline, glutamine, logical formula (I) structure or missing；X₂₁₅It is glutamine, threonine, logical formula (I) structure or missing； X₂₁₆It is threonine, arginine, lysine, logical formula (I) structure or missing；X₂₁₈It is serine or logical formula (I) structure；X₂₁₉It is essence Propylhomoserin or logical formula (I) structure；X₂₂₀It is arginine or logical formula (I) structure；X₂₂₁It is glutamic acid or logical formula (I) structure；X₂₂₂It is asparagus fern Amide or logical formula (I) structure, wherein U_LIt is as defined in claim 13 with logical formula (I).

18. the described in any item compounds of 6-17 according to claim 1, which is characterized in that the compound is selected from:

III -3:

GPETLCGAHLVDALFFVCGDRGFYFNPTGK(N^ε-PEG20K)GSSSRRAPQTGIVDQCCFRSCSLRRLENYCA；

III -4:

GPETLCGAHLVDALFFVCGDRGFYFNPPTGK(N^ε-PEG20K)GSSSAAAPQTGIVDQCCFRSCSLRRLENYCA；

III -5:

GPETLCGAHLVDALFFVCGDRGFYFNDPTGK(N^ε-PEG20K)GSSSAAAPQTGIVDQCCFRSCSLRRLENYCA；

III -12:

GPETLCGAHLVDALFFVCGDRGFYFNPTGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] GSSSAAPQTGIVDQCCFRSCSLRRLENYCA；

III -13:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] SSAAPQTGIVDQCCFRSCSLRRLENYCA；

III -14:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA；

III -15:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] APQTGIVDQCCFRSCSLRRLENYCA；

III -16:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA；

III -17:

GPETLCGAHLVDALFFVCGDRGFYGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] AAPQTGIVDQCCFRSCSLRRLENYCA；

III -18:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] SSRGRGIVDQCCFRSCSLRRLENYCA；

III -20:

GPETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSK[N^ε-(N^α-(HOOC(CH₂)₁₄CO)-γ-Glu)] GRGIVDQCCFRSCSLRRLENYCA；

III-35:G(N^α-PEG20K)PETLCGAHLVDALFFVCGDRGFYFNPTGSGSSSAAAPQTGIVDQCCFRSCSLRR LENYCA；

III-37:GPEHLCGAHLVDALFFVCGDRGFYFNPTGK[N^ε-CO-(CH₂CH₂O)₅-(CH₂)₂-NH-(N^α-(HOOC (CH₂)₁₆CO)-γ-Glu)]GSSSAAAPQTGIVDQCCHRSCSLRRLENYCA；

N^αIndicate the alpha-amido of amino acid or amino acid residue；N^εIt indicates the epsilon-amino of amino acid or amino acid residue, such as relies ammonia The epsilon-amino of sour side chain.

19. acceptable load in a kind of pharmaceutical composition, including the described in any item compounds of claim 1-18 and pharmaceutics Body.

20. pharmaceutical composition according to claim 19, further comprise Semilente Insulin or insulin analog and/or Additive.

21. -18 described in any item compounds are in the drug of preparation treatment diabetes or hyperglycemia according to claim 1 Using.

22. the described in any item compositions of 9-20 are in the drug of preparation treatment diabetes or hyperglycemia according to claim 1 Application.