WO2020134717A1 - Glucagon analogue, preparation method therefor, and use thereof - Google Patents

Abstract

A glucagon analogue, a preparation method therefor, and a use thereof. Provided is a glucagon analogue, comprising a glucagon-like polypeptide fragment with a long-acting carrier cross-linked thereon. The polypeptide chain in the glucagon analogue has only 2-3 amino acids mutated on the basis of a natural glucagon sequence, and has an extremely low risk of immunogenicity. Furthermore, introduction of a C-terminal sequence of the marketed drug Exenatide (trade name Bydureon) on this basis increases safety. In addition, the glucagon analogue has extremely high GLP-1R and GCGR agonistic activity, and the glucagon analogue has significant changes in in vitro activity before and after cross-linking of a fatty acid.

Description

Glucagon analogue, preparation method and use thereof Technical field

The present invention relates to the field of biotechnology, in particular to a glucagon analog, its preparation method and use.

Background technique

Diabetes can be divided into type 1 diabetes and type 2 diabetes according to pathological characteristics. Type 1 diabetes is mainly manifested by insufficient insulin secretion and requires daily insulin injection; while type 2 diabetes is caused by the body's inability to effectively use insulin. The majority of patients with type 2 diabetes. It is estimated that approximately 80-90% of patients with type 2 diabetes are significantly obese (Center for disease control and prevention (CDC) National Diabetes Fact Sheet, 2014).

Among the protein drugs currently on the market, GLP-1R (GLP-1 receptor) agonists, such as Liraglutide (trade name), are mainly used to treat type 2 diabetes

and

), Somalutide (Semeglutide, trade name

)Wait. Liraglutide is a chemically modified GLP-1 analogue. Fatty acid (hexadecanoic acid) is connected to the 26th lysine of GLP-1 protein backbone through γGlu. The fatty acid can bind to serum albumin. Clinical It is administered once a day for two indications of hypoglycemic and weight loss. From the structural point of view, somalutide is that Aib at the 8th position of the GLP-1 (7-37) chain replaces Ala, Arg at the 34 position replaces Lys, and Lys at the 26 position is connected to the octadecane fatty acid chain. Compared with liraglutide, somalutide has a longer fatty acid chain and a higher affinity for serum albumin. It is clinically injected subcutaneously once a week.

Diabetics are generally obese, and weight loss significantly improves diabetes. Therefore, for GLP-1 analogs, weight loss is an important indicator. Although liraglutide is approved for the treatment of obesity, its weight loss is actually only 5.6 kg. The clinical average weight loss of Somatoglutide (0.5 mg) and Somalutide (1.0 mg) treatment groups was 4.2 kg and 5.5 kg. Current weight loss drugs for obesity are generally around 5–10% (compared to placebo), that is, the overall average weight loss rate does not exceed 10% of the patient’s weight (Rudolph L. Leibel et al., Biologic Responses to Weight Loss and Weight Regain: Report From American Diabetes Association Research Symposium, Diabetes, 64(7): 2299-2309, 2015). Therefore, the weight loss effect of such GLP-1 analogues needs to be improved.

Summary of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a glucagon analog, its preparation method and use, for solving the problems in the prior art.

In order to achieve the above object and other related objects, the present invention provides an aspect to provide a glucagon analog, including a glucagon-like polypeptide fragment, the glucagon-like polypeptide fragment is:

a) The polypeptide fragment whose amino acid sequence is shown in SEQ ID No. 3;

HSQGT FTSDX ₁₀ SKYX ₁₄ D X ₁₆ X ₁₇ AAX ₂₀ DFVX ₂₄ W LMNGG PSSGA PPPSX ₄₀ (SEQ ID No. 3)

X ₁₀ = Y, K or C, X ₁₄ = L, K or C, X ₁₆ = S or E, X ₁₇ = Q or E, X ₂₀ = Q, K or C, X ₂₄ = Q, K or C, X ₄₀ is K, C, or deletion, and at least one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ , X ₄₀ is K or C;

Or, b) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity with SEQ ID NO. 3 and having the function of the polypeptide fragment defined in a);

A long-acting carrier is cross-linked on the glucagon-like polypeptide fragment.

In some embodiments of the present invention, one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ and X ₄₀ is K or C.

In some embodiments of the invention, the long-acting carrier is selected from fatty acids, fatty chains or PEG.

In some embodiments of the invention, amino acid residue K and/or amino acid residue C are cross-linked with a long-acting carrier.

In some embodiments of the invention, the C-terminus of the glucagon-like polypeptide is amidated.

In some embodiments of the invention, the glucagon analog is artificially designed.

In some embodiments of the present invention, the fatty acid is selected from C8 to C30 fatty acids.

In some embodiments of the present invention, the fatty acid is a monocarboxylic acid and/or dicarboxylic acid.

In some embodiments of the invention, the fatty acid is linear.

In some embodiments of the present invention, the fatty acid is cross-linked to form a fatty acid group, and the fatty acid group is selected from the groups having the chemical structural formula as follows:

In some embodiments of the present invention, a linker is provided between the glucagon polypeptide fragment and the long-acting carrier.

In some embodiments of the invention, the linker is selected from -Abu-(-L-2-aminobutyryl-), -GABA-(-γ-aminobutyryl-), -EACA-(-ε-aminocaproyl -), -β-Ala-(-β-alanyl-), -γGlu-(-γ-glutamyl), -D-γGlu-(-D-γ-glutamyl-) or its dipeptide , Such as -β-Ala-β-Ala-, -γGluγGlu- and its stereoisomeric forms (S and R enantiomers), -5-Aminopentanoyl-(-5-aminopentanoyl-), -8-Aminooctanoyl -(-ω-aminooctanoyl-), -9-Aminononanoyl-(-9-aminononanoyl-), -10-Aminodecanoyl-(-10-amino-n-decanoyl-), -OEG-(2-(2 -(-2-aminoethoxy)ethoxy)acetyl-), -2xOEG-, -γGlu-OEG-, -γGlu-2xOEG-, -D-γGlu-2xOEG-, -2xOEG-γGlu-, -γGlu -3xOEG-, -γGlu-8xPEG-(-3-((γ-glutamine)-8x polyethylene glycol)-propionyl-), -γGlu-3xOEG-γ-Glu-8xPEG-.

In a preferred embodiment of the present invention, the glucagon analogue comprises SEQ ID No. 3 at position 10 or 14 is K; preferably, the glucagon analogue is at SEQ ID No. 3 The K at the 10-position or the 14-position is crosslinked; preferably, the linker is -γGlu-2xOEG- or -γGlu-; more preferably, the long-acting carrier is selected from C16 to C20 fatty acids; most preferably Specifically, the glucagon analog is selected from any of the following sequences: SEQ ID No. 16-33, SEQ ID No. 49-52, SEQ ID No. 57-60, SEQ ID No. 65-68, SEQ ID No. 73-76, SEQ ID No. 96-99.

Another aspect of the present invention provides a method for preparing the glucagon analog, including: preparing the glucagon analog using a chemical synthesis method.

Another aspect of the present invention provides the use of the glucagon analogue in the preparation of a medicament for treating metabolic diseases and GCGR/GLP-1R multi-effect agonist.

In some embodiments of the present invention, the metabolic disease is selected from diabetes, dyslipidemia, nonalcoholic fatty liver disease, other metabolic syndromes associated with diabetes, high triglycerides, low HDL cholesterol and high LDL cholesterol, insulin resistance Sex, obesity or glucose intolerance.

Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the glucagon analog.

BRIEF DESCRIPTION

Figure 1 shows the results of FC382K14D21 mass spectrometry analysis;

Figure 2 shows the results of FC382K10D21 mass spectrometry analysis;

Figure 3 shows the results of FC382K20D21 mass spectrometry analysis;

Figure 4 shows the results of FC382K24D21 mass spectrometry analysis;

Figures 5-8 show the residual activity results of glucagon derivatives in vitro;

9 to 14 show the results of random blood glucose test after administration of glucagon derivatives in diabetic mice;

Figures 15-17 show the percentage of blood glucose after administration of glucagon derivatives in diabetic mice

Figure 18 shows the results of changes in ALT after administration of glucagon derivatives in NASH model mice;

Figure 19 shows the results of changes in AST after administration of glucagon derivatives in NASH model mice;

Figure 20 shows the results of changes in TG after administration of glucagon derivatives in NASH model mice;

Figure 21 shows the results of changes in HDL after glucagon derivatives were administered in NASH model mice;

Fig. 22 shows the results of NAS scoring after administration of glucagon derivatives in NASH model mice.

detailed description

The inventor of the present invention has provided a glucagon analog and further cross-linked fatty acids, fat chains or PEGs, thereby greatly enhancing the agonistic activity of the glucagon analog. In addition, due to glucagon The peptide fragment in the analog is closer to the natural Glucagon sequence, so the risk of immunogenicity is extremely low, and the present invention has been completed on this basis.

A first aspect of the present invention provides a glucagon analog, including a glucagon-like polypeptide fragment, and the glucagon-like polypeptide fragment is:

a) The polypeptide fragment whose amino acid sequence is shown in SEQ ID No. 3;

HSQGT FTSDX ₁₀ SKYX ₁₄ D X ₁₆ X ₁₇ AAX ₂₀ DFVX ₂₄ W LMNGG PSSGA PPPSX ₄₀ (SEQ ID No. 3)

Where X ₁₀ = Y, K or C, X ₁₄ = L, K or C, X ₁₆ = S or E, X ₁₇ = Q or E, X ₂₀ = Q, K or C, X ₂₄ = Q, K or C, X ₄₀ is K, C or deletion, and at least one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ , X ₄₀ is K or C;

Or, b) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity with SEQ ID NO. 3 and having the function of the polypeptide fragment defined in a);

A long-acting carrier is cross-linked on the glucagon-like polypeptide fragment.

In the glucagon analogs provided by the present invention, the glucagon-like polypeptide fragment is selected from natural Glucagon (abbreviated as GCG herein, the amino acid sequence is shown in SEQ ID NO. 1) or other analogs Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) agonistic active polypeptide fragments. The glucagon analog is artificially designed, and can generally be derived from a polypeptide fragment whose amino acid sequence is shown in SEQ ID NO.1. In order to achieve the purpose of cross-linking, amino acids K or C are introduced on the basis of the original sequences of these polypeptides, and fatty acids, fatty chains or PEG are cross-linked to K or C. For example, the Y-position at position 10 may be mutated to K or C. For example, the L-position at position 14 may be mutated to K or C. For example, the Q-position at position 20 may be mutated to K or C. For example, it may be Q at position 24 is mutated to K or C. For another example, C or K can be added to the C-terminus. At least one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ , and X ₄₀ is K or C, so that fatty acids, fatty chains, or PEG can be cross-linked to the glucagon polypeptide fragment through K or C. In some specific embodiments of the present invention, one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ and X ₄₀ is K or C.

The glucagon-like polypeptide fragment may specifically be: a) a polypeptide fragment having an amino acid sequence as shown in SEQ ID No. 3, or b) an amino acid sequence having more than 90% and more than 93% of SEQ ID NO. 3 , 95% or more, 97% or more, or 99% or more sequence identity, and a polypeptide fragment having the function of the polypeptide fragment defined in a). The amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID No. 3 is substituted, deleted or added one or more (specifically 1-50, 1-30, 1-20, 1 -10, 1-5, or 1-3) amino acids, or add one or more (specifically 1-50, 1-30, N-terminal and/or C-terminal) 1-20, 1-10, 1-5, or 1-3) amino acids, and the encoded polypeptide fragments have the polypeptide fragments encoded by the amino acid sequence shown in SEQ ID No. 3, respectively The functional amino acid sequence.

In some specific embodiments of the present invention, in the SEQ ID No. 3, X ₁₆ =S and X ₁₇ =Q.

In some specific embodiments of the present invention, in the SEQ ID No. 3, X ₁₆ =S and X ₁₇ =E.

In some specific embodiments of the present invention, in the SEQ ID No. 3, X ₁₆ =E and X ₁₇ =Q.

In some specific embodiments of the present invention, in the SEQ ID No. 3, X ₁₆ =E and X ₁₇ =E.

In some specific embodiments of the present invention, the glucagon-like polypeptide fragment may be a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID Nos. 4 to 13, the specific sequence is shown in Table 1, and Table 1 , SEQ ID NO.1 shows the amino acid sequence of glucagon.

Table 1

As described above, the C-terminal amino acid of the glucagon analog provided by the present invention may be modified, such as amidation, which generally refers to the conversion of the -COOH group at the C-terminus to the -CONH ₂ group.

The glucagon analog provided by the present invention may further include a long-acting carrier, and the long-acting carrier may play a role in prolonging the half-life of the glucagon analog in vivo, and the long-acting carrier may be It includes but is not limited to a combination of one or more of fatty acids, fatty chains and polyethylene glycol (PEG). The long-acting carrier can be cross-linked with the glucagon-like polypeptide fragment, usually through the amino acid residue K and/or amino acid residue C on the glucagon-like polypeptide fragment and the active group on the long-acting carrier ( For example, fatty acids may include reactive groups such as carboxyl groups, fatty chains and PEG may include reactive groups such as carboxyl groups, maleimides) to react to achieve cross-linking of long-acting carriers and glucagon-like polypeptide fragments, For example, it can be various types of condensation reactions.

In the glucagon analogs provided by the present invention, a linker may also be provided between the glucagon-like polypeptide fragment and the long-acting carrier. The linker can usually be separated from the lysine residue K and/or cysteine residue C on the glucagon polypeptide fragment and the active group on the long-acting carrier (for example, the linker may include a carboxyl group , Maleimide and other active groups) to react, so that the two ends of the linker are connected to the long-acting carrier and the glucagon peptide fragment, so as to achieve the cross-linking of the long-acting carrier and the glucagon peptide fragment, For example, it can be various types of condensation reactions.

The linker may be various linkers suitable for connecting glucagon-like polypeptide fragments and long-acting carriers in the art. In some specific embodiments of the present invention, the linker may include but is not limited to -Abu-(- L-2-aminobutyryl-), -GABA-(-γ-aminobutyryl-), -EACA-(-ε-aminohexanoyl-), -β-Ala-(-β-alanyl-) , -ΓGlu-(-γ-glutamyl-), -D-γGlu-(-D-γ-glutamyl-) or its dipeptide, such as -β-Ala-β-Ala-, -γGlu-γGlu -And its stereoisomeric forms (S and R enantiomers), -5-Aminopentanoyl-(-5-aminopentanoyl-), -8-Aminooctanoyl-(-ω-aminooctanoyl-), -9- Aminononanoyl-(-9-aminononanoyl-), -10-Aminodecanoyl-(-10-amino-n-decanoyl-), -OEG-(-2-(2-(2-aminoethoxy)ethoxy) Acetyl-), -2xOEG-, -γGlu-OEG-, -γGlu-2xOEG-, -D-γGlu-2xOEG-, -2xOEG-γGlu-, -γGlu-3xOEG-, -γGlu-8xPEG-(-3-( A combination of one or more of (γ-glutamine)-8x polyethylene glycol)-propionyl-), -γGlu-3xOEG-γ-Glu-8xPEG-, and the like. In some other specific embodiments of the present invention, the linker may be a group including but not limited to the chemical structural formula as shown below:

In the glucagon analogue provided by the present invention, the fatty acid may be C8-C30, C8-C12, C12-C16, C16-C20, or C20-C30 fatty acid, and the fatty acid may be monocarboxylic acid and/or Or dicarboxylic acid, the fatty acid may be linear or branched. The fatty acid may specifically include but not limited to caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), or stearic acid (C18), etc., or The corresponding dibasic acids can be, for example, including but not limited to cetyl diacid, octadecyl diacid, eicosyl diacid, behenyl diacid, etc.; the fatty chain can be It is a C8-C30, C8-C12, C12-C16, C16-C20, or C20-C30 fatty chain. The fatty chain may be linear or branched. In a specific embodiment of the present invention, the group formed by the cross-linking may include, but is not limited to, a group with the chemical structural formula shown below:

In some specific embodiments of the present invention, the glucagon analogs may be the compounds listed in Table 2:

Table 2

In Table 2, -γE- is -γGlu(-γ-glutamyl-), for example, “K(palmitoyl-γE)” means that the palmitoyl group is conjugated to the epsilon nitrogen through the -γ-glutamyl-linker Lysine. "K(((octadecanedioyl monoacyl)-γE)-2xOEG)" means that the octadecanedioic monoacyl is conjugated to the epsilon nitrogen via a linker connected to two OEG molecules via -γ-glutamyl- On the lysine. X40=C (mPEG2-maleimide) and X14=C (mPEG2-maleimide) have the structure as shown in the figure below, where the MW of mPEG2-maleimide is 40KD:

According to a second aspect of the present invention, there is provided an isolated polynucleotide encoding the aforementioned glucagon-like polypeptide fragment.

In a third aspect of the present invention, a recombinant expression vector is provided, comprising the isolated polynucleotide provided in the second aspect of the present invention.

According to a fourth aspect of the present invention, there is provided a host cell containing the recombinant polynucleotide provided in the third aspect of the present invention or the isolated polynucleotide provided in the second aspect of the present invention in which a foreign source is integrated into the genome.

A fifth aspect of the present invention provides a method for preparing a glucagon analog provided by the first aspect of the present invention, the preparation method may include: preparing the glucagon analog using a chemical synthesis method; the preparation method also The method may include: cultivating the host cell provided in the fourth aspect of the present invention under suitable conditions, expressing the glucagon-like polypeptide fragment, isolating and purifying the glucagon-like polypeptide fragment, and then removing the A long-acting carrier is chemically cross-linked to the glucagon-like polypeptide fragment. The glucagon analogs of the present invention can be prepared by standard peptide synthesis methods, for example, by standard solid-phase or liquid-phase methods, step-by-step or by fragment assembly, and isolation and purification of the final glucagon-like polypeptide fragments, pancreas Glucagon analog products, or any combination by recombinant and synthetic methods.

The sixth aspect of the present invention provides the use of the glucagon analogue in the preparation of a medicament for treating metabolic diseases and GCGR/GLP-1R multi-effect agonist. The metabolic disease may be specifically selected from diabetes, obesity, dyslipidemia, non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH), other metabolic syndromes associated with diabetes, including hypertriglyceride, Low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity or glucose intolerance.

The seventh aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the glucagon analog provided by the first aspect of the present invention.

An eighth aspect of the present invention provides a method for treating a disease, comprising the steps of: administering to a subject the glucagon analog provided by the first aspect of the present invention, or the pharmaceutical composition provided by the seventh aspect of the present invention. The researchers of the present invention have found that the glucagon analogs provided by the present invention have sufficient water solubility at neutral pH or slightly acidic pH and have improved chemical stability. In the random blood glucose test, the diabetic model mice to which part of the glucagon analogs of the present invention were administered had a blood sugar level lower than that of the control saline group and the liraglutide group after 24 hours, showing extremely stable blood sugar fluctuations . The diabetes model mice to which another part of the glucagon derivative of the present invention was administered had a blood glucose value lower than that of the control saline group and the liraglutide group after 72 hours. In addition, after administration of the glucagon derivative of the present invention in DIO mice, a significant weight loss was induced. After administration of the glucagon derivative in NASH model mice, the NAS histological score, liver TG, AST , ALT content decreased significantly, HDL content increased significantly.

Although in the prior art, research and development of molecules with multiple agonistic activities has become a research hotspot in the field and has very clinical prospects, it is actually very difficult to actually obtain an ideal drug of this type. The first is the safety issue, especially the immunogenicity issue. Hypoglycemic and weight-reducing drugs need to be used for a long time, and they have extremely high safety requirements. In order to design a polypeptide with high multiple agonistic activity and stable in vivo, the existing technical solutions often introduce more mutation sites, and often introduce unnatural amino acids and other modifications. These mutations and the introduction of unnatural amino acids increase the risk of potential immunogenicity. In general, the higher the homology with human sequences, the lower the risk of immunogenicity in humans. The GLP-1 receptor agonist, the hypoglycemic drug Taspoglutide (only 2 unnatural amino acids Aib was introduced) developed by Roche and Ipsen, has an antibody production rate of 49%. Currently, all clinical Phase III studies have been suspended ( JULIO, ROSENSTOCK, etc., The Fate of Taspoglutide, a Weekly GLP-1 Receptor Agonist, Versus Wice-Daily Exenatide for Type 2, DIABETES CARE, 36:498-504, 2013). PHIL AMBERY et al. (THEENDOCRINOLOGIST, SPRING, 2017: 12-13) screened more than 500 structures based on the sequence of GCG, and obtained a candidate peptide MEDI0382. Among them, in order to maintain high GLP-1 and GCG dual activity and in vivo stability, compared with GCG, MEDI0382 introduced 9 mutation sites, the mutation rate reached about 30%; Similarly, Andreas Evers et al. (J Med Med .2017 May 25;60(10):4293-4303) 9 mutation sites were introduced based on the structure of Exendin-4, the mutation rate reached about 23%, and the fatty acid chain modification was carried out to obtain both Hybrid peptide with higher dual activity of GLP-1 and GCG; GLP-1/GCG dual active peptide designed by Brian Finan et al. (Brian Finan et al. Nat. Med. 21:27-36, 2015) was added at the C-terminus of GCG The GPSSGAPPPS sequence was introduced, and 7 mutant amino acids were introduced, including the second mutation to the unnatural amino acid Aib. Therefore, the existing technical solutions often introduce more mutation sites, and often introduce unnatural amino acids and other modifications in order to obtain polypeptides with high activity of both GLP-1 and GCG. These mutations, modifications and the introduction of unnatural amino acids all increase the risk of potential immunogenicity. The safety of drugs for the treatment of diseases such as diabetes and obesity is extremely important. In addition, for small peptides of 30 amino acid lengths such as GLP-1 and Glucagon, sequence changes are extremely sensitive to changes in their activity; for multiple active polypeptides, due to the agonism of multiple different receptors, the changes are Even more complicated, it is impossible to predict the consequences of any amino acid change on receptor agonistic activity.

However, the polypeptide chain in the glucagon analogue provided by the present invention has only 2-3 amino acids mutated based on the natural Glucagon sequence (such as S16E, R17Q, R18A), and the risk of immunogenicity is extremely low. Based on the introduction of the C-terminal sequence of the marketed drug Exenatide (trade name Bydureon), the safety is higher. In addition, the glucagon analogues provided by the present invention have extremely high GLP-1R and GCGR agonistic activity. Surprisingly, the glucagon analogues have significant in vitro activity before and after cross-linking of fatty acids Variety.

The following describes the embodiments of the present invention through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through different specific embodiments, and the details in this specification can also be based on different viewpoints and applications, and various modifications or changes can be made without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention.

When the numerical ranges are given in the embodiments, it should be understood that unless the present invention indicates otherwise, the two endpoints of each numerical range and any one value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, the methods, equipment, and materials described in the embodiments of the present invention can also be used according to the master of the prior art and the description of the present invention by those skilled in the art. Similar or equivalent prior art methods, devices and materials are used to implement the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the technical field. Conventional technology. These technologies have been well described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989 and Third Edition, 2001; Ausubel, etc., CURRENT PROTOCOLS IN MOLECULAR & BIOLOGYs, John Wiley , New York, 1987 and periodic updates; the series METHODS INZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INZYMar, Vol. and APWolffe, eds.), Academic, Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.

The meanings of the abbreviations involved in the examples are as follows:

RT: room temperature

DMF: N,N-dimethylformamide

Fmoc: 9H-fluoren-9-ylmethoxycarbonyl

Trt: trityl

Boc: tert-butoxycarbonyl

HOBt: 1-hydroxybenzotriazole

tBu: tert-butyl

DCM: methylene chloride

DBLK: 20% N,N-dimethylformamide piperidine

DIC: N,N’-diisopropylcarbodiimide

MeOH: methanol

TFA: trifluoroacetic acid

Fmoc-Lys(Pal-Glu-OtBu)-OH: N ^α -fluorenylmethoxycarbonyl-(N ^ε -(γ-glutamyl (N ^α -hexadecyl, α-tert-butyl ester))) lysine acid

Decanoyl: Decanoyl

Stearoyl: octadecanoyl

OEG: 2-(2-(2-aminoethoxy)ethoxy)acetic acid-γGLu-:-γglutamyl-

-γGlu-: -γ-glutamyl-

PEG: polyethylene glycol

DMAP: dimethylaminopyridine

TFEA: 2,2,2-trifluoroethanol

DIEA: N,N-diisopropylethylamine

MTBE: methyl tert-butyl ether

Pd(PPh3)4: tetrakis(triphenylphosphine)palladium

Alloc: allyloxycarbonyl

As a general method, in the example, a method for preparing a glucagon derivative by mutating a specific amino acid position to K or C based on C382 (or its amidated modified polypeptide C381). Similarly, the preparation of glucagon derivatives based on other amino acid sequence polypeptides, such as C462 and C495 (or their amidated modified polypeptides), is consistent with this.

The manufacturers and commodity models of the commercially available amino acids and amino acid fragments and the commercially available resins involved in the examples are as follows:

Fmoc protecting amino acid raw materials, 2-CTC resin and Wang resin are all conventional commercially available reagents (protecting amino acid manufacturer: Chengdu Zhengyuan Biochemical Technology Co., Ltd., resin manufacturer: Tianjin Nankai Hecheng Technology Co., Ltd.);

The sources of organic solvents and other raw materials are all commercially available products (manufacturer: Sinopharm Group Chemical Reagent Co., Ltd.; chemically pure).

In addition, the conditions of HPLC and mass spectrometry, the type of equipment used and the manufacturer's description are as follows:

Instrument: HPLC UltiMate3000; detection conditions are shown in Table 3 below.

table 3

Preparation of liquid phase: Beijing Innovation Tongheng, LC3000.

Mass spectrometer: the instrument model is 5800MALDI-TOF-TOF (AB SCIEX), the analysis software is T0F/TOF Explorer, Data Explorer, MS uses Reflector Positive parameters: CID (OFF), massrang (700-6500Da) Focus Mass (1200Da) Fixed laserintensity(5600)Digitizer: BinSize(0.5ns)

Example 1

Preparation of glucagon derivative FC382K14D21:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(palmitoyl-γE) (SEQ ID NO.16)

The Fmoc-protected amino acids were purchased from Chengdu Zhengyuan Biochemical Technology Co., Ltd. The following amino acids were used in the peptide extension synthesis process: Fmoc-L-Ala-OH, Fmoc-L-Asn(Trt)-OH, Fmoc-L-Asp( OtBu)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-L-His( Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Met-OH, Fmoc-L-Phe-OH, Fmoc- L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

Fmoc-Ser(tBu)-king resin synthesis:

Weigh 12.95g of Wang resin (Tianjin Nankai Hecheng Technology Co., Ltd.) with a substitution degree of 0.58mmol/g, add it to the solid phase reaction column, add 100mL of DCM swollen resin for 30 minutes, and wash with DMF 3 times, 100mL each time . Take another 5.76g Fmoc-Ser(tBu)-OH, 2.43g HOBt and 0.19g DMAP dissolved in DMF, add 2.6mL DIC at 5-8°C and activate for 5min, then add to the above reaction column with resin and react for 16 hours . After Kaiser test was negative, DMF was washed twice, MeOH was washed twice, DCM was washed twice, and MeOH was washed twice, each time the solvent was 100 mL. The material was collected and dried under reduced pressure at room temperature to obtain 14.18 g of uncapped Fmoc-Ser(tBu)-king resin.

The above resin was added to the reaction column. After adding 100 mL of DCM and swollen for 30 minutes, it was drained and washed with DMF 3 times, 100 mL each time. Then add 100 mL of DMF and 13 mL of blocking solution to the reaction column (blocking solution is V acetic anhydride: V pyridine = 1:1), and react for 2 hours, followed by DMF washing twice, MeOH washing twice, DCM washing twice and MeOH washing 2 times, each time the solvent is 100mL. The material was collected and dried under reduced pressure at room temperature to obtain 15.26 g of Fmoc-Ser(tBu)-king resin.

Synthesis of peptide resin:

Weigh 4.48 g (1.0 mmol) of the Fmoc-Ser(tBu)-king resin after capping, add it to the reaction column and swell with 20 mL of DCM for 30 minutes, then wash with DMF 3 times, 20 mL each time. After washing, add 10mL DBLK solution (20% piperidine/DMF (V/V)) to the reaction column, react for 5 minutes, filter with suction, wash once with 20mL DMF, then add 10mL DBLK solution (20% piperidine/ DMF (V/V)), react for 10 minutes, Kaiser test is positive. Filter with suction and wash with DMF 3 times, 20 mL each time. Take another Fmoc-Pro-OH (1.69g, 5.0eq), HOBt (0.81g, 6.0eq) into 10mL DMF to dissolve, add DIC (0.69g, 5.5eq) at 5-8 ℃ to activate for 5min, add to the reaction column During the reaction for 1 hour, Kaiser test was negative, the reaction was complete, and washed with DMF 3 times, 20 mL each time. Repeat the above deprotection and coupling operations to complete the coupling of other amino acids in sequence according to the polypeptide sequence. Among them, K14 uses Fmoc-Lys (Pal-Glu-OtBu)-OH (Chengdu Zhengyuan Biochemical Technology Co., Ltd.) coupling. After the coupling of the last amino acid was completed, deprotection was performed as described above. After deprotection was complete, DMF was washed twice, MeOH was washed twice, DCM was washed twice, and MeOH was washed twice, each washing solvent was 20 mL. The material was collected and dried under reduced pressure at normal temperature to obtain 12.26 g of the target peptide resin.

Crude peptide cleavage:

Weigh 6.01g of the above peptide resin, and slowly add it to 60mL of lysate (trifluoroacetic acid: anisole: anisole: ethanedithiol=90:5:3:2) at 20-30°C, and then complete the reaction 2 hours. After the reaction was completed, the resin was removed by filtration, and the filtrate was poured into pre-cooled methyl tertiary ether (600 mL) under vigorous stirring, and the resulting mixed solution was allowed to settle in an ice bath for 2 hours. The supernatant was removed and centrifugally washed with pre-cooled methyl tertiary ether 5 times, 400 mL each time. After completion, it was collected and dried under reduced pressure at room temperature to obtain 3.00 g of crude peptide.

Crude peptide purification:

The crude peptide was purified through multi-step purification: the first step: stationary phase: C18 (Daisogel: sp-120-40/60-C18-RPS), linear gradient of mobile phase 20-60% B (mobile phase A: 0.1 %TFA, mobile phase B: acetonitrile), 40 minutes, flow rate 15mL/min, ultraviolet (UV) detection at 220nm; second step: stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase Linear gradient 20-60% B (mobile phase A: 0.5% phosphoric acid, mobile phase B: acetonitrile), 40 minutes, flow rate 15mL/min, ultraviolet (UV) detection at 220nm Freeze dryer: Freeze dryer , FD-2A.

Finally lyophilized to obtain sperm peptide (95.6%). MS: m/z 4541.29 (M+H) ⁺ .

Example 2

Preparation of glucagon derivative FC382K14W07:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(octadecanoyl-γE) (SEQ ID NO.81)

The synthesis method is the same as that in Example 1, wherein K14 is coupled by Fmoc-Lys(Stearoyl-Glu-OtBu)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the obtained crude peptide is purified by RP-HPLC, and finally obtained by lyophilization Sperm peptide (97.3%). MS: m/z 4569.02 (M+H) ⁺ .

Example 3

Preparation of glucagon derivative FC382K14W09:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(Eicosanedioyl-γE) (SEQ ID NO.82)

The synthesis method is the same as that in Example 1, in which K14 is coupled by Fmoc-Lys(N-(tBuOCO(CH ₂ ) ₁₈ CO)-Glu-OtBu)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the obtained crude peptide is used Purified by RP-HPLC, and finally lyophilized to obtain sperm peptide (96.7%). MS: m/z 4627.12 (M+H) ⁺ .

Example 4

Preparation of glucagon derivative FC382K14D17:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(decanoyl-γE) (SEQ ID NO.83)

The synthesis method is the same as that in Example 1, wherein K14 is coupled by Fmoc-Lys(Decanoyl-Glu-OtBu)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the obtained crude peptide is purified by RP-HPLC, and finally obtained by lyophilization Sperm peptide (96.1%). MS: m/z 4456.96 (M+H) ⁺ .

Example 5

Preparation of glucagon derivative FC382K14D26:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(((palmitoyl)-γE)-2xOEG) (SEQ ID NO.84)

The synthesis method is the same as in Example 1, in which K14 is coupled using Fmoc-Lys(N-(CH ₃ (CH ₂ ) ₁₄ CO)-Glu-OtBu)-OEG-OEG)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.) The crude peptide obtained was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (96.90%). MS: m/z 4831.18 (M+H) ⁺ .

Example 6

Preparation of glucagon derivative FC382K14W13:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(((octadecanoyl)-γE)-2xOEG) (SEQ ID NO.85)

The synthesis method is the same as in Example 1, in which K14 is coupled by Fmoc-Lys((N-(CH ₃ (CH ₂ ) ₁₆ CO)-Glu-OtBu)-OEG-OEG)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.) The crude peptide obtained was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (97.2%). MS: m/z 4859.20 (M+H) ⁺ .

Example 7

Preparation of glucagon derivative FC382K14W14:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(((Eicosanoyl)-γE)-OEG) (SEQ ID NO.86)

The synthesis method is the same as that in Example 1, wherein K14 is coupled by Fmoc-Lys((N-(CH3(CH2)18CO)-Glu-OtBu)-OEG)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.) The peptide was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (96.6%). MS: m/z 4742.17 (M+H) ⁺ .

Example 8

Preparation of glucagon derivative FC382K10D21:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10=K(palmitoyl-γE) (SEQ ID NO.24)

The synthesis method is the same as in Example 1, in which Y10 is replaced with K10 and coupled with Fmoc-Lys(Pal-Glu-OtBu)-OH (Chengdu Zhengyuan Biochemical Technology Co., Ltd.). The crude peptide obtained is purified by RP-HPLC, and finally Freeze-dried to obtain sperm peptide (96.0%). MS: m/z 4492.02 (M+H) ⁺ .

Example 9

Preparation of glucagon derivative FC382K10D24:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10=K(((palmitoyl)-γE)-γE) (SEQ ID NO.87)

The synthesis method is the same as in Example 1, in which L10 is replaced by K10 and coupled with Fmoc-Lys(N-(N-Pal-Glu-OtBu)-Glu-OtBu)-OH, and the resulting crude peptide is purified by RP-HPLC , And finally lyophilized to obtain sperm peptide (97.0%). MS: m/z 4620.11 (M+H) ⁺ .

Example 10

Preparation of glucagon derivative FC382K20D21:

HSQGT FTSDY SKYLD SQAAX DFVQW LMNGG PSSGA PPPS-OH

X20=K(palmitoyl-γE) (SEQ ID NO.34)

The synthesis method is the same as in Example 1, in which Q20 is replaced with K20 and coupled with Fmoc-Lys(Pal-Glu-OtBu)-OH (Chengdu Zhengyuan Biochemical Technology Co., Ltd.), the crude peptide obtained is purified by RP-HPLC, and finally Freeze-dried to obtain sperm peptide (95.8%). MS: m/z 4525.94 (M+H) ⁺ .

Example 11

Preparation of glucagon derivative FC382K20W07:

HSQGT FTSDY SKYLD SQAAX DFVQW LMNGG PSSGA PPPS-OH

X20=K(octadecanoyl-γE) (SEQ ID NO.89)

The synthesis method is the same as in Example 1, in which Q20 is replaced with K20 and coupled with Fmoc-Lys(Stearoyl-Glu-OtBu)-OH. The obtained crude peptide is purified by RP-HPLC, and finally lyophilized to obtain refined peptide. MS: m/z 4554.02 (M+H) ⁺ .

Example 12

Preparation of glucagon derivative FC382K24D21:

HSQGT FTSDY SKYLD SQAAQ DFVXW LMNGG PSSGA PPPS-OH

X24=K(palmitoyl-γE) (SEQ ID NO.39)

The synthesis method is the same as that in Example 1, in which Q24 is replaced with K24 and coupled with Fmoc-Lys(Pal-Glu-OtBu)-OH (Chengdu Zhengyuan Biochemical Technology Co., Ltd.). The resulting crude peptide is purified by RP-HPLC, and finally Freeze-dried to obtain sperm peptide (96.9%). MS: m/z 4526.12 (M+H) ⁺ .

Example 13

Preparation of glucagon derivative FC382K24W07:

HSQGT FTSDY SKYLD SQAAQ DFVXW LMNGG PSSGA PPPS-OH

X24=K(octadecanoyl-γE) (SEQ ID NO.90)

The synthesis method is the same as in Example 1, in which Q24 is replaced with K24 and coupled with Fmoc-Lys(Stearoyl-Glu-OtBu)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the resulting crude peptide is purified by RP-HPLC. Finally lyophilized to obtain sperm peptide (96.3%). MS: m/z 4554.16 (M+H) ⁺ .

Example 14 Preparation of glucagon derivative FC381K14D21:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-NH ₂

X14=K(palmitoyl-γE) (SEQ ID NO.17)

Synthesis of Fmoc-Ser(tBu)-Rink amide MBHA resin:

Weigh 3.03g of Rink amide MBHA resin (Tianjin Nankai Hecheng Technology Co., Ltd.) with a substitution degree of 0.38mmol/g, add it to the solid phase reaction column, add 10mL of DCM swollen resin for 30 minutes, wash with DMF 3 times, each 10mL. Add 15mL of DBLK solution to the reaction column, react for 5 minutes, filter with suction, wash once with 20mL of DMF, then add 15mL of DBLK solution, react for 10 minutes, Kaiser test is positive. Filter with suction and wash with DMF 3 times, 20 mL each time.

Another 2.03g Fmoc-Ser(tBu)-OH and 1.31g HOBt were dissolved in 10mL DMF, added 1mL DIC at 5-8°C and activated for 5min, then added to the above reaction column with resin and reacted for 2 hours. After Kaiser test is negative, it is directly used in the next step of peptide resin synthesis.

Synthesis of peptide resin:

Weigh the above resin Fmoc-Ser(tBu)-Rink amide MBHA resin (1.0 mmol), add it to the reaction column and swell with 20 mL DCM for 30 minutes, then wash with DMF 3 times, 20 mL each time. After washing, add 10mL DBLK solution (20% piperidine/DMF (V/V)) to the reaction column, react for 5 minutes, filter with suction, wash once with 20mL DMF, then add 10mL DBLK solution (20% piperidine/ DMF (V/V)), react for 10 minutes, Kaiser test is positive. Filter with suction and wash with DMF 3 times, 20 mL each time. Take another Fmoc-Pro-OH (1.69g, 5.0eq), HOBt (0.81g, 6.0eq) into 10mL DMF to dissolve, add DIC (0.69g, 5.5eq) at 5-8 ℃ to activate for 5min, add to the reaction column During the reaction for 1 hour, Kaiser test was negative, the reaction was complete, and washed with DMF 3 times, 20 mL each time. Repeat the above deprotection and coupling operations to complete the coupling of other amino acids in sequence according to the peptide sequence, of which K14 uses Fmoc-Lys(Pal-Glu-OtBu)-OH coupling. After the coupling of the last amino acid is completed, deprotect it according to the above deprotection method. After deprotection is complete, wash with DMF 2 times, MeOH 2 times, DCM 2 times, and MeOH 2 times, each with 20 mL of solvent. It is collected and dried under reduced pressure at normal temperature to obtain the target peptide resin.

Crude peptide cleavage:

Weigh 6.01g of the above peptide resin, and slowly add it to 60mL of lysate (trifluoroacetic acid: anisole: anisole: ethanedithiol=90:5:3:2) at 20-30°C, and then complete the reaction 2 hours. After the reaction was completed, the resin was removed by filtration, and the filtrate was poured into pre-cooled methyl tertiary ether (600 mL) with vigorous stirring, and the resulting mixed solution was allowed to settle in the refrigerator for 2 hours. The supernatant was removed and centrifugally washed with pre-cooled methyl tertiary ether 5 times, 400 mL each time. After completion, it was collected and dried under reduced pressure at room temperature to obtain 3.00 g of crude peptide.

Crude peptide purification:

Using a preparative liquid phase (Beijing Chuangxintongheng, LC3000), the crude peptide was refined through multi-step purification: the first step: stationary phase: C18 (Daisogel: sp-120-40/60-C18-RPS), mobile phase 0.1 %TFA, acetonitrile; second step: stationary phase: C8 (Daisogel: sp-120-10-C8-P), mobile phase: 0.5% phosphoric acid, acetonitrile, third step: stationary phase: C8 (Daisogel: sp-120 -10-C8-P), mobile phase: 50mM ammonium acetate, 0.3% acetic acid, acetonitrile, and finally lyophilized (lyophilizer Beijing Boyi Kang, FD-2A) to obtain sperm peptide (0.120g, 97.8%). Finally, MS was used to determine the molecular weight of sperm peptide: m/z 4540.35 (M+H) ⁺ .

Example 15

Preparation of glucagon derivative FC381K10D21:

HSQGT FTSDXSKYLD SQAAQ DFVQW LMNGG PSSGA PPPS-NH ₂

X10=K(palmitoyl-γE) (SEQ ID NO.25)

The synthesis method is the same as that in Example 14, in which K10 is coupled by Fmoc-Lys(Pal-Glu-OtBu)-OH, the crude peptide obtained is purified by RP-HPLC, and finally lyophilized to obtain refined peptide (98.3%). MS: m/z 4590.55 (M+H) ⁺ .

Example 16

Preparation of glucagon derivative FC382K14W15:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.18)

Solid-phase synthesis of branched-chain protected amino acid W1: Alloc-Lys((OctadecanedioicAcid mono-tert-butylester)-Glu-OtBu)-OEG-OEG)-OH, as shown below:

Synthesis of W1:

Weigh 20 g of 2-CTC resin with a substitution degree of 1.0 mmol/g, add it to the solid-phase reaction column, add it to the solid-phase reaction column, wash once with DMF, and after swelling the resin with DMF for 30 minutes, take 8.53 g of Alloc -Lys(Fmoc)-OH (20mmol) was dissolved in DMF. After 7.5ml DIEA (45mmol) was added to the ice water bath for activation, it was added to the above reaction column with resin. After 2 hours of reaction, 30ml of anhydrous methanol was added to block for 1 hour. , Washed 3 times with DMF. Use a mixed solution of DMF:pyridine volume ratio of 4:1 to remove Fmoc protection, then wash with DMF 6 times, weigh 15.42g [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, 5.41g HOBt was dissolved in DMF, 6.2ml DIC was added under ice water bath to activate, then added to the above reaction column with resin, and reacted at room temperature for 2 hours. Repeat the above steps to remove the Fmoc protection and add the corresponding material coupling, in order to complete the branch sequence, [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid, Fmoc-Glu-OtBu, Monotert-butyl octadecanedioate. After coupling, the resin was washed 3 times with DMF, 5 times with MeOH, and drained. The resin was added to 400mlTFEA/DCM=1:4 and reacted at room temperature for 4h. After filtering the resin, the filtrate was vortexed out of DCM, added to 500 ml of MTBE and settled. After centrifugal drying, the target compound was 19.43 g, yield 95.1%, m/Z1059.41 (M+H).

The synthesis of the polypeptide is the same as that in Example 1, K14 is coupled with W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (96.7%). MS: m/z 4889.57 (M+H) ⁺ .

Example 17

Preparation of glucagon derivative FC382K10W15:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.30)

The branched-chain protected amino acid W1 was synthesized as in Example 16. The synthesis of the polypeptide is the same as in Example 1, in which K10 is coupled using W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (97.5%). MS: m/z 4839.73 (M+H) ⁺ .

Example 18

Preparation of glucagon derivative FC382K20W15:

HSQGT FTSDY SKYLD SQAAX DFVQW LMNGG PSSGA PPPS-OH

X20=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.36)

The branched-chain protected amino acid W1 was synthesized as in Example 16. The synthesis of the polypeptide is the same as in Example 1, in which K20 is coupled using W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (98.3%). MS: m/z 4873.93 (M+H) ⁺ .

Example 19

Preparation of glucagon derivative FC382K24W15:

HSQGT FTSDY SKYLD SQAAQ DFVXW LMNGG PSSGA PPPS-OH

X24=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.41)

The branched-chain protected amino acid W1 was synthesized as in Example 16. The synthesis of the polypeptide is the same as in Example 1, in which K24 is coupled using W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (96.4%). m/z 4874.32 (M+H) ⁺ .

Example 20

Preparation of glucagon derivative FC382K10W09:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10=K(Eicosanedioyl-γE) (SEQ ID NO.91)

The synthesis method is the same as that in Example 1, wherein K10 is coupled by Fmoc-Lys(N-(tBuOCO(CH2)18CO)-Glu-OtBu)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the crude peptide obtained is RP- Purified by HPLC, and finally lyophilized to obtain sperm peptide (96.5%). MS: m/z 4577.20 (M+H)+.

Example 21

Preparation of glucagon derivative FC382K10W03:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10 = K (hexadecanedioic acid mono-GABA) (SEQ ID NO.92)

The synthesis method is the same as that in Example 1, wherein K14 is coupled using Fmoc-Lys(N-(tBuOCO(CH ₂ ) ₁₄ CO)-GABA)-OH (Hangzhou Hesu Chemical Technology Co., Ltd.), and the crude peptide obtained is RP- Purified by HPLC, and finally lyophilized to obtain sperm peptide (96.7%). MS: m/z 4477.12 (M+H)+.

Example 22

Preparation of polypeptide FC382C14D22:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=C (hexadecyl-maleimide) (SEQ ID NO. 48)

Synthesis of N-hexadecyl-2,5-dioxopyrrolidine:

Take hexadecylamine salt (4.10g, 0.017mol), add 60ml of acetic acid to dissolve, add maleic anhydride (2g, 0.02mol), raise the temperature to 120°C and reflux with acetic acid. After refluxing for 8h, add 100ml of water to wash and stir, filter and use water The filter cake was washed, and the filter cake was dried to obtain 3.6 g of cetylamino-2,5-dioxopyrrolidine.

Peptide synthesis:

The synthesis of the polypeptide is the same as in Example 1, in which C14 is coupled by Fmoc-Cys(Trt)-OH, the crude peptide obtained is purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (96.5%).

Fatty acid modification of peptides:

Weigh the polypeptide compound (10mg, 2.3μmol), add 5ml of 50mM PB buffer solution (pH7), add cetylamino-2,5-dioxopyrrolidine (1.30mg, 5.75μmol), stir at room temperature for 3h, RP-HPLC monitored the end of the reaction. After the reaction, the reaction solution was purified by RP-HPLC to obtain sperm peptide (96.7%). MS: m/z4470.10 (M+H)+.

Example 23

Preparation of glucagon derivative FC382C10D22:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-OH

X10=C (hexadecyl-maleimide) (SEQ ID NO.45)

The synthesis of N-hexadecyl-2,5-dioxopyrrolidine is the same as in Example 22.

The synthesis of the polypeptide is the same as in Example 1, in which C10 is coupled using Fmoc-Cys(Trt)-OH, the crude peptide obtained is purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (0.073g, 98.5%). The fatty chain modification of the polypeptide is the same as in Example 22, RP-HPLC monitoring the reaction end point. After the reaction, the reaction solution was purified by RP-HPLC to obtain sperm peptide (95.3%). MS: m/z 4420.30 (M+H) ⁺ .

Example 24

Preparation of glucagon derivative FC381C10D22:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH2

X10 = C (hexadecyl-maleimide) (SEQ ID NO. 47)

The synthesis of N-hexadecyl-2,5-dioxopyrrolidine is the same as in Example 22. The synthesis of the polypeptide is the same as in Example 14, wherein C10 is coupled with Fmoc-Cys(Trt)-OH, and the resulting crude peptide is purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (0.036g, 98.4%). The fatty chain modification of the polypeptide is the same as in Example 22, RP-HPLC monitoring the reaction end point. After the reaction, the reaction solution was purified by RP-HPLC to obtain sperm peptide (97.3%). m/z4419.40(M+H)+.

Example 25

Preparation of glucagon derivative FC381K10W15:

HSQGTFTSDXSKYLDSQAAQDFVQWLMNGGPSSGAPPPS-NH2

X10=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.31)

The branched-chain protected amino acid W1 was synthesized as in Example 16. The synthesis of the polypeptide is the same as in Example 14, in which K10 is coupled using W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC to obtain sperm peptide (95.6%). MS: m/z 4838.80 (M+H) ⁺ .

Example 26

Preparation of glucagon derivative FC381K14W15:

HSQGT FTSDY SKYXD SQAAQ DFVQW LMNGG PSSGA PPPS-NH2

X14=K(((octadecanedioic acid monoacyl)-γE)-2xOEG) (SEQ ID NO.19)

The branched-chain protected amino acid W1 was synthesized as in Example 16. The synthesis of the polypeptide is the same as in Example 14, in which K14 is coupled using W1, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC. Sperm peptide (95.4%) was obtained. MS: m/z 4888.33 (M+H) ⁺ .

Example 27

Preparation of glucagon derivative FC462K14W12:

HSQGT FTSDY SKYXD EEAAQ DFVQW LMNGG PSSGA PPPS-OH

X14=K(((Eicosanedioyl)-γE)-2xOEG) (SEQ ID NO.96)

The synthesis of branched chain protected amino acids is the same as in Example 16, and the solid phase method is used to synthesize branched chain protected amino acids W2: Alloc-Lys ((Eicosanedioic Acid mono-tert-butylester)-Glu-OtBu)-OEG-OEG)- OH (where fatty acid coupling uses eicosanedioic acid mono-tert-butyl ester), as shown below:

Polypeptide synthesis was carried out. The peptide synthesis was the same as in Example 1, in which K14 was coupled using W2, and Pd(PPh3)4 was used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC to obtain sperm peptide (96.4%). MS: m/z 4960.43 (M+H) ⁺ .

Example 28

Preparation of glucagon derivative FC462K10W12:

HSQGTFTSDXSKYLDEEAAQDFVQWLMNGGPSSGAPPPS-OH

X10=K(((Eicosanedioyl)-γE)-2xOEG) (SEQ ID NO.98)

The synthesis of the branched-chain protected amino acid W2 and the synthesis of the polypeptide are the same as in Example 27, in which K10 is coupled using W2 and the Alloc group is removed using Pd(PPh3)4. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain a refined peptide (97.0%). m/z 4910.32(M+H) ⁺ .

Example 29

Preparation of glucagon derivative FC463K14W12:

HSQGT FTSDY SKYXD EEAAQ DFVQW LMNGG PSSGA PPPS-NH2

X14=K(((Eicosanedioyl)-γE)-2xOEG) (SEQ ID NO.97)

The synthesis of branched-chain protected amino acid W2 is the same as in Example 27, and the synthesis of the polypeptide is the same as in Example 14, in which K14 is coupled using W2, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (96.3%). MS: m/z 4959.33 (M+H) ⁺ .

Example 30

Preparation of glucagon derivative FC463K10W12:

HSQGTFTSDXSKYLDEEAAQDFVQWLMNGGPSSGAPPPS-NH2

X10=K(((Eicosanedioyl)-γE)-2xOEG) (SEQ ID NO.99)

The synthesis of branched-chain protected amino acid W2 is the same as in Example 27, and the synthesis of the polypeptide is the same as in Example 14, in which K14 is coupled using W2, and Pd(PPh3)4 is used to remove the Alloc group. The obtained crude peptide was purified by RP-HPLC, and finally lyophilized to obtain refined peptide (96.3%). MS: m/z 4909.45 (M+H) ⁺ .

The preparation of glucagon derivatives in the above examples only uses the C382 and C381 series of polypeptides as an example. The reaction conditions of other glucagon derivatives in Table 2 refer to the above method, that is: the C-terminal group of the polypeptide part -OH group Blocked glucagon derivatives, such as FC495K14D21 (SEQ ID NO. 49) or FC462K14D21 (SEQ ID NO. 65). The synthesis is the same as in Example 1, and the glucagon derivative modified by amidation at the C-terminus of the polypeptide part, For example, FC496K14D21 (SEQ ID NO. 50) or FC463K14D21 (SEQ ID NO. 66) is synthesized in the same manner as in Example 14. As for the fatty acid chain part, the crosslinking of palmitoyl-γE is the same as in Example 1; the crosslinking of octadecanoyl-γE is the same as in Example 2; the crosslinking of eicosanedioic acid-γE is the same as in Example 3 ; Crosslinking of decanoyl-γE is the same as in Example 4; ((palmitoyl)-γE)-2xOEG is the same as in Example 5; ((octadecanoyl)-γE)-2xOEG is crosslinked and implemented Example 6 is the same; ((Eicosanoyl)-γE)-OEG is cross-linked as in Example 7; Palmitoyl-γE-γE is cross-linked as in Example 9; ((octadecanedioic acid monoacyl) -γE)-2xOEG is the same as Example 16; hexadecanedioic acid mono-GABA is the same as Example 21; cetyl-maleimide is the same as Example 22 ; ((Eicosanedioic acid monoyl)-γE)-2xOEG is the same as in Example 27.

Example 31

In vitro cytological activity determination:

(1) Determination of GLP-1R agonistic activity:

GLP-1R agonistic activity was detected by luciferase reporter gene detection method (Jonathan W Day etc.: Nat Chem Biol. 2009 Oct; 5(10):749-57). The human GLP-1R gene was cloned into mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-1R, and the full-length luciferase gene was cloned into pCRE plasmid to obtain pCRE-Luc Recombinant plasmid. The pcDNA3.1-GLP-1R and pCRE-Luc plasmids were transfected into CHO-K1 cells at a molar ratio of 1:10 to screen for stable transfected expression strains.

In a 9-cm cell culture dish, the cells were cultured in DMEM/F12 medium containing 10% FBS and 300 μg/ml G418. When the confluence reached about 90%, the culture supernatant was discarded and digested with 2 ml trypsin for 3 min. Add 2ml DMEM/F12 medium containing 10% FBS and 300μg/ml G418 to neutralize, transfer to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, add 2ml DMEM containing 10% FBS and 300μg/ml G418 /F12 medium was resuspended and counted. Dilute the cells with DMEM/F12 medium containing 10% FBS to 1×10 ⁵ /ml. Spread 100 μl of each well in a 96-well plate, that is, 1×10 ⁴ /well. After attaching, replace with 0.2% FBS in DMEM/ F12 medium culture. After discarding the supernatant on the cells plated in 96-well plates, the purified recombinant protein was diluted with DMEM/F12 medium containing 1% BSA to a series of specified concentrations, added to the cell culture wells, 100 μl/well, after stimulation for 6h Detection. The detection was performed according to the instructions of lucifersae reporter kit (Ray Biotech, Cat: 68-LuciR-S200). The measurement activity of each sample was repeated 3 times.

(B) GCGR agonistic activity detection method:

GCGR agonistic activity detection also uses the luciferase reporter gene detection method. The human-derived GCGR gene was cloned into mammalian cell expression plasmid pcDNA3.1, constructed into a recombinant expression plasmid pCDNA3.1-GCGR, transfected with CHO-K1 and stably transformed cell lines. The screening construction was the same as above. The measurement activity of each sample was repeated 3 times.

Table 4

Glucagon: HSQGT FTSDY SKYLD SRRAQ DFVQW LMNT-OH (SEQ ID NO.1).

Liraglutide: HAEGTFTSDVSSYLEGQAA X EFIAWLVRGRG, X=K (palmitoyl-γE) (SEQ ID NO. 2).

Example 32

Serum stability:

(1) The corresponding glucagon derivative in the figure is prepared into a solution with a concentration of 1.0 mg/ml using 5 mM Tris-HCl, pH 8.5, 0.02% TWEEN80 solution, and after sterilization and filtration (0.22 μm, Millipore SLGP033RB), use Dilute rat serum 10 times, mix well, and divide into sterile centrifuge tubes;

(2) Take 3 tubes of each of the above samples and freeze at -20°C as a control, and place the rest in a 37°C incubator and take samples at 0, 24 and 72 hours to test the activity;

(3) Detect the glucagon derivative GCGR agonistic activity.

Relative activity: The activity value at 0 hours is 100%, and the value measured at the subsequent time point is compared with it.

Figures 5-8 are the results of residual activity of glucagon derivatives over time.

Example 33

Random blood glucose test after administration of db/db mice:

Hypoglycemic experiment in leptin receptor-deficient type 2 diabetes (db/db) mice. The db/db mice were screened and divided into groups based on body weight, non-fasting blood glucose, and pre-drug OGTT response. Six mice in each group were excluded. Excessive or small individuals were excluded. The non-fasting blood glucose was greater than 15 mM. The glucagon derivative was dissolved in 50mM phosphate buffer (pH7.4), 5% sorbitol, 0.02% v/v Tween-80, subcutaneously injected with liraglutide or the glucagon derivative in Table 2 (single (Dose), the dosage is 10 nmol/Kg body weight, blood glucose value was measured before and at 0, 1, 3, 6, 24, 72 hours after administration. The change trend of blood glucose at 0-24 hours is shown in Fig. 9 to Fig. 14, and Fig. 15 is the blood glucose content percentage of the glucagon derivative after crosslinking with different long-acting carriers at 24 hours (percentage compared with the blood glucose content at 0 hours). The percentage of the 72-hour blood glucose content (percentage compared to the 0-hour blood glucose content) is shown in Figures 16 and 17. The blood glucose content of the glucagon derivative shown in Figure 16 and Figure 17 at 72 hours is significantly lower than that at 0 hours, while the other glucagon derivatives have recovered (or are close) to the initial value of 0 hours (the result is not (Display), indicating that the active half-life of the glucagon derivatives in Figs. 16-17 in the body is significantly longer than other glucagon derivatives, and the onset time is longer.

Example 34

Weight loss experiments in diet-induced obese (DIO) mice:

Preparation of DIO mouse model: Male C57BL/6J male mice of about 7 weeks old were given high-fat diet (60% kcal from fat) and kept feeding for about 16 weeks (total 23 weeks), and the experiment was conducted when the body weight was about 45 g. DIO mice were randomly divided into groups, with 6 mice in each group. There was no difference in basis weight, and they were weighed daily. Glucagon derivatives, liraglutide, or PBS were injected subcutaneously. Liraglutide and the glucagon derivative in Table 5 are administered at a dose of 20 nmol/Kg of body weight once a day; the glucagon derivative in Table 6 is administered at a dose of 40 nmol/Kg of body weight every 4 days Medicine once.

table 5

Glucagon derivatives	Weight change (%)	SEM	Glucagon derivatives	Weight change	SEM
Liraglutide	-8.4	2.4	FC495K10D21	-39.2	2.6
PBS	+1.7	1.1	FC496K10D21	-39.7	5.3
FC382K14D21	-36.5	1.7	FC495K20D21	-15.8	1.6
FC381K14D21	-37.2	3.8	FC496K20D21	-14.9	1.5
FC382K10D21	-42.3	3.9	FC495K24D21	-20.1	2.8
FC381K10D21	-41.5	5.3	FC496K24D21	-18.5	3.0
FC382K20D21	-15.6	1.9	FC462K14D21	-40.7	2.6
FC381K20D21	-17.2	2.3	FC463K14D21	-39.4	2.9
FC382K24D21	-16.7	3.2	FC462K10D21	-42.1	3.8
FC381K24D21	-15.9	4.7	FC463K10D21	-42.3	4.5
FC884K14D21	-12.4	2.4	FC462K20D21	-26.5	3.2
FC885K14D21	-21.9	3.2	FC463K20D21	-24.5	3.8
FC495K14D21	-38.9	1.9	FC462K24D21	-14.9	5.4
FC496K14D21	-41.0	2.3	FC463K24D21	-16.4	1.9

Table 6

Example 35

The efficacy of glucagon derivatives in non-alcoholic fatty liver (NASH) mouse model:

C57BL/6 male mice weighing 8-30 weeks old and weighing 25-30 grams were selected to induce NASH model with CDA-HFD feed. The blood glucose level was measured before the start of the experiment and before the end of the experiment. At the end of the experiment, the serum AST, ALT, liver TG content, serum HDL-C content (Hitachi 7060 automatic biochemical detector) and other parameters were detected; liver histopathological analysis: H&E, SR. Statistical methods: t-test or One-way ANOVA was used to test the significance of differences. P<0.01 means significant statistical difference, and P<0.001 means extremely significant statistical difference. Glucagon derivatives were injected subcutaneously. The dosage of glucagon derivatives and FC384K14D21 in Table 5 is 20 nmol/Kg body weight, once a day; the dosage of glucagon derivatives and FC386K10W15 in Table 6 is 40 nmol/Kg body weight, every 4 days once. A total of 7 weeks. The NAS scoring criteria are shown in Table 7. The results are shown in Figures 18-22. The data corresponding to the model mouse group and the normal mouse group in the figure are obtained after subcutaneous injection of PBS, and the rest of the drug group is completed in the model mouse.

Table 7

In summary, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-mentioned embodiments only exemplarily illustrate the principle and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. A glucagon analog, including a glucagon-like polypeptide fragment, the glucagon-like polypeptide fragment is:

   a) The polypeptide fragment whose amino acid sequence is shown in SEQ ID No. 3;

   HSQGT FTSDX ₁₀ SKYX ₁₄ D X ₁₆ X ₁₇ AAX ₂₀ DFVX ₂₄ W LMNGG PSSGA PPPSX ₄₀ (SEQ ID No. 3)

   X ₁₀ = Y, K or C, X ₁₄ = L, K or C, X ₁₆ = S or E, X ₁₇ = Q or E, X ₂₀ = Q, K or C, X ₂₄ = Q, K or C, X ₄₀ is K, C, or deletion, and at least one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ , X ₄₀ is K or C;

   Or, b) a polypeptide fragment having an amino acid sequence having more than 90% sequence identity with SEQ ID NO. 3 and having the function of the polypeptide fragment defined in a);

   A long-acting carrier is cross-linked on the glucagon-like polypeptide fragment.
2. The glucagon derivative according to claim 1, wherein one of X ₁₀ , X ₁₄ , X ₂₀ , X ₂₄ and X ₄₀ is K or C.
3. The glucagon derivative according to claim 1, wherein the long-acting carrier is selected from fatty acids, fatty chains or PEG;

   And/or, amino acid residue K and/or amino acid residue C are cross-linked with a long-acting carrier;

   And/or, the C-terminus of the glucagon-like polypeptide is amidated;

   And/or, the glucagon analog is artificially designed.
4. The glucagon derivative according to claim 3, wherein the fatty acid is selected from C8 to C30 fatty acids;

   And/or, the fatty acid is a monocarboxylic acid and/or dicarboxylic acid;

   And/or, the fatty acid is linear;

   And/or, the fatty acid is cross-linked to form a fatty acid group, and the fatty acid group is selected from the groups shown in the chemical structural formulas as follows:

   
5. The glucagon derivative according to claim 1, wherein a linker is further provided between the glucagon-like polypeptide fragment and the long-acting carrier.
6. The glucagon derivative according to claim 5, wherein the linker is selected from -Abu-(-L-2-aminobutyryl-), -GABA-(-γ-aminobutyryl-) , -EACA-(-ε-aminocaproyl-), -β-Ala-(-β-alanyl-), -γGlu- (-γ-glutamyl), -D-γGlu-(-D- γ-glutamyl-) or its dipeptide, such as -β-Ala-β-Ala-, -γGluγGlu- and its stereoisomeric forms (S and R enantiomers), -5-Aminopentanoyl-(-5 -Aminovaleryl-),-8-Aminooctanoyl-(-ω-aminooctanoyl-),-9-Aminononanoyl-(-9-aminononanoyl-),-10-Aminodecanoyl-(-10-amino-n-decanoyl -), -OEG-(2-(2-(-2-aminoethoxy)ethoxy)acetyl-), -2xOEG-, -γGlu-OEG-, -γGlu-2xOEG-, -D-γGlu- 2xOEG-, -2xOEG-γGlu-, -γGlu-3xOEG-, -γGlu-8xPEG-(-3-((γ-glutamine)-8x polyethylene glycol)-propionyl-), -γGlu-3xOEG- γ-Glu-8xPEG-.
7. The method for preparing the glucagon analogue according to any one of claims 1 to 6, comprising: preparing the glucagon analogue by using a chemical synthesis method.
8. Use of the glucagon analogue according to any one of claims 1 to 6 in the preparation of a medicament for treating metabolic diseases and GCGR/GLP-1R multi-effect agonist.
9. The use according to claim 8, wherein the metabolic disease is selected from diabetes, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, other metabolic syndromes associated with diabetes, triglycerides High and low HDL cholesterol and high LDL cholesterol, insulin resistance, obesity or glucose intolerance.
10. A pharmaceutical composition comprising a therapeutically effective amount of the glucagon analogue according to any one of claims 1 to 6.

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