Glycated peptide glp-1

FIELD: chemistry.

SUBSTANCE: invention refers to gene engineering, more specifically to producing the peptide GLP-1, modified by an oligosaccharide chain, and can be used in medicine for treating or preventing diseases associated with GLP-1. In the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3 two amino acid peptides are substituted by an amino acid modified by a complex bi-antennal oligosaccharide chain, and wherein each of the centres is specified in a group consisting of positions 18, 22, 26, 30, 34 and 36 in the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3. The above modified peptide GLP-1 can involve the deletion, substitution or attachment of 1-5 amino acids, except for the amino acids modified by the oligosaccharide chain.

EFFECT: invention enables producing the peptide GLP-1 modified by the oligosaccharide chain, which shows the stronger activity of blood glucose suppression and twice increased half lifetime as compared to GLP-1 with SEQ ID NO: 3.

24 cl, 5 dwg, 6 tbl, 16 ex

 

The technical field to which this invention

The present invention relates to a peptide GLP-1, the modified oligosaccharide chain.

The level of technology

GLP-1 (like peptide-1) is a peptide of intestinal origin, which plays an important role in the regulation of glucose homeostasis. GLP-1 is synthesized in L-cells of the gastrointestinal tract in the tissue-specific posttranslational processing of preproglucagon, which is the predecessor of glucagon and released into the bloodstream when eating. This peptide plays the role of a primary mediator anterointernal axis and functions by binding to its specific receptors.

It is known that GLP-1 acts mainly on the pancreas and promotes the release of insulin from β-cells, the speed of which depends on the concentration of glucose. Also, consider that GLP-1 apparently suppresses the secretion of glucagon, delays gastric emptying and increases the removal of peripheral glucose.

The introduction of GLP-1 to patients with non-insulin-dependent diabetes mellitus can normalize glucose levels after a meal, which indicates that GLP-1 may be used as therapeutic drugs. GLP-1 also improves glycemic control in patients with insuli zavisimym diabetes. Since the effect of the promotion of insulin secretion caused by GLP-1, depends on the concentration of plasma glucose, GLP-1 acts as a mediator in reducing insulin secretion at low glucose concentrations in the plasma and, therefore, not primarily cause significant hypoglycemia. Thus, high-security treatment of diabetes, if necessary, can be achieved through the control of GLP-1 in blood. However, the half-life of GLP-1 in the blood is so low and equal to from 2 to 6 minutes, which creates a problem of its limited use as a therapeutic drug.

To solve this problem, an attempt was made to carry out the modification of GLP-1. For example, patent document 1 discloses derived paglinawan GLP-1 containing compound GLP-1 conjugated with at least 1 molecule of polyethylene glycol (PEG). In connection paglinawan derived GLP-1, each PEG molecule is attached to the connection GLP-1 to Cys or Lys amino acid or the terminal carboxyl group of the amino acids. Connection paglinawan derived GLP-1 is characterized by an increase of the half-period, at least 1 hour.

In accordance with the patent document 1 derived biologically active peptide is characterized by a longer half-life and much more is izlenim excretion compared with the corresponding parameters for naegeliana peptides. It was also shown that paglierani derived GLP-1 and the composition used for the treatment of such chronic conditions as diabetes, obesity, and irritable bowel syndrome, as well as to reduce blood sugar levels, suppression of gastric and/or intestinal motility, gastric and/or intestinal emptying and control over food intake (for example, non-patent document 1).

However, PEG is a connection that is not metabolized in vivo. Therefore, with continuous introduction connections paglinawan derived GLP-1 PEG is accumulated in vivo and can cause adverse reactions in living organisms (non-patent document 1).

Moreover, to increase half-life, was also a method of joining oligosaccharide chain to GLP-1 or a modified GLP-1 (for example, patent documents 3 and 4). Patent document 3 discloses a method of representing an amino acid, a modified oligosaccharide chain at positions 26, 34 and/or 37 of GLP-1, and so on, But the type of oligosaccharide chains and malls, which are attached to the oligosaccharide chains are less than optimal. On the other hand, patent document 4 discloses a method that represents the binding of the modified hyaluronic acid having a molecular weight of approximately 200 kDa, with an analogue of GLP-1. However, when such large molecules of hyaluronic acid produced in large quantities, it is difficult to make them equal in length or structure. So the real hyaluronic acid can vary significantly in structure and length. For pharmaceutical applications require peptides, modified oligosaccharide chain having the same length or structure.

The basis-4, isolated from the saliva of a lizard (Heloderma), is a molecule that is structurally similar to GLP-1, and has similar activity and high stability in blood (non-patent document 2), which was released on the market in the United States. However, on the basis 4 has an amino acid sequence that differs from the human, and can induce the production of neutralizing antibodies, which necessitates long-term introduction, leading to reduced efficiency (non-patent documents 3-5).

On the other hand, it became obvious that the oligosaccharide chains have different functions in vivo. They are less studied in detail because of their complexity and diversity of structures, although the importance of their study there is no doubt. An attempt was made to develop a method of obtaining glycopeptide with a constant composition (patent document 2). However, this method of obtaining is less than perfect from the point of view of the advantages or large-scale production and is not a constructive way, especially for DL is niebotycznych oligosaccharide chains, existing in vivo.

[Patent document 1] National Publication of International Patent Application No. 2006-520818

[Patent document 2] WO 2005-095331

[Patent document 3] National Republication of International Patent Application No. 2006-095775

[Patent document 4] WO 2007/063907

[Non-patent document 1] Toxicological Science, 42, 152-157 (1998)

[Non-patent document 2] J Biol Chem. 267, 7402-5 (1992)

[Non-patent document 3] Vascular Health and Risk Management 2, 69-77 (2006)

[Non-patent document 4] JAMA. 298, 194-206 (2007)

[Non-patent document 5] Endocrine Reviews 28, 187-218 (2007)

The invention

The problem addressed by the invention

The purpose of this invention is the provision of peptide GLP-1 modified oligosaccharide chain having higher stability in blood than that of GLP-1 and, more preferably, which is a stronger regulator of blood sugar levels, compared to GLP-1.

Resolving problems

The present invention may have the following characteristic features to solve this problem.

Namely, the present invention provides peptide GLP-1, a modified oligosaccharide chain having the activity of GLP-1, where at least two amino acids are replaced by amino acid-modified oligosaccharide chain,

(a) GLP-1;

(b) a peptide having the amino acid sequence of GLP-1 with deletion, substitution or addition of one or several amino acids, sludge is in

(c) the analogue of GLP-1.

The present invention also provides a peptide GLP-1, a modified oligosaccharide chain having the activity of GLP-1, where at least two amino acids are replaced by amino acid-modified oligosaccharide chain,

(a) GLP-1, or

(b) a peptide having the amino acid sequence of GLP-1 with deletion, substitution or addition of one or several amino acids, and having the activity of GLP-1.

The present invention also provides a peptide GLP-1, a modified oligosaccharide chain having the activity of GLP-1, where the peptide GLP-1, a modified oligosaccharide chain 1, is a

(a) peptide GLP-1, a modified oligosaccharide chain, where at least each of the two amino acids of GLP-1 is substituted by an amino acid-modified oligosaccharide chain and at least one of the centers of substitution is in position 18, 20, 22, 26, 30, 34 or 36 in GLP-1; or

(b) peptide GLP-1, a modified oligosaccharide chain having the amino acid sequence of the peptide GLP-1 attached to the oligosaccharide chain, which is characterized (a) by deletion, substitution or addition of one or several amino acids, with the exception of conjugates of oligosaccharide chains with amino acids.

The present invention also provides a peptide GLP-1, a modified oligosaccharide chain is, having the activity of GLP-1, where the peptide GLP-1, a modified oligosaccharide chain, is a

(a) peptide GLP-1, a modified oligosaccharide chain, where at least each of the two amino acids of GLP-1 is substituted by an amino acid-modified oligosaccharide chain, substituted and each of the centers is in position 18, 20, 22, 26, 30, 34 or 36 in GLP-1; or

(b) peptide GLP-1, a modified oligosaccharide chain having the amino acid sequence of the peptide GLP-1 conjugate with oligosaccharide chain, which is characterized (a) by deletion, substitution or addition of one or several amino acids, with the exception of conjugates of oligosaccharide chains with amino acids.

In the present invention the peptide GLP-1, a modified oligosaccharide chain can be a preferably, but not be limited to conjugate oligosaccharide chain with Asn or conjugate oligosaccharide chain with Cys depending on the implementation.

In the present invention, amino acids, modified oligosaccharide chain attached to the peptide GLP-1 may be the same or different type oligosaccharide chains or amino acids.

In the present invention the amino acid-modified oligosaccharide chain oligosaccharide chain may be linked to the amino acid via a linker or without Linke is and. Preferably, when the oligosaccharide chain is linked to the amino acid without a linker (i.e., directly), depending on the implementation.

In the present invention, the oligosaccharide chain is usually preferably represents oligosaccharide chain, consisting of four or more residues of sugars. However, the oligosaccharide chain, consisting of five to eleven residues of sugars, is preferable, depending on the implementation.

In the present invention, the oligosaccharide chain can be a preferably, but not be limited to ventennale oligosaccharide chain complex type, depending on the implementation. Oligosaccharide chain can be a preferably, but not be limited oligosaccharide chain selected from the group consisting of disialo, monomial, asialo, GlcNAc and demonopolisation circuits, depending on the implementation.

In the present invention, the oligosaccharide chain can be a preferably, but not be limited to the oligosaccharide chain, represented by the following formula, depending on the implementation.

Formula 1

where

R1and R2are the same or different and each group presents

Formula 2

and

The AC is an acetyl group.

The present invention also provides a peptide GLP-1, a modified oligosaccharide chain, in which at least one amino acid of the original peptide is substituted by an amino acid-modified oligosaccharide oligosaccharide chain and the chain is oligohaline acid. Examples oligohaline acid may include oligosaccharide chain containing two (tetrachoric) or more and eight or less units, consisting of N-acetylglucosamine and glucuronic acid. Aigagaranca acid can have 2 tetrasaccharide or 4 octasaccharide link.

The present invention also provides a peptide GLP-1, a modified oligosaccharide chain, where the oligosaccharide chain is associated with at least one amino acid linker. Examples of amino acids of the peptide GLP-1 associated linker may include Lys. In this case, the linker can contain terminal amino acid associated with oligosaccharide chain. Example amino acids, located at the end of the linker associated with oligosaccharide chain may include Asn.

In the present invention, the oligosaccharide chain is preferably homogeneous and preferably has, for example, at least 90% or at least 99% homogeneity.

The peptide GLP-1, modificirovannihkh chain the present invention preferably has a higher stability in blood flow than the stability of GLP-1.

The peptide GLP-1, a modified oligosaccharide chain of the present invention may be a stronger regulator of blood sugar levels, preferably at least 5 times, more preferably at least 10 times, more preferably at least 20 times than GLP-1 in OGTT (oral tolerance test glucose).

The peptide GLP-1, a modified oligosaccharide chain of the present invention can have a greater resistance to dipeptidyl peptidase-4 (DPP-IV) is preferably at least 20 times, more preferably at least 30 times, more preferably at least 50 times than GLP-1.

The peptide GLP-1, a modified oligosaccharide chain of the present invention can be used as a new active ingredient in medical application. Such medical applications include the treatment or prevention of diseases associated with GLP-1. A typical example of such diseases is, for example, diabetes.

Of course, one or any combination of the properties of the present invention, described above, are also characteristic of the peptide GLP-1 modified oligosaccharide chain of the present invention.

The contents of the present inventions

GLP-1, modified oligosaccharide chain of the present invention has higher stability in blood flow than GLP-1. In one aspect of the present invention the peptide GLP-1, a modified oligosaccharide chain of the present invention is a stronger regulator of blood sugar levels than GLP-1. Accordingly, the peptide GLP-1, a modified oligosaccharide chain of the present invention can be introduced in a lower dose and with fewer doses than GLP-1.

Oligosaccharide chains which are attached to the peptide GLP-1, the modified oligosaccharide chain of the present invention is easily broken down in vivo and therefore do not cause adverse reactions leading to their accumulation in living organisms.

Some or all of the oligosaccharide chains which are attached to the peptide GLP-1, the modified oligosaccharide chain, the present invention represents oligosaccharide chain, in vivo included in mammals, including humans, birds and so on, or their modified oligosaccharide chain. Perhaps they can cause adverse reactions or antigenicity when introduced into a living organism. Therefore, they do not represent problems in terms of allergic reactions, antibody production or the associated loss of activity.

In most cases, the oligosaccharide chain used is presented in the present invention, are relatively short. Therefore, those that have a homogeneous structure can be obtained without the use of stages, causing complications. Thus, high-quality peptide GLP-1, a modified oligosaccharide chain, pharmaceutical level can be easily obtained in large quantities.

Brief description of drawings

In Fig.1 shows the measurement results using oral samples for glucose tolerance (OGTT) effect of suppressing the increase of blood sugar level with the introduction of peptide GLP-1 modified oligosaccharide chain (conjugate GLP-1 with 26 and 34Cys-disialogangliosides chain or conjugate of GLP-1 with 18 and 36Cys-disialogangliosides chain) or GLP-1. Conjugate GLP-1 with 26 and 34Cys-disialogangliosides chain or conjugate of GLP-1 with 18 and 36Cys-disialogangliosides circuit is administered in a dose of 0.9 nmol/kg, while GLP-1 is administered at a dose of 9 nmol/kg;

in Fig.2 shows the measurement results using oral samples for glucose tolerance (OGTT) effect of suppressing the increase of blood sugar level with the introduction of peptide GLP-1 modified oligosaccharide chain (conjugate GLP-1 with 22 and 30Cys-disialogangliosides chain conjugate GLP-1 with 22 and 36Cys-disialogangliosides chain or conjugate of GLP-1 with 30 and 36Cys-disialogangliosides chain) or GLP-1. Conjugate GLP-1 with 22 and 30Cys-divalerio charidee chain and conjugate GLP-1 with 22 and 36Cys - disialogangliosides chain or conjugate of GLP-1 with 30 and 36Cys-disialogangliosides circuit is administered at a dose 0.9 nmol/kg, while GLP-1 is administered at a dose of 9 nmol/kg;

in Fig.3 shows the measurement results using oral samples for glucose tolerance (OGTT) effect of suppressing the increase of blood sugar level with the introduction of peptide GLP-1 modified oligosaccharide chain (conjugate GLP-1 with 36Cys-tetrasaccharide hyaluronic acid or conjugate of GLP-1 with 36Cys-octasaccharide hyaluronic acid) or GLP-1. Conjugate GLP-1 with 36Cys-tetrasaccharide hyaluronic acid or conjugate of GLP-1 with 36Cys-octasaccharide hyaluronic acid, respectively, is administered at a dose of 9 nmol/kg;

in Fig.4 shows the measurement results using oral samples for glucose tolerance (OGTT) effect of suppressing the increase of blood sugar level with the introduction of peptide GLP-1 modified oligosaccharide chain (conjugate Asn-linkermakeexternalimage GLP-1 with 26Lys-asialoglycoprotein chain) or GLP-1. Conjugate Asn-linkermakeexternalimage GLP-1 with 26Cys-asialoglycoprotein chain and GLP-1 is administered at a dose of 9 nmol/kg; and

in Fig.5 shows the measurement results using oral samples for glucose tolerance (OGTT) conducted to study the effect of the dose of the peptide GLP-1 modified oligosaccharide chain on the effect of suppressing the increase of blood sugar levels. Conjugate GLP-1 with 8 and 36Cys-disialogangliosides the ne circuit is administered at a dose 0.9 nmol/kg, while GLP-1 is administered at a dose of 9 nmol/kg

A detailed study of the preferred embodiments

The term "GLP-1" used here denotes the like peptide-1 and is called GLP-1 (7-37).

GLP-1 (7-37) has an amino acid sequence

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly (SEQ ID no:2).

In the present invention the analogue of GLP-1 is a peptide structurally similar to GLP-1, and/or a peptide structurally overlapping with GLP-1. Examples of such peptides include a peptide having the amino acid sequence of GLP-1 with deletion, substitution or addition of one or several amino acids; peptide having the amino acid sequence of GLP-1 with a conservative substitution of one or several amino acids; modified GLP-1; fragment of a GLP-1 having the activity of GLP-1; elongated GLP-1 having the activity of GLP-1; and on the basis of 4 ("Ex 4" means the basis 4 this description of the patent) and its analogue (Curr. Opin. Investig. Drugs 8, 842-8 (2007), J. Pharmacol. Exp. Ther. 307, 490-496 (2003), Diabetes 50, 2530-9 (2001), etc.).

The term "amino acid" as used here is used in its broadest sense and includes not only natural amino acids, but also non-natural amino acids, for example varieties of amino acids and their derivatives. Given this broad definition, with ecialist in this field can understand examples of amino acids used herein include natural proteinogenic L-amino acids; D-amino acids; chemically modified amino acids, such as varieties of amino acids and their derivatives, natural aprotinine amino acids, such as norleucine, β-alanine and ornithine; and chemically synthesized compounds having properties characteristic of amino acids, known in this area. Examples of such non-natural amino acids include α-methylaminomethyl (α-methylalanine and so on), D-amino acids, histidinate amino acids (2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten and α-methylhistidine and so on), amino acids having an extra methylene group in the side chain ("Homo"amino acids) and amino acids in which the carboxyl functional group in the side chain substituted by a group of sulfonic acids (cysteine acid, etc.,). It is known that some analogs of GLP-1 having the activity of GLP-1, contain unnatural amino acids. In a preferred aspect, the amino acids included in the compounds of the present invention, contain only natural amino acids.

In the sentence "division, substitution or addition of one or several amino acids", as used here, the number of substituted amino acids, etc. are not necessarily limited to, if the activity of LP-1 is preserved. The number of substituted amino acids, etc. is in the range from approximately 1 to 9, preferably from 1 to about 5, more preferably from approximately 1 to 3 or is within 20%, comprising preferably within 10% of the total length. Substituted or added in the amino acid can be a natural amino acids, unnatural amino acids or analogs of amino acids and preferably are natural amino acids. Examples of peptide GLP-1, containing a deletion, substitution or addition of one or several amino acids" include BIM51077, where each of 8Ala and 35Gly in GLP-1 is substituted non-natural amino acid α-methylalanine (also referred to as aminoadamantane acid or Aib; his 37Gly removed from the chain; and it 36Arg converted into amide (Curr. Opin. Investig. Drugs 8, 842-8 (2007)).

The term "conservative substitution of one or several amino acids" used herein refers to amino acid substitution in which the original amino acid is substituted at amino acid having indexes hydrophilicity and/or hydrophobicity, similar belonging to the original amino acid, and does not result in significant reduction or loss of activity of GLP-1 after substitution.

"A modified GLP-1" used here is a compound in which GLP-1 is exposed to natural or artificial mo is eficacia. Examples of such modifications include alkylation, acylation (e.g., acetylation, amidation, carboxylation, etherification, the formation of disulfide bonds, glycosylation, limitirovanie, phosphorylation, hydroxylation and tagging one or more amino acid residues of GLP-1.

"A fragment of GLP-1 having the activity of GLP-1", used here, is a peptide, which is missing one or more amino acids from N-Terminus and/or C-end GLP-1 and which retains the activity of GLP-1.

"Extended GLP-1 having the activity of GLP-1", used herein, is a peptide in which the added one or more amino acids from N-Terminus and/or C-end GLP-1 and which retains the activity of GLP-1 (see, for example, Endocrinology, 125, 3109-14 (1989)).

In the sentence "a peptide containing one or more amino acids, optionally attached to the C-end (position 37) GLP-1" used here, amino acids attached to the C-end GLP-1, consistently refer to the amino acid at position 38 the amino acid at position 39, etc., In the sentence "a peptide containing one or more amino acids, optionally attached to the N-end (position 7) GLP-1, the amino acids attached to the N-end GLP-1, respectively, belong to the amino acid at position 6, the amino acid in position 5 and so on, examples of the peptide, with whom containing a series of one amino acid, additionally attached to the C-end (position 37) GLP-1 include peptides containing Asn or Cys attached to 37Gly in GLP-1.

"Peptide GLP-1, a modified oligosaccharide chain (glycosylated peptide GLP-1 conjugate peptide GLP-1 carbohydrate chain)" of the present invention differs in that at least one amino acid is substituted by oligosaccharide chain attached to the amino acid.

"Peptide GLP-1, a modified oligosaccharide chain", as used here, includes a peptide in which at least one amino acid of GLP-1 is substituted by an amino acid-modified oligosaccharide chain and a peptide in which at least one amino acid analog of GLP-1 is substituted by an amino acid-modified oligosaccharide chain. Such peptides are introduced into the peptide GLP-1, a modified oligosaccharide chain, even when they additionally contain a deletion, substitution or addition of one or several amino acids with the exception of amino acids associated with oligosaccharide chain. The peptide, which is the end of any of these peptides subjected to amidation (e.g., GLP-1(7-36)NH2having the amino acid sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2(SEQ ID no:3), where at least one amino acid is substituted by an amino acid, a modified ol hosharian chain also embedded in the peptide GLP-1, a modified oligosaccharide chain. Salts of these peptides also bound peptide GLP-1, a modified oligosaccharide chain.

Salts used herein may be an additive salt of the acid and salt additive base. Acids used to obtain additive salts of the acid are inorganic acids such as hydrochloric acid, Hydrobromic acid, uudistoodetena acid, sulfuric acid and phosphoric acid, and organic acids, for example p-toluensulfonate, methanesulfonate, oxalic acid, p-bromophenylacetate, carboxylic acid, succinic acid, citric acid and acetic acid. Examples of the additive base salts include salts derived from ammonium hydroxide or hydroxides of alkaline or alkaline-earth metals, and salts derived from inorganic bases such as carbonate and bicarbonate. Especially preferred are pharmaceutically acceptable salts.

The term "amino acid, a modified oligosaccharide chain", used here, is an amino acid associated with oligosaccharide chain. In the context of this work oligosaccharide chain can be attached to the amino acid via the linker. Place on the oligosaccharide is of the second circuit, attached amino acid, is not strictly limited. Preferably, the amino acid is linked to reducing end group of the oligosaccharide chain.

The type of amino acids associated with oligosaccharide chain is not strictly limited and can be used both natural and unnatural amino acids. Based on the fact that the amino acid associated with oligosaccharide chain is structurally the same or similar to that which is in the form of glycopeptide (glycoprotein) in vivo, an amino acid, a modified oligosaccharide chain, preferably represents Asn associated with the oligosaccharide chain, for example with N-linked oligosaccharide chain, or Ser, associated with, or Thr, associated with oligosaccharide chain, for example with O-linked oligosaccharide chain, especially preferred is the Asn associated with the oligosaccharide chain.

When the oligosaccharide chain attached to the amino acid via a linker, the amino acid in conjugate with the oligosaccharide chain is: an amino acid containing two or more carboxyl groups in the molecule, such as aspartic acid or glutamic acid; amino acid containing two or more amino groups in the molecule, such as lysine, arginine, histidine, or tryptophan; amino acid containing hydroxyl the th group in the molecule, for example, serine, threonine or tyrosine, an amino acid containing Tilney group in the molecule, such as cysteine; or amino acid containing an amide group in the molecule, such as asparagine or glutamine, from the viewpoint of more easily bind to the linker. Especially, from the viewpoint of reactivity, preferred are aspartic acid, glutamic acid, lysine, arginine, serine, threonine, cysteine, asparagine or glutamine.

The conjugates of oligosaccharide chains with peptides GLP-1 of the present invention do not significantly differ as regulators increase blood sugar levels from conjugate Asn with oligosaccharide chain (without linker) and Cys conjugate with oligosaccharide chain (via the linker) as amino-modified oligosaccharide chain when they contain oligosaccharide chains of identical structure, the same structure except for the structure of the oligosaccharide chains, the same attaching centres of oligosaccharide chains and the same number attached oligosaccharide chains.

When the oligosaccharide chain attached to the amino acid via the linker, can be used any linkers commonly used in this field. Examples of such linkers may include: -NH-(CO)-(CH2)a-CH2- where "a" is an integer, preferably an integer of is 0 to 4, but are not limited to these numbers except when suppressed interest to us linker functions; C1-10polietilen and-CH2-R-, where R is a group formed by removal of one hydrogen atom from a group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, quinil, substituted quinil, aryl, substituted aryl, carbocyclic groups, substituted carbocyclic groups, heterocyclic groups and substituted heterocyclic groups; and -(CO)-(CH2)and-(CO)-, where "a" is an integer, preferably an integer from 0 to 4, but are not limited to these numbers except when suppressed interest to us linker options.

In the amino acid-modified oligosaccharide chain, when the oligosaccharide chain attached to the amino acid that is incorporated into GLP-1 using the linker, the linker also preferably contains the amino acid at the end associated with oligosaccharide chain. Preferred examples of the types of amino acids may include, but not be limited to Asn.

The peptide GLP-1, a modified oligosaccharide chain containing amino acid, a modified oligosaccharide chain, in which the oligosaccharide chain is linked to the amino acid without a linker, may have a lower antigenic what thew, than the peptide GLP-1, a modified oligosaccharide chain, in which the oligosaccharide chain is linked to the amino acid via a linker. The peptide GLP-1, a modified oligosaccharide chain containing amino acid, a modified oligosaccharide chain, in which the oligosaccharide chain is linked to the amino acid via a linker, may have a higher stability in blood flow than the peptide GLP-1, a modified oligosaccharide chain, in which the oligosaccharide chain is linked to the amino acid without a linker.

A method of obtaining a peptide GLP-1 modified oligosaccharide chain of the present invention is not limited in any way (for example, the description stating "peptide GLP-1, a modified oligosaccharide chain, where the substituted amino acid amino acid-modified oligosaccharide chain"). The peptide GLP-1, a modified oligosaccharide chain obtained by any of the following processes a - C, included in the peptide GLP-1, a modified oligosaccharide chain, which amino acid is substituted by an amino acid-modified oligosaccharide chain". Moreover, for example: peptide GLP-1, a modified oligosaccharide chain, where the oligosaccharide chain is not associated with amino acid, is connected directly or via a linker with the amino acid in the peptide; the peptide GLP-1 modified oligosacharide the chain, where is already attached oligosaccharide chain is additionally extended by the accession of sugar or oligosaccharide chain; peptide GLP-1, a modified oligosaccharide chain, in which one or more amino acids associated with amino and/or carboxyl groups of the amino-modified oligosaccharide chain, optionally attached to one or more fragments of GLP-1; and the peptide GLP-1, a modified oligosaccharide chain, which is associated with oligosaccharide chain amino acid attached through the linker with the amino acid in the peptide, also introduced in the peptide GLP-1, a modified oligosaccharide chain of the present invention, provided the final patterns are consistent with this.

The number of substitutions that correspond to the substitution of amino acids of GLP-1 amino acid-modified oligosaccharide chain can be coordinated accordingly with stability in the bloodstream, biological activity (e.g., the ability to regulate blood sugar levels), the number of amino acids found in the target peptide GLP-1, a modified oligosaccharide chain, the molecular weight of the peptide GLP-1 modified oligosaccharide chain before and after joining the oligosaccharide chain, and so on, for Example, are preferred from 1 to 5 substitutions, and more preferred from up to 3 substitutions. In one aspect of the present invention, preferred are at least 2 of the substitution, for example, it is preferable from 2 to 5 substitutions, and more preferred is 2 to 3 substitutions. Preferably, from the point of view can be selected by one substitution, if it is one substitution leads to the desired activity. Typically, the peptide GLP-1, a modified oligosaccharide chain, in which one amino acid of the peptide GLP-1 is substituted by an amino acid-modified oligosaccharide chain, has increased stability in blood flow and a reduced ability to regulate blood sugar levels, if one or several amino acids with the exception of amino acids, modified oligosaccharide chain, optionally substituted amino acid, a modified oligosaccharide chain (however, the reduced capacity to regulate blood sugar levels can be compensated by increased stability in the blood stream).

In the peptide GLP-1, a modified oligosaccharide chain of the present invention the center of substitution of amino acids amino acid-modified oligosaccharide chain may be appropriately adapted to the stability in blood flow or ability to regulate blood sugar levels.

In one aspect of the present invention the center of substitution aminoxy is lots of GLP-1 amino acid, modified oligosaccharide chain may be selected from any centers of GLP-1 in accordance with the desired activity and is, for example, at least one of the centers selected from the provisions 8, 9, 12, 18, 19, 20, 22, 26, 30, 34, 36 and 38 (= connection amino acids, modified oligosaccharide chain to the amino acid at position 37) in GLP1, preferably at least one of the centers selected from the provisions 18, 20, 22, 26, 30, 34, 36 and 38 e.g., for example, one of the centers selected from the provisions of 18, 26, 30, 34 and 36, and is particularly one of the centers selected from positions 30 and 36.

In one aspect of the present invention from the viewpoint of stability of the peptide GLP-1 modified oligosaccharide chain, blood center of substitution of amino acids amino acid-modified oligosaccharide chain may be selected from any centers of GLP-1 and is, for example, at least one of the centers selected from the provisions 9, 10, 11, 12, 14, 16, 18, 19, 20, 22, 24, 25, 26, 27, 28, 30, 32, 34, 36 and 38 (= connection amino acids, modified oligosaccharide chain to the amino acid at position 37) in GLP1, preferably at least one of the centers selected from positions 9, 10, 11, 12, 14, and 28, and is particularly one of the centers selected from positions 9, 10, 11 and 12. In particular, the substitution of amino acids in the centre, close to the N-end, GLP-1 is also preferred. In particular, examples cent the s substitution at least two amino acids of GLP-1 amino acids, modified oligosaccharide chain may include a substitution at position 18 and 36, the substitution at position 26 and 34, the substitution at position 22 and 30, the substitution at position 22 and 36 and the substitution at position 30 and 36 of GLP-1.

In one aspect of the present invention from the viewpoint of the effect of control of blood sugar peptide GLP-1 modified oligosaccharide chain, the center of substitution of amino acids amino acid-modified oligosaccharide chain, for example, is one of the centers selected from the provisions 18, 19, 20, 22, 26, 30, 34, 36 and 38 (= connection amino acids, modified oligosaccharide chain to the amino acid at position 37) in GLP1, preferably at least one of the centers selected from the provisions of 18, 26, 30, 34 and 36, and is particularly one of the centers selected from positions 30 and 36. In particular, examples of centers of substitution of at least two amino acids of GLP-1 amino acids, modified oligosaccharide chain may include a substitution at position 18 and 36, the substitution at position 26 and 34, the substitution at position 22 and 30, the substitution at position 22 and 36 and the substitution at position 30 and 36 in GLP-1, from the point of view of their effect on controlling blood sugar peptide GLP-1 modified oligosaccharide chain.

In one aspect of the present invention from the viewpoint of the ability to synthesize camp, the functionality of the active centers of the peptide GLP-1, modified oligosaccharide chain, for example, is one of the centers of substitution of amino acids amino acid-modified oligosaccharide chain, preferably at least selected from the provisions 22, 26, 27, 30, 34, 36 and 38 (= connection amino acids, modified oligosaccharide chain to the amino acid at position 37), and more preferably at least one of the centers selected from the provisions of 22, 26, 30, 34, 36 and 38.

In one aspect of the present invention the center of substitution of amino acids amino acid-modified oligosaccharide chain is at least one centre, selected from centres with the exception of regulations 8, 9 and 12 in GLP-1.

In one aspect of the present invention the center of substitution of amino acids amino acid-modified oligosaccharide chain is at least one centre, selected from the centres except for the provisions 7, 10, 13, 15, 19, 21, 28 and 29 in GLP-1, and especially one center, selected from the centres except 7, 10, 15 and 28.

In one aspect of the present invention the center of substitution of amino acids amino acid-modified oligosaccharide chain may be chosen from the binding of GLP-1 receptor GLP-1.

In one aspect of the present invention, when two or more amino acids, substituted amino acids, modificirowan the mi oligosaccharide chain, centers substitution of amino acids amino acids, modified oligosaccharide chain can be selected from, but not limited to any combination of facilities described above. For example, a combination in which one selected from the preferred centers, and other centers choose from any centers of GLP-1 and a combination in which one selected from the preferred centers, and other centers choose from any of the centers of one or several amino acids, optionally attached to the C-end (position 37 of GLP-1 is also included in a preferred aspect of the present invention.

In one aspect of the present invention, preferred examples of deletions, substitutions or accession, one or several amino acids with the exception of conjugates of oligosaccharide chains with amino acid(s) in GLP-1 can be included, but are not limited to:

substitution 8Ala amino acid selected from the group consisting of Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 9Gly amino acid selected from the group consisting of Asp and Lys;

substitution 11 Thr amino acid selected from the group consisting of Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp and Lys;

substitution 12Phe amino acid selected from the group consisting of Trp and Tyr;

substitution 13Thr on Ser;

substitution 14Ser amino acid selected from the group consisting of Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 15Ap on Glu;

substitution 16Val amino acid selected from the group consisting of Phe, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp and Lys;

substitution 17Ser amino acid selected from the group consisting of Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 18Ser amino acid selected from the group consisting of Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 19Tyr amino acid selected from the group consisting of Phe, Trp, Glu, Asp and Lys;

substitution 20Leu amino acid selected from the group consisting of Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 21Glu amino acid selected from the group consisting of Asp and Lys;

substitution 22Glu amino acid selected from the group consisting of Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 23Gln amino acid selected from the group consisting of Asn, Arg, Glu, Asp and Lys;

substitution 24Ala amino acid selected from the group consisting of Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp and Lys;

substitution 25Ala amino acid selected from the group consisting of Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 26Lys amino acid selected from the group consisting of Arg, Gln, Glu, Asp, and His;

substitution 27Glu amino acid selected from the group consisting of Asp, Ile, and Lys;

substitution 28Phe on Trp;

substitution 29Ile amino acid selected from the group consisting of Leu, Val, and Ala;

substitution 30Ala amino acid selected from the group consisting of Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 31Trp amino acid, vibrancies group, consisting of Phe, Tyr, Glu, Asp and Lys;

substitution 32Leu amino acid selected from the group consisting of Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp and Lys;

substitution 33Val amino acid selected from the group consisting of Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp and Lys;

substitution 34Lys amino acid selected from the group consisting of Arg, Glu, Asp, and His;

substitution 35Gly amino acid selected from the group consisting of Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys;

substitution 36Arg amino acid selected from the group consisting of Lys, Glu, Asp, and His; and/or

substitution 37Gly amino acid selected from the group consisting of Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp and Lys.

In one aspect of the present invention the center of deletion, substitution or accession, one or several amino acids with the exception of amino acids, modified oligosaccharide chain is preferably at least one of the centers selected from the centres except for the provisions 7, 10, 13, 15, 19, 21, 28 and 29 in GLP-1, such as at least one of the centers selected from the centres except 7, 10, 15 and 28 (Structure-Activity Studies of Glucagon-like Peptide-1, THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.269, No. 9, Issue of March 4, pp. 6276-6278, 1994).

Examples of peptides GLP-1 modified oligosaccharide chain of the present invention include peptide GLP-1, a modified oligosaccharide chain represented by the General formula (I):

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa18-Xaa9 -Leu-Glu-Xaa22-Gln-Ala-Ala-Xaa26-Glu-Phe-Ile-Xaa30-Trp-Leu-Val-Xaa34-Gly-Xaa36-Xaa37

where:

Xaa18represents Ser, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

Xaa19represents Tyr, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA22represents Gly, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA26represents Lys, Cys conjugate with oligosaccharide chain conjugate Asn with oligosaccharide chain or Lys conjugate with oligosaccharide chain;

XAA30represents Ala, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA34represents Lys, Cys conjugate with oligosaccharide chain conjugate Asn with oligosaccharide chain or Lys conjugate with oligosaccharide chain;

XAA36represents Arg, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA37represents Gly, NH2the conjugate Cys to Gly-oligosaccharide chain or conjugate with Asn Gly-oligosaccharide chain, and

when Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34is the Wallpaper Lys, and XAA 36represents Arg, XAA37is a conjugate Cys to Gly-oligosaccharide chain or conjugate with Asn Gly-oligosaccharide chain, where: at least every two amino acids of the original peptide is substituted by an amino acid-modified oligosaccharide chain; oligosaccharide chain is oligohaline acid; and/or oligosaccharide chain represents oligosaccharide chain on the basis of high mannose. Each of the conjugates of Cys with oligosaccharide chain conjugates Asn with oligosaccharide chain and Lys conjugates with oligosaccharide chain may contain a linker between the oligosaccharide chain and the amino acid. The peptide represented by the General formula (1), represented here by SEQ ID no:1.

Specific examples of peptides GLP-1 modified oligosaccharide chain of the present invention include:

(A1) a peptide represented by the General formula (1), where Xaa18represents Cys conjugate with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Gly, XAA26represents represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:4);

(A2) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, the AA 22represents Cys conjugate with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:5);

(A3) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Cys conjugate with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:6);

(A4) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Cys conjugate with oligosaccharide chain, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:7);

(A5) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Cys conjugate with oligosaccharide chain, a Lys, XAA36is Arg, and XAA37 represents Gly (SEQ ID no:8);

(A6) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Cys conjugate with oligosaccharide chain and XAA37represents Gly (SEQ ID no:9);

(A7) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is Arg, and XAA37is a conjugate Cys to Gly-oligosaccharide chain (SEQ ID no:10);

(A8) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Cys conjugate with oligosaccharide chain, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is Arg, and XAA37represents Gly (SEQ ID no:11);

(A9) the peptide represented by the General formula (1), where Xaa18is a conjugate Asn with oligosaccharide chain, Xaa19represents Tyr, Xaa22represents Gly, XAA 26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is Arg, and XAA37represents Gly (SEQ ID no:12);

(a10) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22is a conjugate Asn with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is Arg, and XAA37represents Gly (SEQ ID no:13);

(a11) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26is a conjugate Asn with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36is Arg, and XAA37represents Gly (SEQ ID no:14);

(A12) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30is a conjugate Asn with oligosaccharide chain, XAA34represents Lys, XAA36is Arg, and XAA37represents Gly (SEQ ID no:15);

(A13) a peptide represented by the General formula (1), where the Xaa 18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34is a conjugate Asn with oligosaccharide chain, XAA36is Arg, and XAA37represents Gly (SEQ ID no:16);

(A14) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is a conjugate Asn with oligosaccharide chain and XAA37represents Gly (SEQ ID no:17);

(A15) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37is a conjugate with Asn Gly-oligosaccharide chain (SEQ ID no:18);

(A16) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19is a conjugate Asn with oligosaccharide chain, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34 represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:19);

(A17) the peptide represented by the General formula (1), where Xaa18represents Cys conjugate with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Gly, XAA36represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:20);

(A18), the compound represented by General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Cys conjugate with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:21);

(A19), the compound represented by General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Cys conjugate with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:22);

(A20), the compound represented by General formula (1), where Xaa18pre who is Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Cys conjugate with oligosaccharide chain, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:23);

(A21) a compound represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Cys conjugate with oligosaccharide chain, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:24);

(A22) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Cys conjugate with oligosaccharide chain and XAA37represents NH2(SEQ ID no:25);

(A23) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Cys conjugate with oligosaccharide chain, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34/sub> represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:26);

(A24) the peptide represented by the General formula (1), where Xaa18is a conjugate Asn with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:27);

(A25) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22is a conjugate Asn with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:28);

(A26) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26is a conjugate Asn with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:29);

(A27) the peptide represented by the General formula (1), where Xaa18presented yet a Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30is a conjugate Asn with oligosaccharide chain, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID no:30);

(A28) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34is a conjugate Asn with oligosaccharide chain XAA36represents Arg, and XAA37represents NH2(SEQ ID no:31);

(A29) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is a conjugate Asn with oligosaccharide chain and XAA37represents NH2(SEQ ID no:32); and

(A30) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19is a conjugate Asn with oligosaccharide chain, XAA22represents Gly, Xaa26represents Lys, XAA30represents Ala, XAA34predstavljaet a Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID NO:33).

In these examples, the peptide GLP-1 modified oligosaccharide chain oligosaccharide chain is preferably, for example, oligohaline acid or oligosaccharide chain on the basis of high mannose.

Examples of peptides GLP-1 modified oligosaccharide chain, the present invention also include:

(A31) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, Xaa22represents Gly, XAA26represents Lys conjugate with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:34); and

(A32) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys conjugate with oligosaccharide chain, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:35).

In these examples, peptides GLP-1 modified oligosaccharide chain oligosaccharide chain is preferably linked via a linker, such as, for example, Lys conjugate with oligosacharides chain.

Examples of peptides GLP-1 modified oligosaccharide chain, also include:

(A33) a peptide represented by the General formula (1), where Xaa18represents Cys conjugate with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Cys conjugate with oligosaccharide chain and XAA37represents Gly (SEQ ID no:36);

(A34) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Cys conjugate with oligosaccharide chain, XAA26represents Lys, XAA30represents Cys conjugate with oligosaccharide chain, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:37);

(A35) the peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Cys conjugate with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Cys conjugate with oligosaccharide chain and XAA37represents Gly (SEQ ID no:38);

(A36) peptide, the sight of the purposes of the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Cys conjugate with oligosaccharide chain, XAA30represents Ala, XAA34represents Cys conjugate with oligosaccharide chain, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:39);

(A37) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Cys conjugate with oligosaccharide chain, XAA34represents Lys, XAA36represents Cys conjugate with oligosaccharide chain and XAA37represents Gly (SEQ ID no:40);

(A38) a peptide represented by the General formula (1), where Xaa18is a conjugate Asn with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is a conjugate Asn with oligosaccharide chain and XAA37represents Gly (SEQ ID no:41);

(A39) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents nyugat Asn with oligosaccharide chain, XAA26represents Lys, XAA30is a conjugate Asn with oligosaccharide chain, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:42);

(A40) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22is a conjugate Asn with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36is a conjugate Asn with oligosaccharide chain and XAA37represents Gly (SEQ ID no:43);

(A41) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26is a conjugate Asn with oligosaccharide chain, XAA30represents Ala, XAA34is a conjugate Asn with oligosaccharide chain, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:44);

(a) a peptide represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26represents Lys, XAA30is a conjugate Asn with oligosaccharide chain, XAA34represents Ly, XAA36is a conjugate Asn with oligosaccharide chain and XAA37represents Gly (SEQ ID no:45).

In these examples, peptides GLP-1 modified oligosaccharide chain oligosaccharide chain preferably represents, for example, biancanello oligosaccharide chain complex type.

Examples of peptides GLP-1 modified oligosaccharide chain, the present invention also include:

(A43) a peptide represented by the General formula (1), where Xaa18represents Cys conjugate with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Cys conjugate with oligosaccharide chain, XAA26represents Lys, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37is a conjugate Cys to Gly-oligosaccharide chain (SEQ ID no:46);

(A44) a peptide represented by the General formula (1), where Xaa18represents Cys conjugate with oligosaccharide chain, Xaa19represents Tyr, XAA22represents Cys conjugate with oligosaccharide chain, XAA26represents Cys conjugate with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents Gly (SEQ ID no:47);

(A45) is epted, represented by the General formula (1), where Xaa18represents Ser, Xaa19represents Tyr, XAA22represents Gly, XAA26is a conjugate Asn with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36is a conjugate Asn with oligosaccharide chain and XAA37is a conjugate with Asn Gly-oligosaccharide chain (SEQ ID no:48); and

(A46) a peptide represented by the General formula (1), where Xaa18is a conjugate Asn with oligosaccharide chain, Xaa19represents Tyr, XAA22is a conjugate Asn with oligosaccharide chain, XAA26is a conjugate Asn with oligosaccharide chain, XAA30represents Ala, XAA34represents Lys, XAA36represents Arg, and XAA37represents NH2(SEQ ID NO:49).

In these examples, peptides GLP-1 modified oligosaccharide chain oligosaccharide chain preferably represents, for example, biancanello oligosaccharide chain complex type.

Examples of conjugates analogue peptide GLP-1 with oligosaccharide chain of the present invention may include the basis having the amino acid sequence

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-er-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2(SEQ ID no:50)

and having attached to it oligosaccharide chain.

The conjugate of the basis 4 with oligosaccharide chain, for example, General formula (2):

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa12-Gln-Xaa14-Glu-Xaa16-Glu-Ala-Val-Xaa20-Leu-Phe-Ile-Xaa24-Trp-Leu-Lys-Xaa28-Gly-Xaa30-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2

where:

Xaa12represents Lys, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

Xaa14represents Met, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

Xaa16represents Glu, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA20represents Arg, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA24represents Glu, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA28represents Asn, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA30represents Gly, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

at least one of Xaa12, Xaa14, Xaa16, XAA20, XAA24, Xaa28and XAA30represents Cys conjugate with oligosaccharide chain or conjugate Asn is oligosaccharide chain (SEQ ID no:51).

Among them XAA24and/or XAA30are preferably Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain. Especially XAA30preferably is conjugate with Cys oligosaccharide chain.

Examples of peptide GLP-1 modified oligosaccharide chain containing a deletion, substitution or addition of one or several amino acids, which is a peptide GLP-1, a modified oligosaccharide chain of the present invention, may include BIM51077 having the amino acid sequence

His-R2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-R2-Arg-NH2,

where R2represents an α-methylamin (SEQ ID no:52),

and attached to it oligosaccharide chain.

Conjugate oligosaccharide chain with BIM51077 represented by the General formula (3):

His-R2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa18-Tyr-Xaa20-Glu-Xaa22-Gln-Ala-Ala-Xaa26-Glu-Phe-Ile-Xaa30-Trp-Leu-Val-Xaa34-R2-Xaa36-NH2,

where:

R2represents an α-methylalanine;

Xaa18represents Ser, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA20represents Leu, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA22represents Gly, Cys conjugate the oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA26represents Lys, Cys conjugate with oligosaccharide chain conjugate Asn with oligosaccharide chain or Lys conjugate with oligosaccharide chain;

XAA30represents Ala, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain;

XAA34represents Lys, Cys conjugate with oligosaccharide chain conjugate Asn with oligosaccharide chain or Lys conjugate with oligosaccharide chain;

XAA36represents Arg, Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain, and

at least one of Xaa18, XAA20, XAA22, XAA26, XAA30, XAA34and XAA36represents Cys conjugate with oligosaccharide chain or conjugate Asn with oligosaccharide chain (SEQ ID no:53).

When synthesized amino acid, a modified oligosaccharide chain, in which the oligosaccharide chain attached to the amino acid on the C-terminal fragment of the peptide, initially containing aminirovanie To-end, for example on the basis of 4 and BIM51077, the end may be neomedievalism.

The term "oligosaccharide chain" used herein refers to a compound containing at least one carbohydrate link (monosaccharide and/or its derivative). When connected to two or more carbohydrate units, such carbohydrate chains bind deshidratarse the condensation due to the formation of glycosidic linkages between them. Examples of such oligosaccharide chains include, but are not limited to monosaccharides and polysaccharides (glucose, galactose, mannose, pocosol, xylose, N-acetylglucosamine, N-atsetilgalaktozamin, sialic acid and their complexes and derivatives) located in living organisms, as well as a wide range of oligosaccharide chains, such as degraded polysaccharides and polysaccharides degraded or formed from complex biological molecules, such as glycoproteins, proteoglycans, glycosaminoglycans and glycolipids. Oligosaccharide chains can be linear or branched.

The term "oligosaccharide chain", as used here, also includes derived oligosaccharide chain. Examples of derived oligosaccharide chains include, but are not limited to oligosaccharide chains composed of sugars containing carboxyl group (for example, aldonova acid, which represents a carboxylic acid formed by oxidation at position C-1 (for example, D-gluconic acid formed by the oxidation of D-glucose and uronic acid in which the terminal carbon atom is oxidized to a carboxyl group (D-glucuronic acid formed by the oxidation of D-glucose)), sugar, contains an amino group or a derivative of an amino group (for example, the acetylated amino group (for example, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine); sugar, containing both amino-and carboxyl group (for example, N-acetylneuraminic acid (sialic acid) and N-acetylmuramyl acid); deoxysugars (for example, 2-deoxy-D-ribose); sulfonated sugar, contains the group of sulfuric acid, and phosphorylated sugar, contains the group of phosphoric acid.

In the present invention the preferred oligosaccharide chains increase the stability of GLP-1 in blood and, more preferably, not lower activity of GLP-1 as a regulator of blood sugar levels when they are attached to the GLP-1 (for example, when the amino acid of GLP-1 is substituted by an amino acid-modified oligosaccharide chain). In one aspect of the present invention the preferred oligosaccharide chains increase the activity of GLP - as a regulator of blood sugar levels when they are attached to the GLP-1 (for example, when the amino acid of GLP-1 is substituted by an amino acid-modified oligosaccharide chain).

Oligosaccharide chains in the peptide GLP-1, a modified oligosaccharide chain of the present invention are very limited and may or may not be present in vivo in the form of complex carbohydrate (glycopeptide (or glycoprotein), proteoglycan, glycolipid, and so on).

Oligosaccharide chains that are found in vivo in the form of complex carbohydrates, are PR is doctitle, because peptides GLP-1, modified oligosaccharide chain of the present invention is introduced into living organisms. Examples of such oligosaccharide chains include N-linked oligosaccharide chains and O-linked oligosaccharide chains, which in vivo is connected to the peptide (or protein) with the formation of glycopeptide (or glycoprotein). It is preferable to use N-linked oligosaccharide chains. Examples of N-linked oligosaccharide chains may include oligosaccharide chain on the basis of high mannose, oligosaccharide chain complex type and oligosaccharide chain of the hybrid type. Especially preferred are the oligosaccharide chain complex type.

Examples of preferred oligosaccharide chains of the complex type used in the present invention include oligosaccharide chain, represented by the following General formula:

Formula 3

where

R1and R2are the same or different and each represent

Formula 4

and

AC represents an acetyl group.

In the peptide GLP-1, a modified oligosaccharide chain of the present invention oligosaccharide chain may be in the form of complex carbohydrate or may be linked to the peptide GLP-1 way that is different from Ouazane and N-bonding. For example, peptides GLP-1, modified oligosaccharide chain, in which the oligosaccharide chain is linked to Cys or Lys via a linker, as described above, is also included in the peptide GLP-1, a modified oligosaccharide chain of the present invention.

In a preferred aspect of the present invention, the oligosaccharide chain is a relatively low molecular weight glycosaminoglycan, such as hyaluronic acid, chondroitin, And chondroitin sulfates, heparin, heparansulfate and keratinolytic. Such oligosaccharide chains contain linearly related repeating disaccharide glycosides of links consisting of amino sugars (N-acetylglucosamine or N-atsetilgalaktozamin) and uronic acid (glucuronic acid or L-iduronovoy acid). The term "relatively low molecular weight glycosaminoglycan" used here means that the molecular weight, for example approximately 10 kDa or less, preferably approximately 6 kDa or less, more preferably approximately 4 kDa or less, or the number of sugar residues is equal to approximately 50 or below, preferably 30 or less, more preferably 20 or lower.

In one aspect of the present invention, the oligosaccharide chain in the peptide GLP-1, a modified oligosaccharide chain of the present invention is a preferably of Oleg sharenow chain, consisting of four or more sugar residues, such as five or more, seven or more, especially nine or ten or eleven or more sugar residues.

In a preferred aspect of the present invention, the oligosaccharide chain in the peptide GLP-1, a modified oligosaccharide chain of the present invention is a contains from five to eleven, from nine to eleven or eleven residues of sugar.

In a preferred aspect of the present invention, the oligosaccharide chain in the peptide GLP-1, a modified oligosaccharide chain of the present invention is biancanello oligosaccharide chain complex type. Oligosaccharide chain complex type is characterized by the fact that consists of two or more types of monosaccharides and has the following basic structure and the structure of lactosamine represented by Galβ1-4GlcNAc:

Formula 5

Bianconera oligosaccharide chain complex type refer to such structures in which manantenina oligosaccharide chain containing from 0 to 3 sugars, associated with each of the both ends of the mannose core structure. Bianconera oligosaccharide chain complex type represents preferably, for example, the following devalorisation chain:

Formula 6

monosialoganglioside chain

Formula 7

belolipskaia chain:

Formula 8

GlcNAc-oligosaccharide chain:

Formula 9

demonopolisation chain:

Formula 10

and so on

Devalorisation chain is more preferred.

The term "Devalorisation chain", "monosialoganglioside chain", "belolipskaia chain", "GlcNAc-oligosaccharide chain" and "demonopolisation chain", as used here, also indicate along with the above chemical formulas oligosaccharide chain with link types that are different from those represented by chemical formulas. Such oligosaccharide chain is also preferably used as the oligosaccharide chains of the present invention. Examples of such oligosaccharide chains include devalorisation chain or asialoglycoprotein a circuit in which sialic acid linked to galactose (α2→3) bond.

Oligosaccharide chain on the basis of high mannose used in the present invention, represents oligosaccharide chain, in which two or more mannose residues optionally linked to the basic structure of the oligosaccharide chain of the integrated is of the first kind. Since the oligosaccharide chain on the basis of high mannose is voluminous, attach the oligosaccharide chain on the basis of high mannose to the peptide can significantly stronger increase stability in the blood. It is preferable oligosaccharide chain containing 5-9 mannose, for example oligosaccharide chain on the basis of high mannose mammals. Can be used oligosaccharide chain containing an increasing number of mannose, for example oligosaccharide chain on the basis of high mannose yeast. Examples of the oligosaccharide chains on the basis of high mannose, preferably used in the present invention, may include:

high mannose-5 (M-5):

Formula 11

and high mannose-9 (M-9):

Formula 12

In the present invention, examples of preferred oligosaccharide chains may include structurally the same oligosaccharide chain (oligosaccharide chain having the same types of sugars that make up their composition and the same types of links between sugars) that oligosaccharide chains that are attached to the protein during the formation of glycoprotein in the human body (e.g., oligosaccharide chain, described in "FEBS LETTERS Vol.50, No. 3, Feb. 1975"), or oligosaccharide chain, which lack one or more residues of sugar with their newos tanalysis all, which are described in the following tables 1-4.

Table 1

Table 2

Table 3

Table 4

In one aspect of the present invention, the oligosaccharide chain is preferably oligosaccharide chain having a linear structure. Examples of such oligosaccharide chains include oligohaline acid. Aigagaranca acid used here, refers to the oligosaccharide chain in which N-acetylglucosamine and glucuronic acid alternately linearly related to education from di - to dotriacontanoic, preferably di - to hexadecasaccharide, more preferably from Tetra - to octasaccharide.

Examples of particularly preferred oligohaline acid in the present invention include oligosaccharide chain, 2 (tetrachoric) or more units and 8 (hexadecasaccharide) or fewer units each of which consists of N-acetylglucosamine and glucuronic acid. Oligosaccharide chain having from 2 (tetrachoric) to 4 (octasaccharide) links is more preferable, and the oligosaccharide chain having 2 (tetrachoric) level, is the most preferred.

Examples of hyaluronic acid, predpochtitelno used in the present invention, include oligohaline acid in the form of tetrasaccharide:

Formula 13

and oligohaline acid in the form of octasaccharide:

Formula 14

In a preferred aspect of the present invention glikopeptid the present invention has a homogeneous oligosaccharide structure. The structure of homogeneous oligosaccharide chain glycopeptide used here, refers to the same oligosaccharide chain attached to the center of the peptide, to the same type included in the sugar oligosaccharide chain, to the same order of joining sugars and the same type of linkage between sugars when compared glycopeptides and means that the structure of the oligosaccharide chain has at least 90%, preferably at least 95%, more preferably at least 99% homogeneity. Glycopeptides having a homogeneous structure of the oligosaccharide chain, have a constant quality and especially preferred in such areas as pharmaceuticals or quantitative analysis. Share homogeneous oligosaccharide chains can be measured using this method, such as HPLC, capillary electrophoresis, NMR, mass spectrometry, etc.

In the present invention, examples of preferred peptides GLP-1, modificirowan the x oligosaccharide chain, may include peptides GLP-1, modified oligosaccharide chain (SEQ ID no:54-66) obtained in examples 1-15, described below, i.e., peptides GLP-1, modified oligosaccharide chain having the sequence

His7-Ala8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Lys34-Gly35-Arg36-Gly37(SEQ ID no:2, GLP-1), where:

(b1) each of 26Lys and 34Lys replaced by Cys conjugate with disialogangliosides circuit (example 1) (SEQ ID no:54);

(b2) each of 18Ser and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 2) (SEQ ID no:55);

(b3) each of 22Gly and 30Ala replaced by Cys conjugate with disialogangliosides circuit (example 3) (SEQ ID no:56);

(b4) each of 22Gly and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 4) (SEQ ID no:57);

(b5) each of 30Ala and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 5) (SEQ ID no:58);

(b6) 30Ala replaced by Cys conjugate with tetrasaccharide oligohaline acid (4) (example 6) (SEQ ID no:59);

(b7) 30Ala replaced by Cys conjugate with octasaccharide oligohaline acid (8) (example 7) (SEQ ID no:60);

(b8) 36Arg replaced by Cys conjugate with tetrasaccharide oligohalob the new acid (4) (example 8) (SEQ ID no:61);

(b9) 36Arg replaced by Cys conjugate with octasaccharide oligohaline acid (8) (example 9) (SEQ ID no:62);

(b10) 30Ala replaced by Cys conjugate with hexadecasaccharide oligohaline acid (16) (example 10) (SEQ ID no:63);

(b11) 36Arg replaced by Cys conjugate with hexadecasaccharide oligohaline acid (16) (example 11) (SEQ ID no:64);

(b12) 36Arg replaced by Cys conjugate with oligosaccharide chain on the basis of high mannose (M5) (example 12) (SEQ ID no:65); and

(b13) conjugate Asn with asialoglycoprotein chain attached to 26Lys through a linker (example 13) (SEQ ID no:66), and

additionally includes:

(b14) the peptide GLP-1, a modified oligosaccharide chain having the sequence H-His1-Gly2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Leu10-Ser11-Lys12-Gln13-Met14-Glu15-Glu16-Glu17-Ala18-Val19-Arg20-Leu21-Phe22-Ile23-Glu24-Trp25-Leu26-Lys27-Asn28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2(SEQ ID no:50, on the basis of 4), where

30Gly replaced by Cys conjugate with disialogangliosides circuit (example 14) (SEQ ID no:67);

(b15) the peptide GLP-1, a modified oligosaccharide chain having the sequence

His7-R28-Glu9-Gly10-Thr11-Phe12 -Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Lys34-R235-Arg36-NH2where R2 represents an α-methylalanine (SEQ ID no:52, BIM51077), where 26Lys replaced by Cys conjugate with disialogangliosides circuit (example 15) (SEQ ID no:68); and

(b16) the peptide GLP-1, a modified oligosaccharide chain having the sequence of basis-4 (SEQ ID no:50), where 30Gly replaced by Cys conjugate with oligosaccharide chain on the basis of high mannose (M5) (example 16).

The peptide GLP-1, a modified oligosaccharide chain of the present invention can be obtained by the inclusion of the addition step oligosaccharide chains in peptide synthesis, known to specialists in this field. To attach the oligosaccharide chain can also be used a method of using the reverse reaction catalyzed by the enzyme, which is a typical representative of transglutaminase. However, this method involves problems such as the large number of oligosaccharide chains, which you want to attach, complex cleanup after conducting the final stage, a limited number of centres, to which are attached oligosaccharide chains, and limited types oligosacharide the x circuits which can be attached. Therefore, this method is not suitable for large-scale, for example, pharmaceutical products, although it can be used for the synthesis of small amounts, for example for the quantitative analysis.

A convenient way to obtain the peptide GLP-1 modified oligosaccharide chain of the present invention and for targeted obtaining peptides GLP-1 modified oligosaccharide chain having a homogeneous structure of the oligosaccharide chains, will be specifically confirmed the following example describes a method of producing peptides GLP-1 modified oligosaccharide chain with amino acids, modified oligosaccharide chain, and the method of peptide synthesis known as solid-phase synthesis or liquid-phase synthesis (method A) and the method of obtaining peptides GLP-1 modified oligosaccharide chain, which consists of obtaining the peptide, in which any amino acid of GLP-1 is substituted by Cys, in accordance with well-known specialists in this field by the method of peptide synthesis and subsequent merger of oligosaccharide chains to Cys by chemical synthesis (method). The method of producing peptides GLP-1 modified oligosaccharide chain comprising the amino acid, a modified oligosaccharide chain, W is an amino acid associated with oligosaccharide chain via a linker and the linker contains the amino acid at the end of the chain, attached to the oligosaccharide chain is confirmed by the method of producing peptides GLP-1 modified oligosaccharide chain, according to which, first, one end of the linker is attached to the conjugate Asn with oligosaccharide chain, and then N-hydroxysuccinimide group is attached to the other end of the linker and N-hydroxysuccinimide group interacts with the side-amino group of a Lys residue of the peptide GLP-1 (method C). Specialists in this field can synthesize various peptides GLP-1, modified oligosaccharide chain, focusing on these methods of obtaining. The resulting peptides GLP-1, modified oligosaccharide chain and the methods for their preparation are very useful in the field of pharmaceutical production. Two or more of these methods from a to C can be implemented in combination. For the synthesis of small quantities, for example for the quantitative analysis, these methods can also be combined with elongation reaction catalyzed by a transferase. Methods a and b described in the instructions WO 2004/005330 (US2005222382 (A1)) and WO 2005/010053 (US2007060543 (A1)), respectively. The disclosure is entirely incorporated here by reference. Obtaining oligosaccharide chains having a homogeneous structure of the oligosaccharide chains used in methods a through C, described in the instructions WO 03/008431 (US2004181054 (A1)), WO 2004/58984 (US2006228784 (A1)), WO 2004/058824 (US2006009421 (A1)), WO 2004/070046 (US2006205039 (A1)), WO 2007/011055 etc., the disclosure is entirely incorporated here by reference.

A method of obtaining a peptide GLP-1 modified oligosaccharide chain (Method A)

First, (1) hydroxyl group of a resin containing hydroxyl group and carboxyl group of amino acids containing the nitrogen of the amino group of fat-soluble protective group, is subjected to the esterification reaction. Because the amino group of this amino acid protected at the nitrogen of the fat-soluble protective group, a hydroxyl group of the resin interacts with the carboxyl group of the amino acids and samarangense amino acids is prevented.

Secondly, (2) fat-soluble protective group is removed from the resulting complex ester, which leads to the formation of free amino groups,

(3) the free amino group lidiruyut carboxyl group of the necessary amino acids, containing the nitrogen of the amino group of fat-soluble protective group,

(4) a fat-soluble protective group is removed, which leads to the formation of free amino groups, and

(5) stage (3) and (4) is repeated at least once and thereby gain a peptide containing the desired number of amino acids, connected and thus one end attached to the resin, and at the other end containing a free amino group.

Then (6) is free and is infogruppu lidiruyut carboxyl group of the asparagine residue, linked oligosaccharide (asparagine conjugate with oligosaccharide chain) containing the nitrogen of the amino group of fat-soluble protective group,

(7) a fat-soluble protective group is removed, which leads to the formation of free amino groups,

(8) the free amino group lidiruyut carboxyl group of the necessary amino acids, containing the nitrogen of the amino group of fat-soluble protective group,

(9) stage (7) and (8) repeating at least one more time

(10) the fat-soluble protective group is removed, which leads to the formation of free amino groups, and thus receive a glycolipid containing the desired number of amino acids, connected and thus one end attached to the resin, at the other end containing a free amino group and a conjugate of asparagine with oligosaccharide chain in an intermediate position.

(11) the Resin is separated by acid, so it may be derived glycolipid containing asparagine conjugate with oligosaccharide chain in the desired position of the peptide chain.

Alternative conjugate asparagine with oligosaccharide chain can be entered at the end of the peptide chain.

The resin containing a hydroxyl group is typically a resin containing hydroxyl group to be used for solid-phase syntheses. Examples of the resins used are resin Amino-PEGA (PR is the product of Merck), the Wang resin (product of Merck), resin HMPA-PEGA (product of Merck), and so on

All amino acids are used as such. Examples of the amino acids used are natural amino acids, such as serine (Ser), asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine (Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), threonine (Thr), cysteine (Cys), methionine (Met), phenylalanine (Phe), tryptophan (Trp) and Proline (Pro).

Examples of fat-soluble protective group are 9-fluorenylmethoxycarbonyl (Fmoc) group, tert-butyloxycarbonyl (Boc) group, benzyl group, allyl group, allyloxycarbonyl group, acetyl group, etc., that are protective groups of the carbonate type or amide type. Fat-soluble protective group, such as Fmoc group can be introduced by the accession 9-fluorenylmethyl-N-Succinimidyl carbonate and sodium bicarbonate to an alleged connection for this reaction. Preferably conduct the reaction at a temperature of from 0 to 50°C, preferably at room temperature, for about 1 to about 5 hours.

The above amino acids can be protected with a fat-soluble protective group as described above. The above protected amino acids can be those that I have are commercially available. Examples are Fmoc-Ser, Fmoc-Asn, Fmoc-Val, Fmoc-Leu, Fmoc-Ile, Fmoc-Ala, Fmoc-Tyr, Fmoc-Gly, Fmoc-Lys, Fmoc-Arg, Fmoc-His, Fmoc-Asp, Fmoc-Glu, Fmoc-Gln, Fmoc-Thr, Fmoc-Cys, Fmoc-Met, Fmoc-Phe, Fmoc-Trp and Fmoc-Pro.

Catalysts for the esterification are dehydrating condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIPCI). Dehydrating condensing agent is used in an amount of from 1 to 10 wt.%, preferably from 2 to 5 wt.% per 1 wt.% the amino acids.

The esterification reaction is conducted preferably by placing the resin, for example, in solid column, washing the resin with a solvent and then attach solution of amino acids in a solvent to the resin. Examples of solvents for washing are dimethylformamide (DMF), 2-propanol, methylene chloride, etc. Examples of solvents for dissolving amino acids are dimethyl sulfoxide (DMSO), DMF, methylene chloride etc., the Reaction is carried out at a temperature from 0 to 50°C, preferably at room temperature for from about 10 to about 30 hours, preferably from about 15 minutes to about 24 hours.

Preferably, unreacted hydroxyl groups remaining in the solid phase during this time acetimidoyl, for example, acetic anhydride to block.

Georaster who may protective group may be removed, for example, by treatment with a base. Examples used bases are piperidine, morpholine, etc. Such treatment is carried out preferably in the presence of a solvent. Examples of the used solvents are DMSO, DMF, methanol and so on

The amidation reaction of the free amino group carboxyl group of the necessary amino acids containing an amino group protected at the nitrogen of the fat-soluble protective group, preferably carried out in the presence of an activator and a solvent.

Examples used activators are dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/hydrochloride (WSC/HCl), diphenylphosphoryl (DPPA), carbonyldiimidazole (CDI), diethylthiophosphate (DEPC), diisopropylcarbodiimide (DIPCI), benzotriazol-1 yloxy-Tris-pyrrolidinedithiocarbamate (Rubor), 1-hydroxybenzotriazole (HOBt), hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP), 1-hydroxy-7-asobancaria (HOAt), hydroxyphthalimide (HOPht), pentafluorophenol (Pfp-OH), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylhexadecane (HBTU), O-(7-asobancaria-1-yl)-1,1,3,3-getrelativeposition (HATU), O-benzotriazol-1-yl-1,1,3,3-tetramethylethylenediamine (TBTU), 3,4-dihydro-3-hydrody-4-oxo-1,2,3-benzotriazine (Dhbt).

The activator is used in an amount of from 1 to 20 equivalents, preferably 1 to 10 equivalents, more PR is doctitle from 1 to 5 equivalents per amino acid, having the amino group, substituted on the nitrogen of the fat-soluble protective group.

Examples of the used solvents are DMSO, DMF, methylene chloride etc., Preferably, at a temperature from 0 to 50°C, preferably at room temperature for from about 10 to about 30 hours, preferably from about 15 minutes to about 24 hours. Fat-soluble protective group can be removed in the same way as described above.

The peptide chain is separated from the resin, preferably by treatment with an acid. Examples of acids used are triperoxonane acid (TFU), hydrogen fluoride (HF), and so on

Glikopeptid containing at least two asparagine conjugate with oligosaccharide chain in the desired position of the peptide chain can be obtained by the relevant Association stage (6), leading to the amidation of the free amino group of the peptide carboxyl group residue of asparagine in the asparagine conjugate with oligosaccharide chain having the amino group, substituted on the nitrogen of the fat-soluble protective group, and stage (7), corresponding to the removal of fat-soluble protective group, which leads to the formation of free amino groups. At this stage glikopeptid containing at least two types of conjugates of asparagine with oligosaccharide chain in the correct put and its peptide chain, can also be obtained through various conjugates of asparagine with oligosaccharide chain.

Glikopeptid containing at least one conjugate of asparagine with oligosaccharide chain in the desired position of the peptide chain can be obtained by carrying out the quality of the final stages, i.e., instead of the steps (9) and (10), stage (6), leading to the amidation of the free amino group of the peptide carboxyl group residue of asparagine in the asparagine conjugate with oligosaccharide chain having the amino group, substituted on the nitrogen of the fat-soluble protective group, and stage (7), corresponding to the removal of fat-soluble protective group, which leads to the formation of free amino groups.

Glikopeptid containing asparagine conjugate with oligosaccharide chain at the C-end, can be obtained by carrying out stage (1) in which a hydroxyl group of the resin is subjected to the esterification reaction carboxyl group residue of asparagine in the asparagine conjugate with oligosaccharide chain containing an amino group protected at the nitrogen of the fat-soluble protective group, instead of amino acids containing an amino group protected at the nitrogen of the fat-soluble protective group. In this case, stage (6) can additionally conduct or not to conduct.

Thus, it can be derived peptide GLP-1, a modified oligosaccharide chain, Sames the config in the right position conjugate with asparagine oligosaccharide chain.

A method of obtaining a peptide GLP-1 modified oligosaccharide chain (Method)

First, the peptide containing Cys, get in the way of solid-phase synthesis, liquid phase synthesis, cellular synthesis and separation and extraction of peptides existing in nature. Oligosaccharide chain can be attached in the desired position by changing the position of the Cys.

Next haloacetonitriles derived oligosaccharide chain complex type is reacted with the thus obtained peptide containing Cys, for large-scale production. The reaction can be conducted at temperatures from 0 to 80°C, preferably from 10 to 60°C., more preferably from 15 to 35°C. the reaction Time is usually from about 30 minutes to about 5 hours. After completion of the reaction, the reaction product can be purified appropriate method known in the field [e.g., high performance liquid column chromatography (HPLC)].

Haloacetonitriles derived oligosaccharide chain complex type, i.e. a compound in which a hydroxyl group linked to the carbon atom in position 1, which is connected with asparagine oligosaccharide chain complex type, substituted-NH-(CO)-(CH2)a-CH2X, where X represents a halogen atom and a is an integer from 0 to 4, but is not limited to these Chi the Lamy as long while interest linker options are not suppressed.

Especially haloacetonitriles derived oligosaccharide chain complex type is introduced into the reaction with the peptide containing Cys in phosphate buffer at room temperature. After completion of the reaction the peptide GLP-1, a modified oligosaccharide chain, substituted by Cys conjugate with oligosaccharide chain can be obtained by HPLC purification.

A method of obtaining a peptide GLP-1 modified oligosaccharide chain (Method C)

First, the peptide containing Lys, get in the way of solid-phase synthesis, liquid phase synthesis, cellular synthesis and separation and extraction of peptides existing in nature.

Next, glutaric acid attached to the amino-modified oligosaccharide chain. For example, an amino acid, a modified oligosaccharide chain is dissolved in a solution of DMSO. To this solution was added a solution of DMSO containing a mixture of glutaric acid - EDC and stirred at room temperature for 1 day. The reaction mixture may be appropriately diluted, and then fractionated by high-performance gel chromatography and so on, to obtain amino acid, a modified oligosaccharide chain, which glutaric acid is linked to the α-amino group.

Subsequently, to a solution of DMSO amino acids, modifitsirovannoi oligosaccharide chain, related glutaric acid, add a solution of N-hydroxysuccinimide and the EDC solution in DMSO and stirred at room temperature for 6 hours. Then EDC can be inactivated for the synthesis of amino-modified oligosaccharide chain associated with a complex ester of N-hydroxysuccinimidyl glutaric acid.

Subsequently, to a solution of DMSO peptide GLP-1 in DMSO added DIPEA and amino acid, a modified oligosaccharide chain associated with N-hydroxysuccinimidyl glutaric acid, and stirred at room temperature for 2 hours. Then the reaction is finished by attaching an aqueous solution of glycine. The reaction solution can be subjected to appropriate treatment to attach amino acids, modified oligosaccharide chain of the Lys residue in the peptide GLP-1 via a linker, glutaric acid. Thus obtained peptide GLP-1, a modified oligosaccharide chain containing amino acid, a modified oligosaccharide chain, in which the oligosaccharide chain is linked to the amino acid (Lys) via the linker and the linker contains the amino acid (Asn) at the end of the oligosaccharide chain.

The peptide GLP-1, a modified oligosaccharide chain containing amino acid, a modified oligosaccharide chain associated with the desired center can be obtained from the Deputy is the group of amino acids in the right center of the peptide GLP-1 lysine (Lys) or by substitution of Lys residue, contained in the peptide GLP-1 wild-type, another amino acid. In accordance with the method, when the oligosaccharide chain attached to Lys, located in GLP-1 wild type, can be obtained peptide GLP-1, a modified oligosaccharide chain having the same peptide skeleton, and the skeleton of the wild type.

Conjugate GLP-1 with oligosaccharide chain of the present invention has the activity of GLP-1.

The term "activity of GLP-1" used here refers to some or all types of biological activity known in the field as GLP-1. It is known that GLP-1 has in addition to the ability to regulate blood sugar levels, for example, the secretion of insulin that is associated with the induction of the synthesis of camp, the protection of the pancreatic islets (inhibition of apoptosis) and the growth of pancreatic islets as a result of action on the pancreatic islets, and also affects the appetite suppression, the suppression of the motility of the gastrointestinal tract, promotion of secretion of calcitonin and cardioprotective effects in ischemia as extrapancreatic effects. Thus, the activity of GLP-1 applies to all or some types of biological activity associated with these effects, and these activities can be measured, respectively, using an approach known specialists in this field.

From GLP-1, for example, the activity of controlling blood sugar levels can be measured by the ability to regulate blood sugar levels in diabetic mice (mice db/db) or measuring the effect of inhibiting the growth of blood sugar test, oral glucose tolerance (OGTT). The phrase "ability to regulate blood sugar levels", as used here, includes both the concept of inhibiting the growth of blood sugar levels and reduce blood sugar levels. In particular, the effect of controlling blood sugar levels in diabetic mice (mice db/db) also refers in this document to "the effect of reducing the level of sugar in the blood, and the ability to regulate blood sugar levels in OGTT also refer in this document to "the effect of inhibiting the growth of blood sugar levels".

Activity regulation of blood sugar levels in the OGTT can be determined by measuring the inhibition of growth of blood sugar levels in mice who were forced to drink sugar. For example, when using the approach of the next test case, where the test compound is first injected mouse starving during the night. After 30 minutes after administration of glucose solution was orally administered mouse. The blood sugar levels in mice is enhanced by glucose, reaches its maximum after approximately 30 minutes after injection and gradually decreased. UB is the tier of sugar in the blood can be measured 30 minutes after administration of glucose and compared, which is obtained after administration of GLP-1, which thus allows to measure the effect of regulating blood sugar levels caused by peptide GLP-1, a modified oligosaccharide chain. When comparing the levels of sugar in the blood, measured after 30 minutes, obtained after administration of GLP-1, it was found that the peptide GLP-1, a modified oligosaccharide chain, is the effect, equal to, preferably, 80% or lower, more preferably 60% or lower, more preferably 40% or lower, especially preferably 20% or below the control of blood sugar levels. The intensity of regulation of blood sugar levels of the peptide GLP-1 modified oligosaccharide chain of the present invention can be determined by comparing the doses validated OGTT, cause equivalent effects of inhibiting the growth of blood sugar levels. When, for example, 10 doses of GLP-1 and 1 dose of the peptide GLP-1 modified oligosaccharide chain, lead to the same effects of the regulation of sugar levels in the blood, regulation of blood sugar levels caused by peptide GLP-1, a modified oligosaccharide chain, 10 times higher than the activity of GLP-1. The peptide GLP-1, a modified oligosaccharide chain of the present invention is the regulation of blood sugar levels, preferably at least 5 times, more FAV is preferably at least 10 times greater than GLP-1.

When the peptide GLP-1, a modified oligosaccharide chain is a peptide, a modified oligosaccharide chain containing a deletion, substitution or addition of one or several amino acids of GLP-1 or a conjugate of an analog of GLP-1 with oligosaccharide chain analogue of GLP-1 or peptide GLP-1 having the amino acid sequence identical to the peptide GLP-1, the modified oligosaccharide chain except for the amino acid-modified oligosaccharide chain, can be used as comparative material for determining the activity of GLP-1.

The effectiveness of the regulation of sugar levels in the blood of mice db/db can be determined by measuring the blood sugar level of diabetic mice after the injection of the test compounds. For example, when the measurement of blood sugar level after administration of the test compounds is carried out in time. The effect of reducing the level of sugar in the blood can be confirmed if the blood sugar level measured, for example, within 120 minutes after administration, is lower compared with the level measured during the injection. Alternatively, the duration of effect of lowering the blood sugar level can be determined by measuring the level of sugar in the blood, for example, for 300 minutes after administration. For example, when the level of sugar in the blood, izmerennogo is within 120 minutes after administration, compare with the results obtained with the introduction of GLP-1, peptide GLP-1, a modified oligosaccharide chain of the present invention exhibits preferably 80% or lower, more preferably 70% or below, particularly preferably 60% or below the control of blood sugar levels. Alternatively, when the blood sugar level measured during 120 minutes after administration, compared with the results obtained during the injection, the peptide GLP-1, a modified oligosaccharide chain of the present invention shows an effect which is preferably 70% or lower, more preferably 60% or lower, especially preferably 50% or less (for example, 45% or below) the control of blood sugar levels. When the blood sugar level measured during 300 minutes after administration, compared with the results obtained with the introduction of GLP-1, peptide GLP-1, a modified oligosaccharide chain of the present invention shows an effect which is preferably 70% or lower, more preferably 50% or below the control of blood sugar levels. When the blood sugar level measured during 300 minutes after administration, compared with the results obtained during the injection, the peptide GLP-1, a modified oligosaccharide chain of the present invention shows an effect which is preferably 70% or lower, more preferably 50% and is below the reference level of sugar in the blood.

Even if the effectiveness of the regulation of blood sugar levels caused by the peptide GLP-1, a modified oligosaccharide chain of the present invention is lower compared to GLP-1, this low activity may be offset by increased stability in the blood.

Of the activities shown GLP-1, for example, the activity of insulin secretion can be measured using the test in vitro, which is manifested in the ability to synthesize camp. GLP-1 increases the intracellular concentration of camp by binding to its receptor and promotion of insulin secretion. Thus, for example, murine receptor GLP-1 expressing cells Cho-K1, stimulate peptide GLP-1 modified oligosaccharide chain, and then measure the amount of camp synthesized in cells. It is ES can be compared with that obtained in the presence of GLP-1, so as to measure the activity of the peptide GLP-1 modified oligosaccharide chain in relation to insulin secretion.

The peptide GLP-1, a modified oligosaccharide chain of the present invention has higher stability in blood flow than GLP-1. Stability in the bloodstream can be measured using an approach known specialists in this field, and can be determined by measuring, for example, stability in plasma or stability with respect DPP-IV (dipeptidyl peptidase IV) and using the bottom half, AUC (area under the curve of blood concentration against time), etc. as an indicator. Increased renal clearance also contributes to increased stability in the blood stream.

Stability in plasma can be determined, for example, using the approach described in the following test example 1. The peptide GLP-1, a modified oligosaccharide chain of the present invention has a high stability in plasma than GLP-1.

Resistance against DPP-IV can be determined by measuring the half-life in solution of DPP-IV, as shown, for example, in the following test example 1. The peptide GLP-1, a modified oligosaccharide chain of the present invention has a higher resistance to DPP-IV than GLP-1, and has a half-life that is at least 1.2 times (for example, at least 2 times, preferably at least 5 times, more preferably at least 10 times, particularly preferably at least 20 times higher than that of GLP-1 (e.g., at least 100 times), when resistance to DPP-IV is measured by, for example, the approach described in the following test example 1.

The peptide GLP-1, a modified oligosaccharide chain, the present invention also has a half-life in the bloodstream, preferably equal to at least 1 hour, more preferably at least 3, 5, 7, 10, 15 and 20 hours,even more preferably at least 24 hours.

Next will be described a pharmaceutical composition comprising the peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient.

The pharmaceutical composition containing the peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient, is effective for the treatment or prevention of diseases associated with GLP-1. It is known that GLP-1 exerts a different effect, as described above, and these effects are associated with various diseases. It was shown that, for example, GLP-1 stimulates insulin secretion and therefore leads to cell seizure of glucose and lower blood sugar levels. It was also shown that GLP-1 inhibits peristalsis of the stomach and/or intestinal motility, gastric emptying and/or gastric emptying and food intake. Thus, diseases associated with GLP-1 include, for example, ainsliezubaida diabetes mellitus (NIDDM), insulin-dependent diabetes mellitus, stroke (see WO 00/16797 by Efendic), myocardial infarction (see WO 98/08531 by Efendic), obesity (see WO 98/19698 by Efendic), functional dyspepsia, irritable bowel syndrome (see WO 99/64060 by Efendic) and transplantation of islet cells of the pancreas. The pharmaceutical composition containing the peptide GLP-1, a modified oligosaccharide chain, this image is the shadow as an active ingredient, especially effective for the treatment or prevention of diabetes, more specifically to prevent type 1 diabetes type and treatment of diabetes of the 2nd type.

The pharmaceutical composition can be designed in the form of a conventional pharmaceutical composition by using diluents or inert fillers, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and commonly used lubricants.

Examples of such pharmaceutical compositions include tablets, pills, powders, liquid formulations, suspensions, emulsions, granules, capsules, medical candles and injection.

The number of peptides GLP-1 modified oligosaccharide chain of the present invention contained in the pharmaceutical composition is not particularly limited and can be selected appropriately within a wide range. The peptide GLP-1, a modified oligosaccharide chain of the present invention is usually contained in the pharmaceutical composition in a quantity of preferably from 1 to 70 wt.%.

The pharmaceutical composition containing the peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient, and may further comprise an additional active ingredient or can also be used in combination pharmaceutical composition, contains an additional active ingredient. Moreover, the pharmaceutical composition comprising the peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient, and may further comprise at least one distinct peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient, or can also be used in combination with a pharmaceutical composition comprising at least one distinct peptide GLP-1, a modified oligosaccharide chain of the present invention as an active ingredient.

Route of administration of the pharmaceutical compositions according to the present invention is not particularly limited. The pharmaceutical composition according to the present invention is administered in a manner suitable for various dosage forms, age and sex of the patient, severity of disease and other conditions. Examples of routes of administration of tablets, pills, liquid formulations, suspensions, emulsions, granules and capsules include oral administration. Injections can be administered intravenously, intramuscularly, intracutaneously, subcutaneously or intraperitoneally either as a single component or as a mixture with conventional infusion of glucose or amino acids. Medical candles administered rectally.

Dose headlight is aseptically composition can be selected appropriately according to the use, age and sex of the patient, severity of disease and other conditions. The pharmaceutical composition is typically administered at a dose of from 0.1 to 900 nmol, preferably from 1 to 90 nmol, in terms of peptide GLP-1, a modified oligosaccharide chain of the present invention, referred to kg of body weight. The peptide GLP-1, a modified oligosaccharide chain of the present invention has higher stability in blood flow than GLP-1. In one aspect, the peptide GLP-1, a modified oligosaccharide chain of the present invention has a higher efficiency regulation of blood sugar levels than GLP-1. Therefore, the dose can be preferably reduced.

The number of doses of the pharmaceutical compositions can be selected appropriately according to the use, age and sex of the patient, severity of disease and other conditions and is, for example, 3 doses/day 2 doses/day or 1 dose/day. Alternatively, the pharmaceutical composition can be injected less frequently (for example, 1 dose/week or 1 dose/month) depending on its stability in the bloodstream. The number of doses of the pharmaceutical composition is preferably 1 dose or less /day. The peptide GLP-1, a modified oligosaccharide chain of the present invention has higher stability in blood flow than GLP-1. So it dose may preferably be of the mind is isana.

The peptide GLP-1, a modified oligosaccharide chain of the present invention is easily degraded in the metabolic system in vivo. In one aspect of the present invention oligosaccharide chain has a structure which is connected in the form of glycopeptide (or glycoprotein) in vivo. Therefore, the peptide GLP-1, a modified oligosaccharide chain of the present invention and the pharmaceutical composition comprising the peptide as an active ingredient, does not cause any side effects, no antigenicity, even if imposed in a living organism. Therefore, they are preferably lead to no allergic reactions, no efficiency losses associated with the production of antibodies.

Moreover, the peptide GLP-1, a modified oligosaccharide chain of the present invention can be stably and conveniently delivered in large quantities and can be very useful from the point of view of providing high-quality medicinal compounds with high stability.

The present invention also provides a method of treating or preventing diseases associated with GLP-1, consisting in the introduction of effective amounts of the peptide GLP-1 modified oligosaccharide chain of the present invention.

Terms used in this document are used to describe particular embodiments and is not pre is believed to limit the present invention.

The term "contains" or "containing" as used herein, means the existence of the fixed point (member, stages, factors, number and so on) and does not preclude the existence of any other provisions (member, stages, factors, number and so on), if in the context of no other interpretation.

All terms (including technical terms and scientific terms) used herein have the same values as those that are widely used by specialists in the field of the present invention, unless otherwise specified. Terms used in this document should be interpreted as having a meaning that is consistent with their values in the context of the patent specification and refers to this area and should not be interpreted in an idealized or overly formal sense unless agreed their other definitions.

Embodiments of the present invention may be described with reference to schematic diagrams. These schematic diagrams may be exaggerated for illustrative purpose. Terms such as "first" and "second" are used for the expression of various factors. It should be understood that such factors should not be limited by these terms. Such terms are used only to distinguish between one factor and another. For example, p is pout factor can be described as the second and Vice versa, without leaving the scope of the present invention.

Hereinafter the present invention will be described in more detail with reference to examples. However, the present invention can be implemented in various aspects and should not be limited by the examples described below.

Information confirming the possibility of carrying out the invention

Hereinafter the present invention will be described particularly with reference to the examples. However, the present invention is in no case should not be limited.

Example 1

Synthesis of a conjugate of the peptide GLP-1 with 26Cys, 34Cys-disialogangliosides chain

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially Conde is serout in accordance with the method, described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the mixture was placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Gly, Fmoc-Cys(Trt), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Cys(Trt), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Arg(Pbf)-Gly-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Cys(Trt)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt)-Boc (sequence No. 69).

The resulting resin containing the peptide formed on it is partially used in the column for solid-phase synthesis. The mixture triperoxonane acid:water:TIPS (= 95:2.5:2.5) are added so that the resin thoroughly swollen, and stirred at room themes is the temperature value for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 8.0 ml/min; 35→60%B, 20 min linear gradient] to obtain the peptide in which each of 26Lys and 34Lys in GLP-1 is substituted by Cys.

The following bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (10.5 mg) and peptide chain synthesized above (2.1 mg), dissolved in 100 mm phosphate buffer (pH 7.5, 210 μl) and incubated at 37°C for 4 hours.

Formula 15

After using HPLC shows total expenditure of reagents, the reaction solution directly immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.1 mg of peptide GLP-1 modified oligosaccharide chain, in which each of 26Lys and 34Lys in GLP-1 is substituted by Cys conjugate with oligosaccharide chain (26,34 Cys GLP-1-disialo).

Example 2

Synthesis of conjugates of GLP-1 with 18 and 36Cys-disialogangliosides chain

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and give the resin completely swelled the folder in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF, the Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF), amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated by using the amino acids protected by Fmoc group (0.5 mmol) for further con is Ansatie amino acids.

Fmoc-Gly, Fmoc-Cys(Trt), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Cys(Trt), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Cys(Trt)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Cys(Trt)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID no:70) on the solid-phase resin.

The resulting resin containing the peptide formed on it is partially used in the column for solid-phase synthesis. The mixture triperoxonane acid:water:TIPS (= 95:2.5:2.5) are added so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TFA% water/90% AN 8.0 ml/min; 35→60%B, 20 min linear gradient] to obtain the peptide in which each of 18LSer and 36Arg in GLP-1 is substituted by Cys.

Bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (10.5 mg) and peptide chain synthesized above (2.1 mg) was dissolved in 100 mm phosphate buffer (pH 7.5, 210 μl) and incubated at 37°C for 4 hours. After using HPLC is shown on the Noah expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and get 0.8 mg of peptide GLP-1 modified oligosaccharide chain, in which each of 18LSer and 36Arg in GLP-1 is substituted by Cys conjugate with oligosaccharide chain (18,36 Cys GLP-1-disialo).

Example 3

Synthesis of conjugates of GLP-1 with 22 and 30Cys-disialogangliosides chain

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially congenerous accordance with the method, described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the mixture was placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Cys(Trt), Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Cys(Trt), Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(Trt), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Arg(Pbf)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Cys(Trt)-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Cys(Trt)-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID NO:71) on the solid-phase resin.

The resulting resin containing the peptide formed therein, partially used in the column for solid-phase synthesis. The mixture triperoxonane acid:water:TIPS (= 95:2.5:2.5) are added so that the resin thoroughly swollen, and premesis the Ute at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The precipitate purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 8.0 ml/min; 35→60%B, 20 min linear gradient] to obtain the peptide in which each of 22Gly and 30Ala in GLP-1 is substituted by Cys.

Bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (7.9 mg) and peptide chain synthesized above (1.3 mg), dissolved in 100 mm phosphate buffer (pH 7.4, 200 μl) and incubated at 37°C for 4 hours. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C 18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 1.0 mg of peptide GLP-1 modified oligosaccharide chain, in which each of 22Gly and 30Ala in GLP-1 is substituted by Cys conjugate with oligosaccharide chain (22, 30Cys GLP-1-disialo).

Example 4

Synthesis of conjugates of GLP-1 with 22 and 36Cys-disialogangliosides chain

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) of the N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF), amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Cys(Trt), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gl(Trt), Fmoc-Cys(Trt), Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala, Fmoc-His(Trt) used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Cys(Trt)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Cys(Trt)-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt)-Cys(Trt) (SEQ ID NO:72) by solid-phase resin.

The resulting resin containing the peptide formed therein, partially used in the column for solid-phase synthesis. The mixture triperoxonane acid:water:TIPS (= 95:2.5:2.5) are added so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 8.0 ml/min; 35→60%B, 20 min linear gradient] to obtain the peptide in which each of 22Gly and 36Arg in GLP-1 is substituted by Cys.

Bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (to 11.9 mg) and peptide chain synthesized above (2.0 mg), dissolved in 100 mm phosphate buffer (pH 7.4, 400 μl) and incubated at 37°C for 1 hour. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC column: SHISEIDO UG-120 (18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 2.9 mg of peptide GLP-1 modified oligosaccharide chain, in which each of 22Gly and 36Arg in GLP-1 is substituted by Cys conjugate with oligosaccharide chain (22, 36Cys GLP-1-disialo).

Example 5

Synthesis of conjugates of GLP-1 with 30, 36Cys-disialogangliosides chain

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

Aminoxy the lot, containing the amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Cys(Trt), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Cys(Trt), Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Cys(Trt)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Cys(Trt)-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID NO:73) on a solid-phase resin.

The resulting resin containing the peptide formed therein, partially used in the column for solid-phase synthesis. The mixture triperoxonane acid:water:TIPS (= 95:2.5:2.5) are added so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin removes filtrowanie is m and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 8.0 ml/min; 35→60%B, 20 min linear gradient] to obtain the peptide in which each of 30Ala and 36Arg in GLP-1 is substituted by Cys.

Bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (11.4 mg) and peptide chain synthesized above (2.1 mg), dissolved in 100 mm phosphate buffer (pH 7.4, 400 μl) and incubated at 37°C for 1 hour. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and get 1.6 mg of peptide GLP-1 modified oligosaccharide chain, in which each of 30Ala and 36Arg in GLP-1 is substituted by Cys conjugate with oligosaccharide chain (30, 36Cys GLP-1-disialo).

Example 6

Synthesis of a conjugate of the peptide GLP-1 tetrasaccharide 30Cys hyaluronic acid (AT-4)

Tetrasaccharide 30Cys hyaluronic acid (below in the examples on oligohaline acid also referred to as "hyaluronic acid") obtained in synthetic example 1 (12,7 mg) is dissolved in 25,4 ál water and 483 μl of dimethyl sulfoxide (DMSO). Add 200 mg of ammonium bicarbonate and incubated at 37°C for 30 hours and then liof the flax is dried. To the resulting lyophilized product type of 22.4 mg of sodium bicarbonate, 300 μl of water and 34,9 mg bromoxynil anhydride (a product of Sigma-Aldrich Corp.), pre-dissolved in 17 μl of N,N-dimethylformamide (DMF) and incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and receive 11,5 mg tetrasaccharide bromoacetonitrile acid of the following formula (I):

Formula 16

The resulting tetrasaccharide bromoacetonitrile acid (I) (2.4 mg) and a peptide chain in which 30Ala in GLP-1 is substituted by Cys (SEQ ID No. 76) (1.3 mg) obtained in synthetic example 2 was dissolved in 100 mm phosphate buffer (pH 7.5, 130 μl) and incubated at 37°C for 1.5 hours. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 1.0 mg of peptide GLP-1 modified oligosaccharide chain, in which 30Ala in GLP-1 is substituted by Cys conjugate with tetrasaccharide hyaluronic acid (30Cys GLP-1-FOR-4).

Example 7

Synthesis of a conjugate of the peptide GLP-1 octasaccharide 30Cys hyaluronic acid (8)

Octasaccharide g is Euronaval acid, obtained in synthetic example 1 (8,7 mg) dissolved 17.4 μl of water and 314 μl of dimethyl sulfoxide (DMSO). Add 100 mg of ammonium bicarbonate and incubated at 37°C for 45 hours and then freeze-dried. To the resulting lyophilized product add to 7.6 mg of sodium bicarbonate, 180 μl of water and 13.8 mg, bromoxynil anhydride (a product of Sigma-Aldrich Corp.), pre-dissolved in 7 μl of N,N-dimethylformamide (DMF) and incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and gain of 7.3 mg octasaccharide bromoacetonitrile acid of the following formula (II):

Formula 17

The resulting octasaccharide bromoacetonitrile acid (2.9 mg) and a peptide chain in which 30Ala in GLP-1 is substituted by Cys (SEQ ID No. 76) (1.5 mg) obtained in synthetic example 2 was dissolved in 100 mm phosphate buffer (pH 7.5, 150 μl) and incubated at 37°C for 1.5 hours. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.5 mg of peptide GLP-1 modified oligosaccharide chain, the which 30Ala in GLP-1 is substituted by Cys conjugate with octasaccharide hyaluronic acid (30Cys GLP-1-HA-8).

Example 8

Synthesis of a conjugate of the peptide GLP-1 tetrasaccharide 36Cys hyaluronic acid (AT-4)

Tetrasaccharide hyaluronic acid obtained in synthetic example 1 (12,7 mg) is dissolved in 25,4 ál water and 483 μl of dimethyl sulfoxide (DMSO). Add 200 mg of ammonium bicarbonate and incubated at 37°C for 30 hours and then freeze-dried. To the resulting lyophilized product type of 22.4 mg of sodium bicarbonate, 300 μl of water and 34,9 mg bromoxynil anhydride (a product of Sigma-Aldrich Corp.),pre-dissolved in 17 μl of N,N-dimethylformamide (DMF), and incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and receive 11,5 mg tetrasaccharide bromoacetonitrile acid (I).

The resulting tetrasaccharide bromoacetonitrile acid (I) (1.1 mg) and the peptide GLP-1, which 36Arg in GLP-1 is substituted by Cys (SEQ ID No. 78) (1.5 mg) obtained in synthetic example 3 was dissolved in 100 mm phosphate buffer (pH 7.5, 130 μl) and incubated at 37°C for 1.5 hours. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and p is to obtain 0.9 mg of peptide GLP-1, modified oligosaccharide chain, which 36Arg in GLP-1 is substituted by Cys conjugate with tetrasaccharide hyaluronic acid (36Cys GLP-1-FOR-4).

Example 9

Synthesis of a conjugate of the peptide GLP-1 octasaccharide 36Cys hyaluronic acid (8)

Octasaccharide hyaluronic acid (8.7 mg) obtained in synthetic example 1 was dissolved 17.4 μl of water and 314 μl of dimethyl sulfoxide (DMSO). Add 100 mg of ammonium bicarbonate and incubated at 37°C for 45 hours and then freeze-dried. To the resulting lyophilized product add to 7.6 mg of sodium bicarbonate, 180 μl of water and 13.8 mg bromoxynil anhydride (a product of Sigma-Aldrich Corp.), pre-dissolved in 7 μl of N,N-dimethylformamide (DMF) and incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and gain of 7.3 mg octasaccharide bromoacetonitrile acid (II).

The resulting octasaccharide bromoacetonitrile acid (2.4 mg) and the peptide GLP-1, which 36Arg in GLP-1 is substituted by Cys (SEQ ID No. 78) (1.5 mg) obtained in synthetic example 3 was dissolved in 100 mm phosphate buffer (pH 7.5, 130 μl) and incubated at 37°C for 2 hours. After using HPLC shows total expenditure of reagents, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO G-120 (18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.5 mg of peptide GLP-1 modified oligosaccharide chain, which 36Arg in GLP-1 is substituted by Cys conjugate with octasaccharide hyaluronic acid (36Cys GLP-1-FOR-8).

Example 10

Synthesis of a conjugate of the peptide GLP-1 hexadecasaccharide 30Cys hyaluronic acid (16)

Hexadecasaccharide hyaluronic acid (11.6 mg) obtained in synthetic example 1 was dissolved in 35 μl of water and 680 μl of dimethyl sulfoxide (DMSO). Added 260 mg of ammonium bicarbonate and incubated at 37°C for 75 hours and then freeze-dried. To the resulting lyophilized product add to 5.5 mg of sodium bicarbonate, 230 μl of water and 10.2 mg bromoxynil anhydride (a product of Sigma-Aldrich Corp.), pre-dissolved in a 5.1 μl of N,N-dimethylformamide (DMF) and incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and receive 8,7 mg next hexadecasaccharide bromoacetonitrile acid (III):

Formula 18

Hexadecasaccharide bromoacetonitrile acid (III) (3.1 mg) and the peptide chain, obtained in synthetic example 2 (SEQ ID NO:76), dissolved in 100 mm phosphate buffer (pH 7.5, 190 μl) and incu is irout at 37°C for 1.5 hours. After the accession of 10 mm aqueous solution of Tris(2-carboxyethyl)fosphenytoin (10 μl) of the mixture incubated at 37°C for 8 hours. Because by HPLC shows that there is no longer decreasing raw materials, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.4 mg of peptide GLP-1 modified oligosaccharide chain, in which 30Ala in GLP-1 is substituted by Cys conjugate with hexadecasaccharide hyaluronic acid (30Cys GLP-1-FOR-16).

Example 11

Synthesis of a conjugate of the peptide GLP-1 hexadecasaccharide 36Cys hyaluronic acid

Hexadecasaccharide bromoacetonitrile acid (III), obtained in example 10 (4.9 mg) and 36 Cys peptide GLP-1 is synthesized in synthetic example 3 (SEQ ID no:78) (1.2 mg), dissolved in 100 μl of phosphate buffer (pH 7.5, 190 μl). Add a 10 mm aqueous solution of Tris(2-carboxyethyl)fosphenytoin (36 μl) of the mixture and incubated at 37°C for 4 hours. After complete consumption of the reactants confirmed by HPLC, the reaction solution immediately subjected to purification by HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU/water solution: 0,09% TN/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.3 mg of peptide GLP-1 modified oligosaccharide chain, in which 36Arg in GLP-1 is substituted by Cys conjugate with hexadecasaccharide hyaluronic acid (36Cys GLP-1-FOR-16).

Example 12

Synthesis of a conjugate of the peptide GLP-1 with 36Cys-oligosaccharide chain on the basis of high mannose (MS)

The soybean powder (100 g), washed twice with 500 ml of acetone and twice with 500 ml of methanol, and get to 61.4 g of powder low-fat soybeans.

To the obtained powder low-fat soybeans (43,0 g) added 430 ml of water and 4.3 g-thinning enzyme T (product HBI Enzymes Inc.) and incubated at 70°C for 19 hours with stirring. The reaction solution is then precipitated by centrifugation (10000 g, 10 min) and receive 800 ml of supernatant. To the precipitate add additional 430 ml of water and 4.3 g-thinning enzyme T and again incubated at 70°C for 19 hours. The reaction solution is then precipitated by centrifugation (10000 g, 10 min) and receive 600 ml of supernatant. The resulting supernatant unite (total volume of 1400 ml). Add 100 ml of 500 mm phosphate buffer (pH 7.0) and 3.0 g orientati ONS (product HBI Enzymes Inc.) and incubated at 50°C for 19 hours with stirring. The reaction solution is filtered to remove insoluble matter and the filtrate is concentrated to a volume equal to 400 ml using a rotary evaporator. The resulting solution is subjected to ultrafiltration through an ultrafiltration membrane, cut-off molecular weight of 1 kDa (Minimate TFF Capsule 1K embrane, product PALL Corp.).

After treatment for 6 hours selected 230 ml of the solution, not passed through the membrane. To the selected solution add 20 ml of 1 M Tris-HCl buffer (pH 8.0), 250 mg of sodium azide and 423,5 mg actinase E (product of KAKEN PHARMACEUTICAL CO., LTD.) and incubated at 37°C for 82 hours. The reaction solution is filtered to remove insoluble substances, and then the filtrate is concentrated to a volume of 100 ml using a rotary evaporator. The concentrate is divided into two parts, each of which fractionary using column Sephadex-G25 (φ 25 mm × 100 mm). Collect only a fraction containing oligosaccharide chain, concentrate, and get 2,22,

The obtained fraction containing oligosaccharide chain, dissolved in, or 21.0 ml of distilled water and 14.9 ml of ethanol. Added 1.13 g of sodium bicarbonate and 2.02 g of Fmoc-OSu and incubated at room temperature for 16 hours. After completion of the reaction there was added 250 ml of acetone, and filtered through a membrane filter (ϕ 47 mm, pore size: 0.5 µm; the product of Advantec Toyo Kaisha, Ltd.). The insoluble matter remaining on the membrane, dissolved in distilled water and concentrated to a volume of 10 ml or less, using a rotary evaporator. Concentrate fractionary using column Sephadex-G25 (φ 25 mm × 100 mm). Collect the fraction containing oligosaccharide chain, concentrate, and get to 1.37 g

Then this fraction is dissolved in 4 ml of distilled water and fractionary using ODS column (Wakogel 100C18, φ 25 mm × 150 mm). Collect only a fraction containing oligosaccharide chain, concentrate, and get to 48.6 mg of paleoceanic oligosaccharide chains. Procedencia oligosaccharide chain purified on a column of HPLC [YMC-Pack ODS-AM φ 20×250 mm, eluent: acetonitrile/buffer 25 mm ammonium acetate = 82/18, flow rate: 8.0 ml/min] and get 13,0 mg oligosaccharide chains on the basis of high mannose, Man5GlcNAc2(oligosaccharide chain M5).

Derived oligosaccharide chain M5 (11.0 mg) was dissolved in 165 μl of water. Add 200 mg of ammonium bicarbonate and incubated at room temperature for 41 hours and then freeze-dried. To the resulting libfilesystem product add 12.5 mg of sodium bicarbonate, 110 μl of water and 19.9 mg bromoxynil anhydride (a product of Sigma-Aldrich Corp.), pre-dissolved in 10 μl of N,N-dimethylformamide (DMF), incubated for 1 hour while cooling in ice, and then another hour at room temperature. The reaction solution was subjected to purification by gel-filtration and receive 7.9 mg next bromoacetaldehyde circuit M5 (b):

Formula 19

Received bromochlorodifluoromethane circuit M5 (b) (4,1 mg) and the peptide GLP-1, which 36Arg replaced by Cys (SEQ ID no:78) (1.2 mg), Sintesi the bath in synthetic example 3, dissolved in 100 mm phosphate buffer (pH 7.5, 190 μl). Add 100 mm aqueous solution of Tris(2-carboxyethyl)fosphenytoin (24 μl) and incubated at 37°C for 10 hours. After completion of the reaction, the reaction solution was subjected to purification HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09% TFU/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.3 mg of peptide GLP-1 modified oligosaccharide chain, in which 36Arg in GLP-1 is substituted by Cys conjugate with oligosaccharide chain on the basis of high mannose M5 (36Cys GLP-1-M5).

Example 13

Synthesis of a conjugate of the peptide Arg34GLP-1(7-37) with a modified linker Asn 26Lys-asialoglycoprotein chain

(1) Synthesis is associated with the linker Asn 26Lys-asialoglycoprotein chain-modified glutaric acid

In the conical flask 10 ml dissolve associated with asparagine asialoglycoprotein chain (50,6 mg, 28.7 mmol) in a mixture of DMSO-water (4:1, vol/about., 1.5 ml). To this solution was added a solution of DMSO (100 μl, of 51.7 mmol) containing 0.52 M mixture of glutaric acid - EDC (1:1, mol/mol) and stirred at room temperature for 1 day. The reaction mixture is diluted with distilled water (1.5 ml), and then re-fractionary three times gel chromatography (Sephadex G-25, ϕ 1.5×45 cm, distilled water). After freeze drying receive following the Yu-related asparagine asialoglycoprotein chain, modified glutaric acid (C) (51,4 mg) (MALDI TOF Mass calculated for [M+Na]+ is equal to 1891.66 defined equal 1891.78):

Formula 20

(2) Synthesis is associated with asparagine asialoglycoprotein chain-modified N-hydroxysuccinimidyl ester of glutaric acid

In an Eppendorf tube of 1.5 ml add 25 µl of 0.44 M N-hydroxysuccinimide (11.1 mmol) in DMSO 75 μl, 0,37 M EDC (27.6 mmol) in DMSO and 200 ál associated with asparagine asialoglycoprotein chain-modified glutaric acid (C) (17,2 mg, 9.2 μmol) in DMSO. After stirring at room temperature for 6 hours EDC inactivate attach DTT (5.7 mg, to 36.8 mmol) (Grabarek, Z., Gergely, J. Anal. Biochem. 1990, 185, 131-135). The resulting solution containing the conjugate of asparagine with the derived N-hydroxysuccinimidyl ester of glutaric acid and asialoglycoprotein circuit (d) is directly used in peptide condensation as a carbohydrate linker reagent (0.03 M solution):

Formula 21

(3) Synthesis of conjugate Arg34GLP-1(7-37) with a modified linker Asn 26Lys-asialoglycoprotein chain

In an Eppendorf tube of 1.5 ml add to 4.8 μl of DIPEA (27.6 mmol) and 150 μl of 0.03 M carbohydrate linker reagent (4.5 mmol), the receipt of which is described above, and 300 μl of Lys26Arg34GLP-1(7-37), sinisiraan the th in synthetic example 4 (SEQ ID no:80) (2.8 mg, 0.83 µmol) in DMSO. After stirring at room temperature for 2 hours the reaction was stopped by adding 200 μl of an aqueous solution of glycine (2 mg, to 26.6 mmol). Fractions corresponding to the peaks at retention time equal to 15.5 min, immediately collected by separation HPLC [column: Bond 300SB-CN, ϕ is 4.6×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09% TFU/10% water /90% AN 1.0 ml/min; 10→40% (0-8 min) 40→50% (8-20 min linear gradient]. After freeze drying receive the conjugate peptide Arg34GLP-1(7-37) with modified linker Asn 26Lys-asialoglycoprotein chain (0.7 mg) (MALDI TOF Mass calculated for [M (average)+H]+equal 5236.35 defined equal 5236.1):

Formula 22

Example 14

Synthesis of conjugate of expandin-4 with 30-Cys-disialogangliosides chain

39-membered peptide, in which 30Gly in Ex-4 is substituted by Cys (12.0 mg) synthesized in synthetic example 5, and bromochlorodifluoromethane (a) (a product of OTSUKA Chemical Co., Ltd.) (36 mg), incubated in 100 mm phosphate buffer (pH 7.4) and 5 mm Tris(carboxyethyl)phosphine (1 ml) at 37°C for 1 hour. The reaction solution immediately purified HPLC [column: SHISEIDO UG-120 (FROM 18.5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09% TFU/10% water/90% AN 8 ml/min; 35→50% B, 20 min linear gradient] and get to 10.6 mg of the conjugate of Ex-4 with oligosaccharide chain, where 30Gly in Ex-4 substituted for nyugalom Cys with disialogangliosides circuit (30Cys Ex-4-disialo) (M:C271H422N58O123S MALDI-TOP MASS, calculated for [M (average)+H]+equal 6493,63 defined equal 6494.33).

Example 15

Synthesis of conjugate BIM51077 26-Cys-disialogangliosides chain

30-membered peptide, in which 26Lys in BIM51077, substituted Cys (2.4 mg, to 0.72 mmol) synthesized in synthetic example 6, and guanidine (216 mg) dissolved in distilled water (240 μl) and the resulting solution in the following order type: aqueous solution TSER (100 mm, 100 μl), bromochlorodifluoromethane (a) (10 mg/ml, 100 μl, 4.2 mmol) and incubated in 500 mm phosphate buffer (pH 7.4, 100 μl). The mixture is incubated at 37°C for 2 hours. The reaction solution immediately purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 4.6×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09% TFU/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and get 1.9 mg of peptide conjugate BIM51077 with oligosaccharide chain, which 26Lys in BIM51077 replaced by Cys conjugate (26Cys in BIM51077-disialo) with disialogangliosides chain (MALDI TOF Mass calculated for [M (average)+H]+equal 5578,72 defined equal 5578,74).

Example 16

Synthesis of a conjugate of the basis 4 30Cys-M5 oligosaccharide chain

39-membered peptide, in which 30Glu in Ex-4, substituted Cys (1.2 mg) synthesized in synthetic example 5, and bromoacetamide M5 oligosaccharide chain (b) synthesized in example 12 (3.9 mg), incubated in 35 mm phosphate buffer (pH 7.4) and mm Tris(carboxyethyl)phosphine (0.17 ml) at 37°C for 3 hours. The reaction solution immediately purified HPLC [column: SHISEIDO UG-120 (C18, 5 μm), φ 4.6×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09% TFU/10% water/90% AN 0.7 ml/min; 35→60%B, 20 min linear gradient] and receive 0.5 mg of peptide conjugate of Ex-4 with oligosaccharide chain, which 30Glu in Ex-4 substituted by Cys conjugate (30Cys in Ex-4-MS) with disialogangliosides circuit on the basis of the high mannose type M5 (MALDI TOF Mass calculated for [M (average)+H]+equal 5504,74 defined equal 5506,85).

In table 5, below, presents MS spectral data (MALDI-TOF mass) peptides GLP-1 modified oligosaccharide chain obtained in the following examples 1-16.

Table 5
Example 126,34 Cys GLP-1-disialodesigned forC317H492N52O171S2[M+H]+7828,1defined7828,8
Example 218,36 CysGLP-1-disialodesigned forC320H449N51O170S2[M+H]+ 7841,1defined7845,3
Example 322,30 Cys GLP-1-disialodesigned forC324H508N54O171S2[M+H]+7960,9defined7960,6
Example 422,36 Cys GLP-1-disialodesigned forC321H501N51O171S2[M+H]+7875,8defined7876,6
Example 530,36 Cys GLP-1-disialodesigned forC320H499N51O171S2[M+H]+7861,8defined7860,4
Example 630Cys GLP-1-HA-4designed forC181H274N43O70S[M+H]+42019 defined4203,5
Example 730Cys GLP-1-HA-8designed forC209H316N45O92S[M+H]+4960,1defined4961,9
Example 836Cys GLP-1-HA-4designed forC178H267N40O70S[M+H]+4116,8defined4118,3
Example 936Cys GLP-1-HA-8designed forC206H309N42O92S[M+H]+4875,1defined4876,9
Example 1030Cys GTP-1-HA-16designed forC265H399N49O136S[M+H]+6476,6defined 6477,8
Example 1136Cys GLP-1-HA-16designed forC262H392N46O136S[M+H]+6391,5defined6394,1
Example 1236Gys GLP-1-M5designed forC196H300N40O83S[M+H]+4575,0defined4576,2
Example 1326Lys asialo-Asn-linkerdesigned forC222H342N48O97[M+H]+5236,35defined5236,1
Example 1430Cys Ex-4-disialodesigned forC271H422N58O123S[M+H]+6493,63defined6494,33
Example 1526Cys-BIM51077-disialodesigned forC285H364N48O107S[M+H]+5578,72defined5578,74
Example 1630Cys Ex-4-M5designed forC233H362N54O97S[M+H]+5504,74defined5506,85

Comparative example 1

In a column for solid-phase synthesis put the resin Amino-PEGA (100 Microm), thoroughly washed DHM and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After re is eshiwani resin washed DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in NMP (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol) and placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Arg(Pbf)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID NO:74) on the solid-phase resin.

After washing DHM and DMF the resin, equivalent to 5 µmol 31-membered peptide, transferred into a test tube Appender is.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC (Cadenza column C18 100×10 mm, eluting solution A: 0.1% aqueous solution of TFU, IN: 0,1%TFU acetonitrile:water = 90:10, gradient A:In=95:5→5:95, 15 min, flow rate: 3.0 ml/min) and receive GLP-1.

Synthetic example 1

Synthesis of oligosaccharide chains fractions oligohaline acid

To 500 mg of hyaluronic acid (the product of Shiseido Co., Ltd., average molecular weight: 1,200,000), add 100 ml of acetate buffer (pH 4) and the mixture is well stirred to dissolve. Add hyaluronidase (2500 int. unit; a product of CALBIOCHEM, Bovine Testes-derived) and incubated at 37°C for 2 days. The solution is concentrated and then dissolved again in 45 ml of acetate buffer (pH 4). Add hyaluronidase (4500 int. unit; a product of CALBIOCHEM, Bovine Testes-derived) and incubated at 37°C for another 2 days. The reaction solution fractionary using ultrafiltration membranes, cut-off molecular weight equal to 3 kDa and 1 kDa (Millipore products). After freeze drying receive 268,4 mg factions g is Euronaval acid of molecular weight from 1 to 3 kDa.

Since the fractions obtained hyaluronate acid of molecular weight from 1 to 3 kDa contain several factions oligohaline acids, for their separation was used for preparative HPLC. Faction oligohaline acid of molecular weight from 1 to 3 kDa dissolved in a small amount of water and fractionary into several parts when cleaning HPLC [column: Shodex Asahipak NH2P-90 20F 9 μm, ϕ 20.0×300 mm, mobile phase: 180 mm NaH2PO4waters.] each peak elution. Then the fractions obtained absoluut gel-filtration, freeze-dried and receive oligohaline acid (from tetrasaccharide to octadecenoamide). The output of each oligohaline acid is shown below.

Tetrasaccharide oligohaline acid 22,5 mg (tR=10,0 min)

Hexasaccharide oligohaline acid of 51.1 mg (tR=11,8 min)

Octasaccharide oligohaline acid and 52.7 mg (tR=14,0 min)

Decesare oligohaline acid 27.0 mg (tR=min 17,0)

Dodecanoate oligohaline acid 9.6 mg (tR=21,4 min)

Tetradecanoate oligohaline acid 7.8 mg (tR=27,6 min)

Hexadecasaccharide oligohaline acid to 4.1 mg (tR=36.0 min)

Octadecanoid oligohaline acid 2.0 mg (tR=47,4 min)

Synthetic example 2

Synthesis of a peptide in which the position 30 in GLP-1 replacement is prohibited Cys

In a column for solid-phase synthesis put the resin Amino-PEGA (100 mmol) (product of Merck), washed thoroughly with DHM and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in NMP (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/D is f (2 ml) for 15 minutes. This operation is repeated using the amino acids protected by Fmoc group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Cys(Trt), Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Arg(Pbf)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Cys(Trt)-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID NO:75) on the solid-phase resin.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09%TFU/10% water/90% AN 8.0 ml/min; 35→60% B, 20 min linear gradient] and get a 31-membered peptide, in which 30Ala in GLP-1 is substituted by Cys (SEQ ID no:76).

Synthetic example 3

Synthesis of a peptide, in which position 36 in GLP-1 is substituted by Cys

In a column for tverdova the aqueous synthesis put the resin Amino-PEGA (100 mmol) (product of Merck), washed thoroughly with dichloromethane (DHM) and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM, to obtain a resin HMPB-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation Provo is Yat re-use amino acids, protected Fmoc-group (0.5 mmol) for further condensation of amino acids.

Fmoc-Gly, Fmoc-Cys(Trt), Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Cys(Trt)-Gly-Lys(Boc)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID NO:77) for solid-phase resin.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09%TFU/10% water/90% AN 8.0 ml/min; 35→60% B, 20 min linear gradient] and get a 31-membered peptide, in which 36Arg in GLP-1 is substituted by Cys (SEQ ID no:78).

Synthetic example 4

Synthesis of a peptide, in which position 34 in GLP-1 is substituted by Arg

In a column for solid-phase synthesis put the resin Amino-PEGA (100 Microm), thoroughly washed On The M and DMF and allow the resin to fully swell in DMF. 4-hydroxymethyl-3-methoxyphenoxy acid (NMRW) (0.25 mmol), TBTU (0.25 mmol) and N-ethylmorpholine (0.25 mmol) dissolved in DMF (2 ml), placed in a column and stirred at room temperature for 4 hours. The resin is washed thoroughly with DMF and DHM and get HMPB resin-PEGA, which is used as a solid phase for solid-phase synthesis.

Fmoc-Glu (0.50 mmol), MSNT (0.50 mmol) and N-Mei (the 0.375 mmol) dissolved in DHM (2 ml), placed in the column for solid-phase synthesis and stirred at 25°C for 3 hours.

After mixing, the resin was washed with DHM and DMF. Fmoc group is removed with 20% solution of piperidine/DMF (2 ml) for 15 minutes. After washing DMF amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

The amino acid containing an amino group protected with Fmoc group, dissolved in N-organic (NMP) (1 ml). Add 0.45 M HCTU·HOBT/NMP (0.4 mmol), the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (0.8 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated by using the amino acids protected by Fmoc group (0.5 mmol) for further Conde the compensation of amino acids.

Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Lys(Boc), Fmoc-Ala, Fmoc-Ala, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc - Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Ala and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 31-membered peptide Gly-Arg(Pbf)-Gly-Arg(Pbf)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Lys(Boc)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Ala-His(Trt) (SEQ ID no:79) on the solid-phase resin.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09%TFU/10% water/90% AN 8.0 ml/min; 35→60% B, 20 min linear gradient] and get a 31-membered peptide, in which 34Lys in GLP-1 is substituted by Arg (SEQ ID no:80).

Synthetic example 5

Synthesis of a peptide in which the position 30 in the basis-4 substituted Cys

In a column for solid-phase synthesis put the resin Amino-PEGA (100 mmol) (product of Merck) and washed with DMF. Then amino acids sequentially condense in appropriate is under way, described below for increasing peptide chain.

Amino acid (0.5 mmol), containing the amino group protected with Fmoc group, dissolved in 0.45 M HCTU·HOBT/NMP (2.5 mmol) and the resulting mixture is placed in a column for solid-phase synthesis. Then there is added 0.9 M DIPEA/NMP (2.5 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated for successive condensation of amino acids.

Fmoc-Ser(tBu), Fmoc-Pro, Fmoc-Pro, Fmoc-Pro, Fmoc-Ala, Fmoc-Gly, Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Pro, Fmoc-Cys(Trt), Fmoc-Gly, Fmoc-Asn(Trt), Fmoc-Lys(Boc), Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Glu(OtBu), Fmoc-Ile, Fmoc-Phe, Fmoc-Leu, Fmoc-Arg(Pbf), Fmoc-Val, Fmoc-Ala, Fmoc-Glu(OtBu), Fmoc-Glu(OtBu), Fmoc-Glu(OtBu), Fmoc-Met, Fmoc-Gln(Trt), Fmoc-Lys(Boc), Fmoc-Ser(tBu), Fmoc-Leu, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Gly and Fmoc-His(Trt) is used as the amino acids protected by Fmoc group to obtain a 39-membered peptide Ser(tBu)-Pro-Pro-Pro-Ala-Gly-Ser(tBu)-Ser(tBu)-Pro-Cys(Trt)-Gly-Asn(Trt)-Lys(Boc)-Leu-Trp(Boc)-Glu(OtBu)-Ile-Phe-Leu-Arg(Pbf)-Val-Ala-Glu(OtBu)- Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Lys(Boc)-Ser(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Gly-His(Trt) (SEQ ID NO:81) solid-phase resin.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature in accordance with the s 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column SHISEIDO UG-120 (C 18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09%TFU/10% water/90% AN 8.0 ml/min; 35→60% B, 20 min linear gradient] and get 39-membered peptide, in which 30Glu in Ex-4 is substituted by Cys (MALDI TOF Mass calculated for [M+H]+equal 4230,60 defined equal 4231,27). (SEQ ID NO:82).

Synthetic example 6

Synthesis of a peptide, in which position 26 of GLP-1 is substituted by Cys

In a column for solid-phase synthesis put the resin amino-PEGA (100 mmol) (product of Merck) and washed with DMF. Then amino acids sequentially condense in accordance with the method described below for increasing peptide chain.

Amino acid (0.5 mmol), containing the amino group protected with Fmoc group, dissolved in 0.45 M HCTU·HOBT/NMP (2.5 mmol) and the resulting mixture is placed in a column for solid-phase synthesis. Then in the column for solid-phase synthesis add 0.9 M DIPEA/NMP (2.5 mmol). After stirring at room temperature for 20 minutes, the resin was washed with DHM and DMF and the Fmoc group removed 20% solution of piperidine/DMF (2 ml) for 15 minutes. This operation is repeated for successive condensation of amino acids.

Fmoc-Arg(Pbf), Fmoc-Aminoisobutyric Acid(Aib), Fmoc-Lys(Boc), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(OtBu), Fmoc-Cys(Trt), Fmoc-Ala, FmocAla, Fmoc-Gln(Trt), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Leu, Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Ser(tBu), Fmoc-Val, Fmoc-Asp(OtBu), Fmoc-Ser(tBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Thr(tBu), Fmoc-Gly, Fmoc-Glu(OtBu), Fmoc-Aib and Fmoc-His(Trt) used as the amino acids protected by Fmoc group to obtain a 30-membered peptide Arg(Pbf)-Aib-Lys(Boc)-Val-Leu-Trp(Boc)-Ala-Ile-Phe-Glu(OtBu)-Cys(Trt)-Ala-Ala-Gln(Trt)-Gly-Glu(OtBu)-Leu-Tyr(tBu)-Ser(tBu)-Ser(tBu)-Val-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Glu(OtBu)-Aib-His(Trt) (SEQ ID NO:83) on the solid-phase resin.

Part of the obtained resin containing formed therein peptide, is transferred into a column for solid-phase synthesis. Add the mixture triperoxonane acid:water:TIPS (= 95:2,5:2,5) so that the resin thoroughly swollen, and stirred at room temperature for 3 hours. The resin is removed by filtration and the reaction solution was concentrated under reduced pressure. The resulting residue purified HPLC [column SHISEIDO UG-120 (C18, 5 μm), φ 20×250 mm, gradient: a solution: 0.1% OF TFU water, solution b: 0,09%TFU/10% water/90% AN 8.0 ml/min; 35→60% B, 20 min linear gradient] and receive 12 mg 30-membered peptide, in which 26Lys in BIM51077 replaced by Cys (MALDI TOF Mass calculated for [M (average)-+H]+equal 3315.69 defined equal 3314,72). (SEQ ID NO:84).

Peptides GLP-1, modified oligosaccharide chain obtained in each of the above examples are peptides GLP-1, modified oligosaccharide chain having the sequence His7-Ala8-Glu9-Gly10-Thr11-Ph 12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Lys34-Gly35-Arg36-Gly37(SEQ ID no:2, GLP-1), where:

(b1) each of 26Lys and 34Lys replaced by Cys conjugate with disialogangliosides circuit (example 1) (SEQ ID no:54);

(b2) each of 18Ser and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 2) (SEQ ID no:55);

(b3) each of 22Gly and 30Ala replaced by Cys conjugate with disialogangliosides circuit (example 3) (SEQ ID no:56);

(b4) each of 22Gly and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 4) (SEQ ID no:57);

(b5) each of 30Ala and 36Arg replaced by Cys conjugate with disialogangliosides circuit (example 5) (SEQ ID no:58);

(b6) 30Ala replaced by Cys conjugate with tetrasaccharide hyaluronic acid (AT-4) (example 6) (SEQ ID no:59);

(b7) 30Ala replaced by Cys conjugate with octasaccharide hyaluronic acid (8) (example 7) (SEQ ID no:60);

(b8) 36Arg replaced by Cys conjugate with tetrasaccharide hyaluronic acid (AT-4) (example 8) (SEQ ID no:61);

(b9) 36Arg replaced by Cys conjugate with octasaccharide hyaluronic acid (8) (example 9) (SEQ ID no:62);

(b10) 30Ala replaced by Cys conjugate with hexadecasaccharide hyaluronic acid (16) (example 10) (SEQ ID no:63);

(b11) 36Arg replaced by Cys conjugate with hexadecasaccharide g is Euronaval acid (16) (example 11) (SEQ ID no:64);

(b12) 36Arg replaced by Cys conjugate with oligosaccharide chain on the basis of high mannose (M5) (example 12) (SEQ ID no:65); and

(b13) conjugate Asn with asialoglycoprotein chain associated with 26Lys through a linker (example 13) (SEQ ID no:66), and

are also

(b14) the peptide GLP-1, a modified oligosaccharide chain having the sequence

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2(SEQ ID no:50, on the basis of 4), where

30Gly replaced by Cys conjugate with disialogangliosides circuit (example 14) (SEQ ID no:67);

(b15) the peptide GLP-1, a modified oligosaccharide chain having the sequence His-R2-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-R2-Arg-NH2where R2represents an α-methylalanine (SEQ ID no:52, BIM51077), in which

26Lys replaced by Cys conjugate with disialogangliosides circuit (example 15) (SEQ ID no:68); and

(b16) the peptide GLP-1, a modified oligosaccharide chain having the sequence of basis-4 (SEQ ID no:50), in which 30Gly replaced by Cys conjugate with oligosaccharide chain on the basis of high mannose (M5) (example 16).

Several peptides of examples 1-15 are presented below in test examples 1 and/or 2.

Test example 1 (Test for resistance to dipeptidyl peptidase IV (DPP-IV))

to 17.7 nmol of peptide GLP-1 modified oligosaccharide chain obtained in each of the m sample, or peptide GLP-1 obtained in comparative example 1, add together with 2.2 mU DPP-IV (dipeptidyl peptidase IV from kidney pig, product of SIGMA) in an Eppendorf tube 0.5 ml of Each mixture was adjusted to 100 mm phosphate buffer to a total volume of 100 μl, and incubated at 37°C. an Aliquot of the reaction solution in 10 μl mixed with 15 μl of 10% triperoxonane acid, obtained in advance in another Eppendorf tube. 20 μl of the mixture Inuktitut in column HPLC to monitor the fullness of the consumption of reagents (HPLC conditions: column: SHISEIDO CAPCELPAK C18 UG120, φ 4.6×250 mm, eluting solvent A: 0.1% aqueous TFA solution, eluting solvent B: 0.09% TFA acetonitrile/water = 90/10, gradient a/b=65/30→30/60, 20 min, flow rate: 0.7 ml/min). The half-period (t1/2), which serves as an indicator for resistance to DPP-IV peptide GLP-1 not containing the attached oligosaccharide chains in comparative example 1 is taken as the standard (=1). The average values determined for peptide GLP-1 modified oligosaccharide chain in each of the examples presented in table 6.

Table 6
ConnectionThe relative stability
Example 1 26,34 Cys GLP-1-disialo23,6
Example 218,36 Cys GLP-1-disialoof 128.6
Example 322,30 Cys GLP-1-disialo32,5
Example 422,36 Cys GLP-1-disialo22,5
Example 530,36 Gay GLP-1-disialo7,5
Example 630 Cys GLP-1-HA-42,1
Example 730 Cys GLP-1-HA-83,4
Example 836 Cys GLP-1-HA-42,2
Example 936 Cys GLP-1-HA-83
Example 1030 Cys GLP-1-HA-16a 3.9
Example 1136 Cys GLP-1-HA-163
Example 1226 Lys GLP-1-linker-asialothe 5.7

The peptide GLP-1, a modified oligosaccharide chain each is example, sustainable from 2.1 to 128 times compared with the stability of GLP-1 comparative example 1.

Test example 2 (oral test glucose tolerance (OGTT)

A solution of the peptide GLP-1 modified oligosaccharide chain in PBS obtained in each of examples or GLP-1 obtained in comparative example 1 was injected intraperitoneally at a dose of 10 ml/kg to mice C57BL/6JJcl (10-week-old, male) on an empty stomach at night. After 30 minutes of orally administered glucose solution at a dose of 1 mg/year Taken away the blood from the eye orbit before the introduction of glucose and after 30 minutes, 60 minutes and 120 minutes after administration of glucose. Measure the sugar level in the blood using the ACCU-CHEK Aviva (Roche Diagnostics). The average results are presented in Fig.1-4. For example, the figures below, "26,34 Cys GLP-1-disialo", which represents the peptide GLP-1, a modified oligosaccharide chain in which the amino acids at each position 26 and 34 in GLP-1 is substituted by Cys conjugate with disialogangliosides chain, if necessary, designate as "s,S". The same is true for other oligosaccharide chains and amino acid centers.

Next, 18,36 Cys GLP-1-disialo and GLP-1 is determined by OGTT, using doses equal to 1/10 (0.9 nmol/kg) and 9 nmol/kg, respectively, and carry out the comparison.

The results are presented in Fig.5.

18,36 Cys GLP-1-disialo introduced in a dose of 0.9 nmol/kg, shows pactically same effect of lowering the blood sugar level, as GLP-1, is introduced at a dose of 9 nmol/kg 18,36 Cys GLP-1-disialo shows approximately 10-fold reduction in blood sugar levels compared to GLP-1.

The sequence listing free text

SEQ ID no:1 peptide GLP-1, a modified oligosaccharide chain represented by the General formula (1).

SEQ ID no:2 is a GLP-1 (7-37).

SEQ ID no:3 is a GLP-1 (7-36) NH2.

SEQ ID no:4 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A1).

SEQ ID no:5 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A2).

SEQ ID no:6 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A3).

SEQ ID no:7 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A4).

SEQ ID no:8 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A5).

SEQ ID no:9 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A6).

SEQ ID no:10 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A7).

SEQ ID no:11 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A8).

SEQ ID no:12 represents the peptide GLP-1, a modified oligosaccharide chain, before the purposes of (A9).

SEQ ID no:13 represents the peptide GLP-1, a modified oligosaccharide chain represented by (a10).

SEQ ID no:14 represents the peptide GLP-1, a modified oligosaccharide chain represented by (a11).

SEQ ID no:15 is a peptide GLP-1, a modified oligosaccharide chain represented by (A12).

SEQ ID no:16 is a peptide GLP-1, a modified oligosaccharide chain represented by (A13).

SEQ ID no:17 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A14).

SEQ ID no:18 represents a peptide GLP-1, a modified oligosaccharide chain represented by (A15).

SEQ ID no:19 represents a peptide GLP-1, a modified oligosaccharide chain represented by (A16).

SEQ ID no:20 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A17).

SEQ ID no:21 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A18).

SEQ ID no:22 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A19).

SEQ ID no:23 represents a peptide GLP-1, a modified oligosaccharide chain represented by (A20).

SEQ ID no:24 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A21).

SEQ ID no:25 is a peptide GLP-1, modified oligosacharides chain presents (A22).

SEQ ID no:26 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A23).

SEQ ID no:27 is a peptide GLP-1, a modified oligosaccharide chain represented by (A24).

SEQ ID no:28 is a peptide GLP-1, a modified oligosaccharide chain represented by (A25).

SEQ ID no:29 represents a peptide GLP-1, a modified oligosaccharide chain represented by (A26).

SEQ ID no:30 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A27).

SEQ ID no:31 represents a peptide GLP-1, a modified oligosaccharide chain represented by (A28).

SEQ ID no:32 represents the peptide GLP-1, a modified oligosaccharide chain represented by (A29).

SEQ ID no:33 is a peptide GLP-1, a modified oligosaccharide chain represented by (A30).

SEQ ID no:34 is a peptide GLP-1, a modified oligosaccharide chain represented by (A31).

SEQ ID no:35 is a peptide GLP-1, a modified oligosaccharide chain represented by (A32).

SEQ ID no:36 is a peptide GLP-1, a modified oligosaccharide chain represented by (a33).

SEQ ID no:37 is a peptide GLP-1, a modified oligosaccharide chain represented by (A34).

SEQ ID no:38 is a peptide GP-1, modified oligosaccharide chain represented by (A35).

SEQ ID no:39 is a peptide GLP-1, a modified oligosaccharide chain represented by (A36).

SEQ ID no:40 is a peptide GLP-1, a modified oligosaccharide chain represented by (A37).

SEQ ID no:41 is a peptide GLP-1, a modified oligosaccharide chain represented by (A38).

SEQ ID no:42 is a peptide GLP-1, a modified oligosaccharide chain represented by (A39).

SEQ ID no:43 is a peptide GLP-1, a modified oligosaccharide chain represented by (A40).

SEQ ID no:44 is a peptide GLP-1, a modified oligosaccharide chain represented by (A41).

SEQ ID no:45 is a peptide GLP-1, a modified oligosaccharide chain represented by (a).

SEQ ID no:46 is a peptide GLP-1, a modified oligosaccharide chain represented by (A43).

SEQ ID no:47 is a peptide GLP-1, a modified oligosaccharide chain represented by (A44).

SEQ ID no:48 is a peptide GLP-1, a modified oligosaccharide chain represented by (A45).

SEQ ID no:49 is a peptide GLP-1, a modified oligosaccharide chain represented by (A46).

SEQ ID no:50 is the basis 4.

SEQ ID no:51 is a conjugate of the basis 4 of Oleg sacharides chain represented by the General formula (2).

SEQ ID no:52 is a BIM51077.

SEQ ID no:53 is a conjugate BIM51077 with oligosaccharide chain represented by the General formula (3).

SEQ ID no:54 is a peptide GLP-1, a modified oligosaccharide chain represented by (b1).

SEQ ID no:55 is a peptide GLP-1, a modified oligosaccharide chain represented by (b2).

SEQ ID no:56 is a peptide GLP-1, a modified oligosaccharide chain represented by (b3).

SEQ ID no:57 is a peptide GLP-1, a modified oligosaccharide chain represented by (b4).

SEQ ID no:58 is a peptide GLP-1, a modified oligosaccharide chain represented by (b5).

SEQ ID no:59 is a peptide GLP-1, a modified oligosaccharide chain represented by (b6).

SEQ ID no:60 is a peptide GLP-1, a modified oligosaccharide chain represented by (b7).

SEQ ID no:61 is a peptide GLP-1, a modified oligosaccharide chain represented by (b8).

SEQ ID no:62 is a peptide GLP-1, a modified oligosaccharide chain represented by (b9).

SEQ ID no:63 is a peptide GLP-1, a modified oligosaccharide chain represented by (b10).

SEQ ID no:64 is a peptide GLP-1, a modified oligosaccharide chain represented by (b11.

SEQ ID no:65 is a peptide GLP-1, a modified oligosaccharide chain represented by (b12).

SEQ ID no:66 is a peptide GLP-1, a modified oligosaccharide chain represented by (b13).

SEQ ID no:67 is a conjugate of the basis 4 with oligosaccharide chain represented by (b14).

SEQ ID no:68 is a conjugate BIM51077 with oligosaccharide chain represented by (b15).

SEQ ID no:69 is a 31-membered peptide with a protective group, synthesized in example 1.

SEQ ID no:70 is a 31-membered peptide with a protective group, synthesized in example 2.

SEQ ID no:71 is a 31-membered peptide with a protective group, synthesized in example 3.

SEQ ID no:72 is a 31-membered peptide with a protective group, synthesized in example 4.

SEQ ID no:73 is a 31-membered peptide with a protective group, synthesized in example 5.

SEQ ID no:74 is a 31-membered peptide with a protective group, is synthesized in comparative example 1.

SEQ ID no:75 is a 31-membered peptide with a protective group, is synthesized in synthetic example 2.

SEQ ID no:76 is a 31-membered peptide with a protective group, is synthesized in synthetic example 2.

SEQ ID no:77 is a 31-membered peptide with a protective group, is synthesized in synthetic grass is m example 3.

SEQ ID no:78 is a 31-membered peptide synthesized in synthetic example 3.

SEQ ID no:79 is a 31-membered peptide with a protective group, is synthesized in synthetic example 4.

SEQ ID no:80 is a 31-membered peptide synthesized in synthetic example 4.

SEQ ID no:81 is a 39-membered peptide with a protective group, is synthesized in synthetic example 5.

SEQ ID no:82 is a 39-membered peptide synthesized in synthetic example 5.

SEQ ID no:83 is a 30-membered peptide with a protective group, is synthesized in synthetic example 6.

SEQ ID no:84 is a 30-membered peptide synthesized in synthetic example 6.

Industrial applicability

The present invention provides a peptide GLP-1, a modified oligosaccharide chain, which has a higher stability in blood flow than GLP-1, and preferably is a stronger regulator of blood sugar levels compared to GLP-1. The present invention is particularly applicable in the pharmaceutical field.

1. The peptide GLP-1, intended for the treatment or prevention of diseases associated with GLP-1, a modified oligosaccharide chain, which is
a) the peptide GLP-1 modified oligosaccharide chain, in which two amino acids of the peptide GLP-1 with SEQID NO: 2 or SEQ ID NO: 3 is substituted, where each amino acid is substituted by an amino acid-modified oligosaccharide chain, and where each of the centers of the substitution is selected from the group consisting of positions 18, 22, 26, 30, 34 and 36 in the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3; or
b) the peptide GLP-1 modified oligosaccharide chain having the amino acid sequence of the peptide GLP-1 modified oligosaccharide chain defined in (a) by deletion, substitution or attachment 1-5 amino acids, with the exception of amino acids, modified oligosaccharide chain;
in which each of these oligosaccharide chains represents independently biancanello oligosaccharide chain complex type; and the specified peptide GLP-1, a modified oligosaccharide chain, shows stronger activity suppression of glucose in the blood than GLP-1 with SEQ ID NO: 3; and the half-life of the indicated peptide GLP-1 modified oligosaccharide chain in solution DPP-IV at least 2 times higher than the half-life of GLP-1, GLP-1 means glucoheptonate peptide-1; and
where each of the amino acids, modified oligosaccharide chain, independently is Asn, modified oligosaccharide chain, or Cys modified oligosaccharide chain; and
where these oligosaccharide chains independently represented by the following formula:
[Formula 1]

where
R1 and R2 may be the same or different, and each presents [Formula 2]

and
AC means an acetyl group;
or its pharmaceutically acceptable salt.

2. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which at least one substitution is at position 18, 22, 30 and 36 of the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3.

3. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which each of these oligosaccharide chains selected from the group consisting of disialo, monomial, asialo, GIcNAc and demonopolisation chains.

4. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which the specified oligosaccharide chain has at least 90% homogeneity.

5. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which each of these oligosaccharide chains is disialogangliosides chain.

6. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which two amino acids of GLP-1 is substituted by an amino acid-modified oligosaccharide chain, the provisions of 18 and 36, 26 and 34, 22 and 30, 22 and 36 or 30 and 36 of the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3.

7. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which two amino acids of GLP-1 is substituted by an amino acid-modified Alyosha IGNOU chain the provisions of 18 and 36, 22 and 30, 22 and 36 or 30 and 36 of the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3.

8. The peptide GLP-1, a modified oligosaccharide chain, p. 1, in which the amino acid of GLP-1 is substituted by an amino acid-modified oligosaccharide chain at positions 22 and 30 or 22 and 36 of the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3.

9. The peptide GLP-1, a modified oligosaccharide chain, p. 1, having the following properties:
higher stability in blood stream than that of GLP-1; activity monitoring of blood sugar levels at least 10 times higher compared to GLP-1 in the test OGTT (Oral Test Glucose Tolerance); and
the half-life in solution of DPP-IV, at least 20 times higher than the half-life of GLP-1.

10. The peptide GLP-1, a modified oligosaccharide chain, under item 1, with the half-life in solution of DPP-IV, at least 30 times higher than the half-life of GLP-1.

11. The peptide GLP-1, a modified oligosaccharide chain according to any one of paragraphs. 1-10, in which the disease associated with GLP-1, is diabetes.

12. The peptide GLP-1, intended for the treatment or prevention of diseases associated with GLP-1, a modified oligosaccharide chain, where one or two amino acids are replaced by amino acid-modified oligosaccharide chain,
a) the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3, where the centers of substitution selected from the group consisting of the C provisions of 18, 22, 30, and 36; or
b) a peptide having the amino acid sequence of the peptide GLP-1, defined in (a) by deletion, substitution or attachment 1-5 amino acids;
and the specified peptide GLP-1, a modified oligosaccharide chain,
shows stronger activity suppression of glucose in the blood than GLP-1 with SEQ ID NO: 3; and the half-life of the indicated peptide GLP-1 modified oligosaccharide chain in solution DPP-IV at least 2 times higher than the half-life of GLP-1;
the oligosaccharide chain is a hyaluronic acid;
and the specified aigagaranca acid represents oligosaccharide chain containing 2 (tetrachoric) or more and 8 (hexadecasaccharide) or less units, each of which consists of N-acetylglucosamine and glucuronic acid, where GLP-1 means glucoheptonate peptide-1;
where the specified amino acid, modified oligosaccharide is Cys, modified oligosaccharide; or its pharmaceutically acceptable salt.

13. The peptide GLP-1, a modified oligosaccharide chain, p. 12, in which the two amino acids are substituted on the amino acid, a modified oligosaccharide chain,
a) the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3; or
b) a peptide having the amino acid sequence of GLP-1,
defined in (a) by deletion, substitution or recognize is the group of one or more amino acids;
or its pharmaceutically acceptable salt.

14. The peptide GLP-1, a modified oligosaccharide chain, p. 12, in which the specified oligosiloxane acid is an oligosaccharide chain having 2 (tetrachoric) link, each of which consists of N-acetylglucosamine and glucuronic acid.

15. The peptide GLP-1, a modified oligosaccharide chain according to any one of paragraphs. 12-14, in which the disease associated with GLP-1, is diabetes.

16. The peptide GLP-1, intended for the treatment or prevention of diseases associated with GLP-1, a modified oligosaccharide chain, where one or two amino acids are replaced by amino acid-modified oligosaccharide chain,
a) the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3, where the centers of substitution selected from the group consisting of positions 18, 22, 30 and 36; or
b) a peptide having the amino acid sequence of the peptide GLP-1, defined in (a), with deletion, substitution or attachment 1-5 amino acids,
and in a specified amino acid-modified oligosaccharide chain oligosaccharide chain is linked to the amino acid via the linker and the amino acid that is associated with the specified linker is Lys; and the specified peptide GLP-1, a modified oligosaccharide chain, shows stronger activity suppression of glucose in the blood than GLP-1 with SEQ ID NO:3; and the half-life is defined peptide GLP-1, modified oligosaccharide chain in solution DPP-IV at least 2 times higher than the half-life of GLP-1, GLP-1 means glucoheptonate peptide - 1;
where the amino acid contained in the linker is Asn;
or its pharmaceutically acceptable salt.

17. The peptide GLP-1, a modified oligosaccharide chain, p. 16, in which one amino acid is substituted by an amino acid-modified oligosaccharide chain,
a) the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3; or
b) a peptide having the amino acid sequence of GLP-1, defined in (a) by deletion, substitution or attachment 1-9 amino acids,
or its pharmaceutically acceptable salt.

18. The peptide GLP-1, a modified oligosaccharide chain, p. 16, wherein said linker contains the amino acid at the end of the chain associated with oligosaccharide chain.

19. The peptide GLP-1, a modified oligosaccharide chain on p. 16, in which the oligosaccharide chain has at least 90% homogeneity.

20. The peptide GLP-1, a modified oligosaccharide chain according to any one of paragraphs. 16-19, in which the disease associated with GLP-1, is diabetes.

21. Pharmaceutical composition for treatment or prevention of diseases associated with GLP-1, containing as active ingredient an effective amount of the peptide GLP-1, a modified oligosaccharide which the second circuit, according to any one of paragraphs. 1-20, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to p. 21, where the disease associated with GLP-1, is diabetes.

23. Method for the treatment or prevention of disease associated with GLP-1, including the introduction of an effective amount of the peptide GLP-1 modified oligosaccharide chain according to any one of paragraphs. 1-20.

24. The method according to p. 23, where the disease associated with GLP-1, is diabetes.



 

Same patents:

Oxyintomoduline // 2542362

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to peptide analogues of oxyintomoduline (OXM, glucagon-37), which can be modified for providing the stability of cleavage and inactivation with dipeptidyl peptidase IV (DPP-IV) for increasing a half-life time in vivo of the peptide analogue alongside with enabling the peptide analogue acting as a double agonist GLP-1/glucagon receptor (GCGR).

EFFECT: peptide analogues are applicable for treating metabolic disorders, such as diabetes and obesity.

16 cl, 16 dwg, 11 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a GLP-1 peptide having an attached oligosaccharide chain, and can be used in medicine. Said GLP-1 peptide, having GLP-1 activity, has (a1) one amino acid further attached to the C end (position 37), wherein said attached amino acid is replaced with an amino acid with an attached oligosaccharide chain; or (a2) one or two amino acids, replaced with an amino acid with an attached oligosaccharide chain, where the replacement site is selected from positions 18, 20, 22, 30 and 36, and can further include 1 to 5 amino acid deletions, replacements or inserts, where said oligosaccharide chain contains five or more sugars and is represented by Formula 1. The invention also relates to a pharmaceutical composition and a method of treating or preventing diseases which can be treated or prevented by administering GLP-1, for example diabetes, which comprises use of said GLP-1 peptide having an attached oligosaccharide chain.

EFFECT: invention enables to obtain a GLP-1 peptide having an attached oligosaccharide chain, having improved stability and higher activity in controlling blood sugar level compared to GLP-1.

14 cl, 22 dwg, 10 tbl, 49 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a glucagon-like peptide-1 (GLP-1) analogues presented by general formula I

wherein X represents glycine or glycinamide. The GLP-1 analogue is resistant to the action of dipeptidyl-peptidase IV and thereby has the prolonged half-life in vivo. What is also presented is using the GLP-1 for reducing blood sugar.

EFFECT: preparing the glucagon-like peptide-1.

9 cl, 3 ex, 3 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: present invention provides a method of controlling conditions for site-specific binding of a polypeptide and a non-peptide polymer by controlling pH and alcohol content of the reaction medium.

EFFECT: method is intended to prevent formation of secondary conjugates, wherein a non-peptide polymer binds with a physiologically vital amino acid residue.

15 cl, 36 dwg, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel peptide analogues of oxyntomodulin, their pharmaceutical compositions and their application for treatment and/or prevention of excessive weight, as well as disorders and diseases, accompanying obesity.

EFFECT: peptide finds special application as appetite suppressor and in treatment of obesity.

30 cl, 41 dwg, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: compounds of the present invention are novel peptide analogues of oxyntomodulin (oxm) wherein one or more amino acid residue of the sequence oxm are replaced. The replacement of amino acid residues 15-24 of the peptide oxm either by amino acid residues 968-977 of the α-latroxin peptide (and versions thereof), or by amino acid residues 15-24 of extendin-4 (and versions thereof), or combining the amino acid residues of these sources, and/or the replacement of amino acid residues 27-33 of the peptide oxm by amino acid residues 27-33 of extendin-4, and/or adding the amino acid residues to an C-terminal of the peptide, enables producing a number of the analogues oxm presenting oxm-like activity to reduce food consumption, and according to some other aspects, more evident ability to reduce food consumption.

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32 cl, 779 dwg, 106 ex

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SUBSTANCE: invention relates to biotechnology and specifically to obtaining glucagon analogues and can be used in medicine. The peptide amino acid sequence includes a negatively charged amino acid at position 28 and a negatively charged amino acid at position 29 or 30 of the native glucagon sequence (SEQ ID NO: 1). The amino acid sequence can also include up to 7 additional amino acid modifications with respect to SEQ ID NO: 1, which enable the glucagon peptide to retain glucagon activity and improved solubility or stability. The obtained peptide is used to treat hypoglycaemia and for temporary paralysis of the intestinal tract.

EFFECT: invention enables to obtain a glucagon peptide with improved solubility or stability with respect to the native glucagon.

15 cl, 10 dwg, 4 tbl, 15 ex

FIELD: chemistry.

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EFFECT: obtaining a purified peptide product.

11 cl, 4 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and chemical technology. What is offered is a method for producing the peptide Exenatide. The peptide Exenatide prepared by synthesis may be used for producing drugs for type 2 diabetes mellitus.

EFFECT: higher yield of the unpurified peptide (42%-60%), as well as higher purity (58%-75%) and content of the end product in a mixture (33%-42%); besides, a cheaper condensing agent (DIC) is used at all the condensation stages, while the synthesis of more than one short fragments simultaneously enables faster end peptide process.

3 cl, 2 tbl, 8 ex

FIELD: chemistry.

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EFFECT: use of pseudo-proline in one of the fragments makes solid-phase synthesis of said fragment easier and also makes easier subsequent linking of said fragment with other fragments in liquid phase.

20 cl, 1 dwg, 14 tbl, 15 ex

FIELD: chemistry.

SUBSTANCE: matrix can be used in purification of proteins, where protein represents antibody, fragment of antibody or antibody-containing fused protein. Ligand corresponds to the following formula (I): R1-R2-N(R3)-R4-R5, where R1 represents non-substituted phenyl group; R2 represents hydrocarbon chain, containing 0-4 carbon atoms, preferably 1-4 carbon atoms; R3 represents hydrocarbon chain, containing 1-3 carbon atoms; R4 represents hydrocarbon chain, containing 1-5 carbon atoms; and R5 represents OH or H. As base matrix contains particles, in fact representing spherical particles, or has membranous or porous structure. Method of obtaining separation matrix includes immobilisation of said ligand on base mainly through amine group. Obtained matrix is placed into chromatographic column and after that sterilised if necessary. In order to separate one or more antibodies from one or more other compounds in liquid sample mobile phase, containing said antibodies and compound(s), are brought into contact with separation matrix. Liquid sample can contain supernatant, obtained in cell fermentation or unprocessed nutritional substance. In the process of application of chromatographic column mobile phase passes through column under impact of gravity and/or rocking, and antibodies are obtained in flow liquid of column. Invention also described set for purification of antibodies from one or more other components in liquid, containing in separate compartments chromatographic column, filled with separation matrix, one or more than one buffer and written instructions.

EFFECT: claimed invention relates to novel separation matrix, containing ligand, bound to base.

20 cl, 6 dwg, 4 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: method includes steps of: (1) extracting fermentation broth containing a compound of formula I or a salt thereof to obtain an extract 1 after filtering and centrifuging; (2) diluting or concentrating the extract 1 in a vacuum with reduction of organic solvent content to obtain an extract 2; (3) feeding the extract 2 into a macroporous adsorption resin; (4) washing the macroporous adsorption resin with water or a mixture of water and an organic solvent as a washing solution and (5) eluting the compound of formula I from the macroporous adsorption resin with the mixture of water and organic solvent as an eluent.

EFFECT: method enables to use a smaller amount of organic solvent and improves purity of the collected compound of formula I.

8 cl, 2 dwg, 2 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns recombinant plasmid pPA-OPRFI DNA coding hybrid recombinant F-I protein of Pseudomonas aeruginosa outer membrane, of the bacterial strain E.coli PA-OPRFI producing this hybrid protein, and a method for producing this recombinant protein. The presented plasmid DNA contains a DNA fragment containing a sequence of modified promoter of bacteriophage T5 and two lactose operons; a DNA fragment containing a ribosome entry site, an initiation ATG-codon and a sequence coding six histidines; a DNA fragment containing the full-size sequences of oprF and opri genes of P.aeruginosa; a DNA fragment containing a ribosome entry site, and a DNA fragment containing a lambda t0 transcription stop region.

EFFECT: presented inventions enables producing the hybrid recombinant F-I protein of Paeruginosa outer membrane for carrying out an immunobiological assay in developing a Pseudomonas aeruginosa vaccine, and also for producing donor immunoglobulins for therapy of active forms of Paeruginosa infection.

3 cl, 3 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

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EFFECT: obtaining pharmaceutically acceptable crystalline or amorphous salts of D-isoglutamyl-D-tryptophan.

22 cl, 15 dwg, 13 ex

FIELD: food industry.

SUBSTANCE: invention relates to fodder additives containing dipeptides or their salts; one amino acid residue of dipeptide is represented by DL-methionine residue; the other amino acid residue of dipeptide is represented by amino acid in L-configuration chosen from the group including lysine, threonine, tryptophan, histidine, valine, leucine, isoleucine, phenylalanine, arginine, cysteine and cystine.

EFFECT: described are fodder mixtures containing such additives and the said dipeptides production method.

31 cl, 17 dwg, 10 tbl, 25 ex

FIELD: chemistry.

SUBSTANCE: method includes steps of: (1) feeding a raw compound of formula I into a macroporous adsorption resin; (2) washing the macroporous adsorption resin with water, an organic solvent or a mixed solution of an organic solvent and water as the washing liquid; and (3) elution of the compound of formula I from the macroporous adsorption resin with water, an organic solvent or a mixed solution of an organic solvent and water as an eluent, where at step (1) the solution containing a raw compound of formula I contains ionisable salts; and the macroporous adsorption resin is selected from a nonpolar aromatic adsorption resin polymerised from styrene and divinyl benzene, or a methacrylic adsorption resin of medium polarity with methacrylate residues in the structure.

EFFECT: purification method has the advantages of using a small amount of organic solvents without using silica gel and low environmental impact; the collected compound also has better purity compared to previous methods.

13 cl, 2 tbl, 2 dwg, 8 ex

FIELD: metallurgy.

SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.

EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).

17 cl, 4 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention represents a method of obtaining collagen from biological material, which includes milling of a raw material, liquid processing of the biological material with obtaining a collagen-containing substance, separated into a sediment and liquid fraction, characterised by the fact that as the biological material applied is a medusa, preferably Rhopilema, preferably its cupola, which is crushed preferably to 1-2 mm, and for obtaining the collagen-containing substance the material prepared in such a way is mixed with drinking water with a ratio by the raw material weight to water as 1:2 and extracted at a temperature preferably of 15-18°C for 6-12 hours with periodical mixing, after that, the obtained extract is separated into the liquid fraction and collagen-containing sediment, which is after that dehydrated to moisture weight in it not more than 10%, after which it is pre-packed and packed.

EFFECT: extension of an arsenal of methods for obtaining neutral collagen.

FIELD: chemistry.

SUBSTANCE: invention relates to a method for chemical conversion of a peptide chain into a peptide thioether. A -C(=X)-R1 group is incorporated into a thiol group of a cysteine residue and the obtained peptide then reacts in an organic solvent with a compound having a substituted group of formula: NH-C(=Y)NHR3, and a -NH-C(=Y)NHR3 group binds in an addition reaction with the carboxyl group of the peptide bond at the N-terminal side of the cysteine residue, through which the peptide bond is broken and the peptide moiety at the C-terminal end is cut off. When the obtained peptide chain, having a -NH-C(=Y)NHR3 group, reacts with thiol in a buffer solution, a thiol exchange reaction occurs, specifically the thiol group of the thiol compound binds with the carbon of the carbonyl to which the -NH-C(=Y)NHR3 was bound, thereby removing the -NH-C(=Y)NHR3 group.

EFFECT: achieving conversion to peptide thioether.

14 cl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.

EFFECT: improvement of the method.

2 cl, 1 tbl, 5 ex

Oxyintomoduline // 2542362

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to peptide analogues of oxyintomoduline (OXM, glucagon-37), which can be modified for providing the stability of cleavage and inactivation with dipeptidyl peptidase IV (DPP-IV) for increasing a half-life time in vivo of the peptide analogue alongside with enabling the peptide analogue acting as a double agonist GLP-1/glucagon receptor (GCGR).

EFFECT: peptide analogues are applicable for treating metabolic disorders, such as diabetes and obesity.

16 cl, 16 dwg, 11 tbl, 12 ex

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