JP2020029433A - Peptide modified with cinnamic acid family - Google Patents

Abstract

To provide a new component in which cinnamic acid or a derivative thereof is bonded to another component, where the new component has a new additional function while retaining activity of the cinnamic acid or the derivative thereof.SOLUTION: In an oligopeptide modified with a cinnamic acid family, an oligopeptide comprising 2-10 amino acid residues is bonded to cinnamic acid or a derivative thereof represented by the general formula (1) in the figure via an amide bond or ester bond. (In the formula (1), p represents an integer from 0 to 5 being the number of substituents on the aromatic ring; and each R independently represents a hydrogen atom or a C1-3 hydrocarbon group.)SELECTED DRAWING: None

Description

  The present invention relates to a method for producing an oligopeptide modified with cinnamic acid or a derivative thereof, a polypeptide modified with cinnamic acid or a derivative thereof, and a polypeptide modified with cinnamic acid or a derivative thereof obtained by the method. .

  Cinnamic acid and its derivatives have been used widely in cosmetics and foods because they have useful effects such as antioxidant action, antibacterial action, tyrosinase activity inhibitory action (whitening action), and ultraviolet absorption action. In addition, cinnamic acid and its derivatives exhibit various pharmacological actions such as anti-proliferative action of cancer cells, ameliorating arteriosclerosis, hypoglycemic action, hypotensive action, and cerebral function improving action based on antioxidant activity. Therefore, application to medicine is also expected.

  Here, if a new material in which other useful functions are added to the physiologically active component is obtained, it can be used as a new component for cosmetics, food, and medicine. Cinnamic acid and its derivatives are also required to combine with other components to add new functions without impairing their physiological activities.

  For example, cinnamic acid and its derivatives are often insoluble in water. For example, ferulic acid, isoferulic acid, sinapinic acid, cinnamic acid and the like are hardly soluble in water, and caffeic acid is soluble in hot water but is hardly soluble in water at room temperature. Such poorly water-soluble cinnamic acids may not be able to be incorporated into cosmetics, foods and medicines in a sufficient amount. Therefore, there is a need for derivatives of cinnamic acids with improved water solubility.

  In an attempt to improve the water solubility of cinnamic acids, U.S. Pat. No. 6,059,064 teaches turning ferulic acid into an ester with glycerol, fructose, or trehalose. However, it is unknown from Patent Document 1 to what extent the water solubility is improved by converting ferulic acid into the above ester.

  Patent Document 2 discloses that, in the presence of an aqueous medium, a protein such as whey protein, soybean protein, and gelatin is added to poorly water-soluble hydroxycinnamic acid such as ferulic acid, and then heat-treated in a specific temperature range. A composition in which the solubility of hydroxycinnamic acid is improved by spray-drying or freeze-drying, or after heat-treating poorly water-soluble hydroxycinnamic acid in a specific temperature range, and then adding the protein and spray-drying or freeze-drying. Teach that you can get. However, Patent Literature 2 only teaches that ferulic acid is complexed with the protein to form a composition, and does not provide a new component containing ferulic acid. Further, the production method of Patent Document 2 is complicated because it requires a heating step and a drying step.

JP 2005-224177 A JP 2014-217369 A

The main object of the present invention is to provide a new component in which cinnamic acid or a derivative thereof is combined with another component, wherein a new function is added while maintaining the activity of the cinnamic acid or a derivative thereof. And
In particular, an object of the present invention is to provide a new component in which cinnamic acid or a derivative thereof is combined with another component, which has improved water solubility as compared with cinnamic acid or a derivative thereof.

（式（１）中、ｐは芳香環上の置換基の数であって０〜５の整数を表す。Ｒは、それぞれ独立して、水素原子、又は炭素数１〜３の炭化水素基を表す。）
で示される桂皮酸又はその誘導体（以下、「桂皮酸類」ということもある。）を、アミノ酸残基２〜１０個からなるオリゴペプチドと、アミド結合又はエステル結合を介して結合させることにより、一般式（１）で示される桂皮酸類の活性を損なうことなく、その水溶解性を向上させることができる。
(ii) (i)の桂皮酸類修飾オリゴペプチドを、それ自体重合させるか、又はアミノ酸、オリゴペプチド、及び／若しくはポリペプチドと重合させることにより、桂皮酸類の導入位置や導入量を容易に制御でき、所望の桂皮酸類修飾ポリペプチドを再現性良く製造することができる。 In order to solve the above-mentioned problems, the present inventor has repeated studies and obtained the following findings.
(i) General formula (1)

(In the formula (1), p is the number of substituents on the aromatic ring and represents an integer of 0 to 5. Each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms. Represents.)
By combining the cinnamic acid or a derivative thereof (hereinafter sometimes referred to as “cinnamic acids”) with an oligopeptide consisting of 2 to 10 amino acid residues via an amide bond or an ester bond. The water solubility of the cinnamic acids represented by the formula (1) can be improved without impairing the activity.
(ii) By introducing the cinnamic acid-modified oligopeptide of (i) itself or by polymerizing it with an amino acid, oligopeptide and / or polypeptide, the introduction position and amount of cinnamic acid can be easily controlled. And a desired cinnamic acid-modified polypeptide can be produced with good reproducibility.

（式（１）中、ｐは芳香環上の置換基の数であって０〜５の整数を表す。Ｒは、それぞれ独立して、水素原子、又は炭素数１〜３の炭化水素基を表す。）
で示される桂皮酸又はその誘導体とアミノ酸残基２〜１０個からなるオリゴペプチドとがアミド結合又はエステル結合を介して結合した桂皮酸類修飾オリゴペプチド。
〔２〕 一般式（１）で示される桂皮酸又はその誘導体が、フェルラ酸、イソフェルラ酸、カフェ酸、シナピン酸、ｐ−クマル酸、及び／又は桂皮酸である〔１〕に記載の桂皮酸類修飾オリゴペプチド。
〔３〕 オリゴペプチドが、側鎖にアミノ基、水酸基又はカルボキシル基を有するアミノ酸残基を含むものであり、一般式（１）で示される桂皮酸又はその誘導体が、オリゴペプチドの側鎖のアミノ基、水酸基、又はカルボキシル基とアミド結合又はエステル結合を形成している〔１〕又は〔２〕に記載の桂皮酸類修飾オリゴペプチド。
〔４〕 オリゴペプチドが、さらに、２０℃における水溶解度が５ｇ／１００ｍＬ以上であるアミノ酸の残基と、主鎖２級アミンを有するアミノ酸残基を含む〔３〕に記載の桂皮酸類修飾オリゴペプチド。
〔５〕 オリゴペプチドが、３アミノ酸残基ごとに１個のグリシン残基を含む〔１〕〜〔４〕の何れかに記載の桂皮酸類修飾オリゴペプチド。
〔６〕 〔１〕〜〔５〕の何れかに記載の桂皮酸類修飾オリゴペプチドを重合させるか、又は〔１〕〜〔５〕の何れかに記載の桂皮酸類修飾オリゴペプチドと、アミノ酸、オリゴペプチド、及び／又はポリペプチドとを重合させる工程を含む桂皮酸類修飾ポリペプチドの製造方法。
〔７〕 〔１〕〜〔５〕の何れかに記載の桂皮酸類修飾オリゴペプチドと、下記式（３）〜（１２）

Ｈ − （Ｘ − Ｙ − Ｇｌｙ）ｎ− ＯＨ （３）
Ｈ − （Ｇｌｙ − Ｘ − Ｙ）ｎ− ＯＨ （４）
Ｈ − （Ｙ − Ｇｌｙ − Ｘ）ｎ− ＯＨ （５）

（式（３）〜（５）中、Ｘ及びＹは、それぞれ独立して、Ｈｙｐ又はＰｒｏを表し、ｎは１〜１０の整数である。）

Ｈ − （ Ｚ^１ ） − ＯＨ （６）

（式（６）中、Ｚ^１は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｃｙｓ、Ｇｌｎ、Ｇｌｕ、Ｇｌｙ、Ｈｉｓ、Ｈｙｐ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｍｅｔ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、Ｔｙｒ、Ｖａｌ、Ａｒｇ−Ｇｌｙ−Ａｓｐ、及びε-ポリリジンからなる群より選択されるアミノ酸残基、オリゴペプチド残基、又はポリペプチド残基を表す。）

Ｈ − （ Ｐｒｏ−Ｌｙｓ−Ｇｌｙ ）ｐ− ＯＨ （７）
Ｈ − （ Ｇｌｙ−Ｐｒｏ−Ｌｙｓ ）ｐ− ＯＨ （８）
Ｈ − （ Ｌｙｓ−Ｇｌｙ−Ｐｒｏ ）ｐ− ＯＨ （９）

（式（７）〜（９）中、ｐは１〜１０の整数である。）

Ｈ − （ Ｚ^２−Ｈｙｐ−Ｇｌｙ ）ｒ− ＯＨ （１０）
Ｈ − （ Ｇｌｙ−Ｚ^２−Ｈｙｐ ）ｒ− ＯＨ （１１）
Ｈ − （ Ｈｙｐ−Ｇｌｙ−Ｚ^２ ）ｒ− ＯＨ （１２）

（式（１０）〜（１２）中、Ｚ^２は、Ａｌａ、Ａｒｇ、Ａｓｎ、Ａｓｐ、Ｃｙｓ、Ｇｌｎ、Ｇｌｕ、Ｇｌｙ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｍｅｔ、Ｐｈｅ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、Ｔｙｒ、及びＶａｌからなる群より選択されるアミノ酸残基を表し、ｒは１〜１０の整数である。）

の何れかで示されるアミノ酸、オリゴペプチド、又はポリペプチドの１種以上を重合させる工程を含む〔６〕に記載の製造方法。
〔８〕 〔１〕〜〔５〕の何れかに記載の桂皮酸類修飾オリゴペプチドの１ｍｏｌに対して、アミノ酸、オリゴペプチド、及び／又はポリペプチドの使用量が１〜１００ｍｏｌである〔６〕又は〔７〕に記載の製造方法。
〔９〕 〔６〕〜〔８〕の何れかに記載の製造方法で得られる桂皮酸類修飾ポリペプチド。 The present invention has been completed based on this finding, and provides the following cinnamic acid-modified oligopeptides, methods for producing cinnamic acid-modified polypeptides, and cinnamic acid-modified polypeptides of the following [1] to [9].
[1] The following general formula (1)

(In the formula (1), p is the number of substituents on the aromatic ring and represents an integer of 0 to 5. Each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms. Represents.)
The cinnamic acid-modified oligopeptide in which the cinnamic acid or a derivative thereof represented by (1) and an oligopeptide consisting of 2 to 10 amino acid residues are bonded via an amide bond or an ester bond.
[2] The cinnamic acids according to [1], wherein the cinnamic acid or a derivative thereof represented by the general formula (1) is ferulic acid, isoferulic acid, caffeic acid, sinapinic acid, p-coumaric acid, and / or cinnamic acid. Modified oligopeptide.
[3] The oligopeptide contains an amino acid residue having an amino group, a hydroxyl group or a carboxyl group in a side chain, and the cinnamic acid represented by the general formula (1) or a derivative thereof is substituted with an amino acid in the side chain of the oligopeptide. The cinnamic acid-modified oligopeptide according to [1] or [2], which forms an amide bond or an ester bond with a group, a hydroxyl group, or a carboxyl group.
[4] the oligopeptide modified with cinnamic acid according to [3], wherein the oligopeptide further comprises an amino acid residue having a water solubility of 5 g / 100 mL or more at 20 ° C. and an amino acid residue having a main chain secondary amine; .
[5] The cinnamic acid-modified oligopeptide according to any of [1] to [4], wherein the oligopeptide contains one glycine residue for every three amino acid residues.
[6] The cinnamic acid-modified oligopeptide of any of [1] to [5] is polymerized, or the cinnamic acid-modified oligopeptide of any of [1] to [5], and an amino acid or an oligopeptide. A method for producing a cinnamic acid-modified polypeptide, comprising a step of polymerizing a peptide and / or a polypeptide.
[7] Cinnamic acid-modified oligopeptide according to any of [1] to [5], and the following formulas (3) to (12)

H- (XY-Gly) n-OH (3)
H- (Gly-XY) n-OH (4)
H- (Y-Gly-X) n-OH (5)

(In the formulas (3) to (5), X and Y each independently represent Hyp or Pro, and n is an integer of 1 to 10.)

^{H - (Z 1) - OH} (6)

(In the formula (6), Z ¹ represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr , Val, Arg-Gly-Asp, and ε-polylysine, representing an amino acid residue, oligopeptide residue, or polypeptide residue.)

H- (Pro-Lys-Gly) p-OH (7)
H- (Gly-Pro-Lys) p-OH (8)
H- (Lys-Gly-Pro) p-OH (9)

(In the formulas (7) to (9), p is an integer of 1 to 10.)

^{H - (Z 2 -Hyp-Gly} ) r- OH (10)
^{H - (Gly-Z 2 -Hyp} ) r- OH (11)
^{H - (Hyp-Gly-Z} 2) r- OH (12)

(In the formulas (10) to (12), Z ² represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr. , And Val represent an amino acid residue, and r is an integer of 1 to 10.)

[6] The production method according to [6], comprising a step of polymerizing at least one of the amino acids, oligopeptides and polypeptides represented by any of the above.
[8] The amount of the amino acid, oligopeptide and / or polypeptide used is 1 to 100 mol per 1 mol of the cinnamic acid-modified oligopeptide according to any one of [1] to [5] [6] or The production method according to [7].
[9] A cinnamic acid-modified polypeptide obtained by the production method according to any one of [6] to [8].

  The cinnamic acid-modified oligopeptide of the present invention has new functions such as improved water solubility as compared with the cinnamic acids before binding the oligopeptide, while having the activity of the cinnamic acids represented by the general formula (1). have. When the water solubility of the cinnamic acid-modified oligopeptide of the present invention is improved, it becomes easy to be incorporated into compositions such as cosmetics, foods, and medicines. In addition, an amount in which sufficient activity can be obtained can be added to cosmetics, foods, medicines, and the like.

In addition, the use of the cinnamic acid-modified oligopeptide of the present invention facilitates the production of a cinnamic acid-bound polypeptide represented by the general formula (1).
More specifically, even if cinnamic acids are directly introduced into a polypeptide, it is difficult to bind cinnamic acids to the amino acid residue of interest, and the amount of cinnamic acids introduced into the polypeptide or the amount of the polypeptide due to cinnamic acids It is difficult to set the modification rate to a desired value.
In this regard, the cinnamic acid-modified oligopeptide of the present invention can easily bind cinnamic acids to the target amino acid residue by using a protecting group. And the rate of introduction or modification of cinnamic acids can be easily controlled. Therefore, the intended cinnamic acid-modified polypeptide can be obtained by polymerizing the cinnamic acid-modified oligopeptide. Further, the cinnamic acid-modified oligopeptide is polymerized with an amino acid, an oligopeptide and / or a polypeptide, and the polymerization ratio is adjusted to obtain a poly (cinnamic acid or a derivative thereof bound to a target amino acid residue). A peptide is obtained. That is, a desired cinnamic acid-modified polypeptide can be obtained with good reproducibility.

（式（１）中、ｐは芳香環上の置換基の数であって０〜５の整数を表す。Ｒは、それぞれ独立して、水素原子、又は炭素数１〜３の炭化水素基を表す。）
で示される桂皮酸又はその誘導体とアミノ酸残基２〜１０個からなるオリゴペプチドとがアミド結合又はエステル結合を介して結合した化合物である。 Hereinafter, the present invention will be described in detail.
(1) Cinnamic acid-modified oligopeptide The cinnamic acid-modified oligopeptide of the present invention has the following general formula (1)

(In the formula (1), p is the number of substituents on the aromatic ring and represents an integer of 0 to 5. Each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms. Represents.)
Is a compound in which cinnamic acid or a derivative thereof represented by (1) and an oligopeptide consisting of 2 to 10 amino acid residues are bonded via an amide bond or an ester bond.

In the general formula (1), cinnamic acid or a derivative thereof represented by the general formula (1), p is preferably 0 to 3, more preferably 1 to 2, and even more preferably 2.
When p is an integer of 1 to 5, especially 1 to 3, and particularly 1 to 2, one or more of R is preferably a hydrogen atom, and one is a hydrogen atom. Is more preferable.
Further, when p is an integer of 1 to 5, particularly 1 to 3, especially 1 to 2, one or more of R is preferably a hydrocarbon group, and 1 to 2 It is more preferably a hydrogen group, and even more preferably one is a hydrocarbon group.
The hydrocarbon group having 1 to 3 carbon atoms in R includes a methyl group, an ethyl group, a propyl group, or an isopropyl group, and among them, a methyl group is preferable.

で表される官能基に対してｐ位、又は１〜２個のｍ位（中でも１個のｍ位）に水酸基が存在することが好ましい。また、ｐ位、及び１〜２個のｍ位（中でも１個のｍ位）に水酸基が存在することも好ましい。
また、上記式（２）で表される官能基に対して、少なくとも１個のｍ位に炭素数１〜３のアルコキシ基、中でもメトキシ基が存在することが好ましく、１個のｍ位に炭素数１〜３のアルコキシ基、中でもメトキシ基が存在することがより好ましい。また、ｐ位に炭素数１〜３のアルコキシ基、中でもメトキシ基が存在することも好ましい。
また、上記式（２）で表される官能基に対してｐ位に水酸基が存在し、かつ１〜２個のｍ位（中でも１個のｍ位）に炭素数１〜３のアルコキシ基、中でもメトキシ基が存在することが好ましい。
また、上記式（２）で表される官能基に対して、ｐ位に炭素数１〜３のアルコキシ基、中でもメトキシ基が存在し、かつ少なくとも１〜２個のｍ位（中でも１個のｍ位）に水酸基が存在することも好ましい。 In the general formula (1), when p is 1 to 5, the following formula (2)

It is preferable that a hydroxyl group is present at the p-position or at one or two m-positions (particularly at one m-position) with respect to the functional group represented by It is also preferable that a hydroxyl group is present at the p-position and at one or two m-positions (among them, one m-position).
Further, with respect to the functional group represented by the above formula (2), at least one m-position alkoxy group having 1 to 3 carbon atoms, particularly a methoxy group, is preferably present, It is more preferable that an alkoxy group of Formulas 1 to 3, especially a methoxy group, be present. It is also preferable that an alkoxy group having 1 to 3 carbon atoms, particularly a methoxy group, be present at the p-position.
In addition, a hydroxyl group is present at the p-position with respect to the functional group represented by the above formula (2), and an alkoxy group having 1 to 3 carbon atoms is present at one to two m-positions (particularly at one m-position); Among them, a methoxy group is preferably present.
Further, with respect to the functional group represented by the above formula (2), an alkoxy group having 1 to 3 carbon atoms, especially a methoxy group, is present at the p-position, and at least 1 to 2 m-positions (among which, It is also preferred that a hydroxyl group is present at the (m-position).

  Cinnamic acids represented by the general formula (1) (hereinafter, also referred to as “compounds represented by the general formula (1)”) include a trans-form and a cis-form. The trans form is preferred.

  Preferred specific examples of the cinnamic acids represented by the general formula (1) include ferulic acid, isoferulic acid, caffeic acid, sinapinic acid, p-coumaric acid, and cinnamic acid.

  Ferulic acid (3-methoxy-4-hydroxycinnamic acid) is a component abundantly contained in cell walls of grasses. Many physiological activities of ferulic acid have been reported, for example, antioxidant action by eliminating free radicals and active oxygen (Milchwissenschaf 34, 205 (1979), Plant. Med. 57, 8 (1991)), fading of food or Discoloration prevention effect (Monthly Food Chemical 8, 76-79 (1999), J. Oleo Sci. 57, 133-137 (2008)), whitening effect by tyrosinase inhibition (Fragrance Journal No. 45, 92 (1980), Fragrance) Journal No. 9, 19 (1997)), UV absorption (fragrance journal No. 129, 41 (1991), J. Am. Acad. Dermatol. 59 418-425 (2008)), Synthesis of somatotropin, a growth hormone Growth-promoting action by promotion (J. Dairy Sci. 66, 624-629 (1983)), action to suppress cancer cell proliferation (Anticancer Res. 19, 3775-3778 (1999), Cancer Epidemiol Biomarkers Prev. 9, 1163-1170 (2000)), Improvement of diabetes by promoting insulin secretion (J. Agric. Food Chem. 55, 9800-9804 (2007), 2001) Wakayama Prefectural Institute of Technology, Research Report, pp. 17-19 (2001)), hypotensive effect based on decreased angiotensin converting enzyme activity (J. Agric. Food Chem. 56, 2825-2830 (2008)), blood total cholesterol For improving lipid abnormalities by lowering serum and triglyceride concentrations (J. Agric. Food Chem. 56, 2825-2830 (2008)), and for protecting cranial nerves from oxidative stress-related apoptosis (Brain Res. 13, 136-150 ( 2008)), an effect of improving learning and memory ability by protecting the brain from β-amyloid peptide (Biochem. Biophys. Acta. 1741, 140-148 (2005), Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 901-907) (2005), Acta. Pharmacol. Sin. 26, 943-951 (2005), J. Neurosci. Res. 84, 418-426 (2006), Br. J. Pharmacol. 133, 89-96 (2001)), Anti-inflammatory and analgesic effects (Pharmaceutical Magazine 97, 911 (1977)) are known.

  Isoferulic acid (3-hydroxy-4-methoxycinnamic acid) is a component widely contained in plants. Numerous physiological activities of isoferulic acid have been reported, for example, anti-inflammatory and analgesic effects (Pharmaceutical Journal 97, 911 (1977)), whitening effect by inhibiting tyrosinase (“Synthesis and Application of Functional Glycosides”, Volume 2, Chapter 4, CMC Publishing, June 28, 2013), antioxidant action by scavenging active oxygen ("Synthesis and Application of Functional Glycosides", Volume 2, Chapter 4, CMC Publishing, June 2013) 28) is known.

  Caffeic acid (3,4-dihydroxycinnamic acid) is contained in coffee and is the source of coffee aroma and color. It is also found in herbs and fruit peels. Numerous physiological activities of caffeic acid have been reported, for example, antibacterial activity (Journal of the Research Institute for Biological Function 6: 1-19 (2006)), antioxidant activity such as suppression of glutathione depletion and suppression of lipid peroxidation (Japanese foods) Journal of the Japan Society for Science and Technology 58 (1), 16-20), suppresses the growth and invasion of cancer cells (Cancer Lett. 2011 Sep 1, 308 (1), 43-53), inhibits LDL oxidation, The effect of suppressing hardening (Free Radic. Med. 1998 Dec, 25 (9), 1098-1105), the anti-inflammatory effect (Pharmaceutical Journal 97, 911 (1977)) and the like are known.

  Sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) is widely present in plants as a precursor of lignin and lignan, which are tree components. Many physiological activities of sinapic acid have been reported, for example, antibacterial activity (Japanese Patent Application Laid-Open No. Hei 7-194356), cerebral protection effect and cognitive improvement effect of rodents (Kanazawa University Juzen Medical Association Journal 117 (1), 2 -9 (2008)), brain function improving activity (JP-A-7-179338), anti-radical activity, anti-mutagenic activity ("Science of Food Functionality", page 343, Scientific Editing Committee of Food Functionality), etc. Are known.

  p-Coumaric acid (4-hydroxycinnamic acid), which is contained in various edible plants, is known for a large number of physiological activities of p-coumaric acid, and includes, for example, an osteoporosis ameliorating action, a diabetes ameliorating action, and a fat. It is known to have an inhibitory effect on cell formation and an inhibitory effect on cancer cell proliferation (Food Materials Research Society, 1959, 4 (1)).

  Cinnamic acid is a component widely existing in plants and is used as a scented oil of bark scent. Also, cinnamic acid is known to have antibacterial activity (Journal of the Research Institute for Biological Functions 6: 1-19 (2006)).

  The cinnamic acid represented by the general formula (1) contained in the cinnamic acid-modified oligopeptide is usually one type, but may be two or more types.

Oligopeptide In the present invention, the number of amino acid residues constituting the oligopeptide is 2 to 10. Within this range, a cinnamic acid-modified oligopeptide having high water solubility can be obtained. The number of amino acid residues constituting the oligopeptide may be 2, 3, 4, 5, 6, 7, 8, 9, or 10. Among them, 2 to 5 are preferred, 2 to 4 are more preferred, and 3 are even more preferred.

  The compound represented by the general formula (1) and the oligopeptide are bound by an amide bond or an ester bond. Examples of the amide bond or ester bond include an amide bond between a carboxyl group of the compound represented by the general formula (1) and a side chain amino group or an N-terminal amino group of the oligopeptide, and a carboxyl group of the compound represented by the general formula (1). Bond between the hydroxyl group and the side chain hydroxyl group of the oligopeptide, or when the compound represented by the general formula (1) has a hydroxyl group on the aromatic ring, the ester bond between the hydroxyl group and the side chain carboxyl group or the C-terminal carboxyl group of the oligopeptide Is mentioned.

The oligopeptide preferably contains amino acid residues having high water solubility. Examples of amino acids having high water solubility include Arg, Ser, Gly, Ala, Val, Met, and Hyp having a water solubility of 5 g / 100 mL or more at 20 ° C. Among them, an amino acid having a water solubility of 10 g / 100 mL or more at 20 ° C. Certain Arg, Ser, Gly, Ala, Hyp are preferable, Arg, Ser, Gly, Hyp having a water solubility at 20 ° C. of 20 g / 100 mL or more are more preferable, and Gly is even more preferable.
In addition, when the oligopeptide has a Gly residue, a collagen-like polypeptide can be easily obtained by producing the cinnamic acid-modified polypeptide, so that one Gly residue exists for every three amino acid residues. Is preferred. Oligopeptides consisting of a total of 3, 6, or 9 amino acid residues in which one Gly residue is present for every three amino acid residues can more efficiently produce a cinnamic acid-modified collagen-like polypeptide.

  The compound represented by the general formula (1) may or may not be bound to such a highly water-soluble amino acid residue, but the cinnamic acid-modified oligopeptide has a high water solubility. In this respect, it is preferred that they are not bonded.

The position of such a highly water-soluble amino acid residue in the oligopeptide is not limited, but is preferably present at the N-terminus or C-terminus of the oligopeptide in that the synthesis of the polypeptide by polycondensation is facilitated, More preferably, it is at the C-terminus.
When such a highly water-soluble amino acid residue (especially, glycine residue) is contained, the number thereof is one for an oligopeptide consisting of two amino acid residues, and 1 to one for an oligopeptide consisting of three amino acid residues. Oligopeptides consisting of two, especially one, four amino acid residues, one to two, especially ones for oligopeptides consisting of one, five amino acid residues, one to two, and especially oligos consisting of one, six amino acid residues 1 to 2 peptides, especially 1 to 2 oligopeptides consisting of 7 amino acid residues; 1 to 3 oligopeptides consisting of 2 and 8 amino acid residues; For oligopeptides consisting of groups, 1-3 are preferred, especially for 3, and for oligopeptides consisting of 10 amino acid residues, 1-4 are preferred, and especially 3 are preferred.

The oligopeptide preferably has an amino acid residue having a main chain secondary amine such as Pro or Hyp. In the present invention, the main chain secondary amine of an amino acid refers to a secondary amine that forms a main chain amide bond upon polymerization of an amino acid. Since Pro and Hyp are common in that they are abundant in collagen, by having Pro and / or Hyp residues, a material containing cinnamic acid-modified oligopeptide is polymerized to produce cinnamic acid-modified collagen-like. Polypeptides are easier to produce.
When the oligopeptide has one Gly residue for every three amino acid residues, the oligopeptide further has a Pro residue and / or a Hyp residue to produce a cinnamic acid-modified collagen-like peptide more efficiently. be able to.

  The compound represented by the general formula (1) may or may not be bound to the amino acid residue having a main chain secondary amine, but it is possible to synthesize a polypeptide by condensation polymerization. It is preferred that they are not bound. Secondary amines are less reactive than amino groups and are less likely to form an amide bond with the carboxyl group of the compound represented by the general formula (1). Therefore, the main chain secondary amine is a compound represented by the general formula (1). It is preferably not used for bonding with

The position of the amino acid residue having the main chain secondary amine in the oligopeptide is not limited, but is present at the N-terminus or C-terminus of the oligopeptide in that the reactivity when synthesizing the polypeptide by polycondensation increases. Preferably, it is more preferably present at the N-terminus.
In the case of including an amino acid residue having a main chain secondary amine, the number thereof is one for an oligopeptide consisting of two amino acid residues, and one or two, especially one for an oligopeptide consisting of three amino acid residues, 1 to 2 for oligopeptides consisting of 4 amino acid residues, 1 to 2 for oligopeptides consisting of 2 and 5 amino acid residues, and 2 to 2 for oligopeptides consisting of 6 amino acid residues; Among them, an oligopeptide consisting of 2 to 3 and 7 amino acid residues has 1 to 3, especially an oligopeptide consisting of 2 to 3 and 8 amino acid residues has 1 to 3, especially 2 to 3 and 9 amino acid residues. Of oligopeptides consisting of 1 to 4, especially 2 to 3, and 10 to 10 amino acid residues, and more preferably 3 to 4 oligopeptides.

The oligopeptide preferably contains an amino acid residue having an amino group, a hydroxyl group, or a carboxyl group in a side chain. Thereby, the compound represented by the general formula (1) can form a bond using these functional groups. When the compound represented by the general formula (1) forms a bond using the side chain functional group of the oligopeptide, the cinnamic acid-modified oligopeptide has a carboxyl group or an amino group of the main chain, and thus is modified with cinnamic acid. It can be used for the production of polypeptide.
Examples of the amino acid having an amino group in the side chain include Gln, Lys, Arg, Asn, Gln, ornithine, citrulline, hydroxylysine and the like. Examples of the amino acid having a hydroxyl group in the side chain include Ser, Thr, Tyr, Hyp, hydroxylysine, O-phosphoserine and the like. Examples of amino acids having a carboxyl group in the side chain include Asp and Glu.
Among them, an amino acid residue having an amino group or a carboxyl group in a side chain is preferable, and an amino acid residue having an amino group in a side chain is more preferable in terms of good reactivity with the compound represented by the general formula (1). Specifically, Arg, Lys, and ornithine are preferable, and Lys is particularly preferable.

The position of the amino acid residue having an amino group, a hydroxyl group, or a carboxyl group in the side chain in the oligopeptide is not limited, but the oligopeptide other than the N-terminal or C-terminal of the oligopeptide is easy to synthesize a cinnamic acid-modified polypeptide. It is preferably present inside the peptide.
When an amino acid residue having an amino group, a hydroxyl group, or a carboxyl group is contained in a side chain, the number of the amino acid residue is one for an oligopeptide consisting of two amino acid residues, and is one to two for an oligopeptide consisting of three amino acid residues. Oligopeptides consisting of 1, 2, or 1 or 4 amino acid residues, and especially 1-2 oligopeptides consisting of 1, 5 or 5 amino acid residues, and especially oligopeptides consisting of 1 or 6 amino acid residues In an oligopeptide consisting of 1 to 2, especially 2, 7 amino acid residues, 1 to 3, especially an oligopeptide consisting of 2, 8 amino acid residues, 1 to 3, especially 2, 9 amino acid residues Of oligopeptides consisting of 1 to 4, especially 3 and 10 amino acid residues, preferably 1 to 4 and especially 3 oligopeptides.

  The compound represented by the general formula (1) may be bound to any amino acid residue including the C-terminal or N-terminal amino acid residue of the oligopeptide. From the viewpoint that steric hindrance at the time of polymerization can be alleviated, it is preferable to bond to amino acid residues inside the oligopeptide other than the C-terminal amino acid residue and the N-terminal amino acid residue of the oligopeptide. In this case, the compound represented by the general formula (1) forms an amide bond or an ester bond with the amino group, hydroxyl group, or carboxyl group of the side chain of the oligopeptide.

  When the cinnamic acid-modified oligopeptide itself is blended in cosmetics, food, medicine, etc., the cinnamic acid-modified oligopeptide may or may not have a main chain amino group and a main chain carboxyl group. . Therefore, the compound represented by the general formula (1) may form a bond with an amino group, a hydroxyl group, or a carboxyl group on the side chain of any amino acid residue, or may form a bond with the main chain carboxyl group of the oligopeptide. Alternatively, the bond may be formed with a main chain amino group. However, when a cinnamic acid-modified oligopeptide is used for producing a cinnamic acid-modified polypeptide, the cinnamic acid-modified oligopeptide must have a main chain amino group and a main chain carboxyl group. The compound represented by (1) may be bonded to the amino group, hydroxyl group, or carboxyl group of the side chain of the oligopeptide.

The number of compounds represented by the general formula (1) bonded to one oligopeptide is not particularly limited. The smaller the number of the compounds of the general formula (1) relative to the number of amino acid residues, the higher the water-solubility of the cinnamic acid-modified oligopeptide, but if too small, the compounds of the general formula (1) of the cinnamic acid-modified oligopeptide May be insufficient in physiological activity. On the other hand, the larger the number of the compounds of the general formula (1) relative to the number of amino acid residues, the higher the physiological activity of the cinnamic acid-modified oligopeptide derived from the compound of the general formula (1). It is difficult to sufficiently improve the water solubility of the acid-modified oligopeptide, and it is difficult to polymerize the cinnamic acid-modified polypeptide using the cinnamic acid-modified oligopeptide.
Therefore, the number of compounds represented by the general formula (1) bonded to an oligopeptide consisting of two amino acid residues is preferably one. The number of compounds represented by the general formula (1) bonded to an oligopeptide consisting of three amino acid residues is preferably one. The number of compounds represented by the general formula (1) bonded to the oligopeptide consisting of four amino acid residues is preferably 1 to 2, and more preferably 1. The number of compounds represented by the general formula (1) bonded to an oligopeptide consisting of 5 amino acid residues is preferably 1 to 2, and more preferably 1. The number of the compounds represented by the general formula (1) bonded to the oligopeptide consisting of 6 amino acid residues is preferably 1 to 3, and more preferably 1 to 2. The number of the compounds represented by the general formula (1) bonded to the oligopeptide consisting of 7 amino acid residues is preferably 1 to 3, and more preferably 1 to 2. The number of compounds represented by the general formula (1) bonded to the oligopeptide consisting of 8 amino acid residues is preferably 1 to 3, more preferably 1 or 2. The number of compounds represented by the general formula (1) bonded to an oligopeptide consisting of 9 amino acid residues is preferably 1 to 3, and more preferably 1 to 2. The number of compounds represented by the general formula (1) bonded to an oligopeptide consisting of 10 amino acid residues is preferably 1 to 3, and more preferably 1 to 2.

Preferred examples of the oligopeptide include a dipeptide comprising an amino acid having an amino group in a side chain such as Lys-Gly, Arg-Gly, ornithine-Gly, Gly-Lys, Gly-Arg, and Gly-ornithine; and glycine; Pro-Lys- Gly, Pro-Gly-Lys, Lys-Pro-Gly, Lys-Gly-Pro, Gly-Pro-Lys, Gly-Lys-Pro, Hyp-Lys-Gly, Hyp-Gly-Lys, Lys-Hyp-Gly, Lys-Gly-Hyp, Gly-Hyp-Lys, Gly-Lys-Hyp, Pro-Arg-Gly, Pro-Gly-Arg, Arg-Pro-Gly, Arg-Gly-Pro, Gly-Pro-Arg, Gly- Arg-Pro, Hyp-Arg-Gly, Hyp-G y-Arg, Arg-Hyp-Gly, Arg-Gly-Hyp, Gly-Hyp-Arg, Gly-Arg-Hyp, Pro-ornithine-Gly, Pro-Gly-ornithine, Ornithine-Pro-Gly, Ornithine-Gly- Pro, Gly-Pro-ornithine, Gly-ornithine-Pro, Hyp-ornithine-Gly, Hyp-Gly-ornithine, ornithine-Gly-Hyp, ornithine-Hyp-Gly, Gly-Hyp-ornithine, Gly-ornithine-Hyp, etc. A tripeptide comprising an amino acid having a main chain secondary amine, an amino acid having an amino group in the side chain, and glycine (among which, in order from the N-terminus, an amino acid having a main chain secondary amine, an amino acid having an amino group in a side chain, And a glycine tripeptide) It is below.
The compound represented by the general formula (1) may be bound to any of the above-described dipeptide or tripeptide amino acid residues. Among them, the compound represented by the general formula (1) can be bonded to an amino acid having an amino group in a side chain of the dipeptide or tripeptide exemplified above.

Method for Producing Cinnamic Acid-Modified Oligopeptide Oligopeptide can be synthesized by a peptide synthesis method by a general dehydration condensation reaction.
In binding the compound represented by the general formula (1) to the oligopeptide, a functional group (particularly, a main chain functional group) in the oligopeptide capable of forming the same kind of bond is protected, and then the general peptide is protected. After the compound represented by the general formula (1) is bonded to the target functional group of the oligopeptide by an amide bond or an ester bond by a dehydration condensation reaction, the compound may be deprotected.

  When the amino group of the oligopeptide is used for bonding to the compound represented by the general formula (1), another amino group or a secondary amine (particularly, an amino group or a secondary amine in the main chain) is replaced with a tert-butoxy group. (-Boc), deprotection with trifluoroacetic acid or hydrochloric acid-ethyl acetate solution, protection with a benzyloxycarbonyl group (-Cbz), and deprotection by a hydrogenation reaction using a palladium catalyst. No. When the hydroxyl group of the oligopeptide is used for bonding to the compound represented by the general formula (1), the other hydroxyl group is protected with a methyl group and deprotected with boron tribromide, protected with a benzyl group, Examples include a method of deprotecting by a hydrogenation reaction using a catalyst. When the carboxyl group of the oligopeptide is used for bonding to the compound represented by the general formula (1), other carboxyl groups (particularly, carboxyl groups of the main chain) are set as methyl esters or ethyl esters, and sodium hydroxide is used. And the like.

  The amount of the compound represented by the general formula (1) is preferably 1 mol or more, more preferably 2 mol or more, even more preferably 3 mol or more, based on 1 mol of the total amount of the oligopeptide. Within this range, the compound represented by the general formula (1) can be sufficiently bonded to the target functional group in the oligopeptide. The amount of the compound represented by the general formula (1) is preferably 20 mol or less, more preferably 15 mol or less, still more preferably 10 mol or less, based on 1 mol of the total amount of the oligopeptide. In this range, the compound represented by the general formula (1) is not wasted.

The dehydration condensation reaction may be performed in a solvent. The solvent can be appropriately selected depending on the solubility of the compound represented by the general formula (1). For example, if the compound is hardly soluble in water such as ferulic acid, it may be performed in an organic solvent or a mixture of an organic solvent and water, and if the compound is easily soluble in water such as p-coumaric acid, water or It may be performed in a mixture of water and an organic solvent. When a mixed solution of water and an organic solvent is used, the mixing ratio can be appropriately selected depending on the solubility of the compound represented by the general formula (1).
Examples of the organic solvent include amide solvents such as dimethylformamide, diethylformamide, dimethylacetamide, tetramethylurea, and hexamethylphosphoramide; polyvalent solvents such as ethylene glycol, propylene glycol, diethylene glycol, dipropylene glycol, and glycerin. Alcohol-based solvents; sulfoxide-based solvents such as dimethylsulfoxide; nitrogen-containing cyclic compound-based solvents such as N-methylpyrrolidone and pyridine; nitrile-based solvents such as acetonitrile and propionitrile; ether-based solvents such as dioxane and tetrahydrofuran Alcohol-based solvents such as methanol, ethanol, propyl alcohol and isopropyl alcohol; carboxylic acid-based solvents such as formic acid and acetic acid.

The dehydration condensation reaction may be usually performed in the presence of a condensing agent or in the presence of a condensing agent and a condensation auxiliary.
Examples of the condensing agent include 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (WSCD (EDC)) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSCD.HCl). , N, N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC), N-cyclohexyl-N '-(2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate (CME-carbodiimide) A carbodiimide-based condensing agent such as N; N'-carbonyldiimidazole; an imidazole-based condensing agent such as 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmol Honium chloride n-hydrate (DMT-MM), trifluoromethanesulfonic acid (4,6- Triazine-based condensing agent such as methoxy-1,3,5-triazin-2-yl)-(2-octoxy-2-oxoethyl) dimethylammonium (DMT-MM); {{[(1-cyano-2-ethoxy) -2-oxoethylidene) amino] oxy} -4-morpholinomethylene} dimethylammonium hexafluorophosphate (COMU), O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetra Methyluronium hexafluorophosphate (HBTU), O- (7-azabenzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU), 1H-benzotriazole-1 -Yl-oxytripyrrolidinophosphonium hexafluorophosphate, 1H-benzotriazole-1 A fluorophosphate-based condensing agent such as yl-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP); O- (7-succinimidyl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TSTU), O- (3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl) -N, N, N ', Fluoroborate-based condensing agents such as N′-tetramethyluronium tetrafluoroborate (TDBTU); diphenylphosphoryl azide (DPPA);
Among them, carbodiimide-based condensing agents are preferable, and WSCD, WSCD.HCl, DCC, and DIC are more preferable.
As the condensing agent, one type can be used alone, or two or more types can be used in combination.
The amount of the condensing agent used can be 1 to 5 mol per 1 mol of the oligopeptide. Within this range, the reaction proceeds efficiently.

The condensation aid may be any as long as it promotes the condensation reaction. For example, N-hydroxysuccinimide (HONSu), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) such as NON -Hydroxydicarboxylic imide (N-hydroxy polycarboxylic imides) -based condensation aid; N-hydroxybenzotriazole-based condensation aid such as 1-hydroxybenzotriazole (HOBt) and 1-hydroxyazabenzotriazole (HOAt) Triazine-based condensation aids such as 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt); 2-hydroxyimino-2-cyanoacetic acid ethyl ester; Can be
Among them, N-hydroxybenzotriazole-based condensation aids are preferred, and HOBt is more preferred.
As the condensation aid, one kind can be used alone, or two or more kinds can be used in combination.
The amount of the condensation aid used can be 1 to 5 mol per 1 mol of the oligopeptide. Within this range, it can be uniformly dissolved in a solvent to function as an auxiliary.

The pH of the solvent for the condensation reaction (the reaction solution before the addition of the condensing agent or the condensing aid to the raw materials) is determined by using, for example, an organic amine (preferably a tertiary amine such as triethylamine or N-methylmorpholine). , About 7-9.
The reaction temperature can be 4 to 50C. Within this range, the condensation reaction proceeds efficiently. The reaction time can be, for example, 8 to 24 hours.

(2) Cinnamic acid-modified polypeptide The above-described cinnamic acid-modified oligopeptide of the present invention is polymerized, and the cinnamic acid-modified oligopeptide of the present invention is combined with an amino acid, an oligopeptide (which may be a peptide oligomer), and / or Alternatively, a cinnamic acid-modified polypeptide can be obtained by polymerizing the polypeptide with a polypeptide.
One or more cinnamic acid-modified oligopeptides may be used, and one or more amino acids, oligopeptides and polypeptides may be used. Amino acids, oligopeptides, and polypeptides are not particularly limited, and those depending on the purpose can be used without limitation.

When polymerizing a cinnamic acid-modified oligopeptide and an oligopeptide, the following formulas (3) to (5) are used as the oligopeptide.

H- (XY-Gly) n-OH (3)
H- (Gly-XY) n-OH (4)
H- (Y-Gly-X) n-OH (5)

(In the formulas (3) to (5), X and Y each independently represent Hyp or Pro, and n is an integer of 1 to 10.)
It is preferable to use one or more oligopeptides represented by any of the above.

  Among them, cinnamic acid-modified oligopeptides having an oligopeptide consisting of 3, 6, or 9 amino acid residues having one Gly residue for every three amino acid residues, and the above-mentioned formulas (3) to (5) By polymerizing the oligopeptide represented by any of them, a collagen-like polypeptide modified with cinnamic acids can be obtained.

  Examples of the combination of X and Y in the formulas (3) to (5) include a combination in which one is Hyp and the other is Pro, a combination in which both are Hyp, and a combination in which both are Pro. When at least one is Hyp, the resulting cinnamic acid-modified polypeptide has improved hydrophilicity and can be suitably applied to, for example, biomaterials that require water retention, such as cosmetic raw materials. Both may be Hyp.

In the formulas (3) to (5), n is preferably 1 to 5, and more preferably 1 to 3. Further, n may be 1 or 2.
The oligopeptides represented by the formulas (3) to (5) can be used alone or in combination of two or more.

In addition, it is also possible to polymerize at least one of the cinnamic acid-modified oligopeptide and an amino acid, oligopeptide or polypeptide represented by the following formulas (6) to (12).

^{H - (Z 1) - OH} (6)

(In the formula (6), Z ¹ represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr , Val, Arg-Gly-Asp, and ε-polylysine, representing an amino acid residue, oligopeptide residue, or polypeptide residue.)

H- (Pro-Lys-Gly) p-OH (7)
H- (Gly-Pro-Lys) p-OH (8)
H- (Lys-Gly-Pro) p-OH (9)

(In the formulas (7) to (9), p is an integer of 1 to 10.)

^{H - (Z 2 -Hyp-Gly} ) r- OH (10)
^{H - (Gly-Z 2 -Hyp} ) r- OH (11)
^{H - (Hyp-Gly-Z} 2) r- OH (12)

(In the formulas (10) to (12), Z ² represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr. , And Val, and r is an integer of 1 to 10.)

Among the amino acids, oligopeptides or polypeptides represented by formulas (6) to (12), acidic amino acids, bases and the like can be used to adjust the polarity and the balance between hydrophilicity and hydrophobicity of the resulting cinnamic acid-modified polypeptide. Acidic amino acids and hydrophobic amino acids are preferred, acidic polar amino acids or basic polar amino acids are more preferred, and Asp, Glu and Lys are even more preferred.
Also, in that it can adjust the hydrophobicity of the polypeptide, ^{Z 2} in Formula (10) to (12) is Ala, Val, Leu, or oligopeptide is Ile is preferred.
In addition, since Arg-Gly-Asp is an RGD motif which is a cell adhesion activity sequence, it is expected that the use of Arg-Gly-Asp can impart an adhesion promoting activity of cells to the obtained polypeptide.
The ε-polylysine in the formula (6) is not particularly limited. For example, ε-polylysine consisting of about 25 to 30 L-Lys and commercially available as a food preservative or the like can be used.

The cinnamic acid-modified oligopeptide and the oligopeptides represented by the formulas (3) to (5) and (7) to (12) are those having the same Gly position relative to the total amount thereof. It is preferable to use 35 to 100 mol%, especially 50 to 100 mol%, especially 70 to 100 mol%, especially 80 to 100 mol%, especially 90 to 100 mol%, particularly 100 mol%. When this ratio is 100 mol%, the polypeptide obtained by polymerization has one glycine for every three amino acid residues. For example, a cinnamic acid-modified oligopeptide consisting of three amino acid residues having Gly at the C-terminus and an oligopeptide represented by the formula (3) are oligopeptides having the same Gly position.
When the use ratio of the oligopeptide having the same Gly position is within the above range, the obtained polypeptide easily adopts a triple helix structure, or the triple helix portion in the obtained polypeptide becomes long. .

  The total amount of amino acids, oligopeptides and polypeptides (among others, amino acids, oligopeptides and polypeptides represented by formulas (3) to (12)) is 1 mol per mol of cinnamic acid-modified oligopeptide. It is preferably at least 3 mol, more preferably at least 3 mol, even more preferably at least 5 mol. Within this range, the polymerization reaction can easily proceed. Moreover, 100 mol or less is preferable, 50 mol or less is more preferable, and 20 mol or less is still more preferable. Within this range, the cinnamic acid-modified polypeptide will exhibit sufficient cinnamic acid activity.

When one or more of the amino acids, oligopeptides or polypeptides represented by the formulas (6) to (12) are used, one or more of the oligopeptides represented by the formulas (3) to (5) are further used. Is preferred. In this case, the total amount of the amino acids, oligopeptides or polypeptides represented by the formulas (6) to (12) is based on 1 mol of the total amount of the oligopeptides represented by the formulas (3) to (5). Thus, it can be 0.001 mol or more, 0.002 mol or more, 0.01 mol or more, 0.02 mol or more, or 0.05 mol or more. Within this range, molecular design such as adjusting the reactivity of the cinnamic acid-modified polypeptide obtained according to the application can be performed.
Further, it can be 5 mol or less, 3 mol or less, 2 mol or less, or 1 mol or less. Within this range, the resulting cinnamic acid-modified polypeptide will easily form a triple helix.

  The cinnamic acid-modified oligopeptides, amino acids, oligopeptides or polypeptides represented by formulas (3) to (12) can be subjected to a condensation reaction in the form of a salt, whereby the reaction proceeds more smoothly. . The salt is preferably an organic acid salt, more preferably a hydrochloride or a trifluoroacetate, and even more preferably a trifluoroacetate.

  The polymerization can be carried out by a general method in which a raw material compound is subjected to a dehydration condensation reaction in an aqueous solvent.

  As the aqueous solvent, water or a mixture of water containing 50% by volume or more of water and an organic solvent can be used. As the organic solvent, for example, the water-miscible organic solvent exemplified for the synthesis of the cinnamic acid-modified oligopeptide can be used.

The dehydration condensation reaction may be usually performed in the presence of a condensing agent or in the presence of a condensing agent and a condensation auxiliary. By using a condensation aid, a condensation reaction is promoted and racemization is suppressed.
The condensing agent may be any one capable of performing dehydration condensation in an aqueous solvent, and for example, those exemplified for the synthesis of cinnamic acid-modified oligopeptides can be used. Among them, carbodiimide-based condensing agents are preferable, and WSCD, WSCD.HCl, DCC, and DIC are more preferable.
As the condensing agent, one type can be used alone, or two or more types can be used in combination.
The amount of the condensing agent used is 0.1 to 1 mol of the total of the raw material compounds (the total of the cinnamic acid-modified oligopeptides or the total of the cinnamic acid-modified oligopeptides and the amino acids, oligopeptides and / or polypeptides). ２０20 mol.

As the condensation aid, for example, those exemplified for the synthesis of cinnamic acid-modified oligopeptides can be used. Among them, N-hydroxybenzotriazole-based condensation aids are preferred, and HOBt is more preferred.
As the condensation aid, one kind can be used alone, or two or more kinds can be used in combination.
The amount of the condensing aid used is 0.1 to 1 mol of the total of the raw material compounds (total of the cinnamic acid-modified oligopeptide or the total of the cinnamic acid-modified oligopeptide and the amino acid, oligopeptide and / or polypeptide). 001 to 10 mol. Within this range, it can be uniformly dissolved in an aqueous solvent to function as an auxiliary.

  The pH of the aqueous solvent for the condensation reaction (the reaction solution before adding the condensing agent or the condensing aid to the raw material) may be adjusted to about 3 to 10. The reaction temperature can be -10 to 80C. Within this range, the condensation reaction proceeds efficiently. The reaction time can be, for example, 1 to 100 hours.

（式（１）中、ｐは芳香環上の置換基の数であって０〜５の整数を表す。Ｒは、それぞれ独立して、水素原子、又は炭素数１〜３の炭化水素基を表す。）
で示される桂皮酸類とポリペプチドとがアミド結合又はエステル結合を介して結合した化合物である。
本発明の桂皮酸類修飾ポリペプチドは、一般式（１）で示される桂皮酸類が結合しているため、桂皮酸類に由来する種々の活性が付与されたものとなる。 The cinnamic acid-modified polypeptide thus obtained is represented by the following general formula (1)

(In the formula (1), p is the number of substituents on the aromatic ring and represents an integer of 0 to 5. Each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms. Represents.)
Is a compound in which the cinnamic acids represented by are bonded to a polypeptide via an amide bond or an ester bond.
The cinnamic acid-modified polypeptide of the present invention has various activities derived from the cinnamic acids, since the cinnamic acids represented by the general formula (1) are bound thereto.

The weight-average molecular weight of the cinnamic acid-modified polypeptide is, for example, 3,000 or more, 5,000 or more, or 10,000 or more, and 1.5 million or less, 1.3 million or less, 1.2 million or less, 1.1 million or less, 1 million or less, 80 million or less. It can be less than 10,000, less than 600,000, less than 300,000, less than 200,000, or less than 100,000.
In the present invention, the weight average molecular weight is a value measured by gel permeation chromatography (GPC), and specifically, a value measured by the method described in Examples. The weight average molecular weight measured by GPC is a value that hardly changes depending on the measuring device and measuring conditions.
The length or weight-average molecular weight of the cinnamic acid-modified polypeptide can be controlled by adjusting the types and amounts of the raw materials, the reaction conditions, and the like, in particular, the amounts of the condensing agent and the condensing aid.

In the present specification, amino acids or amino acid residues are represented by the following abbreviations.
Ala: L-alanine (residue)
Arg: L-arginine (residue)
Asn: L-asparagine (residue)
Asp: L-aspartic acid (residue)
Cys: L-cysteine (residue)
Gln: L-glutamine (residue)
Glu: L-glutamic acid (residue)
Gly: glycine (residue)
His: L-histidine (residue)
Hyp: L-hydroxyproline (residue)
Ile: L-isoleucine (residue)
Leu: L-leucine (residue)
Lys: L-lysine (residue)
Met: L-methionine (residue)
Phe: L-phenylalanine (residue)
Pro: L-proline (residue)
Ser: L-serine (residue)
Thr: L-threonine (residue)
Trp: L-tryptophan (residue)
Tyr: L-tyrosine (residue)
Val: L-valine (residue)

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(1) Synthesis of Feruloyl Group-Containing Oligopeptide According to the following scheme, a tripeptide having a feruloyl group was synthesized.

Synthesis of Compound (a) Boc-Pro-Lys-Gly-OH (compound (a)) was obtained by peptide synthesis by a general dehydration condensation reaction.

Synthesis of Compound (b) Compound (a) (20 g, 50 mmol), trans-ferulic acid (64 g, 330 mmol), triethylamine (8 ml, 55 mmol), N, N'-dicyclohexylcarbodiimide were added to a four-necked eggplant flask (11 g). , 55 mmol) in tetrahydroxyfuran (200 ml), stirred and dissolved, and reacted at room temperature for 16 hours. After completion of the reaction, the mixture was filtered, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in tetrahydroxyfuran and purified by silica gel column chromatography to obtain 20 g of compound (b) as a white solid (yield 69%).
The structure of this compound was confirmed from the following LC-MS spectrum.
LC-MS (ES): Calcd for C28H41N4O9 577.7 [M + H] +, Found.:577.8

Compound (b) (12 g, 21 mmol) and trifluoroacetic acid (13 ml, 168 mmol) were stirred and dissolved in chloroform (60 ml) in an eggplant flask and reacted at room temperature for 16 hours. After completion of the reaction, the solvent was distilled off under reduced pressure. Heptane (60 ml) was added to the obtained residue and stirred, the solvent was distilled off under reduced pressure, diethyl ether (180 ml) was added to the obtained residue, and the precipitated white solid was filtered by suction. The solid was vacuum-dried at room temperature to obtain compound (c) (yield).
The structure of this compound was confirmed from the following LC-MS spectrum.
LC-MS (ES): Calcd for C23H34N4O7 477.5 [M + H] +, Found .: 477.7

(2) As raw materials for synthesizing a feruloyl group-containing polypeptide , L- (4-hydroxyprolyl) -L- (4-hydroxyprolyl) -glycine trifluoroacetate (5 g, 13 mmol) and compound (c) (0.6 g, 1 mmol) was dissolved in 22 ml of distilled water, the pH was adjusted to 4 with triethylamine, and the mixture was cooled to 4 ° C. Thereafter, 1-hydroxybenzotriazole (0.4 g, 3 mmol) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (5 g, 28 mmol) were added, and the mixture was stirred for 1 hour, and then heated to 20 ° C. Warmed and stirred overnight. The obtained reaction solution was ultrafiltered and freeze-dried to obtain 2.5 g (yield 45%, weight-average molecular weight 39,000) of white powder.

(Weight average molecular weight measurement)
The weight average molecular weight of the feruloyl group-containing polypeptide was determined by gel permeation chromatography (GPC). The measurement conditions are as follows.
Equipment: LC-20A system manufactured by Shimadzu Corporation Column: TSKgel GMPWxl
Temperature: 40 ° C
Flow rate: 1.0 mL / min
Eluent: 0.2N aqueous solution of sodium nitrate Molecular weight standard: Showdex STD P series

(3) Evaluation of physical properties
Antioxidant power test The free radical scavenging action was evaluated as an antioxidant power test. As a model of free radicals, a stable free radical, 2,2-diphenyl-1-picrylhydrazyl (hereinafter referred to as "DPPH"), reacts with a test sample at a fixed rate for a certain time and decreases. The amount of radicals was measured using the decrease in absorbance at a wavelength of 562 nm as an index.
Ferulic acid, compound (c), and the above feruloyl group-containing polypeptide were used as test samples. Ferulic acid and compound (c) were used as a 1000 μM ethanol solution, and feruloyl group-containing polypeptide was used as a 20 mg / ml aqueous solution, which was used as a test sample solution. 20 µl of each test sample solution was added to a 96-well microplate, and 80 µl of 0.1 M Tris-HCl buffer (pH 7.4) and 100 µl of 500 µM DPPH ethanol solution were added and mixed. Thereafter, the reaction was carried out in a dark place for 20 minutes, and the absorbance (I) was measured with a microplate reader (iMark microplate reader manufactured by Bio-Rad Laboratories, Inc.).
Further, the absorbance (II) was measured in the same manner as above except that 20 μl of ethanol was used instead of the test sample solution.
As a blank, instead of the ethanol solution of DPPH, 100 μL of ethanol containing no DPPH was added to 100 μL of the solvent used for preparing the test sample solution (ethanol for ferulic acid and compound (c) and water for the feruloyl group-containing polypeptide). The absorbance (III) of the solution to which was added was measured.

The free radical capture removal rate was calculated from the following equation.

Free radical capture removal rate (%) = {1- (I-III) / (II-III)} × 100

フェルラ酸をトリペプチドやポリペプチドと結合させても、抗酸化力はほぼ維持されていた。 Table 1 shows the results.

Even when ferulic acid was bound to the tripeptide or polypeptide, the antioxidant activity was almost maintained.

Evaluation of water solubility The solubility of the feruloyl group-containing tripeptide (compound (c)) at 25 ° C. in water was 3.3 mg / mL. Further, it is known that the water solubility of ferulic acid at 25 ° C. is 1.27 mg / ml (the latest research result of Cyclochem Bio HP Co., Ltd., 57th solubilization of ferulic acid by α-cyclodextrin).
By coupling ferulic acid to the tripeptide, the water solubility was improved.

  According to the present invention, there are provided cinnamic acid-modified oligopeptides and cinnamic acid-modified polypeptides having useful effects such as antioxidant action, antibacterial action, tyrosinase activity inhibitory action (whitening action), and ultraviolet absorption action of cinnamic acids. By binding cinnamic acids to oligopeptides, they have new functions such as improved water solubility, and thus can be easily incorporated into cosmetics, foods, medicines, and the like. In addition, when useful polypeptides used in cosmetics, foods, medicines, and the like are used, cinnamic acid-modified polypeptides having the action of cinnamic acids can be obtained.

Claims (9)

The following general formula (1)

(In the formula (1), p is the number of substituents on the aromatic ring and represents an integer of 0 to 5. Each R independently represents a hydrogen atom or a hydrocarbon group having 1 to 3 carbon atoms. Represents.)
The cinnamic acid-modified oligopeptide in which the cinnamic acid or a derivative thereof represented by (1) and an oligopeptide consisting of 2 to 10 amino acid residues are bonded via an amide bond or an ester bond.   The cinnamic acid-modified oligopeptide according to claim 1, wherein the cinnamic acid represented by the general formula (1) or a derivative thereof is ferulic acid, isoferulic acid, caffeic acid, sinapinic acid, p-coumaric acid, and / or cinnamic acid. .   The oligopeptide contains an amino acid residue having an amino group, a hydroxyl group or a carboxyl group in a side chain, and the cinnamic acid or a derivative thereof represented by the general formula (1) is substituted with an amino group, a hydroxyl group in the side chain of the oligopeptide. 3. The cinnamic acid-modified oligopeptide according to claim 1 or 2, which forms an amide bond or an ester bond with a carboxyl group.   The cinnamic acid-modified oligopeptide according to claim 3, wherein the oligopeptide further comprises an amino acid residue having a water solubility of 5 g / 100 mL or more at 20 ° C and an amino acid residue having a main chain secondary amine.   The cinnamic acid-modified oligopeptide according to any one of claims 1 to 4, wherein the oligopeptide contains one glycine residue for every three amino acid residues.   The cinnamic acid-modified oligopeptide according to any one of claims 1 to 5 is polymerized, or the cinnamic acid-modified oligopeptide according to any one of claims 1 to 5, and an amino acid, an oligopeptide, and / or a polypeptide. A method for producing a cinnamic acid-modified polypeptide, comprising the step of polymerizing A cinnamic acid-modified oligopeptide according to any one of claims 1 to 5, and the following formulas (3) to (12).

H- (XY-Gly) n-OH (3)
H- (Gly-XY) n-OH (4)
H- (Y-Gly-X) n-OH (5)

(In the formulas (3) to (5), X and Y each independently represent Hyp or Pro, and n is an integer of 1 to 10.)

^{H - (Z 1) - OH} (6)

(In the formula (6), Z ¹ represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr , Val, Arg-Gly-Asp, and ε-polylysine, representing an amino acid residue, oligopeptide residue, or polypeptide residue.)

H- (Pro-Lys-Gly) p-OH (7)
H- (Gly-Pro-Lys) p-OH (8)
H- (Lys-Gly-Pro) p-OH (9)

(In the formulas (7) to (9), p is an integer of 1 to 10.)

^{H - (Z 2 -Hyp-Gly} ) r- OH (10)
^{H - (Gly-Z 2 -Hyp} ) r- OH (11)
^{H - (Hyp-Gly-Z} 2) r- OH (12)

(In the formulas (10) to (12), Z ² represents Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr. , And Val, and r is an integer of 1 to 10.)

7. The production method according to claim 6, comprising a step of polymerizing one or more of the amino acids, oligopeptides or polypeptides represented by any of the above.   The production according to claim 6 or 7, wherein the amount of the amino acid, oligopeptide and / or polypeptide used is 1 to 100 mol per 1 mol of the cinnamic acid-modified oligopeptide according to any one of claims 1 to 5. Method.   A cinnamic acid-modified polypeptide obtained by the production method according to claim 6.