JP2011020926A - Modified peptide, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide that can be continuously used in an environment of a living body, for example, in a living body or on the surface thereof, and to achieve a high yield in a method for producing the peptide. <P>SOLUTION: The peptide with both ends thereof modified is characterized in that its N terminal is a thiazolidine ring and its C terminal is a lysine ε-lactam ring. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a modified peptide and a method for producing the same.

  In recent years, peptides have attracted attention in various fields such as pharmaceuticals, foods, and cosmetics. For example, various functional peptides exhibiting useful activity including physiological activity and antimicrobial activity are widely used as active ingredients.

  However, the conventional peptide is easily hydrolyzed in a living environment such as in vivo or on the surface of the living body, and there is a problem that it is degraded before achieving the purpose.

  Thus, attempts have been made to impart hydrolysis resistance by chemically modifying functional peptides. For example, an attempt has been made to design and synthesize analogs that maintain the characteristics of structure and function by replacing the peptide bond in the sequence with its structural equivalent to impart hydrolysis resistance (Non-patent Document 1).

  However, it has been reported that the useful activity is dramatically reduced by modifying a functional peptide (Non-patent Document 2). For this reason, it is extremely difficult to impart hydrolysis resistance to functional peptides while maintaining their useful activity, and this problem has not been solved for a long time.

  In addition, two types of peptide production methods are mainly used: “chemical synthesis” and “biological production” using recombinant DNA technology. The chemical synthesis method can produce a peptide comprising any amino acid and a modifying group, but involves a large amount of organic solvent / by-product and is extremely expensive to produce. On the other hand, in the biological production method, although mass production is possible in an aqueous culture solution or the like, it is limited only to peptide sequences composed of amino acids (generally natural amino acids) that can be used by the production organism, In general, the peptide is isolated without modifying groups at both ends. As a method of utilizing the advantages of these production methods, a method of first obtaining a fusion protein or fusion peptide containing a target peptide and then cutting out the target peptide is performed. For example, after obtaining a fusion protein or fusion peptide containing a target peptide in a cultured microorganism by expression, the target peptide is excised by chemical treatment.

  However, the conventional production method has problems such as poor production yield of the target peptide.

Journal of Synthetic Organic Chemistry, 2008, Vol. 66, No. 9, 846-857 Proc. Natl. Acad. Sci. USA. , April 7, 2009, vol. 106, no. 14, pp 5801-5806

  An object of the present invention is to provide a peptide that can be used continuously in a living environment such as in vivo or on the surface of a living body. Another object is to achieve a high yield in the peptide production method.

  In order to solve the above-mentioned problems, the present inventors made extensive trial and error by examining various treatment methods and the like, and as a result, modified peptides exhibiting outstanding hydrolysis resistance (hereinafter referred to as the present specification). The peptide modified in the book was sometimes simply referred to as “modified peptide”.) It was predicted from the prior art that modified peptides obtained by modifying functional peptides would lose their useful activity. We have found that this modification surprisingly does not significantly affect the useful activity of the peptide.

  Furthermore, the present inventors have obtained a surprising finding that this modified peptide can be produced in a high yield by a predetermined method.

  On the basis of this technical idea, the present inventors have succeeded in developing a more excellent modified peptide and a method for producing the same by spending much more time and effort, and completed the present invention.

That is, the present invention is as follows:
Item 1. A peptide modified at both ends, wherein the N-terminus is a thiazolidine ring and the C-terminus is a lysine ε-lactam ring.

  Item 2. Item 2. The modified peptide according to Item 1, wherein the peptide has physiological activity or antimicrobial activity.

  Item 3. Item 3. The modified peptide according to Item 2, wherein the peptide has antiviral activity.

  Item 4. Item 3. The modified peptide according to Item 2, wherein the peptide has anti-HIV activity.

Item 5. The peptide is
(A) an amino acid sequence of any one of SEQ ID NOs: 1 to 3; or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 1 to 3. Item 5. A modified peptide according to Item 4, which has the modified peptide.

  Item 6. Item 6. A pharmaceutical composition comprising the modified peptide according to any one of Items 2 to 5.

  Item 7. Item 6. A food comprising the modified peptide according to any one of Items 2 to 5.

Item 8. A method for producing the peptide according to any one of Items 1 to 5,
(1) The following formula:
_{X 1 -Cys-A-Lys-} Cys-X 2
(Wherein, A is the amino acid sequence of the peptide according to any one of Items 1 to 5 not containing a cysteine residue, and X ₁ and X ₂ are the same or different and are any amino acid residue or any amino acid. Is an array)
And (2) a step of subjecting the S-cyanated peptide obtained by the step (1) to an alkali treatment.

Item 9. When X ₁ is an amino acid residue, the amino acid residue is an asparagine residue or a lysine residue;
The C-terminus of its amino acid sequence when X ₁ is an amino acid sequence is an asparagine residue or a lysine residue A method according to claim 8.

  Item 10. Item 10. The method according to Item 8 or 9, wherein the alkali treatment in the step (2) is performed with a carbonate.

  The modified peptide of the present invention has hydrolysis resistance. In particular, it has high exopeptidase resistance.

  In addition, the modified peptide of the present invention maintains useful activities including physiological activity and antimicrobial activity without significantly impairing the activity before modification.

  As described above, the modified peptide of the present invention can be used continuously in a living environment such as in vivo or on the surface of a living body. In particular, it exhibits sustained biological activity in serum.

  And the manufacturing method of the modified peptide of this invention can cut out a part with high efficiency from the peptide containing the target amino acid sequence, and can acquire the modified peptide which consists of the amino acid sequence.

  Thus, the method for producing a modified peptide of the present invention can achieve a high yield in producing the modified peptide. Therefore, the modified peptide can be produced at a lower cost and in a larger amount. In particular, a high yield can be achieved when attempting to obtain a modified peptide by excising a part from the recombinant protein.

It is drawing which represented typically the method of manufacturing the modified peptide of this invention. In this figure, an example in which the starting peptide is a recombinant protein is shown. In addition, the amino acid sequence part of A is described as Target peptide. A peptide in which both ends of the amino acid sequence of A are cyclized is obtained by specifically cutting out the “amino acid sequence portion of A” at the position of the white arrow and cyclizing both ends of the amino acid sequence of A by a cyclization reaction. It shows that it gets. It is drawing which illustrated the model peptide used in the Example. It shows that these two amino acid residues are arranged on both ends of SC34EK in the order of asparagine or lysine and cysteine from the N-terminal side, and further, a thioredoxin tag is attached to the N-terminal side. In the figure, “X” is the same or different and is asparagine or lysine, and “TRX” indicates a thioredoxin tag. It is drawing which shows the result analyzed from the circular dichroism spectrum about the modified SC34EK about whether the physicochemical characteristic based on (alpha) -helix structure is impaired. It is drawing which shows the result analyzed from the circular dichroism spectrum about whether the thermal stability is not impaired about modified SC34EK. It is drawing which shows the result of having verified the exopeptidase resistance about modified SC34EK.

1. Modified peptide The modified peptide of the present invention is a peptide modified at both ends, characterized in that the N-terminus is a thiazolidine ring and the C-terminus is a lysine ε-lactam ring. More specifically, the modified peptide of the present invention comprises (1) a peptide serving as a nucleus (hereinafter sometimes referred to as “nuclear peptide”), and (2) a modification of the N-terminal of the N-terminal amino acid of the nuclear peptide. And (3) a lysine ε-lactam ring that modifies the C-terminal of the C-terminal amino acid of the nuclear peptide.

(1) As a nuclear peptide , a peptide having useful activity including physiological activity or antimicrobial activity (in this specification, such a peptide may be referred to as “functional peptide”) is used. can do.

  As used herein, “physiological activity” refers to an activity that exhibits some action on the physiological functions of a living body. For example, neurotransmitter-like activity, neurotrophic factor-like activity, hormone-like activity, growth factor-like activity, autocidal-like activity, cytokine-like activity, and pheromone-like activity.

  Antimicrobial activity is not limited to any antimicrobial activity. Examples thereof include antibacterial (bacteria) activity, antibacterial (Fungi) activity, and antiviral activity.

  Examples of the antiviral activity include anti-HIV activity. Examples of the nuclear peptide having anti-HIV activity include a peptide having an activity of inhibiting membrane fusion between the HIV virus and the target cell. Examples of such peptides include peptides derived from the HR2 region of gp41 of HIV-1. Specifically, for example, SC34EK (Otaka A. et al., Angew. Chem. Int. Ed., 2002, 41, No. 16) consisting of the amino acid sequence of SEQ ID NO: 1, and amino acid sequence of SEQ ID NO: 2 SC35EK (same as above), and T-20EK (Oishi A. et al., 2008, 51, No. 3) consisting of the amino acid sequence of SEQ ID NO: 3 are included. The nuclear peptide having anti-HIV-1 activity further comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of SEQ ID NOs: 1 to 3, and anti-HIV- Examples thereof include peptides having one activity. The deleted amino acid sequence is preferably an amino acid sequence in which amino acids within 80% of the entire amino acid sequence are deleted, and more preferably an amino acid sequence in which amino acids within 85% of the entire amino acid sequence are deleted. Preferably, an amino acid sequence in which amino acids within 90% of the entire amino acid sequence are deleted is more preferable.

  The modified peptide of the present invention is maintained without losing its useful activity by modifying both ends. This is considered to be because the terminal modification groups having the ring structures of thiazolidine ring and lysine ε-lactam ring are not easily decomposed by peptidases from the respective ends.

(2) Thiazolidine ring The thiazolidine ring modifies the N-terminal of the N-terminal amino acid of the nuclear peptide. Specifically, a thiazolidine-carbonyl group, for example, 2-iminothiazolidine-4-carbonyl group (formula (I)) is bonded to the N-terminal amino group of the N-terminal amino acid of the nuclear peptide by forming an amide bond. .

  The thiazolidine ring may have various substituents with the thiazolidine ring structure as a basic skeleton, as long as the effects of the present invention are not hindered.

(3) Lysine ε-lactam ring The lysine ε-lactam ring modifies the C-terminal of the C-terminal amino acid of the nuclear peptide. Specifically, the amino group of the lysine ε-lactam ring (formula (II)) is bonded with the C-terminal carboxyl group of the C-terminal amino acid of the nuclear peptide by forming an amide bond.

The lysine ε-lactam ring may have various substituents while using the lysine ε-lactam ring structure as a basic skeleton as long as the effects of the present invention are not hindered.
2. Composition containing modified peptide Examples of the composition containing the modified peptide of the present invention include pharmaceuticals and foods. In the present specification, “food” includes beverages.

  The pharmaceutical or food containing the modified peptide of the present invention may contain only one type of the modified peptide of the present invention, or may contain two or more types in combination.

  The content ratio of the modified peptide of the present invention in the pharmaceutical or food of the present invention can be selected within the range in which the useful action of the modified peptide is exhibited.

  Although it does not specifically limit as pH of the pharmaceutical or foodstuff of this invention, pH 6-8 are preferable at the point of avoiding an unfavorable influence around the administration site | part at the time of peptide stability or administration (injection).

  The form of the pharmaceutical or food of the present invention is not limited. It may be liquid, semi-liquid, or solid.

  The pharmaceutical or food of the present invention can contain various components as necessary in addition to the modified peptide as long as the effects of the present invention are not hindered.

  The modified peptide nuclear peptide contained in the pharmaceutical product of the present invention exhibits a specific pharmacological activity. In this case, the nuclear peptide is preferably one having antiviral activity, more preferably one having anti-HIV activity.

  The form of the pharmaceutical product of the present invention is appropriately set according to the affected part to be applied, the application method, and the like. Specific examples of the form include solid forms such as tablets, granules, powders, suppositories and capsules; semi-solid forms such as creams, gels and ointments; liquids such as liquids and lotions. .

  The dosage and administration frequency of the pharmaceutical product of the present invention are appropriately set according to the purpose of use of the pharmaceutical product, the form, the type of subject case, the degree of symptoms, the age of the recipient, the degree of pharmacological activity of the modified peptide, and the like. The For example, as an average of the daily dose of the pharmaceutical product of the present invention, for example, the dose (90 mg × 2 times / day) of Fuzeon (T-20) is calculated with reference to the amount of the modified peptide, Usually, about 2-200 mg is mentioned.

In addition, the pharmaceutical agent of the present invention may be administered, for example, once per day or divided into two or three times, and the doses for 2 days to 1 week are administered at a time. May be.
3. Method for producing modified peptide The method for producing the modified peptide of the present invention comprises:
(Step 1) The following formula:
_{X 1 -Cys-A-Lys-} Cys-X 2
(In the formula, A is an arbitrary amino acid sequence not containing a cysteine residue, and X ₁ and X ₂ are the same or different, and are arbitrary amino acids or arbitrary amino acid sequences)
(Hereinafter sometimes referred to as “raw peptide”)
And (step 2) a step of subjecting the S-cyanated peptide obtained in step 1 to an alkali treatment (hereinafter referred to as an “alkali treatment”). Sometimes referred to as a "process".)
It is a method including.

  According to this production method, as shown in FIG. 1, a peptide in which both ends of the amino acid sequence of A are modified can be obtained as a final product using a starting peptide as a starting material. Specifically, a peptide in which both ends of the amino acid sequence of A are modified by excising the amino acid sequence portion of A from the raw peptide by specific cleavage reaction and cyclizing both ends of the amino acid sequence of A by cyclization reaction Can be obtained. More details are as follows. The explanation for A is omitted because there is no difference from the explanation for the amino acid sequence of the nuclear peptide in the modified peptide of the present invention.

(1) Raw material peptide The raw material peptide can be obtained, for example, by expressing DNA containing a base sequence encoding it in a cultured microorganism or the like. Specifically, it can be obtained by preparing an expression vector incorporating the DNA, introducing it into Escherichia coli, expressing a polypeptide containing the starting peptide, and purifying it. In this case, in order to purify the expressed polypeptide more efficiently, the polypeptide may be expressed with a tag sequence for purification added. In this case, as a tag sequence for purification, for example, a histidine tag, a glutathione tag, a biotin tag, a FLAG tag, or the like can be used.

  In addition, the starting peptide can be obtained by peptide synthesis or the like.

(2) S-Cyanation Step The S-cyanation step is a step of S-cyanating a cysteine group. Thereby, the site | part specifically cut | disconnected in the alkali treatment process performed subsequently is determined. Specifically, the N-terminal amide bond of the S-cyanoated cysteine group is specifically cleaved.

  The final purpose of this production method is to obtain a peptide in which both ends of the amino acid sequence of A are modified as a final product. Therefore, if A contains a cysteine residue, the cysteine residue in A is also S-cyanated and its N-terminal amide bond is cleaved. If it does so, since it will become impossible to obtain the peptide by which the both ends of the amino acid sequence of A were modified, A needs to be an amino acid sequence which does not contain a cysteine residue.

  The S-cyanation reaction is not limited, but can be performed by, for example, treating in an acetic acid solution containing 0.5 mM tris (2-carboxyethyl) phosphine and CDAP. In addition to this, the S-cyanation reaction is also performed by, for example, reacting 2-nitro-5-thiocyanobenzoic acid (NTCB) in a pH 7-9 buffer solution. be able to.

  The temperature can be performed at 0 to 50 ° C., for example. In view of reaction efficiency, the reaction is preferably performed at 20 to 50 ° C.

  For example, the reaction time can be 10 to 300 minutes. It is preferable to carry out for 30 minutes or more in terms of yield.

(3) Alkali treatment step The alkali treatment step is a step of cleaving the N-terminal amide bond of the S-cyanoated cysteine residue and cyclizing a predetermined cleavage site.

  By the alkali treatment, the cysteine residue S-cyanated in the S-cyanation step on the N-terminal side of the amino acid sequence of A is cyclized to a thiazolidine ring. At the same time, the N-terminal amide bond is specifically cleaved, and the portion A is cut out.

Meanwhile X ₁ of the raw material peptide is any amino acid or any amino acid sequence. X ₁ is linked to A via a cysteine residue that is S-cyanated. The amide bond between the cysteine group and X ₁ is S- cyanation is cleaved by alkaline treatment step. Thus X ₁ is adjacent to the cleavage site. Therefore, when X ₁ is an amino acid, the type thereof, or when X ₁ is a peptide, the type of C-terminal amino acid (amino acid adjacent to the cleavage site) affects the reactivity of the cleavage reaction by the alkali treatment step. give. The cleavage reaction can be carried out without any problem with any amino acid, but if it is asparagine or lysine, the progress of the cyclization reaction based on the properties of the side chain functional groups of these residues (formation of aspartimide or lysine ε-lactam) Is preferable because it promotes the above thiazolidine ring formation reaction and increases the reaction efficiency. Among them, lysine is more preferable in terms of reaction efficiency.

X ₂ of the raw peptides, any amino acid or any amino acid sequence. X ₂ is bonded to A via a lysine group and a cysteine group to be S-cyanoated. Since X ₂ is not adjacent to the cleavage site unlike X ₁ , it does not affect the reactivity of the cleavage reaction by the alkali treatment step.

Although alkali treatment reaction is not limited, For example, it can carry out by processing with various bases. Examples of various bases include, but are not limited to, ammonia, amines, weak acid salts, metal hydroxides, and the like. These may be used alone or in combination of two or more. Examples of amines that can be used include triethylamine and isopropylethanolamine. As the weak acid salt, for example, acetate, bicarbonate, carbonate and the like can be used. As the metal hydroxide, sodium hydroxide, lithium hydroxide, or the like can be used. As the salt, for example, sodium, potassium and the like can be used. From the viewpoint of reaction efficiency, an aqueous solution of carbonate is preferable, and an aqueous solution of K ₂ CO ₃ is more preferable among the carbonates.

  Although the base concentration depends on the type of base, for example, a base of 0.1 to 5M can be used. In the case of an aqueous carbonate solution, for example, 0.1 to 2M carbonate can be used.

  The temperature can be performed at 0 to 50 ° C., for example. In view of reaction efficiency, the reaction is preferably performed at 20 to 50 ° C.

  For example, the reaction time can be 10 to 300 minutes. It is preferable to carry out for 30 minutes or more in terms of yield.

  Hereinafter, the present invention will be specifically described based on test examples and examples. However, the present invention is not limited to these specific examples.

Test Example 1 Study on simultaneous cleavage reaction at both ends of nuclear peptide (1) Synthesis of model peptide The following formula (III):

[Ac is an acetyl group, and X is any amino acid residue (except for a cysteine residue). The N-terminal side of the N-terminal tyrosine is acetylated, and the C-terminal side of the C-terminal lysine is amidated. Y represents tyrosine, E represents glutamic acid, Q represents glutamine, K represents lysine, C represents cysteine, and F represents phenylalanine. ]
Was used as a model peptide.

  The model peptide was synthesized as follows. The peptide was synthesized by the standard Fmoc (fluorenylmethoxycarbonyl) synthesis method by a peptide solid phase synthesis method. As a solid phase, 83 mg and 0.05 mmol of Rink Amide resin (manufactured by Navabiochem) were used. For amino acid side chain protection, t-Bu for tyrosine, serine and threonine; t-Bu ester for aspartic acid and glutamic acid; Boc for lysine; Trt for cysteine, histidine, asparagine and glutamine; As for arginine, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (pbf) was used, respectively.

The construction of the protected peptide resin was carried out by condensing Fmoc amino acid with 5 equivalents of a reagent (Fmoc amino acid, N, N′-diisopropylcarbodiimide, HOBt · H ₂ O) and then in DMF containing 20% piperidine for 1 minute × The removal of the Fmoc group by treating twice for 20 minutes × one time was performed by repeating each amino acid.

After treating the finally obtained protected peptide resin in TFA / H ₂ O / m-cresol / thioanisole / 1,2-ethanedithiol (80: 5: 5: 5: 5) for 2 hours at room temperature The resin was filtered off. Next, 30 mL of ice-cooled dry diethyl ether was added to the filtrate. The produced powder was collected by centrifugation and washed three times with 15 mL of ice-cooled dry diethyl ether. The product was purified by preparative HPLC to obtain a colorless powdery model peptide.

  The model peptide cysteine was S-cyanated as follows. First, 0.58 ml of a 0.1N acetic acid solution containing a model peptide (for example, 5.8 mg when X is a lysine residue) was added to 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [1-cyano-4. -Dimethylaminopyridinium tetrafluoroborate (CDAP); source SIGMA; product number C2776] was added to 0.182 mL of 0.1 N acetic acid solution (10 mg / mL). After stirring at room temperature for 30 minutes, purification by preparative HPLC yielded a lyophilized product of S-cyanated peptide. Purification by preparative HPLC was performed as follows. Cosmosil 5C18-ARII preparative column (20 × 250 mm, flow rate 10 mL / min, manufactured by Nacalai Tesque) was used. When X is a lysine residue, the S-cyanated peptide was 5.7 mg and the yield was 97%.

(2) Study on cleavage reaction Subsequently, 1 mg of the S-cyanated model peptide was treated in 0.1 mL of 3M NH ₃ solution at 20 ° C. for 20 minutes to thereby remove the C-terminal amide bond of S-cyanated X. Cleaved specifically.

  By analyzing the product of this cleavage reaction by reversed-phase high performance liquid chromatography (RP-HPLC), peptides represented by the following formulas (IV) to (VIII) were obtained. RP-HPLC was performed as follows. Cosmosil 5C18-ARII analytical column (4.6 × 250 mm, flow rate 1 mL / min, manufactured by Nacalai Tesque) was used. The eluted product was detected by UV (220 nm). As the solvent system, 0.1 v / v% TFA (solvent A) and 0.1 v / v% TFA-containing MeCN (solvent B) were used. Analysis was performed using MALDI-TOF-MS (AXIMA-CFR plus, Shimadzu Corp.) or QqTof (QSTAR pulsar i, Applied Biosystems). NMR spectra were measured using a Bruker AVANCE500.

  The peptides represented by the formulas (IV) and (V) are N-terminal fragments obtained when X is an amino acid that is neither asparagine nor lysine. The peptide represented by the formula (IV) has a carboxamide group on the C-terminal side, and the peptide represented by the formula (V) has a carboxylic acid on the C-terminal side.

  The peptide represented by the formula (VI) is a C-terminal fragment. The N-terminal side is a 2-thiazolidine ring. Specifically, it is a 2-iminothiazolidine-4-carbonyl group.

  The peptide represented by the formula (VII) is an N-terminal fragment obtained only when X is asparagine. The C-terminal side becomes a cyclic aspartimide group by an intramolecular cyclization reaction.

  The peptide represented by the formula (VIII) is an N-terminal fragment obtained only when X is lysine. The C-terminal side becomes a lysine ε-lactam ring by an intramolecular cyclization reaction.

  By analyzing these peptides by RP-HPLC, the cleavage rate (%) of the peptide represented by the formula (III) was calculated.

  Specifically, the cutting rate was calculated as follows. First, peak areas were measured for all products by RP-HPLC. Next, the cleavage rate (%) is calculated based on the sum of the peak areas of the respective peptides represented by formula (IV) or formula (V), formula (VI), and formula (VII) or formula (VIII). did.

  It was examined whether a higher cleavage rate could be obtained when any of the 19 types of amino acid residues was X. When X is asparagine, the cleavage rate is 79%, and when X is lysine, the cleavage rate is 82%. It was found that the highest cleavage rate was obtained in these two cases. When X is asparagine, the ratio of the peptide represented by formula (IV) to the peptide represented by formula (VII) was 53:47. When X is lysine, the ratio of the peptide represented by formula (IV) to the peptide represented by formula (VIII) was 22:78.

  It has been found that it is particularly advantageous when X is lysine in terms of efficiently obtaining a terminally cyclized peptide.

(2) Examination on simultaneous cleavage reaction at both ends of nuclear peptide From the examination result of (1), the following formula:
_{X 1 -Cys-A-Cys-} X 2
(In the formula, A is a nuclear peptide consisting of an arbitrary amino acid sequence, and X ₁ and X ₂ are the same or different and are arbitrary amino acids or arbitrary amino acid sequences)
First S- cyanation each peptide represented in, followed by the S- cyanation peptide alkali treatment, C-terminal amide bond of the peptide X _1, and nuclear peptide C-terminal amide bond at the same time As a result of the cleavage, it was expected that the nuclear peptide could be cleaved with high efficiency.

And the following formula:
_{X 1 -Cys-A-Lys-} Cys-X 2; or _{X 1 -Cys-A-Asn-} Cys-X 2
(In the formula, A is a nuclear peptide consisting of an arbitrary amino acid sequence, and X ₁ and X ₂ are the same or different and are arbitrary amino acids or arbitrary amino acid sequences)
It was expected that the nuclear peptide could be excised with both ends in a cyclic structure by treating each peptide represented by

  The state in which both ends have a cyclic structure means that the N-terminal side is a 2-iminothiazolidine ring and the C-terminal side is an aspartimide group or a lysine ε-lactam ring, as shown in the examination result of (1). It is in the state.

  Therefore, it is verified whether the reaction of cleaving the nuclear peptide can be actually performed with both ends in a cyclic structure, using the above model peptides (of which X is asparagine or lysine). did.

Specifically, the verification was performed as follows. First, the S-cyanation reaction was performed as described above. Next, the alkali treatment reaction was carried out using various bases, and it was examined which nuclear peptide could be excised with both ends in a cyclic structure when any base was used. Various bases include 3M NH ₃ , 0.5M NH ₃ , 1M Et ₃ N, 1M (i-Pr) ₂ EtN, 1M AcONa, 1M NaHCO ₃ , 1M Na ₂ CO ₃ , 0.3M Na ₂ CO ₃ A total of 10 types of 1M K ₂ CO ₃ and 0.3M K ₂ CO ₃ were examined. All alkali treatments were performed at 20 ° C. for 20 minutes.

As a result of the verification, it was revealed that when an aqueous carbonate solution was used, a peptide of formula (VII) or formula (VIII) having a cyclized terminal can be obtained with particularly high efficiency. It was also revealed that these peptides can be obtained with higher efficiency when using an aqueous solution of K ₂ CO ₃ among carbonates.

  As described above, with respect to the reaction of cutting out the nuclear peptide in a state where both ends have a cyclic structure, a processing method for realizing it was established.

Example 1. Production of modified peptide of the present invention Based on the treatment method established in Test Example 1, it was verified whether the actually modified nuclear peptide could be cut out.
(1) Setting of model peptide SC34EK which is an anti-HIV active peptide (HIV fusion inhibitor) was used as a model peptide of the nuclear peptide. SC34EK is a peptide consisting of the amino acid sequence of SEQ ID NO: 1. This peptide was designed based on the α-helix structure on the C-terminal side of gp41, which is an envelope glycoprotein of HIV. In the membrane fusion process of HIV infection, it is known that antiparallel 6-helical bundle structure formation by NGP and CHR of gp41 is necessary, and SC34EK inhibits not only the wild type but also the enfu. It also exhibits antiviral activity against HIV-1 strains exhibiting resistance to biltide (T-20).

(2) Expression of model peptide thioredoxin fusion protein First, a fusion protein in which SC34EK was fused with thioredoxin was expressed in E. coli (E. coli BL21 strain). As shown in FIG. 2, these two amino acid residues were arranged at both ends of SC34EK so that asparagine or lysine and cysteine were in this order from the N-terminal side (in FIG. 2, X was the same or Differently asparagine or lysine). By doing in this way, it is expected that SC34EK is cut out by S-cyanation and subsequent alkali treatment.

First, as a template for PCR amplification, a nucleic acid having a cDNA base sequence represented by SEQ ID NO: 2 (KKC-SC34EK-KCW) and a nucleic acid having a cDNA base sequence represented by SEQ ID NO: 3 (KNC-SC34EK-NCW) are used. Chemically synthesized. Each of these has a restriction enzyme recognition site (GGATCC) and (CTCGAG) by BamHI and XhoI. It has been replaced with a codon that is frequently used in E. coli. Each fragment cleaved with BamHI and XhoI was inserted into the pET32a vector. Using these plasmids (pET32a-KKC-SC34EK-KCW and pET32a-KNC-SC34EK-NCW) E. coli BL21 (DE3) -RIL strain was transformed for forced expression. The isolated colony was picked up and cultured overnight in 10 mL of LB medium containing 50 μg / ml ampicillin at 30 ° C. with shaking. This culture solution was transferred to an LB culture solution (1 L) containing 50 μg / ml ampicillin. When OD ₆₀₀ reached 0.6-0.8 at 30 ° C., protein expression was initiated by adding 1 mM IPTG. Thereafter, the culture was further continued at 25 ° C. for 6 hours, and the cells were collected by centrifugation at 4,000 rpm for 20 minutes. The cells were suspended in B-PER (PIERCE) solution and disrupted by sonication. Centrifugation was performed at 12,000 rpm for 30 minutes, 0.5 mM TCEP was added to the supernatant, and the mixture was transferred to a Ni-NTA agarose column (QIAGEN). The column was washed with wash buffer (20 mM phosphate, pH 6.0, 0.5 M NaCl, 0.5 mM TCEP). Protein was eluted using 0.5 mM TCEP-containing phosphate buffer (pH 6.0) containing 10-200 mM imidazole. The expression and purification of the fusion protein was analyzed using SDS-PAGE (10-20% gradient gel). The yield of the fusion protein was measured using Protein Assay Kit (BIO-RAD Laboratories, Hercules, CA).

(3) Excision of the modified nuclear peptide The fusion protein was extracted with 0.5 mM tris (2-carboxyethyl) phosphine [tris (2-carboxyethyl) phosphine (TCEP); source SIGMA, product number C4706], and S-cyanation was performed by treatment in 0.1N acetic acid solution containing 10 mM CDAP for 30 minutes. During this process, cysteine residues will remain in a reduced state.

The obtained S-cyanated fusion protein was alkali-treated with 0.3M K ₂ CO ₃ for 30 minutes to cut out SC34EK with both ends in a cyclic structure. The product was analyzed by LC-MS. The product was also purified by preparative HPLC.

  The N-terminal side of the cut out SC34EK is a 2-iminothiazolidine-4-carbonyl group. On the other hand, when asparagine is arranged together with cysteine on both ends of SC34EK, the C-terminal side is a 2-aspartimide group. On the other hand, when lysine is arranged together with cysteine at both ends of SC34EK, the C-terminal side is a lysine ε-lactam ring.

  As for the yield, when asparagine was placed together with cysteine on both ends of SC34EK, 24% of modified SC34EK could be obtained. When lysine was placed together with cysteine at both ends of SC34EK, 21% of modified SC34EK could be obtained.

(4) Evaluation of modified nuclear peptides For these modified SC34EK, (i) anti-HIV activity, (ii) physicochemical properties, and (iii) biological stability, respectively, were evaluated as follows: did. For control, the activity was similarly evaluated for SC34EK and the unmodified form of both ends of SC34EK (N-terminal unprotected and C-terminal carboxylic acid).

First, anti-HIV activity was evaluated for each of these modified SC34EK. Evaluation was performed by the improved MAGI assay as follows (Kodama, EI et al., Antimicrob. Agents Chemother. 2001, 45, 1539-1546; Madea Y. et al., J. Infec. Dis. 1998, 177, 1207-1213). Target cells (Hela-CD4-LTR-β-gal) 104 cells / well were seeded in 96-well flat microtiter culture plates. The next day, HIV-1 clone NL4-3 was seeded so that 60 MAGI U / well, 60 blue cells appeared after 48 hours. Thereafter, the specimen was added and cultured. After 48 hours, the number of cells stained with X-gal (5-bromo-4-chloro-3-indoleyl-β-d-galactopyranoside) was counted. The activity of the analytes was evaluated at a concentration (EC ₅₀ ) that inhibits HIV-1 replication by 50%. As a result, as shown in Table 1, it was revealed that these modified SC34EKs showed the same activity as SC34EK. The introduction of a cyclic structure was expected to impair the activity, but this result was contrary.

  In addition, these modified SC34EK were also examined for whether the physicochemical characteristics based on the α-helix structure were not impaired. This physicochemical feature is an important feature associated with anti-HIV activity. The analysis was performed as follows from the circular dichroism spectrum. 10 μM of the peptide was treated in 5 mM HEPES buffer (pH 7.2) at 37 ° C. for 30 minutes. The CD spectrum was measured with an average of 8 scans at 25 ° C. using a circular dichroism spectrophotometer (J-710 manufactured by JASCO Corporation). After equilibration for 0.25 minutes, after a 1.0 second integration time, thermal denaturation was measured every 0.5 ° C.

  As a result, as shown in FIG. 3, it became clear that the physicochemical characteristics based on the α-helix structure were not impaired.

Furthermore, these modified SC34EKs were also verified to see whether the thermal stability was impaired. A midpoint of thermal denaturation (melting point Tm) was calculated based on [θ] ₂₂₂ value by circular dichroism spectrum analysis.

  As a result, as shown in FIG. 4, it became clear that the thermal stability was not impaired.

  Finally, the resistance to degradation caused by exopeptidase was verified. Each peptide was incubated in mouse serum at 37 ° C. for 24 hours, during which time sampling was performed for RP-HPLC analysis. As a result, as shown in FIG. 5, it was revealed that SC34EK that is not modified at both ends is immediately decomposed. It was also revealed that the modified SC34EK having a thiazolidine ring on the N-terminal side and a 2-aspartimide group on the C-terminal side gradually decomposes. On the other hand, surprisingly, a modified backbone peptide having a thiazolidine ring on the N-terminal side and a lysine ε-lactam ring on the C-terminal side has significantly improved biodegradation resistance, and both ends are amidated or acylated. It became clear that biodegradation resistance equivalent to that of the modified SC34EK was exhibited. This modified backbone peptide was stable after 24 hours of incubation.

Claims (10)

A peptide modified at both ends, wherein the N-terminus is a thiazolidine ring and the C-terminus is a lysine ε-lactam ring. The modified peptide according to claim 1, wherein the peptide has physiological activity or antimicrobial activity. The modified peptide according to claim 2, wherein the peptide has antiviral activity. The modified peptide of claim 2, wherein the peptide has anti-HIV activity. The peptide is
(A) an amino acid sequence of any one of SEQ ID NOs: 1 to 3; or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences of SEQ ID NOs: 1 to 3. The modified peptide according to claim 4, which has a peptide. A pharmaceutical composition comprising the modified peptide according to any one of claims 2 to 5. A food comprising the modified peptide according to any one of claims 2 to 5. A method for producing the peptide according to claim 1,
(1) The following formula:
_{X 1 -Cys-A-Lys-} Cys-X 2
(In the formula, A is an amino acid sequence of the peptide according to any one of claims 1 to 5 which does not contain a cysteine residue, and X ₁ and X ₂ are the same or different, and any amino acid residue or any (It is an amino acid sequence)
And (2) a step of subjecting the S-cyanated peptide obtained by the step (1) to an alkali treatment. When X ₁ is an amino acid residue, the amino acid residue is an asparagine residue or a lysine residue;
The C-terminus of its amino acid sequence when X ₁ is an amino acid sequence is an asparagine residue or a lysine residue The method of claim 8. The method according to claim 8 or 9, wherein the alkali treatment in the step (2) is performed with a carbonate.

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