CN101691395A - Method for solid phase synthesis of bitter gourd MC-JJ62 peptide analogs under microwave irradiation and application of bitter gourd MC-JJ62 peptide analogs - Google Patents

Abstract

The invention relates to new bitter gourd MC-JJ62 peptide analogs capable of efficiently regulating blood sugar level, and a method for solid phase synthesis of bitter gourd MC-JJ62 peptide analogs under microwave irradiation. The method comprises the following steps: obtaining the MC-JJ62 peptide analogs having longer pharmacological effect time by modifying 2, 4, 5, 8 or 9 site of natural bitter gourd analogs; efficiently and quickly realizing the chemical synthesis of the MC-JJ62 peptide analogs by adopting a solid phase synthesis method under microwave irradiation; purifying the crude products by using preparation grade highly effective liquid phase; and freeze-drying the purified products to obtain the MC-JJ62 peptide analogs.

Description

Microwave promotes solid phase synthesis balsam pear MC-JJ62 polypeptide analog and application thereof

Technical field

The present invention relates to MC-JJ62 polypeptide analog and microwave thereof and promote solid phase synthesis process.

Background technology

Diabetes are important diseases of current harm humans health, and the whole world has the people of 5%-7% to be subjected to diabetes mellitus approximately at present, is considered to after cardiovascular and cerebrovascular diseases, malignant tumour, cause human wounded or disabled, dead the third-largest killer.

Diabetes are divided into I type and II type, and type i diabetes is a kind of autoimmune disease, and patient's survival needs insulin injection.Type ii diabetes accounts for about 95% of numerous diabetic subject's sums.The reason of type ii diabetes is an islet cells metabolism generation obstacle, hypoinsulinism, perhaps producing insulin resistant, is to be main companion's insulin secretion relative deficiency with the insulin resistant, serves as the metabolic disease that main companion's insulin resistant causes with the insulin secretion relative deficiency perhaps.Hypoinsulinism perhaps produces insulin resistant and all can cause in the blood glucose excessive, and when blood-sugar content surpasses the kidney presentation time, glucose is just transferred in the urine and discharged, thereby produces dextrosuria.

The several drugs of clinical application at present all can not be cured diabetes, mainly is by different mechanism of action, reduces patient's glucose level.The diabetic subject must take medicine throughout one's life, and medicine itself is threatening diabetics's health equally to the toxic side effect of human body.In China, use at present medicine can controlling blood sugar to reach arm's length standard, 20% (the Zou Dajin that is less than diabetic subject's sum, the chief culprit of diabetes B---insulin resistant, Popular Medicine, 2002, (6): 17), directly caused many diabetic subjects' complication to take place early, progress is fast.Obviously, except screening more safely and effectively the newtype drug, seeking other the effective glucose level control of auxiliary treatment means, is crucial for the health level that improves the diabetics equally.

Balsam pear (Momordica charantia) is that a kind of mankind have the edible historical vegetables of long-term safety.Recent two decades comes, people have found many compositions with hypoglycemic activity in the fruit of balsam pear, the extract of some Fructus Momordicae charantiaes, perhaps directly with the tea drink of making after the Fructus Momordicae charantiae drying, recommended the diabetics and used, some balsam pear composition has been applied the patent as the treatment diabetes medicament.But, from scientific and technical literature, introduce and existing patent or product, mainly be based on in the many blood-sugar decreasing actives of balsam pear to certain understanding of principal component, do not recognize, have in the balsam pear composition of hypoglycemic activity known just have multiple, and chemical property has nothing in common with each other, and in addition, it is unknown also having some to have hypoglycemic activeconstituents in the balsam pear.So, existing document, patent or product, its limitation also is tangible.

(CN 98805001, the Orally active composition in the balsam pear, its bioactive peptide, and the purposes of their treatment diabetes for United States Patent (USP) (US 6391854B1) and Chinese patent;＜applicant〉U.S. Calyx Therapeutics Inc) provide a kind of water soluble extract of balsam pear to be called MC6, it contains three kinds of peptides in the feature of MC6, its preparation method's feature is: the Succus Momordicae charantiae of centrifugal clarification passes through 30kDal, the ultra-filtration membrane of 10kDal and 3kDal successively.The MC6 molecular weight can pass through the 10kDal ultra-filtration membrane, and be the 3kDal membrane retention less than 10kDal.The feature of activated MC6 is to move with single strips in the SDS-PAGE gel electrophoresis, and molecular weight comprises three kinds of peptides less than 10kDal.The peptide component that also has MC6 that provides simultaneously is called MC6.1, and the derivative of MC6.1 and stand-in.Active MC6, MC6.1, MC6.2, MC6.3 has hypoglycemic activity, diabetes especially, wherein preferred oral gives these active medicines.In this patent, the balsam pear hypoglycemic survivor of a murder attempt who is utilized also has this activity after obeying administration.The method composition that utilizes these active medicine treatment hyperglycemia diseases in addition that provides simultaneously only is the water-soluble protein (polypeptide) that enough sees through ultra-filtration membrane.Patent application person describes, and MC6 does not improve the activity of insulin level in the animal pattern blood plasma.

The invention of international patent application (PCT/IN99/00052 1999.9.28) provides a kind of efficient blood sugar lowing polypeptide-k first, and it extracts from balsam pear.Polypeptide-k preparation method that patent application person introduces is: handle balsam pear seed (or fruit) with non-polar solvent, remove unwanted grease, flavonoid and sapogeninium(i), use the protein in moisture 80% the acetone extraction residue then, optionally Crystallization Separation goes out polypeptide-k at last.The applicant describes, and polypeptide-k is water insoluble, and its hypoglycemic mechanism of action is to activate the Regular Insulin of non-activity in the blood.

Clearly, MC6 and polypeptide-k are the protein that has hypoglycemic activity in the balsam pear, and still, MC6 is water-soluble, and polypeptide-k is water insoluble, according to the preparation method of MC6, has then lost the polypeptide-k in the balsam pear; And, lost the MC6 in the balsam pear equally according to the preparation method of polypeptide-k.

Some Chinese patents disclose the content of extracting sugar reducing substance or make Chinese medicine from balsam pear in addition:

1,＜and application number〉99115925＜denomination of invention〉a kind of bitter gourd sugar-reducing capsule and preparation technology thereof, this invention is to be raw material with fresh balsam pear, and is freezing through cleaning, low temperature slurrying, press filtration is crossed 100 mesh sieves with the filter residue drying, makes the balsam pear ground-slag; Filtrate clarification refilters, and stirs 3 hours under 15 ℃ of conditions through acid (pH3.0) ethanol, staticly settled 12 hours, after the filtration with throw out through cryodrying, mistake 100 mesh sieves are made bitter melon protein; Filtered liquid is through 60 ℃ of vacuum concentration, add 2 times of water dissolution, add 2 times of ether defattings, 50 ℃ of vacuum concentration reclaim ether, and 60 ℃ of vacuum reclaim ethanol and make thick balsam pear total saponins, again through anhydrous propanone washing of precipitate several under 4 ℃ of conditions, vacuum concentration below 30 ℃ is made the balsam pear total saponins, with total saponins, bitter melon protein powder and balsam pear slag, incapsulate and make bitter gourd sugar-reducing capsule through mixing by 10: 2: 88 weight ratios at last.It is extensive to have raw material sources, and technology is simple, easy administration, and low price, the advantage that has no side effect can be as the auxiliary agent of treatment diabetes.

2,＜application number 98110802＜Granted publication day 08 month calendar year 2001 15 days＜denomination of invention medicine of reducing blood sugar using natural bitter gourd extract and preparation method thereof, disclosure of the Invention a kind of making method of medicine of reducing blood sugar using natural bitter gourd extract, comprise the steps: that a. cuts into slices ripe balsam pear, and carry out drying treatment; B. the exsiccant balsam pear slice is soaked in the water, and extracts immersion liquid; C. the balsam pear immersion liquid is condensed into concentrated solution; D. concentrated solution is carried out biological enzymolysis, get the high reactivity degradation solution; E. degradation solution is carried out the freeze-day with constant temperature granulation of dusting; F. particle is incapsulated patent medicine; This method processing unit is simple, meets the scale operation requirement, product yield height, and result of treatment is good, has no side effect.

3,＜application number 95103732＜denomination of invention the treatment diabetes Chinese medicine and production method thereof, this invention is a kind of Chinese medicine for the treatment of diabetes, the main component of this Chinese medicine is animal pancreas, balsam pear, pumpkin, the Radix Astragali, Chinese yam, it is thousand dry that production method is that animal pancreas is pulverized, mix with the balsam pear that obtains with water extraction or separating out alcohol method, pumpkin, Radix Astragali extract and to add Rhizoma Dioscoreae powder again and obtain, this medicine has supplementing QI for promoting the production of body fluid, invigorating the spleen is moisturized, nourishing Yin and falling fire power is imitated, and the enhance immunity function is arranged, promote insulin secretion, the lowering blood glucose effect.

4,＜and application number〉93119351.6＜denomination of invention〉new component PA of plant hypoglycemic active protein and preparation thereof, this invention relates to field of biological pharmacy.From the vegetable material balsam pear, extract the new component PA of hypoglycemic active protein, materials purification processing-cryogenic freezing among the preparation technology-fragmentation-acid alcohol dipping-high-speed homogenization-acid alcohol extraction-suction filtration-centrifugal purification-precipitation-dissolving-Sephadex gel is crossed the preparation method of post-ultraviolet detection fraction collection-concentrated-new component PA of exsiccant balsam pear hypoglycemic activated protein again, has been characterized in adopting isoelectric precipitation and gel to cross the method for post, ultraviolet detection fraction collection.The contriver think this material have separation and Extraction easy, consume few, the yield advantages of higher of organic solvent, obvious through animal experiment proof blood sugar decreasing effect.

5,＜and application number〉02102754＜denomination of invention〉a kind of method of rapidly and efficiently extracting plant insuline, the balsam pear seed contains more rich plant insuline, is a kind of effective constituent for the treatment of diabetes at present.The invention technical characterictic is: the shelling of balsam pear seed, plant benevolence and pulverized 60 or 100 mesh sieves, adopt acidic ethanol to extract then, the centrifuging and taking supernatant liquor, add NaCl and account for volume 3% ~ 5%, with 1 ~ 5Mol/L NaOH regulator solution to pH value 7.0 ± 7.5 solution becomes muddinesses, centrifugal, remove liquid, precipitate part three times with washing with acetone, vacuum-drying obtains the plant insuline crude product.Carry out the gel affinity chromatography with 0.1MTris.c1 solution (pH7.5) dissolving, collect elution peak solution, concentrate vacuum-drying with acetone or alcohol and obtain the plant insuline elaboration.This method can be extracted the highly purified plant insuline composition of preparation, can be used for treatment of diabetes.

6,＜and application number〉98102198.0＜denomination of invention〉a kind of balsam pear buccal lozenge and preparation method thereof, this invention is a kind of balsam pear buccal lozenge and preparation method thereof.This balsam pear buccal lozenge mainly contains Fructus Momordicae charantiae powder, dextrin, citric acid and N.F,USP MANNITOL etc., during preparation, earlier with bright balsam pear through squeeze the juice, filtration, lyophilize make balsam pear freeze-dried powder, mix with other composition again, and compacting is in flakes.Adopt preparation method of the present invention scientific and reasonable, fully kept the multiple nutritional components in the balsam pear, do not lose the activity of balsam pear under the made lozenge normal temperature, have the sharp gas of clearing heat and detoxicating, wide chest, promote the production of body fluid to quench thirst, effects such as lowering blood glucose, raising immunizing power, anticancer, it is daily life, particularly in disaster area rescue, warlike operation and tourism, exploring, can regain one's strength of body rapidly, improve the requisite that makes things convenient for of immunizing power.Principal claim (A61K35/78) requires: a kind of balsam pear buccal lozenge is characterized in that containing Fructus Momordicae charantiae powder, dextrin, N.F,USP MANNITOL, spices, pigment and Magnesium Stearate.

Existing studies show that, outside the isolating protein, saponin(e also is a kind of very effectively composition of hypoglycemic activity in the balsam pear.

Some scientific and technical literature reports, the balsam pear saponin(e is Fourth Ring three mushroom compounds, and a plurality of heterogeneities are arranged, and all is insoluble in water, available ethanol extracts (Chang Fenggang: balsam pear chemical constitution study, herbal medicine, 1995,26 (10) 507-510) from the exsiccant Fructus Momordicae charantiae.The balsam pear saponin(e does not influence insulin content in the animal subject model blood plasma, mainly be the synthetic (Wang Xianyuan that promotes to increase muscle glycogen and liver starch, Jin Hong, Xu Zhiqin etc.: balsam pear saponin(e hypoglycemic activity and Initial Study of Mechanism, amino acid and Biological resources, 2001,23 (3): 42-45), thereby have significant hypoglycemic activity (Sun Shuqing, the pharmacology of balsam pear and clinical study overview, a Chinese medicinal materials, 1997, (8): 428--429), animal experiment proves that its blood sugar reduces amplitude even is better than " glyburide " (Sun Baoying etc., the pharmacodynamic experiment research of balsam pear saponin(e, Henan Chinese materia medica academic periodical, 1994,9 (6): 19--21).

Water-soluble polypeptide MC6, water-insoluble polypeptide-k and balsam pear saponin(e, because the difference of their chemical property, thereby belong to the material that differs from one another.

And the balsam pear hypoglycemic activeconstituents not merely is known saponin(e and protein.

From the water cooking liquid of fresh balsam pear, isolated the composition that dissolves in ether and be insoluble to two the unknowns of ether, identified and be not saponin(e, flavones and alkaloid, these two kinds of compositions all show obvious functions of blood sugar activity (Wang Yongqing: balsam pear hypoglycemic effect research, Hunan Chinese materia medica magazine, 1998, C6): 51-52).Prove that thus blood sugar reducing component also has other hypoglycemic activity compound in the balsam pear except that saponin(e and vegetable-protein.

Several different methods extracts of balsam pear hypoglycemic effect to tetraoxypyrimidine inductive chemical diabetes model mice is compared, the result shows, the fruit juice of the not heated direct squeezing of balsam pear is better than the blood sugar decreasing effect of the efficient part that other method is extracted, and infers that its hypoglycemic activity thing may be the water miscible easy ruined composition (Fan Yuling that is heated; Cui Fude, the comparative studies of balsam pear efficient part hypoglycemic activity, Shenyang Pharmaceutical University's journal 2001,18 (1): 50-52).

Experimentation on animals shows, bright Succus Momordicae charantiae can enable tetraoxypyrimidine induce diabetic mice the beta Cell of islet of damaged obtain repairing, and the β cell of regenerating and making new advances, improved the insulin content (Ahmed in the blood plasma, I., Adeghate, E, Sharma, A.K., Pallot, D.J.et al.Effects of Momordica charantia fruit juice on islet morphology in the pancreas of thestreptozotocin-diabetic rat.Diabetes research and clinical practice.1998,40,145-151).What body was interior and stripped further studies show that; this mechanism of action of bright Succus Momordicae charantiae is the pancreas that has alleviated diabetic mice; the lipid peroxidation of RIN cell and pancreas islet; and reduced tetraoxypyrimidine inductive apoptosis in the RIN cell; thereby to the RIN cell; (the Sitasawad that shields of pancreas islet and pancreatic beta cell; S.L.; Shewade.Y e; Bhonde, Ro, Role of bittergourd fruit juice in stz-induced diabeticstate in vivo and in vitro.Journal of Ethno-pharmacology; 2000,73 (1): 71-79).And in the known hypoglycemic activity compound of balsam pear, still fail to determine to have this reparation or the islet cells of regenerating, improve the biological activity of insulin level.

By the above-mentioned fact as can be known, the function of blood sugar reduction actives of balsam pear has knownly, also belongs to unknown in addition, the function of blood sugar reduction of balsam pear, and actual is the wherein contained multiple synergistic result of composition with hypoglycemic activity.With regard to known activeconstituents,, be difficult to they be extracted simultaneously with a kind of solvent because of its chemical property difference.In addition, in the hypoglycemic activity thing of balsam pear, include heat labile composition, the process of its extraction should be heated.

With the way of defibrination that fresh balsam pear pulp organization cell is fully broken, all inclusion of cell comprise all being released to water-soluble and the blood-sugar decreasing active that is insoluble in water in the pulp.With the robust fibre in the pulp (coming from the flesh cell wall) elimination, lyophilize then, the freeze-dried products of this Succus Momordicae charantiae has comprised undoubtedly than more comprehensive blood-sugar decreasing active.But the content of hypoglycemic activity thing is very low in fact in the Succus Momordicae charantiae, direct freeze-drying cost costliness, and on the other hand, the glucide that exists in the Succus Momordicae charantiae is deliquescence very easily, makes this freeze-dried products packing under normal operation, storage and use at all.Though add the deliquescence problem that a large amount of drying aids or non-blushing thinner can solve the freeze-dried products of Succus Momordicae charantiae, can't make high-load balsam pear hypoglycemic goods.

In sum, the preparation of the balsam pear effective constituent of document and patent disclosure, basically be to adopt the physics method and chemical method extracts certain or several clear and definite chemical ingredientss are main, or balsam pear certain solvent under heating condition is (as water, ethanol or its mixture) general extractive, they or the not mentioned blood-sugar decreasing active that how to utilize those chemical property the unknowns in the balsam pear, some thermo-sensitivity hypoglycemic activity thing is subjected to the heat collapse problem in the perhaps not clear and definite balsam pear.

No. 99115925 Chinese invention patents, a kind of bitter gourd sugar-reducing capsule and preparation technology thereof have proposed the utilization of bitter melon protein and balsam pear saponin(e, but have used ethanol in preparation process, acetone, organic solvents such as ether.No. 98110802 patents are mentioned in medicine of reducing blood sugar using natural bitter gourd extract and preparation method thereof the balsam pear concentrated solution are carried out biological enzymolysis, the high reactivity degradation solution, but what enzyme what adopt in the not mentioned biological enzymolysis is.98102198.0 what use in number patent, a kind of balsam pear buccal lozenge and preparation method thereof is lyophilize Succus Momordicae charantiae technology, but it does not consider to remove the known problem that does not have the composition of hypoglycemic activity in the balsam pear.

For these reasons, the different all kinds of hypoglycemic activity things of physico-chemical property in the exploitation one cover energy full use balsam pear, and active component content height are so for fully and reasonably utilizing and develop the balsam pear resource, also be very necessary.

Polypeptide can obtain by the method for engineered method or chemosynthesis.Engineered method has superiority than chemical process obtaining on long peptide (amino-acid residue length is greater than 50) or the protein, but the chemical synthesis process of polypeptide is especially after the solid phase synthesis strategy occurs, handiness, diversity and the high efficiency etc. that have gene engineering method to hardly match on less than 40 polypeptide or little peptide in preparation amino-acid residue length.Merrifield in 1963 found and have developed the method for solid-phase synthetic peptide.Solid-phase polypeptide is synthetic generally two kinds of strategies: i.e. Boc/Bzl orthogonally protect solid phase synthesis strategy and Fmoc/tBu orthogonally protect solid phase synthesis strategy, the solid-phase synthesis that Merrifield foundes is Boc/Bzl orthogonally protect solid phase synthesis strategy.But, have some shortcomings in the Boc/Bzl orthogonally protect solid phase synthesis strategy, many as side reaction, condition is harsh and in the process of prolongation peptide chain polypeptide chain can lose from solid phase etc.The Fmoc/tBu orthogonally protect solid phase synthesis strategy of Chu Xianing reaction conditions gentleness many then subsequently.After microwave technology applies in the solid phase synthesis of polypeptide in recent years, make the synthetic technology of polypeptide produce a leap.Microwave promotes chemical reaction to be because it makes polar molecule fast rotational in microwave field, makes fast 10 to 1000 times of the more conventional heating means of some speed of reaction, and productive rate improves greatly.People's reported first such as Gedye in 1986 with microwave application in organic synthesis, microwave can promote many chemical reactions, as the Diels-Alder reaction, saponification reaction etc. can be accelerated speed of reaction significantly and improve yield.In 1992, Hui-ming Yu etc. reported at first with microwave application that in polypeptide solid phase synthesis field they have finished the synthetic of acyl carrier protein segment decapeptide (acyl carrier protein, ACP 65 ~ 74).

How synthetic efficiently long peptide remain a challenge in the world, because polypeptide is along with the increase of peptide chain, its synthetic difficulty is multiplied.With the synthetic long peptide of traditional solid phase method, the more purification difficult of the often very low impurity of its yield, thereby say from practical angle and to have lost the synthetic meaning.Secondly synthesis cycle is long, and 10 peptides nearly time in a week of general needs just can not finished synthesis cycle under the centre does not encounter difficulties the situation of peptide preface, if difficult peptide preface, often just can't synthesize and obtains target polypeptides.And use microwave to promote solid-phase synthetic peptide, can overcome difficulties the peptide preface, the polypeptide that synthetic traditional solid phase method can't obtain, and generated time shortens greatly, and significantly improve polypeptide crude product purity and yield, make that the purifying of product is convenient greatly, as long as just can obtain the purity high product through a preparation HPLC; Can carry out pointed decoration with D type amino acid and alpha-non-natural amino acid etc. to polypeptide very easily in addition, remove to seek active higher, polypeptide that biological half-life is longer, this especially genetic engineering technique can't accomplish.Therefore, our employing this kind method synthetic MC-JJ62 serial analogs that obtains rapidly and efficiently in balsam pear hypoglycemic (MC-JJ62) polypeptide analog synthetic.

Summary of the invention

It is to obtain polypeptide to modify and obtain the higher MC-JJ62 polypeptide analog of hypoglycemic activity from the medicinal and edible plant balsam pear of tool hypoglycemic activity that the present invention has two first purposes of purpose.

Second purpose of the present invention provided the method for preparing solid phase of MC-JJ62 polypeptide derivative, and the present invention adopts microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy efficiently to synthesize apace and obtains serial MC-JJ62 polypeptide analog.

It is characterized in that: adopt microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy, earlier syntheticly on solid phase carrier obtain being loaded with first Fmoc and protect amino acid whose resin, ninhydrin method detects sloughs the resin that the Fmoc protecting group obtains being loaded with first amino-acid residue after negative; Enter next coupling circulation again; repeat the step of coupling and deprotection with different protection amino acid according to corresponding peptide preface; prolong required aminoacid sequence successively, the synthetic resin that obtains being loaded with corresponding polypeptide cuts down polypeptide with cracking agent at last and obtains the polypeptide crude product from resin.Crude product is through the preparation scale high-efficient liquid phase chromatogram purification, and freeze-drying gets the pure product of polypeptide.

Be loaded with the preparation method that first Fmoc protects amino acid whose resin; it is characterized in that by obtaining with the solid phase carrier coupling again after the activated dose of activation earlier of Fmoc protection amino acid under the microwave irradiation condition; add 1-hydroxyl-benzotriazole (HOBT) or derivatives thereof in the reaction and suppress racemization, and use the acidity of organic bases neutralization reaction.Being loaded with first Fmoc, to protect the employed solid phase carrier of amino acid whose resin be Rink resin, Wang resin or 2-chlorine trityl chloride resin.Activator is dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC), N, N " carbonyl dimidazoles (CDI); 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl); 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester (HBTU) or 1-hydroxyl-benzotriazole (HOBT) derivative that uses are N-hydroxy-succinamide (HOSU), 1-hydroxyl-7-azo benzotriazole (HOAT) or 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone (HOOBT).Organic bases is triethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20 ~ 100 ℃, the reaction times is 5～15min.

Removing of Fmoc protecting group is to contain 0.1molL by use ^-1Hexahydropyridine solution reaction under microwave promotes of 1-hydroxyl-benzotriazole (HOBT), selecting dimethyl formamide (DMF), methyl-sulphoxide (DMSO) or N-Methyl pyrrolidone (NMP) for use is reaction solvent.Microwave promotion condition is: microwave frequency 2450MHz, temperature of reaction: 20 ~ 100 ℃, the reaction times is 1～10min.

The invention has the advantages that:

1. a kind of MC-JJ62 polypeptide analog of Ti Chuing can improve the stability of MC-JJ62 on the basis that keeps hypoglycemic activity, prolongs action time.

2. microwave promotes solid phase synthesis MC-JJ62 polypeptide analog to improve coupled reaction speed greatly, and amino acid of the abundant coupling of conventional solid phase synthesis process goes to resin, often needs do not wait by 20 hours in 2 hours, even longer.Microwave promotes then average to need about 10 minutes; Conventional solid phase synthesis process takes off the Fmoc protecting group, often needs do not wait by 1 hour in 30 minutes, and microwave promotes then on average only to need about 5 minutes, and this has improved polypeptide synthetic efficient greatly, has shortened synthesis cycle.

3. microwave promotes the purity of the synthetic crude product that obtains of solid phase synthesis MC-JJ62 polypeptide analog greater than 60%, more conventional solid phase synthesis process improves greatly, this has made things convenient for follow-up purifying work, only need be through once preparing the liquid phase purifying, and freeze-drying can obtain the pure product of target.

4. microwave promotes the synthetic MC-JJ62 polypeptide analog of solid phase method, and its cost is low because coupling efficiency is higher, required protection amino acid is average only need 2 times excessive, more conventional solid phase synthesis process needs 4 to 5 times excessively greatly to reduce.

5. microwave promotes solid phase synthesis MC-JJ62 polypeptide analog method easily to be automated, to change on a large scale, and this makes it be more suitable for suitability for industrialized production.

Therefore promote the MC-JJ62 polypeptide analog that solid phase synthesis technique prepares with microwave provided by the invention, the yield height, synthesis cycle is short, purifying crude is easy, and production cost is low, be easy to industrial automation production.The MC-JJ62 polypeptide analog for preparing, more stable than natural MC-JJ62, be suitable as the activeconstituents for the treatment of diabetes medicament.

Embodiment

Adopt following abbreviation in this specification:

Et ₃N: triethylamine; The NMM:N-methylmorpholine; DIEA:N, N '-diisopropylethylamine; DMF: dimethyl formamide; DM SO: methyl-sulphoxide; DCM: methylene dichloride; The Fmoc:N-9 fluorenylmethyloxycarbonyl; DIC:N, N '-DIC; CDI:N, N '-carbonyl dimidazoles; The DMAP:4-Dimethylamino pyridine; The HOSU:N-N-Hydroxysuccinimide; EDC.HCl:1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; HATU:2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HBTU: benzotriazole-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; HCTU:6-chlorobenzene and triazole-1,1,3,3-tetramethyl-urea phosphofluoric acid ester; HOAT:1-hydroxyl-7-azo benzotriazole; HOBT:1-hydroxyl-benzotriazole; PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus; HPLC: high performance liquid chromatography; ESI-MS: electrospray ionization mass spectrum; Gly: glycine; Ser: Serine; Ala: L-Ala; Thr: Threonine; Val: Xie Ansuan; Ile: Isoleucine; Leu: leucine; Tyr: tyrosine; Phe: phenylalanine; His: Histidine; Pro: proline(Pro); Asp: aspartic acid; Met: methionine(Met); Glu: L-glutamic acid; Trp: tryptophane; Lys: Methionin; Arg: arginine.Asn: l-asparagine; Gln: glutamine.

The present invention describes by following implementation column, but these embodiment do not do any restriction explanation of the present invention.

Embodiment 1

(D-Lys)-microwave of Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:2) promotes solid phase synthesis

(1) swelling of resin

Take by weighing Fmoc-Ala Resin 50mg (replacement amount 0.4mmol/g), through 7mL DCM swelling 30min, suction filtration removes DCM, uses 10mL NMP swelling 30min again, uses NMP at last respectively, DCM, and NMP 7mL rinses well.

(2) microwave promotes removing of Fmoc protecting group

The resin that swelling is good is put into reactor, adds 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT, reacts 1min in microwave reactor, microwave power is 15W, temperature of reaction is controlled in 50 ℃, uses the cooling of air compressor pressurized air, and reaction finishes back elimination solution; Add 25% piperidines/NMP (V/V) solution that 7mL contains 0.1M HOBT again and react 4min again in microwave reactor, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air compressor pressurized air.Reaction finishes back elimination solution, uses the NMP washes clean.Obtain sloughing the resin of the Fmoc protecting group of initial connection.

(3) microwave promotes synthesizing of Fmoc-Ala-rink amide-MBHA Resin

With Fmoc-Ala-rink (0.04mmol), HBTU (0.04mmol), HOBT (0.04mmol) and DIPEA (0.08mmol) are dissolved among the 10mL NMP, in the resin above again this solution being added, in microwave reactor, react 7min, microwave power is 25W, and temperature of reaction is controlled at 50 ℃, uses the cooling of air compressor pressurized air.Reaction finishes back filtering reaction solution, uses DCM and each 7mL washing resin of NMP 3 times.

(4) detection of coupling efficiency

With the coupling efficiency of ninhydrin method or bromjophenol blue method qualitative detection resin, color reaction is negative can to enter next coupling circulation.

Ninhydrin method: the resin particle washing with alcohol takes a morsel, put into transparent bottle and add respectively 2 of 5% triketohydrindene hydrate ethanol, KCN pyridine solution (2ml 0.001MKCN is diluted in the 98ml pyridine), 80% phenol ethanolic solns, in 100 ℃ of heating 5 minutes, promptly positive if resin shows blueness.The bromjophenol blue method: the resin particle that takes a morsel washs with two formyl ethanamides, puts into the tetrabromophenol sulfonphthalein dimethylacetamide solution that transparent bottle adds 3 1%, and jolting is 3 minutes under the normal temperature, and is promptly positive if resin shows blueness.

(5) prolongation of peptide chain

According to Ala ₃-MC-JJ62-(1 ~ 11)-NH ₂Sequence, the steps in sequence that repeats above-mentioned deprotection and coupling is connected corresponding amino acid, the coupling microwave promotes reaction times 5～20min not wait.Obtain being connected with the resin of Ala3-MC-JJ62-(1 ~ 11)-NH2.

(6) cracking of polypeptide on the resin

With above-mentioned obtain be connected with Ala ₃The resin of-MC-JJ62-(1 ~ 11)-NH2 is put into reaction flask, and each adds cracking agent Reagent K, and (TFA/ thioanisole/water/phenol/EDT, 82.5: 5: 5: 5: 2.5, V/V) 10mL earlier at 0 ℃ of following jolting 30min, reacted 3h more at normal temperatures.Reaction finishes the back suction filtration, adds a small amount of TFA and DCM washing three times, merging filtrate.Filtrate added in a large amount of ice ether separate out white flocks, frozen centrifugation obtains the crude product of target polypeptides.Finally obtain Ala ₃-MC-JJ62-(1 ~ 11)-NH2 crude product 63.2mg, yield is 94.3%.

(7) Ala ₃The purifying of-MC-JJ62-(1 ~ 11)-NH2-NH2 crude product

With the Ala that obtains above ₃-MC-JJ62-(1 ~ 11)-NH2 crude product is dissolved in a spot of water, prepares type reversed-phase HPLC purifying crude product with Tianjin, island.Adopt the anti-phase preparative column of C18 (340mm * 28mm, 5 μ m) in the purifying; Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V); Eluent gradient: Mobile phase B 13%～15%, 30min; Flow velocity is 6mL/min; The detection wavelength is 214nm.The solution freeze-drying of collecting gets pure product, finally obtains pure product 29.7mg.Theoretical relative molecular mass 1079.3.ESI-MS m/z:found[M+3H] ³⁺360.8, [M+4H] ⁴⁺270.8, [M+5H] ⁵⁺216.9; Calu[M+3H] ³⁺360.8, [M+4H] ⁴⁺270.7, [M+5H] ⁵⁺216.9;

According to embodiment 1 described method, according to the synthetic polypeptide that obtains embodiment 2 ~ 54 of corresponding sequence, by electrospray ionization mass spectrum (ESI-MS) conclusive evidence molecular weight separately.

Embodiment 2

(D-Ala) Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:3); Theoretical relative molecular mass 1022.2.ESI-MSm/z:found[M+3H] ³⁺341.7, [M+4H] ⁴⁺256.6, [M+5H] ⁵⁺205.4; Calu[M+3H] ³⁺341.6, [M+4H] ⁴⁺256.6, [M+5H] ⁵⁺205.4;

Embodiment 3

(D-Val)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:4); Theoretical relative molecular mass 1050.3.ESI-MS m/z:found[M+3H] ³⁺351.1, [M+4H] ⁴⁺263.6, [M+5H] ⁵⁺211.1; Calu[M+3H] ³⁺351.1, [M+4H] ⁴⁺263.6, [M+5H] ⁵⁺211.1;

Embodiment 4

(D-Leu)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:5); Theoretical relative molecular mass 1064.2.ESI-MS m/z:found[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.1, [M+5H] ⁵⁺213.9; Calu[M+3H] ³⁺355.7, [M+4H] ⁴⁺267.1, [M+5H] ⁵⁺213.9;

Embodiment 5

(D-Ile)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:6); Theoretical relative molecular mass 1064.3.ESI-MS m/z:found[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.1, [M+5H] ⁵⁺213.9; Calu[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.0, [M+5H] ⁵⁺213.9;

Embodiment 6

(D-Phe)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:7); Theoretical relative molecular mass 1098.3.ESI-MS m/z:found[M+3H] ³⁺367.1, [M+4H] ⁴⁺275.6, [M+5H] ⁵⁺220.7; Calu[M+3H] ³⁺367.0, [M+4H] ⁴⁺275.5, [M+5H] ⁵⁺220.5;

Embodiment 7

(D-Pro) Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:8); Theoretical relative molecular mass 1048.2.ESI-MS m/z:found[M+3H] ³⁺350.4, [M+4H] 4 ⁺263.1, [M+5H] ⁵⁺210.6; Calu[M+3H] ³⁺350.4, [M+4H] ⁴⁺263.0, [M+5H] ⁵⁺210.6;

Embodiment 8

(D-Met)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:9); Theoretical relative molecular mass 1082.3.

ESI-MS?m/z：found[M+3H] ³⁺361.8，[M+4H] ⁴⁺271.6，[M+5H] ⁵⁺217.5；calu[M+3H] ³⁺316.8，[M+4H] ⁴⁺271.7，[M+5H] ⁵⁺217.5；

Embodiment 9

(D-Cys)-Thr-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:10); Theoretical relative molecular mass 1054.3.ESI-MS m/z:found[M+3H] ³⁺352.4, [M+4H] ⁴⁺264.6, [M+5H] ⁵⁺211.9; Calu[M+3H] ³⁺352.2, [M+4H] ⁴⁺264.5, [M+5H] ⁵⁺211.9;

Embodiment 10

Lys-(D-Ala)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:11); Theoretical relative molecular mass 1049.3.ESI-MSm/z:found[M+3H] ³⁺350.8, [M+4H] ⁴⁺263.3, [M+5H] ⁵⁺210.9; Calu[M+3H] ³⁺350.6, [M+4H] ⁴⁺263.8, [M+5H] ⁵⁺210.8;

Embodiment 11

Lys-(D-Val)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:12); Theoretical relative molecular mass 1077.3.ESI-MSm/z:found[M+3H] ³⁺360.1, [M+4H] ⁴⁺270.3, [M+5H] ⁵⁺216.5; Calu[M+3H] ³⁺360.0, [M+4H] ⁴⁺270.3, [M+5H] ⁵⁺216.5;

Embodiment 12

Lys-(D-Leu)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:13); Theoretical relative molecular mass 1091.4ESI-MSm/z:found[M+3H] ³⁺364.8, [M+4H] ⁴⁺273.8, [M+5H] ⁵⁺219.3; Calu[M+3H] ³⁺364.8, [M+4H] ⁴⁺273.7, [M+5H] ⁵⁺219.1;

Embodiment 13

Lys-(D-Ile)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:14); Theoretical relative molecular mass 1091.4.ESI-MSm/z:found[M+3H] ³⁺364.8, [M+4H] ⁴⁺273.8, [M+5H] ⁵⁺219.3; Calu[M+3H] ³⁺364.7, [M+4H] ⁴⁺273.6, [M+5H] ⁵⁺219.3;

Embodiment 14

Lys-(D-Phe)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:15); Theoretical relative molecular mass 1125.4.ESI-MSm/z:found[M+3H] ³⁺367.1, [M+4H] ⁴⁺282.3, [M+5H] ⁵⁺226.1; Calu[M+3H] ³⁺367.0, [M+4H] ⁴⁺282.2, [M+5H] ⁵⁺226.0;

Embodiment 15

Lys-(D-Pro)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:16); Theoretical relative molecular mass 1075.3.ESI-MSm/z:found[M+3H] ³⁺359.4, [M+4H] ⁴⁺269.8, [M+5H] ⁵⁺216.1; Calu[M+3H] ³⁺359.4, [M+4H] ⁴⁺269.8, [M+5H] ⁵⁺216.0;

Embodiment 16

Lys-(D-Met)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:17); Theoretical relative molecular mass 1109.4.ESI-MSm/z:found[M+3H] ³⁺370.8, [M+4H] ⁴⁺278.3, [M+5H] ⁵⁺222.9; Calu[M+3H] ³⁺370.8, [M+4H] ⁴⁺278.2, [M+5H] ⁵⁺222.7;

Embodiment 17

Lys-(D-Cys)-Asn-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:18); Theoretical relative molecular mass 1081.3.ESI-MSm/z:found[M+3H] ³⁺361.4, [M+4H] ⁴⁺271.3, [M+5H] ⁵⁺217.3; Calu[M+3H] ³⁺361.4, [M+4H] ⁴⁺271.3, [M+5H] ⁵⁺217.2;

Embodiment 17

Lys-Thr-(D-Ala)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:19); Theoretical relative molecular mass 1036.3.ESI-MSm/z:found[M+3H] ³⁺346.4, [M+4H] ⁴⁺260.1, [M+5H] ⁵⁺208.3; Calu[M+3H] ³⁺346.3, [M+4H] ⁴⁺260.0, [M+5H] ⁵⁺208.2;

Embodiment 19

Lys-Thr-(D-Val)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:20); Theoretical relative molecular mass 1064.3.ESI-MSm/z:found[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.1, [M+5H] ⁵⁺213.9; Calu[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.0, [M+5H] ⁵⁺213.9;

Embodiment 20

Lys-Thr-(D-Leu)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:21); Theoretical relative molecular mass 1078.4.ESI-MSm/z:found[M+3H] ³⁺360.5, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7; Calu[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.5, [M+5H] ⁵⁺216.5;

Embodiment 21

Lys-Thr-(D-Ile)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:22); Theoretical relative molecular mass 1078.4.ESI-MSm/z:found[M+3H] ³⁺360.5, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7; Calu[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.5, [M+5H] ⁵⁺216.5;

Embodiment 22

Lys-Thr-(D-Phe)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:23); Theoretical relative molecular mass 1112.4.ESI-MSm/z:found[M+3H] ³⁺371.8, [M+4H] ⁴⁺279.1, [M+5H] ⁵⁺223.5; Calu[M+3H] ³⁺371.6, [M+4H] ⁴⁺279.0, [M+5H] ⁵⁺23.4;

Embodiment 23

Lys-Thr-(D-Pro)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:24); Theoretical relative molecular mass 1062.3.ESI-MSm/z:found[M+3H] ³⁺355.1, [M+4H] ⁴⁺266.6, [M+5H] ⁵⁺213.3; Calu[M+3H] ³⁺355.0, [M+4H] ⁴⁺266.6, [M+5H] ⁵⁺213.3;

Embodiment 24

Lys-Thr-(D-Met)-Met-Lys-Gly-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:25); Theoretical relative molecular mass 1096.4.ESI-MSm/z:found[M+3H] ³⁺366.5, [M+4H] ⁴⁺275.1, [M+5H] ⁵⁺220.3; Calu[M+3H] ³⁺366.5, [M+4H] ⁴⁺275.0, [M+5H] ⁵⁺220.2;

Embodiment 25

Lys-Thr-(D-Cys)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:26); Theoretical relative molecular mass 1068.3.ESI-MSm/z:found[M+3H] ³⁺357.1, [M+4H] ⁴⁺268.1, [M+5H] ⁵⁺214.7; Calu[M+3H] ³⁺357.0, [M+4H] ⁴⁺268.0, [M+5H] ⁵⁺214.5;

Embodiment 26

(D-Lys)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:27); Theoretical relative molecular mass 1093.3.ESI-MS m/z:found[M+3H] ³⁺365.4, [M+4H] ⁴⁺274.3, [M+5H] ⁵⁺219.7; Calu[M+3H] ³⁺365.4, [M+4H] ⁴⁺274.5, [M+5H] ⁵⁺219.5;

Embodiment 27

(D-Ala)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:28); Theoretical relative molecular mass 1036.2.ESI-MS m/z:found[M+3H] ³⁺365.4, [M+4H] ⁴⁺274.3, [M+5H] ⁵⁺219.6; Calu[M+3H] ³⁺365.4, [M+4H] ⁴⁺274.2, [M+5H] ⁵⁺219.7;

Embodiment 28

(D-Val)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:29); Theoretical relative molecular mass 1064.3.ESI-MSm/z:found[M+3H] ³⁺355.8, [M+4H] ⁴⁺267.1, [M+5H] ⁵⁺213.9; Calu[M+3H] ³⁺355.7, [M+4H] ⁴⁺267.0, [M+5H] ⁵⁺213.9;

Embodiment 29

(D-Leu)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:30); Theoretical relative molecular mass 1078.3.ESI-MSm/z:found[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7; Calu[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7;

Embodiment 30

(D-Ile)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:31); Theoretical relative molecular mass 1078.3.ESI-MSm/z:found[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7; Calu[M+3H] ³⁺360.4, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.8;

Embodiment 31

(D-Phe)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:32); Theoretical relative molecular mass 1112.3.ESI-MSm/z:found[M+3H] ³⁺371.8, [M+4H] ⁴⁺279.1, [M+5H] ⁵⁺223.5; Calu[M+3H] ³⁺371.7, [M+4H] ⁴⁺279.0, [M+5H] ⁵⁺223.5;

Embodiment 32

(D-Pro)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:33); Theoretical relative molecular mass 1062.3.ESI-MSm/z:found[M+3H] ³⁺355.1, [M+4H] ⁴⁺266.6, [M+5H] ⁵⁺213.5; Calu[M+3H] ³⁺355.1, [M+4H] ⁴⁺266.6, [M+5H] ⁵⁺213.5;

Embodiment 33

(D-Met)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:34); Theoretical relative molecular mass 1096.3.ESI-MSm/z:found[M+3H] ³⁺366.5, [M+4H] ⁴⁺275.1, [M+5H] ⁵⁺220.3, calu[M+3H] ³⁺366.5, [M+4H] ⁴⁺275.0, [M+5H] ⁵⁺220.3;

Embodiment 34

(D-Cys)-Thr-Ans-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:35); Theoretical relative molecular mass 1068.3.ESI-MSm/z:found[M+3H] ³⁺357.1, [M+4H] ⁴⁺268.1.[M+5H] ⁵⁺214.7; Calu[M+3H] ³⁺357.0, [M+4H] ⁴⁺268.0, [M+5H] ⁵⁺214.5;

Embodiment 35

Lys-(D-Ala)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:36); Theoretical relative molecular mass 1063.3.ESI-MSm/z:found[M+3H] ³⁺355.4, [M+4H] ⁴⁺266.8, [M+5H] ⁵⁺213.7; Calu[M+3H] ³⁺355.4, [M+4H] ⁴⁺266.8, [M+5H] ⁵⁺213.7;

Embodiment 36

Lys-(D-Val)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:37); Theoretical relative molecular mass 1091.4.ESI-MSm/z:found[M+3H] ³⁺364.8, [M+4H] ⁴⁺273.8, [M+5H] ⁵⁺219.3; Calu[M+3H] ³⁺364.8, [M+4H] ⁴⁺273.8, [M+5H] ⁵⁺219.3;

Embodiment 37

Lys-(D-Leu)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.IDNO:38); Theoretical relative molecular mass 1105.4.ESI-MSm/z:found[M+3H] ³⁺369.5, [M+4H] ⁴⁺277.3, [M+5H] ⁵⁺222.1; Calu[M+3H] ³⁺369.5, [M+4H] ⁴⁺277.2, [M+5H] ⁵⁺222.2;

Embodiment 38

Lys-(D-Ile)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:39); Theoretical relative molecular mass 1105.4.ESI-MSm/z:found[M+3H] ³⁺369.5, [M+4H] ⁴⁺277.3, [M+5H] ⁵+ 222.1; Calu[M+3H] ³⁺369.6, [M+4H] ⁴⁺277.3, [M+5H] ⁵⁺222.0;

Embodiment 39

Lys-(D-Phe)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.IDNO:40); Theoretical relative molecular mass 1139.4.ESI-MSm/z:found[M+3H] ³⁺380.8, [M+4H] ⁴⁺285.8, [M+5H] ⁵⁺228.9; Calu[M+3H] ³⁺380.8, [M+4H] ⁴⁺285.8, [M+5H] ^5+228.9

Embodiment 40

Lys-(D-Pro)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:41); Theoretical relative molecular mass 1089.3.ESI-MSm/z:found[M+3H] ³⁺364.1, [M+4H] ⁴⁺273.3, [M+5H] ⁵⁺218.9; Calu[M+3H] ³⁺364.0, [M+4H] ⁴⁺273.3, [M+5H] ⁵⁺218.9;

Embodiment 41

Lys-(D-Met)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:42); Theoretical relative molecular mass 1123.4.ESI-MSm/z:found[M+3H] ³⁺375.5, [M+4H] ⁴⁺281.9, [M+5H] ⁵⁺225.7; Calu[M+3H] ³⁺375.7, [M+4H] ⁴⁺281.9, [M+5H] ⁵⁺225.5;

Embodiment 42

Lys-(D-Cys)-Asn-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:43); Theoretical relative molecular mass 1095.4.ESI-MSm/z:found[M+3H] ³⁺366.1, [M+4H] ⁴⁺274.8, [M+5H] ⁵⁺220.1; Calu[M+3H] ³⁺366.1, [M+4H] ⁴⁺274.8, [M+5H] ⁵⁺220.0;

Embodiment 43

Lys-Thr-(D-Ala)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:44); Theoretical relative molecular mass 1050.3.ESI-MSm/z:found[M+3H] ³⁺351.1, [M+4H] ⁴⁺263.6, [M+5H] ⁵⁺211.1; Calu[M+3H] ³⁺351.1, [M+4H] ⁴⁺263.6, [M+5H] ⁵⁺211.1;

Embodiment 44

Lys-Thr-(D-Val)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:45); Theoretical relative molecular mass 1078.4.ESI-MSm/z:found[M+3H] ³⁺360.5, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7; Calu[M+3H] ³⁺360.5, [M+4H] ⁴⁺270.6, [M+5H] ⁵⁺216.7;

Embodiment 45

Lys-Thr-(D-Leu)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:46); Theoretical relative molecular mass 1092.4.ESI-MSm/z:found[M+3H] ³⁺365.1, [M+4H] ⁴⁺274.1, [M+5H] ⁵⁺219.5; Calu[M+3H] ³⁺365.1, [M+4H] ⁴⁺274.0, [M+5H] ⁵⁺219.5;

Embodiment 46

Lys-Thr-(D-Ile)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:47); Theoretical relative molecular mass 1092.4.ESI-MSm/z:found[M+3H] ³⁺365.1, [M+4H] ⁴⁺274.1, [M+5H] ⁵⁺219.5; Calu[M+3H] ³⁺365.1, [M+4H] ⁴⁺274.0, [M+5H] ⁵⁺219.6;

Embodiment 47

Lys-Thr-(D-Phe)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:48); Theoretical relative molecular mass 1126.4.ESI-MSm/z:found[M+3H] ³⁺376.5, [M+4H] ⁴⁺282.6, [M+5H] ⁵⁺226.3; Calu[M+3H] ³⁺376.6, [M+4H] ⁴⁺282.7, [M+5H] ⁵⁺226.3;

Embodiment 48

Lys-Thr-(D-Pro)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:49); Theoretical relative molecular mass 1076.3.ESI-MSm/z:found[M+3H] ³⁺359.8, [M+4H] ⁴⁺270.1, [M+5H] ⁵⁺216.3; Calu[M+3H] ³⁺359.8, [M+4H] ⁴⁺270.0, [M+5H] ⁵⁺216.4;

Embodiment 49

Lys-Thr-(D-Met)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:50); Theoretical relative molecular mass 1110.4.ESI-MSm/z:found[M+3H] ³⁺371.1, [M+4H] ⁴⁺278.6, [M+5H] ⁵⁺223.1; Calu[M+3H] ³⁺371.0, [M+4H] ⁴⁺278.6, [M+5H] ⁵⁺223.1;

Embodiment 50

Lys-Thr-(D-Cys)-Met-Lys-Ala-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:51); Theoretical relative molecular mass 1082.4.ESI-MSm/z:found[M+3H] ³⁺361.8, [M+4H] ⁴⁺271.6, [M+5H] ⁵⁺217.5; Calu[M+3H] ³⁺361.7, [M+4H] ⁴⁺271.7, [M+5H] ⁵⁺217.6;

Embodiment 51

Lys-Thr-Gly-Met-Lys-(D-His)-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:52); Theoretical relative molecular mass 1102.3.ESI-MSm/z:found[M+3H] ³⁺368.4, [M+4H] ⁴⁺276.6, [M+5H] ⁵⁺221.5; Calu[M+3H] ³⁺368.4, [M+4H] ⁴⁺276.6, [M+5H] ⁵⁺221.6;

Embodiment 52

Lys-Thr-Gly-Met-Lys-(D-Lys)-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:53); Theoretical relative molecular mass 1093.4.ESI-MSm/z:found[M+3H] ³⁺365.5, [M+4H] ⁴⁺274.3, [M+5H] ⁵⁺219.7; Calu[M+3H] ³⁺365.6, [M+4H] ⁴⁺274.2, [M+5H] ⁵⁺219.7;

Embodiment 53

Lys-Thr-Gly-Met-Lys-(D-Arg)-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:54); Theoretical relative molecular mass 1121.4.ESI-MS m/z:found[M+3H] ³⁺374.8, [M+4H] ⁴⁺281.3, [M+5H] ⁵⁺225.3; Calu[M+3H] ³⁺347.9, [M+4H] ⁴⁺281.4, [M+5H] ⁵⁺225.1;

Embodiment 54

Lys-Thr-Gly-Met-Lys-(D-Met)-Met-Ala-Gly-Ala-Ala (SEQ.ID NO:55); Theoretical relative molecular mass 1096.4.ESI-MSm/z:found[M+3H] ³⁺366.5, [M+4H] ⁴⁺275.1, [M+5H] ⁵⁺220.3; Calu[M+3H] ³⁺366.6, [M+4H] ⁴⁺275.0, [M+5H] ⁵⁺220.5;

Embodiment 55

Hypoglycemic activity experiment in MC-JJ62 and the similar object thereof

Get 10 the week age male mouse of kunming (body weight 18 ~ 22g), random packet, 6 every group.Tail vein injection tetraoxypyrimidine administration modeling, dosage is 60mg/kg, after 72 hours, mouse fasting 5 ~ 6 hours is surveyed blood sugar and is selected the blood glucose value scope 13 ~ 23 mouse random packet, 6 every group.The continuous 10d abdominal injection of mouse behind Cheng Mo MC-JJ62 polypeptide and analogue thereof.0,30,60,90,120 usefulness blood glucose meter are measured blood glucose value.

As shown in table 1, the hypoglycemic activity of MC-JJ62 polypeptide and analogue thereof is better than N1,N1-Dimethylbiguanide.

Table 1MC-JJ62 and the hypoglycemic effect of analogue thereof

	??0min	??30min	??60min	??90min	??120min
						??SEQ.ID?NO：2	??15.36±1.19*	??18.37±0.79*	??18.01±3.69**	??15.98±2.41**	??15.49±0.52**
??SEQ.ID?NO：4	??18.23±3.21	??19.35±1.58*	??18.33±2.54**	??17.55±2.48**	??16.17±3.26
						??SEQ.ID?NO：7	??17.34±2.42*	??17.22±1.37*	??16.25±1.85***	??15.27±7.83**	??14.54±3.77**
??SEQ.ID?NO：8	??17.54±2.69*	??19.62±9.72*	??18.37±12.15**	??15.81±12.52	??12.27±6.91***
						??SEQ.ID?NO：12	??19.45±10.17	??21.12±15.40*	??18.71±18.57**	??18.84±15.64*	??17.57±15.21*
??SEQ.ID?NO：15	??16.61±2.27*	??17.40±3.94*	??16.96±6.34***	??14.15±6.51***	??13.30±6.53***
						??SEQ.ID?NO：19	??20.15±10.84	??18.86±11.54	??17.24±11.34***	??15.95±13.94**	??11.71±11.05***
??SEQ.ID?NO：24	??17.89±8.74*	??18.45±12.46*	??19.19±21.68*	??18.45±22.44**	??17.82±8.31*
						Positive blank	??22.1±5.54	??22.76±4.41	??22.53±3.39	??21.83±1.33	??20.35±0.45
N1,N1-Dimethylbiguanide	??22.75±8.57	??20.45±7.00	??18.88±2.20	??17.71±5.31	??15.77±5.25
						Negative blank	??7.43±0.58	??8.50±0.62	??7.38±0.90	??7.18±2.26	??6.51±1.49

n＝6，x±s. ^*P＜0.05， ^**P＜0.01， ^***P＜0.001?vs?saline

Sequence table

＜110〉China Medicine University

＜120〉microwave promotes solid phase synthesis balsam pear MC-JJ62 polypeptide analog and application thereof

<160>11

<210>1

<211>116

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(1)..(1)

＜223〉the 1st Xaa is Lys, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

<220>

＜221〉synthetic construct

<222>(2)..(2)

＜223〉the 2nd Xaa is Thr, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

<220>

＜221〉synthetic construct

<222>(3)..(3)

＜223〉the 3rd Xaa is Asn, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

<220>

＜221〉synthetic construct

<222>(4)..(4)

＜223〉the 4th Xaa is Ala or Gly

<400>1

<210>2

<211>116

<212>PRT

＜213〉artificial sequence

<220>

＜221〉synthetic construct

<222>(1)..(1)

＜223〉the 1st Xaa is D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met, D-Cys or Lys;

<400>2

(D-Lys)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>3

<211>11

<212>PRT

＜213〉artificial sequence

<400>3

(D-Ala)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>4

<211>11

<212>PRT

＜213〉artificial sequence

<400>4

(D-Val)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>5

<211>11

<212>PRT

＜213〉artificial sequence

<400>5

(D-Leu)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>6

<211>11

<212>PRT

＜213〉artificial sequence

<400>6

(D-Ile)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>7

<211>11

<212>PRT

＜213〉artificial sequence

<400>7

(D-Phe)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>8

<211>11

<212>PRT

＜213〉artificial sequence

<400>8

(D-Pro)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>9

<211>11

<212>PRT

＜213〉artificial sequence

<400>9

(D-Met)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>10

<211>11

<212>PRT

＜213〉artificial sequence

<400>10

(D-Cys)Thr?Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>11

<211>11

<212>PRT

＜213〉artificial sequence

<400>11

Lys(D-Ala)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>12

<211>11

<212>PRT

＜213〉artificial sequence

<400>12

Lys(D-Val)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>13

<211>11

<212>PRT

＜213〉artificial sequence

<400>13

Lys(D-Leu)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>14

<211>11

<212>PRT

＜213〉artificial sequence

<400>14

Lys(D-Ile)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>15

<211>11

<212>PRT

＜213〉artificial sequence

<400>15

Lys(D-Phe)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>16

<211>11

<212>PRT

＜213〉artificial sequence

<400>16

Lys(D-Pro)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>17

<211>11

<212>PRT

＜213〉artificial sequence

<400>17

Lys(D-Met)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>18

<211>11

<212>PRT

＜213〉artificial sequence

<400>18

Lys(D-Cys)Asn?Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>19

<211>11

<212>PRT

＜213〉artificial sequence

<400>19

Lys?Thr(D-Ala)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>20

<211>11

<212>PRT

＜213〉artificial sequence

<400>20

Lys?Thr(D-Val)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>21

<211>11

<212>PRT

＜213〉artificial sequence

<400>21

Lys?Thr(D-Leu)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>22

<211>11

<212>PRT

＜213〉artificial sequence

<400>22

Lys?Thr(D-Ile)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>23

<211>11

<212>PRT

＜213〉artificial sequence

<400>23

Lys?Thr(D-Phe)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>24

<211>11

<212>PRT

＜213〉artificial sequence

<400>24

Lys?Thr(D-Pro)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>25

<211>11

<212>PRT

＜213〉artificial sequence

<400>25

Lys?Thr(D-Met)Met?Lys?Gly?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>26

<211>11

<212>PRT

＜213〉artificial sequence

<400>26

Lys?Thr(D-Cys)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>27

<211>11

<212>PRT

＜213〉artificial sequence

<400>27

(D-Lys)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>28

<211>11

<212>PRT

＜213〉artificial sequence

<400>28

(D-Ala)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>29

<211>11

<212>PRT

＜213〉artificial sequence

<400>29

(D-Val)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>30

<211>11

<212>PRT

＜213〉artificial sequence

<400>30

(D-Leu)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>31

<211>11

<212>PRT

＜213〉artificial sequence

<400>31

(D-Ile)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>32

<211>11

<212>PRT

＜213〉artificial sequence

<400>32

(D-Phe)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>33

<211>11

<212>PRT

＜213〉artificial sequence

<400>33

(D-Pro)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>34

<211>11

<212>PRT

＜213〉artificial sequence

<400>34

(D-Met)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>35

<211>11

<212>PRT

＜213〉artificial sequence

<400>35

(D-Cys)Thr?Ans?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1??????????????????5???????????????????10

<210>36

<211>11

<212>PRT

＜213〉artificial sequence

<400>36

Lys(D-Ala)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>37

<211>11

<212>PRT

＜213〉artificial sequence

<400>37

Lys(D-Val)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>38

<211>11

<212>PRT

＜213〉artificial sequence

<400>38

Lys(D-Leu)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>39

<211>11

<212>PRT

＜213〉artificial sequence

<400>39

Lys(D-Ile)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>40

<211>11

<212>PRT

＜213〉artificial sequence

<400>40

Lys(D-Phe)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>41

<211>11

<212>PRT

＜213〉artificial sequence

<400>41

Lys(D-Pro)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>42

<211>11

<212>PRT

＜213〉artificial sequence

<400>42

Lys(D-Met)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>43

<211>11

<212>PRT

＜213〉artificial sequence

<400>43

Lys(D-Cys)Asn?Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>44

<211>11

<212>PRT

＜213〉artificial sequence

<400>44

Lys?Thr(D-Ala)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>45

<211>11

<212>PRT

＜213〉artificial sequence

<400>45

Lys?Thr(D-Val)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>46

<211>11

<212>PRT

＜213〉artificial sequence

<400>46

Lys?Thr(D-Leu)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>47

<211>11

<212>PRT

＜213〉artificial sequence

<400>47

Lys?Thr(D-Ile)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>48

<211>11

<212>PRT

＜213〉artificial sequence

<400>48

Lys?Thr(D-Phe)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>49

<211>11

<212>PRT

＜213〉artificial sequence

<400>49

Lys?Thr(D-Pro)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>50

<211>11

<212>PRT

＜213〉artificial sequence

<400>50

Lys?Thr(D-Met)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>51

<211>11

<212>PRT

＜213〉artificial sequence

<400>51

Lys?Thr(D-Cys)Met?Lys?Ala?Met?Ala?Gly?Ala?Ala

1?????????????????5???????????????????10

<210>52

<211>11

<212>PRT

＜213〉artificial sequence

<400>52

Lys?Thr?Gly?Met?Lys(D-His)Met?Ala?Gly?Ala?Ala

1???????????????5???????????????????10

<210>53

<211>11

<212>PRT

＜213〉artificial sequence

<400>53

Lys?Thr?Gly?Met?Lys(D-Lys)Met?Ala?Gly?Ala?Ala

1???????????????5???????????????????10

<210>54

<211>11

<212>PRT

＜213〉artificial sequence

<400>54

Lys?Thr?Gly?Met?Lys(D-Arg)Met?Ala?Gly?Ala?Ala

1???????????????5???????????????????10

<210>55

<211>11

<212>PRT

＜213〉artificial sequence

<400>55

Lys?Thr?Gly?Met?Lys(D-Met)Met?Ala?Gly?Ala?Ala

1???????????????5???????????????????10

Claims (8)

1. balsam pear MC-JJ62 polypeptide analog that contains formula I (SEQ.ID NO:1) structure with hypoglycemic activity, the balsam pear MC-JJ62 polypeptide analog that obtains; It is characterized in that its structure has following form:

Xaa ₁-Xaa ₂-Xaa ₃-Met-Lys-Xaa ₄-Met-Ala-Gly-Ala-Ala(SEQ.ID?NO：1)

Xaa ₁: Lys, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

Xaa ₂: Thr, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

Xaa ₃: Asn, D-Lys, D-Ala, D-Val, D-Leu, D-Ile D-Phe, D-Pro, D-Met or D-Cys;

Xaa ₄: Ala or Gly.

2. polypeptide according to claim 1 has following sequence:

( D-Lys)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：2)

( D-Ala)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：3)

( D-Val)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：4)

( D-Leu)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：5)

( D-Ile)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：6)

( D-Phe)Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：7)

( D-Pro)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：8)

( D-Met)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：9)

( D-Cys)-Thr-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：10)

Lys-( D-Ala)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：11)

Lys-( D-Val)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：12)

Lys-( D-Leu)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：13)

Lys-( D-Ile)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：14)

Lys-( D-Phe)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：15)

Lys-( D-Pro)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：16)

Lys-( D-Met)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：17)

Lys-( D-Cys)-Asn-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：18)

Lys-Thr-( D-Ala)-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：19)

Lys-Thr-( D-Val)-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：20)

Lys-Thr-( D-Leu)-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：21)

Lys-Thr-( D-Ile)-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：22)

Lys-Thr-( D-Phe)-Met-Lys- Gly-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：23)

Lys-Thr-( D-Pro)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：24)

Lys-Thr-( D-Met)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：25)

Lys-Thr-( D-Cys)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：26)

( D-Lys)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：27)

( D-Ala)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：28)

( D-Val)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：29)

( D-Leu)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：30)

( D-Ile)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：31)

( D-Phe)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：32)

( D-Pro)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：33)

( D-Met)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：34)

( D-Cys)Thr-Ans-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala????(SEQ.ID?NO：35)

Lys-( D-Ala)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：36)

Lys-( D-Val)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：37)

Lys-( D-Leu)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：38)

Lys-( D-Ile)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：39)

Lys-( D-Phe)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：40)

Lys-( D-Pro)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：41)

Lys-( D-Met)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：42)

Lys-( D-Cys)-Asn-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：43)

Lys-Thr-( D-Ala)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：44)

Lys-Thr-( D-Val)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：45)

Lys-Thr-( D-Leu)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：46)

Lys-Thr-( D-Ile)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：47)

Lys-Thr-( D-Phe)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：48)

Lys-Thr-( D-Pro)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：49)

Lys-Thr-( D-Met)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：50)

Lys-Thr-( D-Cys)-Met-Lys- Ala-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：51)

Lys-Thr- Gly-Met-Lys-( D-His)-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：52)

Lys-Thr- Gly-Met-Lys-( D-Lys)-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：53)

Lys-Thr- Gly-Met-Lys-( D-Arg)-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：54)

Lys-Thr- Gly-Met-Lys-( D-Met)-Met-Ala-Gly-Ala-Ala???(SEQ.ID?NO：55)。

3. a pharmaceutical composition comprises the MC-JJ62 polypeptide analog described in the claim 1 for the treatment of significant quantity or its pharmacy acceptable salt and pharmaceutically acceptable carrier or thinner.

4. the MC-JJ62 polypeptide analog described in the claim 1 or its pharmacy acceptable salt and pharmaceutically acceptable carrier or thinner are used for the treatment of utilization in the medicine of diabetes in preparation.

5. the preparation method of the MC-JJ62 polypeptide analog described in the claim 1, it is characterized in that: adopt microwave to promote Fmoc/tBu orthogonally protect solid phase synthesis strategy, earlier syntheticly on solid phase carrier obtain being loaded with first Fmoc and protect amino acid whose resin, ninhydrin method detects sloughs the resin that the Fmoc protecting group obtains being loaded with first amino-acid residue after negative; Enter next coupling circulation again; repeat the step of coupling and deprotection with different protection amino acid according to corresponding peptide preface; prolong required aminoacid sequence successively, the synthetic resin that obtains being loaded with corresponding polypeptide cuts down polypeptide with cracking agent at last and obtains the polypeptide crude product from resin.Crude product is through the preparation scale high-efficient liquid phase chromatogram purification, and freeze-drying gets the pure product of polypeptide.

6. promote solid phase synthesis MC-JJ62 polypeptide analog method according to microwave described in the claim 5; it is characterized in that being loaded with the preparation method that first Fmoc protects amino acid whose resin; be by obtaining with the solid phase carrier coupling again after the activated dose of activation earlier of Fmoc protection amino acid under the microwave irradiation condition; add 1-hydroxyl-benzotriazole (HOBT) or derivatives thereof in the reaction and suppress racemization, and use the acidity of organic bases neutralization reaction.

7. according to claim 6ly be loaded with the preparation method that first Fmoc protects amino acid whose resin, it is characterized in that the solid phase carrier that uses is Rink resin, Wang resin or 2-chlorine trityl chloride resin; The activator that uses is dicyclohexylcarbodiimide (DCC), N, N '-DIC (DIC), N, N " carbonyl dimidazoles (CDI), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), 2-(7-azo benzotriazole)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HATU), benzotriazole-N; N; N ', N '-tetramethyl-urea phosphofluoric acid ester (HBTU) or 6-chlorobenzene and triazole-1,1,3,3-tetramethyl-urea phosphofluoric acid ester (HCTU); 1-hydroxyl-benzotriazole (HOBT) derivative that uses is N-hydroxy-succinamide (HOSU), 1-hydroxyl-7-azo benzotriazole (HOAT) or 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone (HOOBT); The organic bases that uses is triethylamine (TEA), N-methylmorpholine (NMM) or diisopropylethylamine (DIEA); Microwave promotion condition is: magnetron frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 5～15min.

8. promote solid phase synthesis MC-JJ62 polypeptide analog synthetic method according to microwave described in the claim 5, it is characterized in that removing of Fmoc protecting group is to contain 0.1mol.L by use ^-1Hexahydropyridine solution reaction under microwave promotes of 1-hydroxyl-benzotriazole (HOBT), selecting dimethyl formamide (DMF), methyl-sulphoxide (DMSO) or N-Methyl pyrrolidone (NMP) for use is reaction solvent; Microwave promotion condition is: magnetron frequency 2450MHz, temperature of reaction: 20～100 ℃, the reaction times is 1～10min.