A kind of GLP-1 derivative DLG3312 and solid-state chemical reaction method method thereof
Technical field
The invention belongs to biology, technical field of chemical engineering, relate to one particularly and there is the active GLP-1 derivative DLG3312 of human glucagon-like-peptide-l (GLP-1) and solid-state chemical reaction method method thereof.
Background technology
Nineteen twenty-nine, that-class is separated by Zunz and Labbare from enteron aisle, that glucose stimulated insulin can be promoted to secrete humoral factor called after intestines hypoglycemic element (incretin), GLP-1 is main component wherein.GLP-1 is that it secretes after food intake by the product of protogene before the hyperglycemic-glycogenolytic factor of enteron aisle L emiocytosis.Main in body exist two kinds of biologically active forms: GLP-1(7 ~ 36) amide and GLP-1(7 ~ 37), the circulation of GLP-1 about 80% is active in the former.GLP-1, as a kind of hormone, reduces postprandial blood sugar by stimulation excreting insulin, simultaneously the secretion of glucagon suppression.Further, its have suppress gi tract food empty the absorption with food, in addition, it can cause the propagation of beta Cell of islet.
GLP-1 is applicable to the treatment of type-II diabetes, it falls hypoglycemic effect is that blood sugar is dependent, when blood sugar concentration is higher than 6mmol/L, GLP-1 significantly promotes insulin secretion, once blood sugar recovery is to normal value, no longer continue to play blood sugar reducing function, so there will not be hypoglycemia in medication process.By long-term treatment, effectively can improve the glycolated hemoglobin amount of type-II diabetes patient, the prevention and therapy of type-II diabetes is had a good application prospect.
In actual applications, according to previous investigation, it is 2 minutes that GLP-1 enters the transformation period after in body, and its reason is that multiple protein enzyme in body is to the degraded of GLP-1, and current DPP-4 is the enzyme of wherein main concern.For resisting the degraded of this enzyme to GLP-1, extend effective drug duration in vivo, existing multiple method, as amino acid whose rite-directed mutagenesis, molecular modification etc.As CJC-1131 makes GLP-1 Increased Plasma Half-life to 18 hour, it utilizes linkers to be connected with plasma proteins by polypeptide by chemosynthesis and prolong half-life greatly; LY315902 connects 1 8C aliphatic chain on GLP-1, is 3-6 hour at dog Half-life in vivo; Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide), its Methionin place of 26 in GLP-1 connects 1 16C fatty acid side chain, and sports Methionin by 34, is 8 hours at people's Half-life in vivo; GLP-1 after PEG modifies is than natural GLP-1 length plasma half-life 40 times etc.
Although can extend effective drug duration by carrying out transformation to the sequence of GLP-1, because the homology of improved molecule and natural GLP-1 is low, differ greatly the side effect caused in various degree, as allergy, vomiting, gastrointestinal upset etc.
In prior art, the existing DNA recombinant technology that adopts prepares GLP-1 derivative, name is called in " a kind of human glucagon-like-peptide-1 derivative and Synthesis and applications thereof " patent of invention (200610024355.X) to there is following shortcoming: utilize the Host Strains that intestinal bacteria are expressed as GLP-1 derivative, in subsequent purification process, need the residual quantity of detection and control escherichia coli endotoxin and e. coli dna, add production stage and cost.
GLP-1 has two kinds of forms in vivo, and one is GLP-1 (7-36) amide, is made up of 30 amino-acid residues, and another kind is GLP-1 (7-37), and be made up of 31 amino-acid residues, the two all has biologic activity.The GLP-1 that the present invention relates to refers to GLP-1 (7-37), and its sequence is as follows: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.
Present invention improves over GLP-1 sequence and structure, propose a kind of GLP-1 derivative DLG3312 and solid-state chemical reaction method method thereof.The GLP-1 derivative DLG3312 that the present invention proposes solves short problem of natural GLP-1 activity in vivo time, and it is active to have good biological, its reason is mainly: in the structure X-Y-X of GLP-1 derivative DLG3312 of the present invention, the mode of connection of X and Y is that the carbon teminal carboxyl of 2 X is connected with 2 amino condensations of Y respectively, forms the U-shaped homodimer of the ad hoc structure be made up of 2 X and 1 Y.In the present invention, the avidity of dimer and acceptor improves a lot relative to monomer, thus can more effectively activated receptor; Comprise the amino acid of sudden change in dimer, meanwhile, dimeric structure in itself, can hinder and multiple enzyme and its combination in body, thus reduce the degradation rate to it; Dimer, compared with monomer, has relatively large molecular weight, relatively extends the time of removing it because of renal glomerular filtration thus, and making it the residence time in vivo increases, prolongation effective drug duration.Further, in security, it also avoid the side effect that the current multiple diabetes medicament used has, as caused hypoglycemia, body weight to increase, causing insulin resistant, beta Cell of islet exhaustion etc.Contrary, DLG3312 of the present invention has the function improved insulin resistant and suppress islet beta-cell apoptosis, from the Sugar metabolism ability improving diabetic at all.In addition, prior art is prepared in GLP-1 derivative method and is had that step is many, high in cost of production defect, and the preparation method that the present invention proposes has the advantages such as easy and simple to handle, with low cost, has larger application potential.
Summary of the invention
The present invention proposes a kind of GLP-1 derivative DLG3312, comprise its tautomer, solvate and pharmacological-acceptable salt; The structure of described GLP-1 derivative DLG3312 is X-Y-X; Wherein, the sequence that X represents is H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: X8 is any one in A, G, dA or V, and dA represents D-Ala; Y represents diamino monocarboxylic acid, comprises Lys, Orn; Described GLP-1 derivative DLG3312 connects into homodimer by the carbon teminal carboxyl of described X and the amino condensation of described Y.
Wherein,
When X8 is A, then sequence X is SeqIDNo.1;
When X8 is G, then sequence X is SeqIDNo.2;
When X8 is dA, then sequence X is SeqIDNo.3;
When X8 is V, then sequence X is SeqIDNo.4.
Wherein,
When sequence X is SeqIDNo.1, when Y is Lys, described GLP-1 derivative is for shown in the following structural formula of DLG3312-1 (1); When Y is Orn, described GLP-1 derivative is for shown in the following structural formula of DLG3312-5 (5):
When sequence X is SeqIDNo.2, when Y is Lys, described GLP-1 derivative is for shown in the following structural formula of DLG3312-2 (2), and when Y is Orn, described GLP-1 derivative is for shown in the following structural formula of DLG3312-6 (6):
When sequence X is SeqIDNo.3, when Y is Lys, described GLP-1 derivative is for shown in the following structural formula of DLG3312-3 (3), and when Y is Orn, described GLP-1 derivative is for shown in the following structural formula of DLG3312-7 (7):
When sequence X is SeqIDNo.4, when Y is Lys, described GLP-1 derivative is for shown in the following structural formula of DLG3312-4 (4), and when Y is Orn, described GLP-1 derivative is for shown in the following structural formula of DLG3312-8 (8):
The invention allows for a kind of solid-state chemical reaction method method of described GLP-1 derivative DLG3312, first resin is inserted Peptide synthesizer, by the diamino monocarboxylic acid Y of band protecting group and described resin-bonded, to the amino acid monomer of protecting group be with according to described X sequence again, be arranged in Peptide synthesizer from C end to N end, the polypeptide resin of anamorphic zone Side chain protective group, then through Deprotection, cut-out resin, HPLC purifying, lyophilize, obtain described GLP-1 derivative DLG3312, wherein, the diamino monocarboxylic acid Y of described band protecting group comprises: Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Orn (Fmoc)-OH, the amino acid monomer of described band protecting group comprises: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Val-OH.
Wherein, when sequence X is SeqIDNo.1,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Ala-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-1,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Orn (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Ala-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-5.
Wherein, when sequence X is SeqIDNo.2,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Gly-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-2,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Orn (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Gly-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-6.
Wherein, when sequence X is SeqIDNo.3,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-D-Ala-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-3,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Orn (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-D-Ala-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-7.
Wherein, when sequence X is SeqIDNo.4,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Val-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-4,
The amino acid monomer of described band protecting group is followed successively by: Fmoc-L-Orn (Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Val-OH, during Fmoc-L-His (Trt)-OH, gained GLP-1 derivative is DLG3312-8.
The invention allows for the pharmaceutical composition of a kind of GLP-1 derivative DLG3312, comprise described GLP-1 derivative DLG3312 and pharmaceutically acceptable vehicle.
The invention allows for the application of GLP-1 derivative DLG3312 in the medicine preparing treatment diabetes and obesity.
GLP-1 derivative DLG3312 of the present invention has structure X-Y-X, wherein, X represents GLP-1 (7-37) and variant amino acid sequence thereof, be H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: X8 is A, G, dA to be D-Ala, V, 2-ME-A be in alpha-Me-Ala, S and L any one amino acid; Y represents a kind of carboxylic acid of diamino, is: Lys or Orn or 2,4-diaminobutyric acid or diaminopimelic acid.In the structure X-Y-X of GLP-1 derivative DLG3312 of the present invention, the mode of connection of X and Y is that the carbon teminal carboxyl of 2 X is connected with 2 amino condensations of Y respectively, forms the U-shaped homodimer of the ad hoc structure be made up of 2 X and 1 Y.The present invention proposes the U-shaped homodimer of node configuration of innovation, its biologic activity, action time are all created to the effect of enhancing, its major cause is: the avidity of dimer and acceptor is greatly improved relative to monomer, thus can more effectively activated receptor; Dimeric structure in itself, possesses the ability hindered with multiple enzyme in body and its combination, thus its degradation rate is reduced; Dimer, compared with monomer, has relatively large molecular weight, relatively extends the time of removing it because of renal glomerular filtration thus, and making it the residence time in vivo increases, prolongation effective drug duration.DLG3312 as GLP-1 acceptor agonist and retain most of biological activity of natural GLP-1.And the degradation capability of opposed body endoproteinase (particularly DPP-4) is better than natural GLP-1, be expected to solve the problem that the prolongation biologic activity time does not at present reach ideal effect.The compounds of this invention can pass through non-oral routes, as subcutaneous or muscle, and oral administration, be mainly used in the treatment of type-II diabetes.The present invention adopts polypeptide solid-state reaction method design and synthesis compound.The compounds of this invention has hypoglycemic activity in body, and for natural GLP-1, significantly enhances its stability in vivo, extends its hypoglycemic time in vivo.
The invention provides a kind of thinking of peptide derivative and the method for solid-state chemical reaction method said derivative is provided transformed.It is very ripe that solid-state chemical reaction method method is less than 40 amino acid whose little peptide techniques in preparation, have fast, the advantage such as simple purification, and there is not the problems such as endotoxin removal in subsequent processes, production stage is few, the technique of purifying is comparatively simple, production cost is low, product with stable quality, is applicable to large-scale mass production.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
The amino acid monomer and other chemical reagent etc. of band protecting group used in specification sheets and following examples; all can buy from associated companies and obtain; the experimental technique of unreceipted actual conditions, condition can carry out routinely, or be undertaken by the condition that goods supplier is advised.The plant and instrument that all embodiments all use the synthetic method in summary of the invention to specify and reagent and operate according to the step that the synthetic method in summary of the invention specifies, here just do not repeat, all embodiments only enumerate the committed step relevant with respective product.
The amino acid monomer of the band protecting group that the present invention adopts has 22, comprise: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-alpha-Me-Ala-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-2, 4-diaminobutyricacid, Fmoc-2, 6-diaminopimelicacid, Fmoc-L-Gln (Trt)-OH, Fmoc-L-Glu (OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Lys (Fmoc)-OH, Fmoc-L-Orn (Fmoc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Val-OH.
Wherein to abridge expression:
Fmoc:9-fluorenylmethyloxycarbonyl;
Boc: tertbutyloxycarbonyl, i.e. tert-butyloxycarbonyl;
Trt: trityl, i.e. trityl;
OtBu: tertiary butyl ester;
TBU: the tertiary butyl, i.e. tert-butyl;
Pbf:2,2,5,7,8-pentamethyl-chroman-6-benzenesulfonyl;
The plant and instrument that the present invention is used and reagent as follows:
Instrument: SYMPHONY type 12 channel polypeptide synthesizer, model: SYMPHONY, U.S.'s product.
Reagent: N-Methyl pyrrolidone, methylene dichloride, hexahydropyridine, methyl alcohol, Dimethylamino pyridine and Dimethylaminopyridinel/DMF, DIPEA and N, N-diisopropylethylamine/NMP, HBTUl00mmol/0.5MHOBTinDMF, N, N-dicyclohexylcarbodiimide and N, N-Dicyclohexylcarbodiimide/NMP
Wherein: DMF is DMF;
NMP is N-Methyl pyrrolidone;
HOBT is I-hydroxybenzotriazole;
HBTU is 2-(1 hydrogen benzotriazole base)-tetramethyl-urea hexafluorophosphate, i.e. 2-(lH-benzotriazol-l-yl)-l, l, 3,3-tetramethyluroniumhexafluorophosphate.
The solid-state chemical reaction method method of GLP-1 derivative DLG3312 of the present invention comprises the following steps:
The synthesis of polypeptide resin: for 0.25mmol scale, take Wang resin 0.25g, insert in the reactor on SYMPHONY type 12 channel polypeptide synthesizer, the diamino monocarboxylic acid of band protecting group is Y in (I), that is: Fmoc-L-Lys (Fmoc)-OH or Fmoc-L-Orn (Fmoc)-OH or Fmoc-2, 6-diaminopimelicacid or Fmoc-L-2, 4-diaminobutyricacid takes 1mmol bottling, by the carboxyl of Y and resin-bonded, and then the amino acid monomer of band protecting group is taken 1mmol bottling, by SeqIDNo.1, SeqIDNo.2, SeqIDNo.3, or the aminoacid sequence of the X of SeqIDNo.4, according to being arranged in described synthesizer from C-end to N-end, at 25 DEG C, controlled automatically to carry out de-Fmoc protection by computer program, activation, connect, then next round circulation is carried out again, so complete synthesis, obtain the polypeptide resin being with Side chain protective group, described synthesizer dries up, weigh.
Deprotection and cut-out resin: the polypeptide resin of the band Side chain protective group the first step obtained is placed in tool plug Erlenmeyer flask, adds following lytic reagent: 0.25ml water, 0.25mlEDT and l, 2-dithioglycol, 1mlTIS i.e. three second propyl silanes, 9.45ml trifluoroacetic acid, then 2 hours are reacted in 30 DEG C of induction stirring, filter, collect filtrate, resin trifluoroacetic acid washs, merge and collect liquid and washings, add ether and produce precipitation, filter, precipitation washed with diethylether, drying, obtains crude product;
HPLC separation and purification, lyophilize: the crude product preparation HPLC obtained by second step carries out separation and purification, then through lyophilize, obtain product DLG3312.
The product DLG3312 obtained according to preparation method of the present invention contains the GLP-1 of above-mentioned proposition and the aminoacid sequence of derivative thereof.
eXAMPLE l
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-1 of the present invention, and its X is SeqIDNo.1, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Lys (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Lys (Fmoc)-OH; press the aminoacid sequence of SeqIDNo.1 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-1, and its structure is as shown in the formula shown in (1).
In DLG3312-1 shown in formula (1), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Lys, be connected to form homodimer, take the shape of the letter U structure.
embodiment 2
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-2 of the present invention, and its X is SeqIDNo.2, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Lys (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Lys (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.2 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-2, and its structure is as shown in the formula shown in (2).
In DLG3312-2 shown in formula (2), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Lys, be connected to form homodimer, take the shape of the letter U structure.
embodiment 3
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-3 of the present invention, and its X is SeqIDNo.3, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Lys (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Lys (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.3 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-D-Ala-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-3, and its structure is as shown in the formula shown in (3).
In DLG3312-3 shown in formula (3), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Lys, be connected to form homodimer, take the shape of the letter U structure.
embodiment 4
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-4 of the present invention, and its X is SeqIDNo.4, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Lys (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Lys (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.4 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Val-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-4, and its structure is as shown in the formula shown in (4).
In DLG3312-4 shown in formula (4), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Lys, be connected to form homodimer, take the shape of the letter U structure.
embodiment 5
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-5 of the present invention, and its X is SeqIDNo.1, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Orn (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Orn (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.1 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-5, and its structure is as shown in the formula shown in (5).
In DLG3312-5 shown in formula (5), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Orn, be connected to form homodimer, take the shape of the letter U structure.
embodiment 6
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-6 of the present invention, and its X is SeqIDNo.2, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Orn (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Orn (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.2 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-6, and its structure is as shown in the formula shown in (6).
In DLG3312-6 shown in formula (6), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Orn, be connected to form homodimer, take the shape of the letter U structure.
embodiment 7
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-7 of the present invention, and its X is SeqIDNo.3, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Orn (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Orn (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.3 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-D-Ala-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-7, and its structure is as shown in the formula shown in (7).
In DLG3312-7 shown in formula (7), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Orn, be connected to form homodimer, take the shape of the letter U structure.
embodiment 8
The present embodiment solid-state chemical reaction method method synthesizes DLG3312-8 of the present invention, and its X is SeqIDNo.4, and the part operation steps identical with aforesaid method repeats no more.
In the present embodiment; Fmoc-L-Orn (Fmoc)-OH is taken 1mmol bottling; make carboxyl and the resin-bonded of Fmoc-L-Orn (Fmoc)-OH; press the aminoacid sequence in SeqIDNo.4 again; be arranged in SYMPHONY type 12 channel polypeptide synthesizer from C-end to N-end, the amino acid monomer of band protecting group by synthesis addition sequence is:
Fmoc-L-Gly-OH、Fmoc-L-Arg(Pbf)-OH、Fmoc-L-Gly-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Val-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Ala-OH、Fmoc-L-Ile-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Lys(Boc)-OH、Fmoc-L-Ala-OH、Fmoc-L-Ala-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Val-OH、Fmoc-L-Asp(OtBu)-OH、Fmoc-L-Ser(tBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Phe-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Gly-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Val-OH、Fmoc-L-His(Trt)-OH。
The present embodiment prepares DLG3312-8, and its structure is as shown in the formula shown in (8).
In DLG3312-8 shown in formula (8), the carbon teminal carboxyl on 2 X respectively with 2 amino condensation reactions of Orn, be connected to form homodimer, take the shape of the letter U structure.
embodiment 9
Experiment material and method:
Female Sexual health kunming mice (cleaning grade, Shanghai Si Laike provides);
35.67% glucose solution;
0.9%NaCl solution;
GLP-1;
DLG3312 is the preparation-obtained product of embodiment 1-8: DLG3312-1, DLG3312-2, DLG3312-3, DLG3312-4, DLG3312-5, DLG3312-6, DLG3312-7, DLG3312-8;
Blood glucose monitoring system (Shanghai Xin Li Medical Devices Co., Ltd. product).
About 30g female Sexual health kunming mice overnight fasting, is divided into 10 groups (n=8).L, glucose control group; 2, GLP-1 administration control group; 3 ~ 10, DLG3312 administration group, in the present embodiment, DLG3312 used is the product that embodiment 1-8 prepares.GLP-1 administration control group 2 presses the GLP-1 of 18mmol/kg body weight abdominal injection glucose solution and 0.337mg/kg body weight, DLG3312 administration group 3 ~ 10, often organize the different DLG3312 respectively by 18mmol/kg body weight abdominal injection glucose solution and 0.337mg/kg body weight, now engraving was zero moment.Carry out mouse tail vein respectively at 30min, 2h, 6h, 9h and get blood about 10 μ l, measure blood sugar concentration with blood glucose monitoring system.For observing the hypoglycemic activity of GLP-1 and DLG3312 for a long time, need again press 18mmol/kg body weight abdominal injection glucose solution, to check the hypoglycemic activity of GLP-1 and DLG3312 in 1.5h, 5.5h and 8.5.Glucose control group only presses 18mmol/kg body weight abdominal injection glucose solution, does not give GLP-1 or DLG3312, by same time measuring space blood sugar.Hypoglycemic rate calculates as follows: hypoglycemic rate (%)=(glucose control group blood glucose value-administration group blood glucose value)/glucose control group blood glucose value × 100%.
Result is as shown in table l, and in table, numerical value is the hypoglycemic rate of DLG3312-1, DLG3312-2, DLG3312-3, DLG3312-4, DLG3312-5, DLG3312-6, DLG3312-7, DLG3312-8, and shown numerical value is the average of n=8.Compared with glucose group mouse, upon administration within 30 minutes, DLG3312 administration group and GLP-1 administration group can reduce mouse blood sugar, and DLG3312 administration group still can reduce mouse blood sugar in 180 minutes, duration of efficacy is 4 ~ 5 times of GLP-1, and can reach 11 hours.Thus, show that the DLG3312 preparing gained by the inventive method all shows the hypoglycemic activity being better than natural GLP-l, and more natural GLP-1 has more long-acting hypoglycemic activity.In addition, when in Mice Body, blood sugar reaches a certain low value, DLG3312 does not demonstrate hypoglycemic activity, shows that DLG3312 can not cause hypoglycemia, is used for the treatment of diabetes and possesses security.
embodiment 10
Comprise the composition of DLG3312
Get the DLG3312 that 1mg solid-state chemical reaction method legal system is standby, be dissolved in 0.5ml deionized water; Separately get 50mg N.F,USP MANNITOL, be dissolved in 0.5ml deionized water; The two mixing is the composition of DLG3312 and N.F,USP MANNITOL, wherein containing the N.F,USP MANNITOL of DLG3312 and 50mg/ml of 1mg/ml.The composition containing DLG3312 prepared by the present embodiment is used for, in the hypoglycemic experiment of embodiment 9, obtaining same result, shows that the composition containing DLG3312 has good hypoglycemic activity and longer effective drug duration.
Above-mentioned embodiment is intended to lock and states preferred forms of the present invention instead of limit the present invention in any form.Those skilled in the art are according to enlightenment of the present invention, and the various changes that the general knowledge in conjunction with this area is done, all drop in the scope of patent application claims of the present invention.
<110> East China Normal University
<120> GLP-1 derivative DLG3312 and solid-state chemical reaction method method thereof
<160>12
<210>1
<211>30
<212>PRT
<213> ethnic group (Homosapiens)
<400>1
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
<210>2
<211>30
<212>PRT
<213> artificial sequence
<400>2
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
<210>3
<211>30
<212>PRT
<213> artificial sequence
<400>3
HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
<210>4
<211>30
<212>PRT
<213> artificial sequence
<400>4
HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG